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Sample records for chaperone full-length dimer

  1. Dimerization and oligomerization of the chaperone calreticulin

    DEFF Research Database (Denmark)

    Jørgensen, Charlotte S; Ryder, L Rebekka; Steinø, Anne; Højrup, Peter; Hansen, Jesper; Beyer, N Helena; Heegaard, Niels H H; Houen, Gunnar

    2003-01-01

    The chaperone calreticulin is a highly conserved eukaryotic protein mainly located in the endoplasmic reticulum. It contains a free cysteine SH group but does not form disulfide-bridged dimers under physiological conditions, indicating that the SH group may not be fully accessible in the native...... calreticulin was oligomerized. Thus, calreticulin shares the ability to self-oligomerize with other important chaperones such as GRP94 and HSP90, a property possibly associated with their chaperone activity....

  2. Nucleocapsid protein-mediated maturation of dimer initiation complex of full-length SL1 stemloop of HIV-1: sequence effects and mechanism of RNA refolding

    OpenAIRE

    Mujeeb, Anwer; Ulyanov, Nikolai B.; Georgantis, Stefanos; Smirnov, Ivan; Chung, Janet; Parslow, Tristram G.; James, Thomas L.

    2007-01-01

    Specific binding of HIV-1 viral protein NCp7 to a unique 35-base RNA stem-loop SL1 is critical for formation and packaging of the genomic RNA dimer found within HIV-1 virions. NCp7 binding stimulates refolding of SL1 from a metastable kissing dimer (KD) into thermodynamically stable linear dimer (LD). Using UV melting, gel electrophoresis and heteronuclear NMR, we investigated effects of various site-specific mutations within the full-length SL1 on temperature- or NCp7-induced refolding in vi...

  3. Architecture of a Full-length Retroviral Integrase Monomer and Dimer, Revealed by Small Angle X-ray Scattering and Chemical Cross-linking

    Energy Technology Data Exchange (ETDEWEB)

    Bojja, Ravi S.; Andrake, Mark D.; Weigand, Steven; Merkel, George; Yarychkivska, Olya; Henderson, Adam; Kummerling, Marissa; Skalka, Anna Marie (Fox Chase); (NWU)

    2012-02-07

    We determined the size and shape of full-length avian sarcoma virus (ASV) integrase (IN) monomers and dimers in solution using small angle x-ray scattering. The low resolution data obtained establish constraints for the relative arrangements of the three component domains in both forms. Domain organization within the small angle x-ray envelopes was determined by combining available atomic resolution data for individual domains with results from cross-linking coupled with mass spectrometry. The full-length dimer architecture so revealed is unequivocally different from that proposed from x-ray crystallographic analyses of two-domain fragments, in which interactions between the catalytic core domains play a prominent role. Core-core interactions are detected only in cross-linked IN tetramers and are required for concerted integration. The solution dimer is stabilized by C-terminal domain (CTD-CTD) interactions and by interactions of the N-terminal domain in one subunit with the core and CTD in the second subunit. These results suggest a pathway for formation of functional IN-DNA complexes that has not previously been considered and possible strategies for preventing such assembly.

  4. Multiple functions of the histone chaperone Jun dimerization protein 2.

    Science.gov (United States)

    Tsai, Ming-Ho; Wuputra, Kenly; Lin, Yin-Chu; Lin, Chang-Shen; Yokoyama, Kazunari K

    2016-09-30

    The Jun dimerization protein 2 (JDP2) is part of the family of stress-responsible transcription factors such as the activation protein-1, and binds the 12-O-tetradecanoylphorbol-13-acetateresponse element and the cAMP response element. It also plays a role as a histone chaperone and participates in diverse processes, such as cell-cycle arrest, cell differentiation, apoptosis, senescence, and metastatic spread, and functions as an oncogene and anti-oncogene, and as a cellular reprogramming factor. However, the molecular mechanisms underlying these multiple functions of JDP2 have not been clarified. This review summarizes the structure and function of JDP2, highlighting the specific role of JDP2 in cellular-stress regulation and prevention. PMID:27041241

  5. Tah1 helix-swap dimerization prevents mixed Hsp90 co-chaperone complexes

    Energy Technology Data Exchange (ETDEWEB)

    Morgan, Rhodri M. L.; Pal, Mohinder; Roe, S. Mark; Pearl, Laurence H., E-mail: laurence.pearl@sussex.ac.uk; Prodromou, Chrisostomos, E-mail: laurence.pearl@sussex.ac.uk [University of Sussex, Falmer, Brighton BN1 9RQ (United Kingdom)

    2015-05-01

    A helix swap involving the fifth helix between two adjacently bound Tah1 molecules restores the normal binding environment of the conserved MEEVD peptide of Hsp90. Dimerization also explains how other monomeric TPR-domain proteins are excluded from forming inappropriate mixed co-chaperone complexes with Hsp90 and Tah1. Specific co-chaperone adaptors facilitate the recruitment of client proteins to the Hsp90 system. Tah1 binds the C-terminal conserved MEEVD motif of Hsp90, thus linking an eclectic set of client proteins to the R2TP complex for their assembly and regulation by Hsp90. Rather than the normal complement of seven α-helices seen in other tetratricopeptide repeat (TPR) domains, Tah1 unusually consists of the first five only. Consequently, the methionine of the MEEVD peptide remains exposed to solvent when bound by Tah1. In solution Tah1 appears to be predominantly monomeric, and recent structures have failed to explain how Tah1 appears to prevent the formation of mixed TPR domain-containing complexes such as Cpr6–(Hsp90){sub 2}–Tah1. To understand this further, the crystal structure of Tah1 in complex with the MEEVD peptide of Hsp90 was determined, which shows a helix swap involving the fifth α-helix between two adjacently bound Tah1 molecules. Dimerization of Tah1 restores the normal binding environment of the bound Hsp90 methionine residue by reconstituting a TPR binding site similar to that in seven-helix-containing TPR domain proteins. Dimerization also explains how other monomeric TPR-domain proteins are excluded from forming inappropriate mixed co-chaperone complexes.

  6. Oxaliplatin Binding to Human Copper Chaperone Atox1 and Protein Dimerization.

    Science.gov (United States)

    Belviso, Benny D; Galliani, Angela; Lasorsa, Alessia; Mirabelli, Valentina; Caliandro, Rocco; Arnesano, Fabio; Natile, Giovanni

    2016-07-01

    Copper trafficking proteins have been implicated in the cellular response to platinum anticancer drugs. We investigated the reaction of the chaperone Atox1 with an activated form of oxaliplatin, the third platinum drug to reach worldwide approval. Unlike cisplatin, which contains monodentate ammines, oxaliplatin contains chelated 1,2-diaminocyclohexane (DACH), which is more resistant to displacement by nucleophiles. In solution, one or two {Pt(DACH)(2+)} moieties bind to the conserved CXXC metal-binding motif of Atox1; in the latter case the two sulfur atoms likely bridging the two platinum units. At longer reaction times, a dimeric species is formed whose composition, Atox12·Pt(2+)2, indicates complete loss of the diamine ligands. Such a dimerization process is accompanied by partial unfolding of the protein. Crystallization experiments aiming at the characterization of the monomeric species have afforded, instead, a dimeric species resembling that already obtained by Boal and Rosenzweig in a similar reaction performed with cisplatin. However, while in the latter case there was only one Pt-binding site (0.4 occupancy) made of four sulfur atoms of the CXXC motifs of the two Atox1 chains in a tetrahedral arrangement, we found, in addition, a secondary Pt-binding site involving Cys41 of the B chain (0.25 occupancy). Moreover, both platinum atoms have lost their diamines. Thus, there appears to be little relationship between what is observed in solution and what is formed in the solid state. Since full occupancy of the tetrahedral cavity is a common feature of all Atox1 dimeric structures obtained with other metal ions (Cu(+), Cd(2+), and Hg(2+)), we propose that in the case of platinum, where the occupancy is only 0.4, the remaining cavities are occupied by Cu(+) ions. Experimental evidence is reported in support of the latter hypothesis. Our proposal represents a meeting point between the initial proposal of Boal and Rosenzweig (0.4 Pt occupancy) and the

  7. Structure of full-length Drosophila cryptochrome

    Energy Technology Data Exchange (ETDEWEB)

    Zoltowski, Brian D.; Vaidya, Anand T.; Top, Deniz; Widom, Joanne; Young, Michael W.; Crane, Brian R. (Cornell); (Rockefeller)

    2011-12-15

    The cryptochrome/photolyase (CRY/PL) family of photoreceptors mediates adaptive responses to ultraviolet and blue light exposure in all kingdoms of life. Whereas PLs function predominantly in DNA repair of cyclobutane pyrimidine dimers (CPDs) and 6-4 photolesions caused by ultraviolet radiation, CRYs transduce signals important for growth, development, magnetosensitivity and circadian clocks. Despite these diverse functions, PLs/CRYs preserve a common structural fold, a dependence on flavin adenine dinucleotide (FAD) and an internal photoactivation mechanism. However, members of the CRY/PL family differ in the substrates recognized (protein or DNA), photochemical reactions catalysed and involvement of an antenna cofactor. It is largely unknown how the animal CRYs that regulate circadian rhythms act on their substrates. CRYs contain a variable carboxy-terminal tail that appends the conserved PL homology domain (PHD) and is important for function. Here, we report a 2.3-{angstrom} resolution crystal structure of Drosophila CRY with an intact C terminus. The C-terminal helix docks in the analogous groove that binds DNA substrates in PLs. Conserved Trp536 juts into the CRY catalytic centre to mimic PL recognition of DNA photolesions. The FAD anionic semiquinone found in the crystals assumes a conformation to facilitate restructuring of the tail helix. These results help reconcile the diverse functions of the CRY/PL family by demonstrating how conserved protein architecture and photochemistry can be elaborated into a range of light-driven functions.

  8. Management of full-length complete ureteral avulsion

    Directory of Open Access Journals (Sweden)

    Kaifa Tang

    2016-02-01

    Full Text Available ABSTRACT Introduction Complete ureteral avulsion is one of the most serious complications of ureteroscopy. The aim of this report was to look for a good solution to full-length complete ureteral avulsion. Case presentation A 40-year-old man underwent ureteroscopic management. Full-length complete avulsion of ureter occurred during ureteroscopy. Pyeloureterostomy plus greater omentum investment outside the avulsed ureter and ureterovesical anastomosis were performed 6 hours after ureteral avulsion. The patient was followed-up during 34 months. Double-J tube was removed at 3 months after operation. Twenty three months after the first operation, the patient developed hydronephrosis because of a new ureter upside stone, then rigid ureteroscopy and holmium laser lithotripsy were used successfully. Conclusion Pyeloureterostomy plus greater omentum investment outside the avulsed ureter and ureterovesical anastomosis may be a good choice for full-length complete ureteral avulsion.

  9. Study on full-length recoverable resin-metal bolts

    Institute of Scientific and Technical Information of China (English)

    孙玉宁; 康全玉

    2003-01-01

    The paper introduces a kind of full-length recoverable resin-metal bolts, expounds its structural principle and stress features, and gives some instances in laboratory tests and underground tests. The results show that full-length recoverable resin-metal bolts can be used for supporting the walls of class Ⅰ~Ⅲ mining gateways, that the anchoring force is 50 kN or so, and that the recoverability rate is more than 80%, thus the supporting effect is better than that of split-set bolts.

  10. Full-length fuel rod behavior under severe accident conditions

    International Nuclear Information System (INIS)

    This document presents an assessment of the severe accident phenomena observed from four Full-Length High-Temperature (FLHT) tests that were performed by the Pacific Northwest Laboratory (PNL) in the National Research Universal (NRU) reactor at Chalk River, Ontario, Canada. These tests were conducted for the US Nuclear Regulatory Commission (NRC) as part of the Severe Accident Research Program. The objectives of the test were to simulate conditions and provide information on the behavior of full-length fuel rods during hypothetical, small-break, loss-of-coolant severe accidents, in commercial light water reactors

  11. Full-length genomic analysis of korean porcine sapelovirus strains

    DEFF Research Database (Denmark)

    Son, Kyu-Yeol; Kim, Deok-Song; Kwon, Joseph;

    2014-01-01

    structural features of PSV genomes, the full-length nucleotide sequences of three Korean PSV strains were determined and analyzed using bioinformatic techniques in comparison with other known PSV strains. The Korean PSV genomes ranged from 7,542 to 7,566 nucleotides excluding the 3' poly(A) tail, and showed...

  12. Recovering full-length viral genomes from metagenomes

    Directory of Open Access Journals (Sweden)

    Saskia eSmits

    2015-10-01

    Full Text Available Infectious disease metagenomics is driven by the question: what is causing the disease? in contrast to classical metagenome studies which are guided by what is out there?. In case of a novel virus, a first step to eventually establishing etiology can be to recover a full-length viral genome from a metagenomic sample. However retrieval of a full-length genome of a divergent virus is technically challenging and can be time-consuming and costly. Here we discuss different assembly and fragment linkage strategies such as iterative assembly, motif searches, k-mer frequency profiling, coverage profile binning and other strategies used to recover genomes of potential viral pathogens in a timely and cost-effective manner.

  13. Development of a full-length human protein production pipeline

    OpenAIRE

    Saul, Justin; Petritis, Brianne; Sau, Sujay; Rauf, Femina; Gaskin, Michael; Ober-Reynolds, Benjamin; Mineyev, Irina; Magee, Mitch; Chaput, John; Qiu, Ji; LaBaer, Joshua

    2014-01-01

    There are many proteomic applications that require large collections of purified protein, but parallel production of large numbers of different proteins remains a very challenging task. To help meet the needs of the scientific community, we have developed a human protein production pipeline. Using high-throughput (HT) methods, we transferred the genes of 31 full-length proteins into three expression vectors, and expressed the collection as N-terminal HaloTag fusion proteins in Escherichia col...

  14. Renal Agenesis with Full Length Ipsilateral Refluxing Ureter

    Science.gov (United States)

    Chandra, Vipin; Banerjee, Manju

    2016-01-01

    Unilateral renal agenesis with vesicoureteral reflux in the ipsilateral full length ureter is a rare phenomenon. Herein we report a case of 10-year old boy who presented with recurrent urinary tract infections. No renal tissue was identified on left side in various imaging studies. Micturating cystourethrogram (MCUG) showed left sided refluxing and blind ending ureter. Left ureterectomy was done because of recurrent UTI in the refluxing system.

  15. QUANTIFYING ELONGATION RHYTHM DURING FULL-LENGTH PROTEIN SYNTHESIS

    OpenAIRE

    Rosenblum, Gabriel; Chen, Chunlai; Kaur, Jaskiran; Cui, Xiaonan; Zhang, Haibo; Asahara, Haruichi; Chong, Shaorong; Smilansky, Zeev; Goldman, Yale E.; Cooperman, Barry S.

    2013-01-01

    Pauses regulate the rhythm of ribosomal protein synthesis. Mutations disrupting even minor pauses can give rise to improperly formed proteins and human disease. Such minor pauses are difficult to characterize by ensemble methods, but can be readily examined by single-molecule (sm) approaches. Here we use smFRET to carry out real-time monitoring of the expression of a full-length protein, the green fluorescent protein variant Emerald GFP. We demonstrate significant correlations between measure...

  16. Resolving hot spots in the C-terminal dimerization domain that determine the stability of the molecular chaperone Hsp90.

    Directory of Open Access Journals (Sweden)

    Emanuele Ciglia

    Full Text Available Human heat shock protein of 90 kDa (hHsp90 is a homodimer that has an essential role in facilitating malignant transformation at the molecular level. Inhibiting hHsp90 function is a validated approach for treating different types of tumors. Inhibiting the dimerization of hHsp90 via its C-terminal domain (CTD should provide a novel way to therapeutically interfere with hHsp90 function. Here, we predicted hot spot residues that cluster in the CTD dimerization interface by a structural decomposition of the effective energy of binding computed by the MM-GBSA approach and confirmed these predictions using in silico alanine scanning with DrugScore(PPI. Mutation of these residues to alanine caused a significant decrease in the melting temperature according to differential scanning fluorimetry experiments, indicating a reduced stability of the mutant hHsp90 complexes. Size exclusion chromatography and multi-angle light scattering studies demonstrate that the reduced stability of the mutant hHsp90 correlates with a lower complex stoichiometry due to the disruption of the dimerization interface. These results suggest that the identified hot spot residues can be used as a pharmacophoric template for identifying and designing small-molecule inhibitors of hHsp90 dimerization.

  17. Technology development for gene discovery and full-length sequencing

    Energy Technology Data Exchange (ETDEWEB)

    Marcelo Bento Soares

    2004-07-19

    In previous years, with support from the U.S. Department of Energy, we developed methods for construction of normalized and subtracted cDNA libraries, and constructed hundreds of high-quality libraries for production of Expressed Sequence Tags (ESTs). Our clones were made widely available to the scientific community through the IMAGE Consortium, and millions of ESTs were produced from our libraries either by collaborators or by our own sequencing laboratory at the University of Iowa. During this grant period, we focused on (1) the development of a method for preferential cloning of tissue-specific and/or rare transcripts, (2) its utilization to expedite EST-based gene discovery for the NIH Mouse Brain Molecular Anatomy Project, (3) further development and optimization of a method for construction of full-length-enriched cDNA libraries, and (4) modification of a plasmid vector to maximize efficiency of full-length cDNA sequencing by the transposon-mediated approach. It is noteworthy that the technology developed for preferential cloning of rare mRNAs enabled identification of over 2,000 mouse transcripts differentially expressed in the hippocampus. In addition, the method that we optimized for construction of full-length-enriched cDNA libraries was successfully utilized for the production of approximately fifty libraries from the developing mouse nervous system, from which over 2,500 full-ORF-containing cDNAs have been identified and accurately sequenced in their entirety either by our group or by the NIH-Mammalian Gene Collection Program Sequencing Team.

  18. Full-length Ebola glycoprotein accumulates in the endoplasmic reticulum

    Directory of Open Access Journals (Sweden)

    Bhattacharyya Suchita

    2011-01-01

    Full Text Available Abstract The Filoviridae family comprises of Ebola and Marburg viruses, which are known to cause lethal hemorrhagic fever. However, there is no effective anti-viral therapy or licensed vaccines currently available for these human pathogens. The envelope glycoprotein (GP of Ebola virus, which mediates entry into target cells, is cytotoxic and this effect maps to a highly glycosylated mucin-like region in the surface subunit of GP (GP1. However, the mechanism underlying this cytotoxic property of GP is unknown. To gain insight into the basis of this GP-induced cytotoxicity, HEK293T cells were transiently transfected with full-length and mucin-deleted (Δmucin Ebola GP plasmids and GP localization was examined relative to the nucleus, endoplasmic reticulum (ER, Golgi, early and late endosomes using deconvolution fluorescent microscopy. Full-length Ebola GP was observed to accumulate in the ER. In contrast, GPΔmucin was uniformly expressed throughout the cell and did not localize in the ER. The Ebola major matrix protein VP40 was also co-expressed with GP to investigate its influence on GP localization. GP and VP40 co-expression did not alter GP localization to the ER. Also, when VP40 was co-expressed with the nucleoprotein (NP, it localized to the plasma membrane while NP accumulated in distinct cytoplasmic structures lined with vimentin. These latter structures are consistent with aggresomes and may serve as assembly sites for filoviral nucleocapsids. Collectively, these data suggest that full-length GP, but not GPΔmucin, accumulates in the ER in close proximity to the nuclear membrane, which may underscore its cytotoxic property.

  19. Conformational states of the full-length glucagon receptor

    Science.gov (United States)

    Yang, Linlin; Yang, Dehua; de Graaf, Chris; Moeller, Arne; West, Graham M.; Dharmarajan, Venkatasubramanian; Wang, Chong; Siu, Fai Y.; Song, Gaojie; Reedtz-Runge, Steffen; Pascal, Bruce D.; Wu, Beili; Potter, Clinton S.; Zhou, Hu; Griffin, Patrick R.; Carragher, Bridget; Yang, Huaiyu; Wang, Ming-Wei; Stevens, Raymond C.; Jiang, Hualiang

    2015-07-01

    Class B G protein-coupled receptors are composed of an extracellular domain (ECD) and a seven-transmembrane (7TM) domain, and their signalling is regulated by peptide hormones. Using a hybrid structural biology approach together with the ECD and 7TM domain crystal structures of the glucagon receptor (GCGR), we examine the relationship between full-length receptor conformation and peptide ligand binding. Molecular dynamics (MD) and disulfide crosslinking studies suggest that apo-GCGR can adopt both an open and closed conformation associated with extensive contacts between the ECD and 7TM domain. The electron microscopy (EM) map of the full-length GCGR shows how a monoclonal antibody stabilizes the ECD and 7TM domain in an elongated conformation. Hydrogen/deuterium exchange (HDX) studies and MD simulations indicate that an open conformation is also stabilized by peptide ligand binding. The combined studies reveal the open/closed states of GCGR and suggest that glucagon binds to GCGR by a conformational selection mechanism.

  20. Simulations of The Dalles Dam Proposed Full Length Spillwall

    Energy Technology Data Exchange (ETDEWEB)

    Rakowski, Cynthia L.; Perkins, William A.; Richmond, Marshall C.; Serkowski, John A.

    2008-02-25

    This report presents results of a computational fluid dynamics (CFD) modeling study to evaluatethe impacts of a full-length spillwall at The Dalles Dam. The full-length spillwall is being designed and evaluated as a structural means to improve tailrace egress and thus survival of juvenile fish passing through the spillway. During the course of this study, a full-length spillwall at Bays 6/7 and 8/9 were considered. The U.S. Army Corps of Engineers (USACE) has proposed extending the spillwall constructed in the stilling basin between spillway Bays 6 and 7 about 590 ft farther downstream. It is believed that the extension of the spillwall will improve egress conditions for downstream juvenile salmonids by moving them more rapidly into the thalweg of the river hence reducing their exposure to predators. A numerical model was created, validated, and applied the The Dalles Dam tailrace. The models were designed to assess impacts to flow, tailrace egress, navigation, and adult salmon passage of a proposed spill wall extension. The more extensive model validation undertaken in this study greatly improved our confidence in the numerical model to represent the flow conditions in The Dalles tailrace. This study used these validated CFD models to simulate the potential impacts of a spillwall extension for The Dalles Dam tailrace for two locations. We determined the following: (1)The construction of an extended wall (between Bays 6/7) will not adversely impact entering or exiting the navigation lock. Impact should be less if a wall were constructed between Bays 8/9. (2)The construction of a wall between Bays 6/7 will increase the water surface elevation between the wall and the Washington shore. Although the increased water surface elevation would be beneficial to adult upstream migrants in that it decreases velocities on the approach to the adult ladder, the increased flow depth would enhance dissolved gas production, impacting potential operations of the project because of

  1. Scalable production in human cells and biochemical characterization of full-length normal and mutant huntingtin.

    Directory of Open Access Journals (Sweden)

    Bin Huang

    Full Text Available Huntingtin (Htt is a 350 kD intracellular protein, ubiquitously expressed and mainly localized in the cytoplasm. Huntington's disease (HD is caused by a CAG triplet amplification in exon 1 of the corresponding gene resulting in a polyglutamine (polyQ expansion at the N-terminus of Htt. Production of full-length Htt has been difficult in the past and so far a scalable system or process has not been established for recombinant production of Htt in human cells. The ability to produce Htt in milligram quantities would be a prerequisite for many biochemical and biophysical studies aiming in a better understanding of Htt function under physiological conditions and in case of mutation and disease. For scalable production of full-length normal (17Q and mutant (46Q and 128Q Htt we have established two different systems, the first based on doxycycline-inducible Htt expression in stable cell lines, the second on "gutless" adenovirus mediated gene transfer. Purified material has then been used for biochemical characterization of full-length Htt. Posttranslational modifications (PTMs were determined and several new phosphorylation sites were identified. Nearly all PTMs in full-length Htt localized to areas outside of predicted alpha-solenoid protein regions. In all detected N-terminal peptides methionine as the first amino acid was missing and the second, alanine, was found to be acetylated. Differences in secondary structure between normal and mutant Htt, a helix-rich protein, were not observed in our study. Purified Htt tends to form dimers and higher order oligomers, thus resembling the situation observed with N-terminal fragments, although the mechanism of oligomer formation may be different.

  2. Quantifying elongation rhythm during full-length protein synthesis.

    Science.gov (United States)

    Rosenblum, Gabriel; Chen, Chunlai; Kaur, Jaskiran; Cui, Xiaonan; Zhang, Haibo; Asahara, Haruichi; Chong, Shaorong; Smilansky, Zeev; Goldman, Yale E; Cooperman, Barry S

    2013-07-31

    Pauses regulate the rhythm of ribosomal protein synthesis. Mutations disrupting even minor pauses can give rise to improperly formed proteins and human disease. Such minor pauses are difficult to characterize by ensemble methods, but can be readily examined by single-molecule (sm) approaches. Here we use smFRET to carry out real-time monitoring of the expression of a full-length protein, the green fluorescent protein variant Emerald GFP. We demonstrate significant correlations between measured elongation rates and codon and isoacceptor tRNA usage, and provide a quantitative estimate of the effect on elongation rate of replacing a codon recognizing an abundant tRNA with a synonymous codon cognate to a rarer tRNA. Our results suggest that tRNA selection plays an important general role in modulating the rates and rhythms of protein synthesis, potentially influencing simultaneous co-translational processes such as folding and chemical modification. PMID:23822614

  3. Molecular cloning and characterization of the full-length Hsp90 gene from Matricaria recutita.

    Science.gov (United States)

    Ling, S P; Su, S S; Zhang, H M; Zhang, X S; Liu, X Y; Pan, G F; Yuan, Y

    2014-01-01

    Heat shock protein 90 (Hsp90) is one of the most abundant and conserved chaperone proteins and plays important roles in plant growth and responses to environmental stimuli. However, little is known regarding the sequence and function of Hsp90s in Matricaria recutita. In the present study, we cloned the full-length cDNA sequence of the hsp90 gene from this species. Using rapid amplification of cDNA ends technologies with 2 degenerate primers that were designed based on the hsp90 gene sequence from other members of Asteraceae, we isolated and characterized an Hsp90 homolog gene from M. recutita (Mr-Hsp90). The full-length Mr-hsp90 cDNA sequence, containing 2097 base pairs, encodes a protein of 698 amino acids. Based on amino acid sequence identity, Mr-Hsp90 showed high similarity to other cloned Hsp90 proteins. The Mr-Hsp90 protein was closely clustered with the Lactuca sativa in a phylogenetic tree. These results indicate that the cloned sequence of Mr-Hsp90 is a member of the Hsp90 family, which is reported for the first time in M. recutita. Next, we conducted a salt stress experiment to determine the protein's function under salt stress conditions. Survival of chamomile seedlings subjected to heat-shock pretreatment was significantly increased compared with groups that had not undergone heat-shock pretreatment in a salt stress environment. This indicates that Mr-Hsp90 plays an important role in the salt resistance of chamomile seedlings. PMID:25526220

  4. Analysis on the anchor mechanism of the full length resin bolt

    Institute of Scientific and Technical Information of China (English)

    宋彦波; 刘洪涛

    2003-01-01

    The purpose of this paper is to reveal the stress distribution characteristic along the full length anchor bolt. Based on the mechanic model set up, the author calculated the anchor mechanism of the full length resin rock-bolt. The stress distribution characteristic is different according to different type of surrounding-rock. The conclusion is important to optimize the roadway bolt support design.

  5. Analysis of proteins encoded by full-length cDNA sequence from IRM-2 mouse

    International Nuclear Information System (INIS)

    Objective: To screen and isolate radioresistance-related genes from IRM-2 mouse. Methods: Full-length cDNA products were amplified by PCR from IRM-2 mouse cDNA library according to twenty-one pieces of expressed sequence tags. The property of proteins encoded by full-length cDNA were analyzed by comparing with GenBank database. Results: Five pieces of full-length cDNA which were not the same source as the known mice genes were found out from IRM-2 mouse cDNA library.Amino acid sequence and property of proteins encoded by these five pieces of full-length cDNA were obtained. Conclusion: Proteins encoded by full-length cDNA imply that unknown radioresistance-related genes may exist in IR M-2 mouse. (authors)

  6. [Analysis of full-length gene sequence of rabies vaccine virus aG strain].

    Science.gov (United States)

    Li, Jia; Cao, Shou-Chun; Shi, Lei-Tai; Wu, Xiao-Hong; Liu, Jing-Hua; Wang, Yun-Peng; Tang, Jian-Rong; Yu, Yong-Xin; Dong, Guan-Mu

    2013-06-01

    To sequence and analyze the full-length gene sequence of rabies vaccine virus aG strain. The full-length gene sequence of aG strain was amplified by RT-PCR by 8 fragments,each PCR product was cloned into vector pGEM-T respectively, sequenced and assemblied; The 5' leader sequence was sequenced with method of 5' RACE. The homology between aG and other rabies vaccine virus was analyzed by using DNAstar and Mega4. 0 software. aG strain was 11 925nt(GenBank accession number: JN234411) in length and belonged to the genotype I . The Bioinformatics revealed that the homology showed disparation form different rabies vaccine virus. the full-length gene sequence of rabies vaccine virus aG strain provided a support for perfecting the standard for quality control of virus strains for production of rabies vaccine for human use in China. PMID:23895005

  7. piggyBac transposons expressing full-length human dystrophin enable genetic correction of dystrophic mesoangioblasts.

    Science.gov (United States)

    Loperfido, Mariana; Jarmin, Susan; Dastidar, Sumitava; Di Matteo, Mario; Perini, Ilaria; Moore, Marc; Nair, Nisha; Samara-Kuko, Ermira; Athanasopoulos, Takis; Tedesco, Francesco Saverio; Dickson, George; Sampaolesi, Maurilio; VandenDriessche, Thierry; Chuah, Marinee K

    2016-01-29

    Duchenne muscular dystrophy (DMD) is a genetic neuromuscular disorder caused by the absence of dystrophin. We developed a novel gene therapy approach based on the use of the piggyBac (PB) transposon system to deliver the coding DNA sequence (CDS) of either full-length human dystrophin (DYS: 11.1 kb) or truncated microdystrophins (MD1: 3.6 kb; MD2: 4 kb). PB transposons encoding microdystrophins were transfected in C2C12 myoblasts, yielding 65±2% MD1 and 66±2% MD2 expression in differentiated multinucleated myotubes. A hyperactive PB (hyPB) transposase was then deployed to enable transposition of the large-size PB transposon (17 kb) encoding the full-length DYS and green fluorescence protein (GFP). Stable GFP expression attaining 78±3% could be achieved in the C2C12 myoblasts that had undergone transposition. Western blot analysis demonstrated expression of the full-length human DYS protein in myotubes. Subsequently, dystrophic mesoangioblasts from a Golden Retriever muscular dystrophy dog were transfected with the large-size PB transposon resulting in 50±5% GFP-expressing cells after stable transposition. This was consistent with correction of the differentiated dystrophic mesoangioblasts following expression of full-length human DYS. These results pave the way toward a novel non-viral gene therapy approach for DMD using PB transposons underscoring their potential to deliver large therapeutic genes. PMID:26682797

  8. 3.5A cryoEM structure of hepatitis B virus core assembled from full-length core protein.

    Directory of Open Access Journals (Sweden)

    Xuekui Yu

    Full Text Available The capsid shell of infectious hepatitis B virus (HBV is composed of 240 copies of a single protein called HBV core antigen (HBc. An atomic model of a core assembled from truncated HBc was determined previously by X-ray crystallography. In an attempt to obtain atomic structural information of HBV core in a near native, non-crystalline environment, we reconstructed a 3.5Å-resolution structure of a recombinant core assembled from full-length HBc by cryo electron microscopy (cryoEM and derived an atomic model. The structure shows that the 240 molecules of full-length HBc form a core with two layers. The outer layer, composed of the N-terminal assembly domain, is similar to the crystal structure of the truncated HBc, but has three differences. First, unlike the crystal structure, our cryoEM structure shows no disulfide bond between the Cys61 residues of the two subunits within the dimer building block, indicating such bond is not required for core formation. Second, our cryoEM structure reveals up to four more residues in the linker region (amino acids 140-149. Third, the loops in the cryoEM structures containing this linker region in subunits B and C are oriented differently (~30° and ~90° from their counterparts in the crystal structure. The inner layer, composed of the C-terminal arginine-rich domain (ARD and the ARD-bound RNAs, is partially-ordered and connected with the outer layer through linkers positioned around the two-fold axes. Weak densities emanate from the rims of positively charged channels through the icosahedral three-fold and local three-fold axes. We attribute these densities to the exposed portions of some ARDs, thus explaining ARD's accessibility by proteases and antibodies. Our data supports a role of ARD in mediating communication between inside and outside of the core during HBV maturation and envelopment.

  9. Insertion of Introns: A Strategy to Facilitate Assembly of Infectious Full Length Clones

    DEFF Research Database (Denmark)

    Johansen, Ida Elisabeth; Lund, Ole Søgaard

    2008-01-01

    Some DNA fragments are difficult to clone in Escherichia coli by standard methods. It has been speculated that unintended transcription and translation result in expression of proteins that are toxic to the bacteria. This problem is frequently observed during assembly of infectious full-length vi......Some DNA fragments are difficult to clone in Escherichia coli by standard methods. It has been speculated that unintended transcription and translation result in expression of proteins that are toxic to the bacteria. This problem is frequently observed during assembly of infectious full......-length virus clones. If the clone is constructed for transcription in vivo, interrupting the virus sequence with an intron can solve the toxicity problem. The AU-rich introns generally contain many stop codons, which interrupt translation in E. coli, while the intron sequence is precisely eliminated from the...... virus sequence in the plant nucleus. The resulting RNA, which enters the cytoplasm, is identical to the virus sequence and can initiate infection...

  10. Full-length high-temperature severe fuel damage test No. 1

    Energy Technology Data Exchange (ETDEWEB)

    Rausch, W.N.; Hesson, G.M.; Pilger, J.P.; King, L.L.; Goodman, R.L.; Panisko, F.E.

    1993-08-01

    This report describes the first full-length high-temperature test (FLHT-1) performed by Pacific Northwest Laboratory (PNL) in the National Research Universal (NRU) reactor at Chalk River, Ontario, Canada. The test is part of a series of experiments being performed for the NRC as a part of their Severe Fuel Damage Program and is one of several planned for PNL`s Coolant Boilaway and Damage Progression Program. The report summarizes the test design and test plan. it also provides a summary and discussion of the data collected during the test and of the photos taken during the post-test examination. All objectives for the test were met. The key objective was to demonstrate that severe fuel damage tests on full-length fuel bundles can be safely conducted in the NRU reactor.

  11. Structure of the full-length TRPV2 channel by cryo-EM

    Science.gov (United States)

    Huynh, Kevin W.; Cohen, Matthew R.; Jiang, Jiansen; Samanta, Amrita; Lodowski, David T.; Zhou, Z. Hong; Moiseenkova-Bell, Vera Y.

    2016-03-01

    Transient receptor potential (TRP) proteins form a superfamily Ca2+-permeable cation channels regulated by a range of chemical and physical stimuli. Structural analysis of a `minimal' TRP vanilloid subtype 1 (TRPV1) elucidated a mechanism of channel activation by agonists through changes in its outer pore region. Though homologous to TRPV1, other TRPV channels (TRPV2-6) are insensitive to TRPV1 activators including heat and vanilloids. To further understand the structural basis of TRPV channel function, we determined the structure of full-length TRPV2 at ~5 Å resolution by cryo-electron microscopy. Like TRPV1, TRPV2 contains two constrictions, one each in the pore-forming upper and lower gates. The agonist-free full-length TRPV2 has wider upper and lower gates compared with closed and agonist-activated TRPV1. We propose these newly revealed TRPV2 structural features contribute to diversity of TRPV channels.

  12. Functional correction of adult mdx mouse muscle using gutted adenoviral vectors expressing full-length dystrophin

    OpenAIRE

    DelloRusso, Christiana; Scott, Jeannine M.; Hartigan-O'Connor, Dennis; Salvatori, Giovanni; Barjot, Catherine; Robinson, Ann S.; Robert W Crawford; Brooks, Susan V; Jeffrey S. Chamberlain

    2002-01-01

    Duchenne muscular dystrophy is a lethal X-linked recessive disorder caused by mutations in the dystrophin gene. Delivery of functionally effective levels of dystrophin to immunocompetent, adult mdx (dystrophin-deficient) mice has been challenging because of the size of the gene, immune responses against viral vectors, and inefficient infection of mature muscle. Here we show that high titer stocks of three different gutted adenoviral vectors carrying full-length, muscle-specific, dystrophin ex...

  13. Structural organization of a full-length gp130/LIF-R cytokine receptor transmembrane complex

    OpenAIRE

    Skiniotis, Georgios; Lupardus, Patrick; Martick, Monika; Walz, Thomas; Garcia, K. Christopher

    2008-01-01

    gp130 is a shared receptor for at least nine cytokines, and can signal either as a homodimer, or as a heterodimer with Leukemia Inhibitory Factor Receptor (LIF-R). Here we biophysically and structurally characterize the full-length, transmembrane form of a quaternary cytokine receptor complex consisting of gp130, LIF-R, the cytokine Ciliary Neurotrophic Factor (CNTF), and its alpha receptor (CNTF-Rα). Thermodynamic analysis indicates that, unlike the cooperative assembly of the symmetric gp13...

  14. Dental Pulp Tissue Engineering in Full-length Human Root Canals

    OpenAIRE

    De Rosa, V; Zhang, Z; Grande, R.H.M.; Nör, J.E.

    2013-01-01

    The clinical translation of stem-cell-based dental pulp regeneration will require the use of injectable scaffolds. Here, we tested the hypothesis that stem cells from exfoliated deciduous teeth (SHED) can generate a functional dental pulp when injected into full-length root canals. SHED survived and began to express putative markers of odontoblastic differentiation after 7 days when mixed with Puramatrix™ (peptide hydrogel), or after 14 days when mixed with recombinant human Collagen (rhColla...

  15. Results of simulated abnormal heating events for full-length nuclear fuel rods

    International Nuclear Information System (INIS)

    Full-length nuclear fuel rods were tested in a furnace to simulate the slow heating rates postulated for commercial pressurized water reactor fuel rods exposed to an overheating event in a storage cask. Fuel rod temperatures and internal gas pressures were monitored during the test and are presented along with mensural data for cladding. Metallography of the cladding provided data on grain growth, hydriding, oxidation, cladding stresses, and the general nature of the failures

  16. Expression of full-length and splice forms of FoxP3 in rheumatoid arthritis

    DEFF Research Database (Denmark)

    Ryder, L R; Woetmann, A; Ødum, N;

    2010-01-01

    (MTX) given to the patients. CONCLUSION: RA patients express more full-length FoxP3 than healthy controls in peripheral blood CD4-positive cells, suggesting an increased number of regulatory T cells (Tregs). However, no concomitant increase in CTLA-4 expression was seen. We therefore propose that the...... Tregs are left unable to suppress the ongoing inflammation due to a deficiency in CTLA-4 needed for cell contact-dependent suppression....

  17. Full-length high-temperature severe fuel damage test No. 2

    International Nuclear Information System (INIS)

    Hazardous conditions associated with performing the Full-Length High- Temperature (FLHT). Severe Fuel Damage Test No. 2 experiment have been analyzed. Major hazards that could cause harm or damage are (1) radioactive fission products, (2) radiation fields, (3) reactivity changes, (4) hydrogen generation, (5) materials at high temperature, (6) steam explosion, and (7) steam pressure pulse. As a result of this analysis, it is concluded that with proper precautions the FLHT- 2 test can be safely conducted

  18. Full-length genomic characterizations of two canine parvoviruses prevalent in Northwest China.

    Science.gov (United States)

    Han, Shi-Chong; Guo, Hui-Chen; Sun, Shi-Qi; Shu, Long; Wei, Yan-Quan; Sun, De-Hui; Cao, Sui-Zhong; Peng, Guang-Neng; Liu, Xiang-Tao

    2015-05-01

    Canine parvovirus (CPV) can cause acute hemorrhagic diarrhea and fatal myocarditis in young dogs. Currently, most studies have focused on the evolution of the VP2 gene, whereas the full-length genome of CPV has been rarely reported. In this study, the whole genomes of CPV-LZ1 and CPV-LZ2 strains prevalent in Northwest China were determined and analyzed in comparison with those of the reference CPVs. The genome sequences of both LZ strains consisted of 5053 nucleotides. CPV-LZ1 and CPV-LZ2 strains were designated as new CPV-2a and CPV-2b, respectively. Sequence alignment analysis results revealed that these two new strains underwent specific unique variations during the process of local adaption. The left non-translated regions of these strains formed a Y-shaped hairpin structure, whereas the right non-translated regions lacked the reiteration of DNA sequence. A phylogenetic tree constructed from 33 whole coding regions of CPVs showed a strong spatial clustering, and these two strains belonged to the Chinese strain cluster lineage. This study provides a method to obtain the full-length genome of CPV. The isolation and characterization of these viruses adds incrementally to the knowledge of the full-length genome of CPV. The results from this study also provide insight into the molecular epidemiology and genetic diversity of the CPV field isolates from Northwest China and can be useful in preventing and controlling CPV infection in this region. PMID:25690604

  19. Triggers of full-length tau aggregation: a role for partially folded intermediates.

    Science.gov (United States)

    Chirita, Carmen N; Congdon, Erin E; Yin, Haishan; Kuret, Jeff

    2005-04-19

    Alzheimer's disease is characterized in part by the accumulation of full-length tau proteins into intracellular filamentous inclusions. To clarify the events that trigger lesion formation, the aggregation of recombinant full-length four-repeat tau (htau40) was examined in vitro under near-physiological conditions using transmission electron microscopy and spectroscopy methods. In the absence of exogenous inducers, tau protein behaved as an assembly-incompetent monomer with little tertiary structure. The addition of anionic inducers led to fibrillization with nucleation-dependent kinetics. On the basis of circular dichroism spectroscopy and reactivity with thioflavin S and 8-anilino-1-naphthalenesulfonic acid fluorescent probes, the inducer stabilized a monomeric species with the folding characteristics of a premolten globule state. Planar aromatic dyes capable of binding the intermediate state with high affinity were also capable of triggering fibrillization in the absence of other inducers. Dye-mediated aggregation was characterized by concentration-dependent decreases in lag time, indicating increased nucleation rates, and submicromolar critical concentrations, indicating a final equilibrium that favored the filamentous state. The data suggest that the rate-limiting barrier for filament formation from full-length tau is conformational and that the aggregation reaction is triggered by environmental conditions that stabilize assembly-competent conformations. PMID:15823045

  20. [Construction and sequencing of full-length cDNA of peste des petits ruminants virus].

    Science.gov (United States)

    Zhai, Jun-Jun; Dou, Yong-Xi; Zhang, Hai-Rui; Mao, Li; Meng, Xue-Lian; Luo, Xuo-Nong; Cai, Xue-Peng

    2010-07-01

    To develop a reverse genetics system of Peste des petits ruminants virus(PPRV), five pairs of oligonucleotide primers were designed on the basis of the full-length genomic sequence of PPRV Nigeria 75/ 1 strain. Using RT-PCR technique, five over-lapping cDNA fragments, designated as JF1, JF2, JF3, JF4 and JF5, respectively, were amplified, followed by cloning into pcDNA3.1(+)vector. An AscI restriction enzyme site and a T7 promoter sequence were introduced immediately upstream of 5'-end, while a PacI restriction enzyme site was engineered downstream of 3'-end. Using pok12 as a plasmid vector, the full-length cDNA clone pok12-PPRV of Nigeria 75/1 was assembled by connecting the five cDNA fragments via the unique restriction endonuclease site of PPRV genome. The resultant nucleotide sequence of the PPRV Nigeria 75/1 strain in the study was compared with other members of genus morbillivirus, and phylogenetic analysis was used to examine the evolutionary relationships. The results showed that PPRV Nigeria 75/ 1 was antigenically closely related to Rinderpest virus and Measles virus. Successful construction of full-length cDNA clone of PPRV Nigeria 75/1 strain lays the basis rescuing PPRV effectively and enables further research of PPRV at molecular level. PMID:20836386

  1. Generation and Analysis of Full-length cDNA Sequences from Elephant Shark (Callorhinchus milii)

    KAUST Repository

    Kodzius, Rimantas

    2009-03-17

    Cartilaginous fishes are the oldest living group of jawed vertebrates and therefore is an important group for understanding the evolution of vertebrate genomes including the human genome. Our laboratory has proposed elephant shark (C. milii) as a model cartilaginous fish genome because of its relatively small genome size (910 Mb). The whole genome of C. milii is being sequenced (first cartilaginous fish genome to be sequenced completely). To characterize the transcriptome of C. milii and to assist in annotating exon-intron boundaries, transcriptional start sites and alternatively spliced transcripts, we are generating full-length cDNA sequences from C. milii.

  2. Structural Organization of a Full-Length Gp130/LIF-R Cytokine Receptor Transmembrane Complex

    Energy Technology Data Exchange (ETDEWEB)

    Skiniotis, G.; Lupardus, P.J.; Martick, M.; Walz, T.; Garcia, K.C.

    2009-05-26

    gp130 is a shared receptor for at least nine cytokines, and can signal either as a homodimer, or as a heterodimer with Leukemia Inhibitory Factor Receptor (LIF-R). Here we biophysically and structurally characterize the full-length, transmembrane form of a quaternary cytokine receptor complex consisting of gp130, LIF-R, the cytokine Ciliary Neurotrophic Factor (CNTF), and its alpha receptor (CNTF-R{alpha}). Thermodynamic analysis indicates that, unlike the cooperative assembly of the symmetric gp130/Interleukin-6/IL-6R{alpha} hexameric complex, CNTF/CNTF-R{alpha} heterodimerizes gp130 and LIF-R via non-cooperative energetics to form an asymmetric 1:1:1:1 complex. Single particle electron microscopic (EM) analysis of the full-length gp130/LIF-R/CNTF-R{alpha}/CNTF quaternary complex elucidates an asymmetric structural arrangement, in which the receptor extracellular and transmembrane segments join as a continuous, rigid unit, poised to sensitively transduce ligand engagement to the membrane-proximal intracellular signaling regions. These studies also enumerate the organizing principles for assembly of the 'tall' class of gp130-family cytokine receptor complexes including LIF, IL-27, IL-12, and others.

  3. Evidence for a Complex Mosaic Genome Pattern in a Full-length Hepatitis C Virus Sequence

    Directory of Open Access Journals (Sweden)

    R.S. Ross

    2008-01-01

    Full Text Available The genome of the hepatitis C virus (HCV exhibits a high genetic variability. This remarkable heterogeneity is mainly attributed to the gradual accumulation of mutational changes, whereas the contribution of recombination events to the evolution of HCV remains controversial so far. While performing phylogenetic analyses including a large number of sequences deposited in the GenBank, we encountered a full-length HCV sequence (AY651061 that showed evidence for inter-subtype recombination and was, therefore, subjected to a detailed analysis of its molecular structure. The obtained results indicated that AY651061 does not represent a “simple” HCV 1c isolate, but a complex 1a/1c mosaic genome, showing five putative breakpoints in the core to NS3 regions. To our knowledge, this is the first report on a mosaic HCV full- length sequence with multiple breakpoints. The molecular structure of AY651061 is reminiscent of complex homologous recombinant variants occurring among other members of the flaviviridae family, e.g. GB virus C, dengue virus, and Japanese encephalitis virus. Our finding of a mosaic HCV sequence may have important implications for many fields of current HCV research which merit careful consideration.

  4. Full-length Dysferlin Transfer by the Hyperactive Sleeping Beauty Transposase Restores Dysferlin-deficient Muscle.

    Science.gov (United States)

    Escobar, Helena; Schöwel, Verena; Spuler, Simone; Marg, Andreas; Izsvák, Zsuzsanna

    2016-01-01

    Dysferlin-deficient muscular dystrophy is a progressive disease characterized by muscle weakness and wasting for which there is no treatment. It is caused by mutations in DYSF, a large, multiexonic gene that forms a coding sequence of 6.2 kb. Sleeping Beauty (SB) transposon is a nonviral gene transfer vector, already used in clinical trials. The hyperactive SB system consists of a transposon DNA sequence and a transposase protein, SB100X, that can integrate DNA over 10 kb into the target genome. We constructed an SB transposon-based vector to deliver full-length human DYSF cDNA into dysferlin-deficient H2K A/J myoblasts. We demonstrate proper dysferlin expression as well as highly efficient engraftment (>1,100 donor-derived fibers) of the engineered myoblasts in the skeletal muscle of dysferlin- and immunodeficient B6.Cg-Dysf(prmd) Prkdc(scid)/J (Scid/BLA/J) mice. Nonviral gene delivery of full-length human dysferlin into muscle cells, along with a successful and efficient transplantation into skeletal muscle are important advances towards successful gene therapy of dysferlin-deficient muscular dystrophy. PMID:26784637

  5. Full-length high-temperature severe fuel damage test No. 5

    Energy Technology Data Exchange (ETDEWEB)

    Lanning, D.D.; Lombardo, N.J.; Hensley, W.K.; Fitzsimmons, D.E.; Panisko, F.E. [Pacific Northwest Lab., Richland, WA (United States); Hartwell, J.K. [EG and G Idaho, Inc., Idaho Falls, ID (United States)

    1993-09-01

    This report describes and presents data from a severe fuel damage test that was conducted in the National Research Universal (NRU) reactor at Chalk River Nuclear Laboratories (CRNL), Ontario, Canada. The test, designated FLHT-5, was the fourth in a series of full-length high-temperature (FLHT) tests on light-water reactor fuel. The tests were designed and performed by staff from the US Department of Energy`s Pacific Northwest Laboratory (PNL), operated by Battelle Memorial Institute. The test operation and test results are described in this report. The fuel bundle in the FLHT-5 experiment included 10 unirradiated full-length pressurized-water reactor (PWR) rods, 1 irradiated PWR rod and 1 dummy gamma thermometer. The fuel rods were subjected to a very low coolant flow while operating at low fission power. This caused coolant boilaway, rod dryout and overheating to temperatures above 2600 K, severe fuel rod damage, hydrogen generation, and fission product release. The test assembly and its effluent path were extensively instrumented to record temperatures, pressures, flow rates, hydrogen evolution, and fission product release during the boilaway/heatup transient. Post-test gamma scanning of the upper plenum indicated significant iodine and cesium release and deposition. Both stack gas activity and on-line gamma spectrometer data indicated significant ({approximately}50%) release of noble fission gases. Post-test visual examination of one side of the fuel bundle revealed no massive relocation and flow blockage; however, rundown of molten cladding was evident.

  6. Highly efficient full-length hepatitis C virus genotype 1 (strain TN) infectious culture system

    DEFF Research Database (Denmark)

    Li, Yi-Ping; Ramirez, Santseharay; Jensen, Sanne B;

    2012-01-01

    Chronic infection with hepatitis C virus (HCV) is an important cause of end stage liver disease worldwide. In the United States, most HCV-related disease is associated with genotype 1 infection, which remains difficult to treat. Drug and vaccine development was hampered by inability to culture...... patient isolates representing HCV genotypes 1-7 and subtypes; only a recombinant 2a genome (strain JFH1) spontaneously replicated in vitro. Recently, we identified three mutations F1464L/A1672S/D2979G (LSG) in the nonstructural (NS) proteins, essential for development of full-length HCV 2a (J6) and 2b (J8......) culture systems in Huh7.5 cells. Here, we developed a highly efficient genotype 1a (strain TN) full-length culture system. We initially found that the LSG substitutions conferred viability to an intergenotypic recombinant composed of TN 5' untranslated region (5'UTR)-NS5A and JFH1 NS5B-3'UTR; recovered...

  7. De novo assembly ofZea nicaraguensis root transcriptome identiifed 5261full-length transcripts

    Institute of Scientific and Technical Information of China (English)

    JIANG Wei; LIU Hai-lan; WU Yuan-qi; ZHANG Su-zhi; LIU Jian; LU Yan-li; TANG Qi-lin; RONG Ting-zhao

    2016-01-01

    Zea nicaraguensis, a wild relative of cultivated maize (Zea mayssubsp. mays), is considered to be a valuable germplasm to improve the waterlogging tolerance of cultivated maize. Use of reverse genetic-based gene cloning and function veriif-cation to discover waterlogging tolerance genes inZ. nicaraguensis is currently impractical, because little gene sequence information forZ. nicaraguensis is available in public databases. In this study,Z. nicaraguensis seedlings were subjected to simulated waterlogging stress and total RNAs were isolated from roots stressed and non-stressed controls. In total, 80 mol L–1 Ilumina 100-bp paired-end reads were generated.De novo assembly of the reads generated 81002 ifnal non-re-dundant contigs, from which 5261 full-length transcripts were identiifed. Among these full-length transcripts, 3169 had at least one Gene Ontology (GO) annotation, 2354 received cluster of orthologous groups (COG) terms, and 1992 were assigned a Kyoto encyclopedia of genes and genomes (KEGG) Orthology number. These sequence data represent a valuable resource for identiifcation ofZ. nicaraguensisgenes involved in waterlogging response.

  8. Full-length genomic characterization and molecular evolution of canine parvovirus in China.

    Science.gov (United States)

    Zhou, Ling; Tang, Qinghai; Shi, Lijun; Kong, Miaomiao; Liang, Lin; Mao, Qianqian; Bu, Bin; Yao, Lunguang; Zhao, Kai; Cui, Shangjin; Leal, Élcio

    2016-06-01

    Canine parvovirus type 2 (CPV-2) can cause acute haemorrhagic enteritis in dogs and myocarditis in puppies. This disease has become one of the most serious infectious diseases of dogs. During 2014 in China, there were many cases of acute infectious diarrhoea in dogs. Some faecal samples were negative for the CPV-2 antigen based on a colloidal gold test strip but were positive based on PCR, and a viral strain was isolated from one such sample. The cytopathic effect on susceptible cells and the results of the immunoperoxidase monolayer assay, PCR, and sequencing indicated that the pathogen was CPV-2. The strain was named CPV-NY-14, and the full-length genome was sequenced and analysed. A maximum likelihood tree was constructed using the full-length genome and all available CPV-2 genomes. New strains have replaced the original strain in Taiwan and Italy, although the CPV-2a strain is still predominant there. However, CPV-2a still causes many cases of acute infectious diarrhoea in dogs in China. PMID:27038801

  9. Full-length high-temperature severe fuel damage test No. 5

    International Nuclear Information System (INIS)

    This report describes and presents data from a severe fuel damage test that was conducted in the National Research Universal (NRU) reactor at Chalk River Nuclear Laboratories (CRNL), Ontario, Canada. The test, designated FLHT-5, was the fourth in a series of full-length high-temperature (FLHT) tests on light-water reactor fuel. The tests were designed and performed by staff from the US Department of Energy's Pacific Northwest Laboratory (PNL), operated by Battelle Memorial Institute. The test operation and test results are described in this report. The fuel bundle in the FLHT-5 experiment included 10 unirradiated full-length pressurized-water reactor (PWR) rods, 1 irradiated PWR rod and 1 dummy gamma thermometer. The fuel rods were subjected to a very low coolant flow while operating at low fission power. This caused coolant boilaway, rod dryout and overheating to temperatures above 2600 K, severe fuel rod damage, hydrogen generation, and fission product release. The test assembly and its effluent path were extensively instrumented to record temperatures, pressures, flow rates, hydrogen evolution, and fission product release during the boilaway/heatup transient. Post-test gamma scanning of the upper plenum indicated significant iodine and cesium release and deposition. Both stack gas activity and on-line gamma spectrometer data indicated significant (∼50%) release of noble fission gases. Post-test visual examination of one side of the fuel bundle revealed no massive relocation and flow blockage; however, rundown of molten cladding was evident

  10. Shear-induced unfolding and enzymatic cleavage of full-length VWF multimers

    CERN Document Server

    Lippok, Svenja; Obser, Tobias; Kleemeier, Lars; Schneppenheim, Reinhard; Budde, Ulrich; Netz, Roland R; Rädler, Joachim O

    2015-01-01

    Proteolysis of the multimeric blood coagulation protein von Willebrand Factor (VWF) by ADAMTS13 is crucial for prevention of microvascular thrombosis. ADAMTS13 cleaves VWF within the mechanosensitive A2 domain, which is believed to open under shear flow. Here, we combine Fluorescence Correlation Spectroscopy (FCS) and a microfluidic shear cell to monitor real-time kinetics of full-length VWF proteolysis as a function of shear stress. For comparison, we also measure the Michaelis-Menten kinetics of ADAMTS13 cleavage of wild-type VWF in the absence of shear but partially denaturing conditions. Under shear, ADAMTS13 activity on full-length VWF arises without denaturing agent as evidenced by FCS and gel-based multimer analysis. In agreement with Brownian hydrodynamics simulations, we find a sigmoidal increase of the enzymatic rate as a function of shear at a threshold shear rate 5522/s. The same flow-rate dependence of ADAMTS13 activity we also observe in blood plasma, which is relevant to predict hemostatic dysf...

  11. Impaired heat shock response in cells expressing full-length polyglutamine-expanded huntingtin.

    Directory of Open Access Journals (Sweden)

    Sidhartha M Chafekar

    Full Text Available The molecular mechanisms by which polyglutamine (polyQ-expanded huntingtin (Htt causes neurodegeneration in Huntington's disease (HD remain unclear. The malfunction of cellular proteostasis has been suggested as central in HD pathogenesis and also as a target of therapeutic interventions for the treatment of HD. We present results that offer a previously unexplored perspective regarding impaired proteostasis in HD. We find that, under non-stress conditions, the proteostatic capacity of cells expressing full length polyQ-expanded Htt is adequate. Yet, under stress conditions, the presence of polyQ-expanded Htt impairs the heat shock response, a key component of cellular proteostasis. This impaired heat shock response results in a reduced capacity to withstand the damage caused by cellular stress. We demonstrate that in cells expressing polyQ-expanded Htt the levels of heat shock transcription factor 1 (HSF1 are reduced, and, as a consequence, these cells have an impaired a heat shock response. Also, we found reduced HSF1 and HSP70 levels in the striata of HD knock-in mice when compared to wild-type mice. Our results suggests that full length, non-aggregated polyQ-expanded Htt blocks the effective induction of the heat shock response under stress conditions and may thus trigger the accumulation of cellular damage during the course of HD pathogenesis.

  12. The first detection and full-length genome sequence of porcine deltacoronavirus isolated in Lao PDR.

    Science.gov (United States)

    Lorsirigool, Athip; Saeng-Chuto, Kepalee; Temeeyasen, Gun; Madapong, Adthakorn; Tripipat, Thitima; Wegner, Matthew; Tuntituvanont, Angkana; Intrakamhaeng, Manakant; Nilubol, Dachrit

    2016-10-01

    Porcine deltacoronavirus (PDCoV) has been reported in many countries, including Hong Kong, the United States, South Korea, China and Thailand. In January 2016, clinical diarrhea similar to that of porcine epidemic diarrhea virus (PEDV) with a lower mortality rate was reported on a swine farm in Lao PDR. Intestine samples were collected from 3-day-old pigs with clinical diarrhea and assayed for the presence of swine enteric coronaviruses. The PCR results were positive for PDCoV but negative for PEDV and TGEV. A phylogenetic tree demonstrated that PDCoV from Lao PDR was grouped separately from PDCoV isolates from China and the USA, but was more closely related to the Chinese isolates than to the US isolates. The full-length genome sequence of the novel PDCoV isolate P1_16_BTL_0116 was determined. PMID:27424024

  13. Cocrystallization studies of full-length recombinant butyrylcholinesterase (BChE) with cocaine

    Energy Technology Data Exchange (ETDEWEB)

    Asojo, Oluwatoyin Ajibola; Asojo, Oluyomi Adebola; Ngamelue, Michelle N.; Homma, Kohei; Lockridge, Oksana (Nebraska-Med)

    2011-09-16

    Human butyrylcholinesterase (BChE; EC 3.1.1.8) is a 340 kDa tetrameric glycoprotein that is present in human serum at about 5 mg l{sup -1} and has well documented therapeutic effects on cocaine toxicity. BChE holds promise as a therapeutic that reduces and finally eliminates the rewarding effects of cocaine, thus weaning an addict from the drug. There have been extensive computational studies of cocaine hydrolysis by BChE. Since there are no reported structures of BChE with cocaine or any of the hydrolysis products, full-length monomeric recombinant wild-type BChE was cocrystallized with cocaine. The refined 3 {angstrom} resolution structure appears to retain the hydrolysis product benzoic acid in sufficient proximity to form a hydrogen bond to the active-site Ser198.

  14. Full-length RNA structure prediction of the HIV-1 genome reveals a conserved core domain

    DEFF Research Database (Denmark)

    Sukosd, Zsuzsanna; Andersen, Ebbe S.; Seemann, Stefan E.;

    2015-01-01

    A distance constrained secondary structural model of the approximate to 10 kb RNA genome of the HIV-1 has been predicted but higher-order structures, involving long distance interactions, are currently unknown. We present the first global RNA secondary structure model for the HIV-1 genome, which...... integrates both comparative structure analysis and information from experimental data in a full-length prediction without distance constraints. Besides recovering known structural elements, we predict several novel structural elements that are conserved in HIV-1 evolution. Our results also indicate that the...... structure of the HIV-1 genome is highly variable in most regions, with a limited number of stable and conserved RNA secondary structures. Most interesting, a set of long distance interactions form a core organizing structure (COS) that organize the genome into three major structural domains. Despite...

  15. Quench start localization in full-length SSC R ampersand D dipoles

    International Nuclear Information System (INIS)

    Full-length SSC R ampersand D dipole magnets instrumented with four voltage taps on each turn of the inner quarter coils have been tested. These voltage taps enable accurate location of the point at which the quenches start and detailed studies of quench development in the coil. Attention here is focused on localizing the quench source. After recalling the basic mechanism of a quench (why it occurs and how it propagates), the method of quench origin analysis is described: the quench propagation velocity on the turn where the quench occurs is calculated, and the quench location is then verified by reiterating the analysis on the adjacent turns. Last, the velocity value, which appears to be higher than previously measured, is discussed

  16. Loss of full length CtBP1 expression enhances the invasive potential of human melanoma

    International Nuclear Information System (INIS)

    The C-terminal binding protein 1 (CtBP1) is a known co-repressor of gene transcription. We recently revealed that CtBP1 expression is lost in melanoma cells and melanoma inhibitory activity (MIA) expression is subsequently increased. The present study was performed to evaluate a more general role of CtBP1 in human melanoma and identify further CtBP1-regulated target genes. Sequence analysis and expression profile of CtBP1 in melanoma cell lines were done by PCR. Boyden Chamber assays and co-immunoprecipitation were performed to investigate the functional role of CtBP1. Gene expression analysis and micro array data were used to define target genes. Interestingly, we detected an alternative splice product of CtBP1 with unknown function whose expression is induced at reduction of full length CtBP1. Overexpression of full length CtBP1 in melanoma cells had no effect on cell proliferation but did influence cell migration and invasiveness. To understand the effect of CtBP1 we identified putative LEF/TCF target genes found to be strongly expressed in melanoma using DNA microarray analysis. We focused on fourteen genes not previously associated with melanoma. Detailed analysis revealed that most of these were known to be involved in tumor metastasis. Eleven genes had expression profiles associated with melanoma cell invasiveness. In summary, this study revealed that reduction of CtBP1 expression is correlated with migratory, invasive potential of melanoma cells

  17. Release of full-length PrPC from cultured neurons following neurotoxic challenges

    Directory of Open Access Journals (Sweden)

    Kevin K W Wang

    2012-10-01

    Full Text Available The susceptibility of the normal cellular prion protein isoform, PrPC, to proteolytic digestion has been well documented. In addition, a link between PrPC and the cytosolic protease, calpain, has been reported although the specifics of the interaction remain unclear. We performed in vitro and in cell-based studies to examine this relationship. We observed that human recombinant PrP (HrPrP was readily cleaved by calpain-1 and -2, and we have identified and defined the targeted cleavage sites. In contrast, HrPrP was resistant to caspase-3 digestion. Unexpectedly, when brain lysates from PrPC-expressing mice were treated with calpain, no appreciable loss of the intact PrPC, nor the appearance of PrPC breakdown products (BDPs were observed, even though alpha II-spectrin was converted to its signature calpain-induced BDPs. In addition, when rat cerebrocortical neuronal cultures (RtCNC were subjected to the two neurotoxins at subacute levels, maitotoxin (MTX and N-methyl-D-aspartate (NMDA, PrPC BDPs were also not detectable. However, a novel finding from these cell-based studies is that apparently full-length, mature PrPC is released into culture media from RtCNC challenged with subacute doses of MTX and NMDA. Calpain inhibitor SNJ-1945 and caspase inhibitor IDN-6556 did not attenuate the release of PrPC. Similarly, the lysosomal protease inhibitor, NH4Cl, and the proteasome inhibitor, lactacystin, did not significantly alter the integrity of PrPC or its release from the RtCNC. In conclusion, rat neuronal PrPC is not a significant target for proteolytic modifications during MTX and NMDA neurotoxic challenges. However, the robust neurotoxin-mediated release of full-length PrPC into the cell culture media suggests an unidentified neuroprotective mechanism for PrPC.

  18. First structure of full-length mammalian phenylalanine hydroxylase reveals the architecture of an autoinhibited tetramer.

    Science.gov (United States)

    Arturo, Emilia C; Gupta, Kushol; Héroux, Annie; Stith, Linda; Cross, Penelope J; Parker, Emily J; Loll, Patrick J; Jaffe, Eileen K

    2016-03-01

    Improved understanding of the relationship among structure, dynamics, and function for the enzyme phenylalanine hydroxylase (PAH) can lead to needed new therapies for phenylketonuria, the most common inborn error of amino acid metabolism. PAH is a multidomain homo-multimeric protein whose conformation and multimerization properties respond to allosteric activation by the substrate phenylalanine (Phe); the allosteric regulation is necessary to maintain Phe below neurotoxic levels. A recently introduced model for allosteric regulation of PAH involves major domain motions and architecturally distinct PAH tetramers [Jaffe EK, Stith L, Lawrence SH, Andrake M, Dunbrack RL, Jr (2013) Arch Biochem Biophys 530(2):73-82]. Herein, we present, to our knowledge, the first X-ray crystal structure for a full-length mammalian (rat) PAH in an autoinhibited conformation. Chromatographic isolation of a monodisperse tetrameric PAH, in the absence of Phe, facilitated determination of the 2.9 Å crystal structure. The structure of full-length PAH supersedes a composite homology model that had been used extensively to rationalize phenylketonuria genotype-phenotype relationships. Small-angle X-ray scattering (SAXS) confirms that this tetramer, which dominates in the absence of Phe, is different from a Phe-stabilized allosterically activated PAH tetramer. The lack of structural detail for activated PAH remains a barrier to complete understanding of phenylketonuria genotype-phenotype relationships. Nevertheless, the use of SAXS and X-ray crystallography together to inspect PAH structure provides, to our knowledge, the first complete view of the enzyme in a tetrameric form that was not possible with prior partial crystal structures, and facilitates interpretation of a wealth of biochemical and structural data that was hitherto impossible to evaluate. PMID:26884182

  19. Isolation and characterization of full-length putative alcohol dehydrogenase genes from polygonum minus

    Science.gov (United States)

    Hamid, Nur Athirah Abd; Ismail, Ismanizan

    2013-11-01

    Polygonum minus, locally named as Kesum is an aromatic herb which is high in secondary metabolite content. Alcohol dehydrogenase is an important enzyme that catalyzes the reversible oxidation of alcohol and aldehyde with the presence of NAD(P)(H) as co-factor. The main focus of this research is to identify the gene of ADH. The total RNA was extracted from leaves of P. minus which was treated with 150 μM Jasmonic acid. Full-length cDNA sequence of ADH was isolated via rapid amplification cDNA end (RACE). Subsequently, in silico analysis was conducted on the full-length cDNA sequence and PCR was done on genomic DNA to determine the exon and intron organization. Two sequences of ADH, designated as PmADH1 and PmADH2 were successfully isolated. Both sequences have ORF of 801 bp which encode 266 aa residues. Nucleotide sequence comparison of PmADH1 and PmADH2 indicated that both sequences are highly similar at the ORF region but divergent in the 3' untranslated regions (UTR). The amino acid is differ at the 107 residue; PmADH1 contains Gly (G) residue while PmADH2 contains Cys (C) residue. The intron-exon organization pattern of both sequences are also same, with 3 introns and 4 exons. Based on in silico analysis, both sequences contain "classical" short chain alcohol dehydrogenases/reductases ((c) SDRs) conserved domain. The results suggest that both sequences are the members of short chain alcohol dehydrogenase family.

  20. Effect of systemically increasing human full-length Klotho on glucose metabolism in db/db mice.

    Science.gov (United States)

    Forsberg, E A; Olauson, H; Larsson, T; Catrina, S B

    2016-03-01

    The metabolic effects of antiaging Klotho were previously investigated in vivo by genetic manipulation. We have here studied the metabolic effect of physiologic levels of circulating full length Klotho in db/db mice. Increasing the full-length human Klotho levels has a positive effect on blood glucose through increasing insulin secretion. PMID:26806457

  1. Construction, characterization and expression of full length cDNA clone of sheep YAP1 gene.

    Science.gov (United States)

    Sun, Wei; Li, Da; Su, Rui; Musa, Hassan H; Chen, Ling; Zhou, Hong

    2014-02-01

    RT-PCR, 5'RACE, 3'RACE were used to clone sheep full length cDNA sequence of YAP1 (Yes-associated protein 1), eukaryotic expression plasmid and a mutant that cannot be phosphorylated at Ser42 was successfully constructed. The amino acid sequence analysis revealed that sheep YAP1 gene encoded water-soluble protein and its relative molecular weight and isoelectric point was 44,079.0 Da and 4.91, respectively. Sub-cellular localization of YAP1 was in the nucleus, it is hydrophilic non-transmembrane and non-secreted protein. YAP1 protein contained 33 phosphorylation sites, seven glycosylation sites and two WW domains. The secondary structure of YAP1 was mainly composed of random coil, while the tertiary structure of domain area showed a forniciform helix structure. YAP1 gene was expressed in different tissues, the highest expression was in kidney and the lowest was in hypothalamus. The CDS of sheep YAP1was amplified by RT-PCR from healthy sheep longissimus dorsi muscle, cloned into pMD19-T simple vector by T/A ligation. YAP1 coding region was further sub-cloned into pEGFP-C1 vector by T4 Ligase to construct a eukaryotic expression plasmid and then make the eukaryotic expression vector as the template to construct the phosphorylation site mutant. PCR, restriction enzyme and sequencing were used to confirm the recombinant plasmid. The sheep full-length YAP1 cDNA sequence is 1712 in length encoding 403 amino acids. It was confirmed that the sheep YAP1 CDS was correctly inserted into eukaryotic expression vector and serine had been mutated to alanine by PCR, restriction digestion and sequencing. The result showed that the recombinant plasmid pEGFP-C1-YAP1 and pEGFP-C1-YAP1 S42A was constructed correctly, this will help for further studies on the YAP1 protein expression and its biological activities. PMID:24381103

  2. Conformational change in full-length mouse prion: A site-directed spin-labeling study

    International Nuclear Information System (INIS)

    The structure of the mouse prion (moPrP) was studied using site-directed spin-labeling electron spin resonance (SDSL-ESR). Since a previous NMR study by Hornemanna et al., [Hornemanna, Korthb, Oeschb, Rieka, Widera, Wuethricha, Glockshubera, Recombinant full-length murine prion protein, mPrP (23-231): purification and spectroscopic characterization, FEBS Lett. 413 (1997) 277-281] has indicated that N96, D143, and T189 in moPrP are localized in a Cu2+ binding region, Helix1 and Helix2, respectively, three recombinant moPrP mutations (N96C, D143C, and T189C) were expressed in an Escherichia coli system, and then refolded by dialysis under low pH and purified by reverse-phase HPLC. By using the preparation, we succeeded in preserving a target cystein residue without alteration of the α-helix structure of moPrP and were able to apply SDSL-ESR with a methane thiosulfonate spin label to the full-length prion protein. The rotational correlation times (τ) of 1.1, 3.3, and 4.8 ns were evaluated from the X-band ESR spectra at pH 7.4 and 20 deg C for N96R1, D143R1, and T189R1, respectively. τ reflects the fact that the Cu2+ binding region is more flexible than Helix1 or Helix2. ESR spectra recorded at various temperatures revealed two phases together with a transition point at around 20 deg C in D143R1 and T189R1, but not in N96R1. With the variation of pH from 4.0 to 7.8, ESR spectra of T189R1 at 20 deg C showed a gradual increase of τ from 2.9 to 4.8 ns. On the other hand, the pH-dependent conformational changes in N96R1 and D143R1 were negligible. These results indicated that T189 located in Helix2 possessed a structure sensitive to physiological pH changes; simultaneously, N96 in the Cu2+ binding region and D143 in Helix1 were conserved

  3. Structure and Histone Binding Properties of the Vps75-Rtt109 Chaperone-Lysine Acetyltransferase Complex

    Energy Technology Data Exchange (ETDEWEB)

    Su, Dan; Hu, Qi; Zhou, Hui; Thompson, James R.; Xu, Rui-Ming; Zhang, Zhiguo; Mer, Georges (Mayo); (Chinese Aca. Sci.)

    2011-11-02

    The histone chaperone Vps75 presents the remarkable property of stimulating the Rtt109-dependent acetylation of several histone H3 lysine residues within (H3-H4){sub 2} tetramers. To investigate this activation mechanism, we determined x-ray structures of full-length Vps75 in complex with full-length Rtt109 in two crystal forms. Both structures show similar asymmetric assemblies of a Vps75 dimer bound to an Rtt109 monomer. In the Vps75-Rtt109 complexes, the catalytic site of Rtt109 is confined to an enclosed space that can accommodate the N-terminal tail of histone H3 in (H3-H4){sub 2}. Investigation of Vps75-Rtt109-(H3-H4)2 and Vps75-(H3-H4)2 complexes by NMR spectroscopy-probed hydrogen/deuterium exchange suggests that Vps75 guides histone H3 in the catalytic enclosure. These findings clarify the basis for the enhanced acetylation of histone H3 tail residues by Vps75-Rtt109.

  4. Chaperoning the Chaperone: A Role for the Co-chaperone Cpr7 in Modulating Hsp90 Function in Saccharomyces cerevisiae

    OpenAIRE

    Zuehlke, Abbey D.; Johnson, Jill L.

    2012-01-01

    Heat-shock protein 90 (Hsp90) of Saccharomyces cerevisiae is an abundant essential eukaryotic molecular chaperone involved in the activation and stabilization of client proteins, including several transcription factors and oncogenic kinases. Hsp90 undergoes a complex series of conformational changes and interacts with partner co-chaperones such as Sba1, Cpr6, Cpr7, and Cns1 as it binds and hydrolyzes ATP. In the absence of nucleotide, Hsp90 is dimerized only at the carboxy-terminus. In the pr...

  5. Full-length RNA structure prediction of the HIV-1 genome reveals a conserved core domain.

    Science.gov (United States)

    Sükösd, Zsuzsanna; Andersen, Ebbe S; Seemann, Stefan E; Jensen, Mads Krogh; Hansen, Mathias; Gorodkin, Jan; Kjems, Jørgen

    2015-12-01

    A distance constrained secondary structural model of the ≈10 kb RNA genome of the HIV-1 has been predicted but higher-order structures, involving long distance interactions, are currently unknown. We present the first global RNA secondary structure model for the HIV-1 genome, which integrates both comparative structure analysis and information from experimental data in a full-length prediction without distance constraints. Besides recovering known structural elements, we predict several novel structural elements that are conserved in HIV-1 evolution. Our results also indicate that the structure of the HIV-1 genome is highly variable in most regions, with a limited number of stable and conserved RNA secondary structures. Most interesting, a set of long distance interactions form a core organizing structure (COS) that organize the genome into three major structural domains. Despite overlapping protein-coding regions the COS is supported by a particular high frequency of compensatory base changes, suggesting functional importance for this element. This new structural element potentially organizes the whole genome into three major domains protruding from a conserved core structure with potential roles in replication and evolution for the virus. PMID:26476446

  6. Improving the diffraction of full-length human selenomethionyl metavinculin crystals by streak-seeding

    Energy Technology Data Exchange (ETDEWEB)

    Rangarajan, Erumbi S.; Izard, Tina (Scripps)

    2012-06-28

    Metavinculin is an alternatively spliced isoform of vinculin that has a 68-residue insert in its tail domain (1134 total residues) and is exclusively expressed in cardiac and smooth muscle tissue, where it plays important roles in myocyte adhesion complexes. Mutations in the metavinculin-specific insert are associated with dilated cardiomyopathy (DCM) in man. Crystals of a DCM-associated mutant of full-length selenomethionine-labeled metavinculin grown by hanging-drop vapor diffusion diffracted poorly and were highly sensitive to radiation, preventing the collection of a complete X-ray diffraction data set at the highest possible resolution. Streak-seeding markedly improved the stability, crystal-growth rate and diffraction quality of DCM-associated mutant metavinculin crystals, allowing complete data collection to 3.9 {angstrom} resolution. These crystals belonged to space group P4{sub 3}2{sub 1}2, with two molecules in the asymmetric unit and unit-cell parameters a = b = 170, c = 211 {angstrom}, {alpha} = {beta} = {gamma} = 90{sup o}.

  7. Molecular cloning of a full-length cDNA for ECBP21 from Angelica dahurica

    Institute of Scientific and Technical Information of China (English)

    2002-01-01

    ECBP21 is an extracellular calmodulin-binding protein which was first detected and purified from extracellular extracts of suspension-cultured cells of Angelica dahurica. The purified protein was electroblotted onto PVDF membrane and the amino acid sequences from 1 to 20 were determined. Using degenerate oligonucleotides of the sequence, a full-length cDNA coding for ECBP21 was isolated by a combination of RT-PCR and 5′-RACE cloning. The cDNA contains 947 nucleotides and codes for a precursor protein of 216 amino acids. The N-terminal 1-25 amino acid sequence is a predicted signal peptide and the other 26-216 amino acid sequence is a mature peptide. The 26-45 amino acid sequence shows identity with the N-terminal amino acid sequence of purified ECBP21 from Angelica dahurica. The fragment of encoding the mature protein was cloned into pET-28b(+) and transformed into E. coli BL21(DE3). A protein with relative molecular mass 21 ku was expressed in E. coli. Using a biotinylated-CaM gel overlay technique, the expression protein was tested for its ability to bind CaM. The results indicated that the expression protein is a Ca2+- dependent CaM-binding protein. Thus, these results further defined the cDNA clone for ECBP21. This work laid a foundation for elucidating biological functions of ECBP21 by using molecular biological means.

  8. Identification and characterization of full-length cDNAs in channel catfish (Ictalurus punctatus and blue catfish (Ictalurus furcatus.

    Directory of Open Access Journals (Sweden)

    Fei Chen

    Full Text Available BACKGROUND: Genome annotation projects, gene functional studies, and phylogenetic analyses for a given organism all greatly benefit from access to a validated full-length cDNA resource. While increasingly common in model species, full-length cDNA resources in aquaculture species are scarce. METHODOLOGY AND PRINCIPAL FINDINGS: Through in silico analysis of catfish (Ictalurus spp. ESTs, a total of 10,037 channel catfish and 7,382 blue catfish cDNA clones were identified as potentially encoding full-length cDNAs. Of this set, a total of 1,169 channel catfish and 933 blue catfish full-length cDNA clones were selected for re-sequencing to provide additional coverage and ensure sequence accuracy. A total of 1,745 unique gene transcripts were identified from the full-length cDNA set, including 1,064 gene transcripts from channel catfish and 681 gene transcripts from blue catfish, with 416 transcripts shared between the two closely related species. Full-length sequence characteristics (ortholog conservation, UTR length, Kozak sequence, and conserved motifs of the channel and blue catfish were examined in detail. Comparison of gene ontology composition between full-length cDNAs and all catfish ESTs revealed that the full-length cDNA set is representative of the gene diversity encoded in the catfish transcriptome. CONCLUSIONS: This study describes the first catfish full-length cDNA set constructed from several cDNA libraries. The catfish full-length cDNA sequences, and data gleaned from sequence characteristics analysis, will be a valuable resource for ongoing catfish whole-genome sequencing and future gene-based studies of function and evolution in teleost fishes.

  9. The function analysis of full-length cDNA sequence from IRM-2 mouse cDNA library

    International Nuclear Information System (INIS)

    Objective: To identify the function of full-length cDNA sequence from IRM-2 mouse cDNA library. Methods: Full-length cDNA products were amplified by PCR from IRM-2 mouse cDNA library according to twenty-one pieces of expressed sequence tag. The expression of full-length cDNAs were detected after mouse embryonic fibroblasts were exposed to 6.5 Gy γ-ray radiation. And the effect on the growth of radiosensitivity cells AT5B1VA transfected with full-length cDNAs was investigated. Results: The expression of No.4, 5 and 2 full-length cDNAs from IRM-2 mouse were higher than that of parental ICR and 615 mouse after mouse embryonic fibroblasts irradiated with γ-ray radiation. And the survival rate of AT5B1VA cells transfected with No.4, 5 and 2 full-length cDNAs was high. Conclusion: No.4, 5 and 2 full-length cDNAs of IRM-2 mouse are of high radioresistance. (authors)

  10. Crystal structure of the full-length bacterial selenocysteine-specific elongation factor SelB.

    Science.gov (United States)

    Itoh, Yuzuru; Sekine, Shun-Ichi; Yokoyama, Shigeyuki

    2015-10-15

    Selenocysteine (Sec), the 21(st) amino acid in translation, uses its specific tRNA (tRNA(Sec)) to recognize the UGA codon. The Sec-specific elongation factor SelB brings the selenocysteinyl-tRNA(Sec) (Sec-tRNA(Sec)) to the ribosome, dependent on both an in-frame UGA and a Sec-insertion sequence (SECIS) in the mRNA. The bacterial SelB binds mRNA through its C-terminal region, for which crystal structures have been reported. In this study, we determined the crystal structure of the full-length SelB from the bacterium Aquifex aeolicus, in complex with a GTP analog, at 3.2-Å resolution. SelB consists of three EF-Tu-like domains (D1-3), followed by four winged-helix domains (WHD1-4). The spacer region, connecting the N- and C-terminal halves, fixes the position of WHD1 relative to D3. The binding site for the Sec moiety of Sec-tRNA(Sec) is located on the interface between D1 and D2, where a cysteine molecule from the crystallization solution is coordinated by Arg residues, which may mimic Sec binding. The Sec-binding site is smaller and more exposed than the corresponding site of EF-Tu. Complex models of Sec-tRNA(Sec), SECIS RNA, and the 70S ribosome suggest that the unique secondary structure of tRNA(Sec) allows SelB to specifically recognize tRNA(Sec) and characteristically place it at the ribosomal A-site. PMID:26304550

  11. Structural Influences: Cholesterol, Drug, and Proton Binding to Full-Length Influenza A M2 Protein.

    Science.gov (United States)

    Ekanayake, E Vindana; Fu, Riqiang; Cross, Timothy A

    2016-03-29

    The structure and functions of the M2 protein from Influenza A are sensitive to pH, cholesterol, and the antiinfluenza drug Amantadine. This is a tetrameric membrane protein of 97 amino-acid residues that has multiple functions, among them as a proton-selective channel and facilitator of viral budding, replacing the need for the ESCRT proteins that other viruses utilize. Here, various amino-acid-specific-labeled samples of the full-length protein were prepared and mixed, so that only interresidue (13)C-(13)C cross peaks between two differently labeled proteins representing interhelical interactions are observed. This channel is activated at slightly acidic pH values in the endosome when the His(37) residues in the middle of the transmembrane domain take on a +2 or +3 charged state. Changes observed here in interhelical distances in the N-terminus can be accounted for by modest structural changes, and no significant changes in structure were detected in the C-terminal portion of the channel upon activation of the channel. Amantadine, which blocks proton conductance by binding in the aqueous pore near the N-terminus, however, significantly modifies the tetrameric structure on the opposite side of the membrane. The interactions between the juxtamembrane amphipathic helix of one monomer and its neighboring monomer observed in the absence of drug are disrupted in its presence. However, the addition of cholesterol prevents this structural disruption. In fact, strong interactions are observed between cholesterol and residues in the amphipathic helix, accounting for cholesterol binding adjacent to a native palmitoylation site and near to an interhelix crevice that is typical of cholesterol binding sites. The resultant stabilization of the amphipathic helix deep in the bilayer interface facilitates the bilayer curvature that is essential for viral budding. PMID:27028648

  12. Sequencing, mapping, and analysis of 27,455 maize full-length cDNAs.

    Directory of Open Access Journals (Sweden)

    Carol Soderlund

    2009-11-01

    Full Text Available Full-length cDNA (FLcDNA sequencing establishes the precise primary structure of individual gene transcripts. From two libraries representing 27 B73 tissues and abiotic stress treatments, 27,455 high-quality FLcDNAs were sequenced. The average transcript length was 1.44 kb including 218 bases and 321 bases of 5' and 3' UTR, respectively, with 8.6% of the FLcDNAs encoding predicted proteins of fewer than 100 amino acids. Approximately 94% of the FLcDNAs were stringently mapped to the maize genome. Although nearly two-thirds of this genome is composed of transposable elements (TEs, only 5.6% of the FLcDNAs contained TE sequences in coding or UTR regions. Approximately 7.2% of the FLcDNAs are putative transcription factors, suggesting that rare transcripts are well-enriched in our FLcDNA set. Protein similarity searching identified 1,737 maize transcripts not present in rice, sorghum, Arabidopsis, or poplar annotated genes. A strict FLcDNA assembly generated 24,467 non-redundant sequences, of which 88% have non-maize protein matches. The FLcDNAs were also assembled with 41,759 FLcDNAs in GenBank from other projects, where semi-strict parameters were used to identify 13,368 potentially unique non-redundant sequences from this project. The libraries, ESTs, and FLcDNA sequences produced from this project are publicly available. The annotated EST and FLcDNA assemblies are available through the maize FLcDNA web resource (www.maizecdna.org.

  13. Identification and nearly full-length genome characterization of novel porcine bocaviruses.

    Directory of Open Access Journals (Sweden)

    Wei-xia Cheng

    Full Text Available The genus bocavirus includes bovine parvovirus (BPV, minute virus of canines (MVC, and a group of human bocaviruses (HBoV1-4. Using sequence-independent single primer amplification (SISPA, a novel bocavirus group was discovered with high prevalence (12.59% in piglet stool samples. Two nearly full-length genome sequences were obtained, which were approximately 5,100 nucleotides in length. Multiple alignments revealed that they share 28.7-56.8% DNA sequence identity with other members of Parvovirinae. Phylogenetic analyses indicated their closest neighbors were members of the genus bocavirus. The new viruses had a putative non-structural NP1 protein, which was unique to bocaviruses. They were provisionally named porcine bocavirus 1 and 2 (PBoV1, PBoV2. PBoV1 and PBoV2 shared 94.2% nucleotide identity in NS1 gene sequence, suggesting that they represented two different bocavirus species. Two additional samples (6V, 7V were amplified for 2,407 bp and 2,434 bp products, respectively, including a partial NP1 gene and the complete VP1 gene; Phylogenetic analysis indicated that 6Vand 7V grouped with PBoV1 and PBoV2 in the genus of bocavirus, but were in the separate clusters. Like other parvoviruses, PBoV1, PBoV2, 6Vand 7V also contained a putative secretory phospholipase A(2 (sPLA(2 motif in the VP1 unique region, with a conserved HDXXY motif in the catalytic center. The conserved motif YXGXF of the Ca(2+-binding loop of sPLA2 identified in human bocavirus was also found in porcine bocavirus, which differs from the YXGXG motif carried by most other parvoviruses. The observation of PBoV and potentially other new bocavirus genus members may aid in molecular and functional characterization of the genus bocavirus.

  14. Full-length cDNA cloning and structural characterization of preproinsulin in Alligator sinensis.

    Science.gov (United States)

    Zhang, R; Zhang, S Z; Li, E; Wang, C; Wang, C L; Wu, X B

    2014-01-01

    Insulin is an important endocrine hormone that plays a critical physiological role in regulating metabolism and glucostasis in vertebrates. In this study, the complete cDNA of Alligator sinensis preproinsulin gene was cloned for the first time by reverse transcription-polymerase chain reaction and rapid amplification of cDNA ends methods; the amino acid sequence encoded and protein structure were analyzed. The full-length of preproinsulin cDNA sequence consists of 528 base pairs (bp), comprising a 34-bp 5'-untranslated region, a 170-bp 3'-untranslated region and an open reading frame that is 324 bp in length. The open reading frame encodes a 107-amino acid preproinsulin with a molecular weight of approximately 12,153.8 Da, theoretical isoelectric point of 5.68, aliphatic index of 92.06, and grand average of hydropathicity of -0.157, from which a signal peptide, a B-chain, a C-peptide, and an A-chain are derived. Online analysis suggested that the deduced preproinsulin amino acid sequence contains a transmembrane region, and that it has a signal peptide whose cleavage site occurs between alanine 24 and alanine 25. Comparative analysis of preproinsulin amino acid sequences indicated that the A-chain and B-chain sequences of preproinsulins are highly conserved between reptiles and birds, and that the preproinsulin amino acid sequence of Alligator sinensis shares 89% similarity to that of Chelonia mydas, but low similarity of 48-63% to those of mammals and fishes. The phylogenetic tree constructed using the neighbor-joining method revealed that preproinsulin of Alligator sinensis had high homology with reptiles and birds, such as Chelonia mydas, Gallus gallus, and Columba livia. PMID:25366775

  15. Cognitive defects are reversible in inducible mice expressing pro-aggregant full-length human Tau

    Science.gov (United States)

    Sydow, Astrid; Hofmann, Anne; Wu, Dan; Messing, Lars; Balschun, Detlef; D'Hooge, Rudi; Mandelkow, Eva-Maria

    2016-01-01

    Neurofibrillary lesions of abnormal Tau are hallmarks of Alzheimer´s disease and frontotemporal dementias. Our regulatable (Tet-OFF) mouse models of tauopathy express variants of human full-length Tau in the forebrain (CaMKIIα promoter) either with mutation ΔK280 (pro-aggregant) or ΔK280/I277P/I308P (anti-aggregant). Co-expression of luciferase enables in vivo quantification of gene expression by bioluminescence imaging. Pro-aggregant mice develop synapse loss and Tau pathology including missorting, phosphorylation and early pretangle formation, whereas anti-aggregant mice do not. We correlated hippocampal Tau pathology with learning/memory performance and synaptic plasticity. Pro-aggregant mice at 16 months of gene expression exhibited severe cognitive deficits in Morris water-maze and in passive-avoidance paradigms, whereas anti-aggregant mice were comparable to controls. Cognitive impairment of pro-aggregant mice was accompanied by loss of hippocampal LTP in CA1 and CA3 areas and by a reduction of synaptic proteins and dendritic spines, although no neuronal loss was observed. Remarkably, memory and LTP recovered when pro-aggregant Tau was switched-OFF for ∼4 months, Tau phosphorylation and missorting were reversed, and synapses recovered. Moreover soluble and insoluble pro-aggregant hTau40 disappeared while insoluble mouse Tau was still present. This study links early Tau pathology without neurofibrillary tangles and neuronal death to cognitive decline and synaptic dysfunction. It demonstrates that Tau-induced impairments are reversible after switching-OFF pro-aggregant Tau. Therefore our mouse model may mimic an early phase of AD when the hippocampus does not yet suffer from irreversible cell death but cognitive deficits are already striking. It offers potential to evaluate drugs with regard to learning and memory performance. PMID:22532069

  16. Structure of the HIV-1 Full-Length Capsid Protein in a Conformationally Trapped Unassembled State Induced by Small-Molecule Binding

    Energy Technology Data Exchange (ETDEWEB)

    Du, Shoucheng; Betts, Laurie; Yang, Ruifeng; Shi, Haibin; Concel, Jason; Ahn, Jinwoo; Aiken, Christopher; Zhang, Peijun; Yeh, Joanne I. (Pitt); (Vanderbilt); (UNC)

    2012-11-26

    The capsid (CA) protein plays crucial roles in HIV infection and replication, essential to viral maturation. The absence of high-resolution structural data on unassembled CA hinders the development of antivirals effective in inhibiting assembly. Unlike enzymes that have targetable, functional substrate-binding sites, the CA does not have a known site that affects catalytic or other innate activity, which can be more readily targeted in drug development efforts. We report the crystal structure of the HIV-1 CA, revealing the domain organization in the context of the wild-type full-length (FL) unassembled CA. The FL CA adopts an antiparallel dimer configuration, exhibiting a domain organization sterically incompatible with capsid assembly. A small compound, generated in situ during crystallization, is bound tightly at a hinge site ('H site'), indicating that binding at this interdomain region stabilizes the ADP conformation. Electron microscopy studies on nascent crystals reveal both dimeric and hexameric lattices coexisting within a single condition, in agreement with the interconvertibility of oligomeric forms and supporting the feasibility of promoting assembly-incompetent dimeric states. Solution characterization in the presence of the H-site ligand shows predominantly unassembled dimeric CA, even under conditions that promote assembly. Our structure elucidation of the HIV-1 FL CA and characterization of a potential allosteric binding site provides three-dimensional views of an assembly-defective conformation, a state targeted in, and thus directly relevant to, inhibitor development. Based on our findings, we propose an unprecedented means of preventing CA assembly, by 'conformationally trapping' CA in assembly-incompetent conformational states induced by H-site binding.

  17. A novel copper(II) coordination at His186 in full-length murine prion protein

    Energy Technology Data Exchange (ETDEWEB)

    Watanabe, Yasuko [Laboratory of Radiation Biology, Graduate School of Veterinary Medicine, Hokkaido University, Sapporo 060-0818 (Japan); Hiraoka, Wakako [Laboratory of Biophysics, School of Science and Technology, Meiji University, Kawasaki 214-8571 (Japan); Igarashi, Manabu; Ito, Kimihito [Department of Global Epidemiology, Hokkaido University Research Center for Zoonosis Control, Sapporo 001-0020 (Japan); Shimoyama, Yuhei [Soft-Matter Physics Laboratory, Graduate School of Emergent Science, Muroran Institute of Technology, Muroran 050-8585 (Japan); Horiuchi, Motohiro [Laboratory of Prion Diseases, Graduate School of Veterinary Medicine, Hokkaido University, Sapporo 060-0818 (Japan); Yamamori, Tohru; Yasui, Hironobu; Kuwabara, Mikinori [Laboratory of Radiation Biology, Graduate School of Veterinary Medicine, Hokkaido University, Sapporo 060-0818 (Japan); Inagaki, Fuyuhiko [Laboratory of Structural Biology, Graduate School of Pharmaceutical Sciences, Hokkaido University, Sapporo 060-0812 (Japan); Inanami, Osamu, E-mail: inanami@vetmed.hokudai.ac.jp [Laboratory of Radiation Biology, Graduate School of Veterinary Medicine, Hokkaido University, Sapporo 060-0818 (Japan)

    2010-04-09

    To explore Cu(II) ion coordination by His{sup 186} in the C-terminal domain of full-length prion protein (moPrP), we utilized the magnetic dipolar interaction between a paramagnetic metal, Cu(II) ion, and a spin probe introduced in the neighborhood of the postulated binding site by the spin labeling technique (SDSL technique). Six moPrP mutants, moPrP(D143C), moPrP(Y148C), moPrP(E151C), moPrP(Y156C), moPrP(T189C), and moPrP(Y156C,H186A), were reacted with a methane thiosulfonate spin probe and a nitroxide residue (R1) was created in the binding site of each one. Line broadening of the ESR spectra was induced in the presence of Cu(II) ions in moPrP(Y148R1), moPrP(Y151R1), moPrP(Y156R1), and moPrP(T189R1) but not moPrP(D143R1). This line broadening indicated the presence of electron-electron dipolar interaction between Cu(II) and the nitroxide spin probe, suggesting that each interspin distance was within 20 A. The interspin distance ranges between Cu(II) and the spin probes of moPrP(Y148R1), moPrP(Y151R1), moPrP(Y156R1), and moPrP(T189R1) were estimated to be 12.1 A, 18.1 A, 10.7 A, and 8.4 A, respectively. In moPrP(Y156R1,H186A), line broadening between Cu(II) and the spin probe was not observed. These results suggest that a novel Cu(II) binding site is involved in His186 in the Helix2 region of the C-terminal domain of moPrP{sup C}.

  18. High avidity antibodies to full-length VAR2CSA correlate with absence of placental malaria.

    Science.gov (United States)

    Tutterrow, Yeung Lo; Salanti, Ali; Avril, Marion; Smith, Joseph D; Pagano, Ian S; Ako, Simon; Fogako, Josephine; Leke, Rose G F; Taylor, Diane Wallace

    2012-01-01

    VAR2CSA mediates sequestration of Plasmodium falciparum-infected erythrocytes in the placenta, increasing the risk of poor pregnancy outcomes. Naturally acquired antibodies (Ab) to placental parasites at delivery have been associated with improved pregnancy outcomes, but Ab levels and how early in pregnancy Ab must be present in order to eliminate placental parasites before delivery remains unknown. Antibodies to individual Duffy-binding like domains of VAR2CSA have been studied, but the domains lack many of the conformational epitopes present in full-length VAR2CSA (FV2). Thus, the purpose of this study was to describe the acquisition of Ab to FV2 in women residing in high and low transmission areas and determine how Ab levels during pregnancy correlate with clearance of placental parasites. Plasma samples collected monthly throughout pregnancy from pregnant women living in high and low transmission areas in Cameroon were evaluated for Ab to FV2 and the proportion of high avidity Ab (i.e., Ab that remain bound in the presence of 3M NH(4)SCN) was assessed. Ab levels and proportion of high avidity Ab were compared between women with placental malaria (PM(+)) and those without (PM(-)) at delivery. Results showed that PM(-) women had significantly higher Ab levels (p = 0.0047) and proportion of high avidity Ab (p = 0.0009) than PM(+) women throughout pregnancy. Specifically, women with moderate to high Ab levels (>5,000 MFI) and those with ≥ 35% high avidity Ab at 5-6 months were found to have 2.3 (95% CI, 1.0-4.9) and 7.6-fold (p = 0.0013, 95% CI: 1.2-50.0) reduced risk of placental malaria, respectively. These data show that high levels of Ab to FV2, particularly those with high avidity for FV2, produced by mid-pregnancy are important in clearing parasites from the placenta. Both high Ab levels and proportion of high avidity Ab to FV2 may serve as correlates of protection for assessing immunity against placental malaria. PMID:22761948

  19. High avidity antibodies to full-length VAR2CSA correlate with absence of placental malaria.

    Directory of Open Access Journals (Sweden)

    Yeung Lo Tutterrow

    Full Text Available VAR2CSA mediates sequestration of Plasmodium falciparum-infected erythrocytes in the placenta, increasing the risk of poor pregnancy outcomes. Naturally acquired antibodies (Ab to placental parasites at delivery have been associated with improved pregnancy outcomes, but Ab levels and how early in pregnancy Ab must be present in order to eliminate placental parasites before delivery remains unknown. Antibodies to individual Duffy-binding like domains of VAR2CSA have been studied, but the domains lack many of the conformational epitopes present in full-length VAR2CSA (FV2. Thus, the purpose of this study was to describe the acquisition of Ab to FV2 in women residing in high and low transmission areas and determine how Ab levels during pregnancy correlate with clearance of placental parasites. Plasma samples collected monthly throughout pregnancy from pregnant women living in high and low transmission areas in Cameroon were evaluated for Ab to FV2 and the proportion of high avidity Ab (i.e., Ab that remain bound in the presence of 3M NH(4SCN was assessed. Ab levels and proportion of high avidity Ab were compared between women with placental malaria (PM(+ and those without (PM(- at delivery. Results showed that PM(- women had significantly higher Ab levels (p = 0.0047 and proportion of high avidity Ab (p = 0.0009 than PM(+ women throughout pregnancy. Specifically, women with moderate to high Ab levels (>5,000 MFI and those with ≥ 35% high avidity Ab at 5-6 months were found to have 2.3 (95% CI, 1.0-4.9 and 7.6-fold (p = 0.0013, 95% CI: 1.2-50.0 reduced risk of placental malaria, respectively. These data show that high levels of Ab to FV2, particularly those with high avidity for FV2, produced by mid-pregnancy are important in clearing parasites from the placenta. Both high Ab levels and proportion of high avidity Ab to FV2 may serve as correlates of protection for assessing immunity against placental malaria.

  20. A novel copper(II) coordination at His186 in full-length murine prion protein

    International Nuclear Information System (INIS)

    To explore Cu(II) ion coordination by His186 in the C-terminal domain of full-length prion protein (moPrP), we utilized the magnetic dipolar interaction between a paramagnetic metal, Cu(II) ion, and a spin probe introduced in the neighborhood of the postulated binding site by the spin labeling technique (SDSL technique). Six moPrP mutants, moPrP(D143C), moPrP(Y148C), moPrP(E151C), moPrP(Y156C), moPrP(T189C), and moPrP(Y156C,H186A), were reacted with a methane thiosulfonate spin probe and a nitroxide residue (R1) was created in the binding site of each one. Line broadening of the ESR spectra was induced in the presence of Cu(II) ions in moPrP(Y148R1), moPrP(Y151R1), moPrP(Y156R1), and moPrP(T189R1) but not moPrP(D143R1). This line broadening indicated the presence of electron-electron dipolar interaction between Cu(II) and the nitroxide spin probe, suggesting that each interspin distance was within 20 A. The interspin distance ranges between Cu(II) and the spin probes of moPrP(Y148R1), moPrP(Y151R1), moPrP(Y156R1), and moPrP(T189R1) were estimated to be 12.1 A, 18.1 A, 10.7 A, and 8.4 A, respectively. In moPrP(Y156R1,H186A), line broadening between Cu(II) and the spin probe was not observed. These results suggest that a novel Cu(II) binding site is involved in His186 in the Helix2 region of the C-terminal domain of moPrPC.

  1. Construction of a full-length enriched cDNA library and preliminary analysis of expressed sequence tags from Bengal Tiger Panthera tigris tigris.

    Science.gov (United States)

    Liu, Changqing; Liu, Dan; Guo, Yu; Lu, Taofeng; Li, Xiangchen; Zhang, Minghai; Ma, Jianzhang; Ma, Yuehui; Guan, Weijun

    2013-01-01

    In this study, a full-length enriched cDNA library was successfully constructed from Bengal tiger, Panthera tigris tigris, the most well-known wild Animal. Total RNA was extracted from cultured Bengal tiger fibroblasts in vitro. The titers of primary and amplified libraries were 1.28 × 106 pfu/mL and 1.56 × 109 pfu/mL respectively. The percentage of recombinants from unamplified library was 90.2% and average length of exogenous inserts was 0.98 kb. A total of 212 individual ESTs with sizes ranging from 356 to 1108 bps were then analyzed. The BLASTX score revealed that 48.1% of the sequences were classified as a strong match, 45.3% as nominal and 6.6% as a weak match. Among the ESTs with known putative function, 26.4% ESTs were found to be related to all kinds of metabolisms, 19.3% ESTs to information storage and processing, 11.3% ESTs to posttranslational modification, protein turnover, chaperones, 11.3% ESTs to transport, 9.9% ESTs to signal transducer/cell communication, 9.0% ESTs to structure protein, 3.8% ESTs to cell cycle, and only 6.6% ESTs classified as novel genes. By EST sequencing, a full-length gene coding ferritin was identified and characterized. The recombinant plasmid pET32a-TAT-Ferritin was constructed, coded for the TAT-Ferritin fusion protein with two 6× His-tags in N and C-terminal. After BCA assay, the concentration of soluble Trx-TAT-Ferritin recombinant protein was 2.32 ± 0.12 mg/mL. These results demonstrated that the reliability and representativeness of the cDNA library attained to the requirements of a standard cDNA library. This library provided a useful platform for the functional genome and transcriptome research of Bengal tigers. PMID:23708105

  2. Construction of a Full-Length Enriched cDNA Library and Preliminary Analysis of Expressed Sequence Tags from Bengal Tiger Panthera tigris tigris

    Directory of Open Access Journals (Sweden)

    Changqing Liu

    2013-05-01

    Full Text Available In this study, a full-length enriched cDNA library was successfully constructed from Bengal tiger, Panthera tigris tigris, the most well-known wild Animal. Total RNA was extracted from cultured Bengal tiger fibroblasts in vitro. The titers of primary and amplified libraries were 1.28 × 106 pfu/mL and 1.56 × 109 pfu/mL respectively. The percentage of recombinants from unamplified library was 90.2% and average length of exogenous inserts was 0.98 kb. A total of 212 individual ESTs with sizes ranging from 356 to 1108 bps were then analyzed. The BLASTX score revealed that 48.1% of the sequences were classified as a strong match, 45.3% as nominal and 6.6% as a weak match. Among the ESTs with known putative function, 26.4% ESTs were found to be related to all kinds of metabolisms, 19.3% ESTs to information storage and processing, 11.3% ESTs to posttranslational modification, protein turnover, chaperones, 11.3% ESTs to transport, 9.9% ESTs to signal transducer/cell communication, 9.0% ESTs to structure protein, 3.8% ESTs to cell cycle, and only 6.6% ESTs classified as novel genes. By EST sequencing, a full-length gene coding ferritin was identified and characterized. The recombinant plasmid pET32a-TAT-Ferritin was constructed, coded for the TAT-Ferritin fusion protein with two 6× His-tags in N and C-terminal. After BCA assay, the concentration of soluble Trx-TAT-Ferritin recombinant protein was 2.32 ± 0.12 mg/mL. These results demonstrated that the reliability and representativeness of the cDNA library attained to the requirements of a standard cDNA library. This library provided a useful platform for the functional genome and transcriptome research of Bengal tigers.

  3. [Rapid construction of full-length MnSOD cDNA of chickens by one-step 3'RACE].

    Science.gov (United States)

    Bu, You-Quan; Luo, Xu-Gang; Liu, Bin; Li, Su-Fen

    2004-07-01

    RACE (rapid amplification of cDNA ends) is a popular technique to rapidly obtain the full-length cDNA. After obtaining the 3' cDNA and 5' cDNA fragments with a overlapped region by 3' RACE and 5' RACE, the full-length cDNA could be generated by end-to-end PCR or subcloning. In this study, 3' RACE combined with touch-down PCR was successfully used for the rapid construction of full-length MnSOD cDNA of chickens. Compared with the conventional end-to-end PCR or subcloning, this method, called one-step 3' RACE, is fast, economical and highly specific. It especially fits the rapid construction of full-length cDNA by RACE method. PMID:15640053

  4. Functional Recombinant Extra Membrane Loop of Human CD20, an Alternative of the Full Length CD20 Antigen

    OpenAIRE

    Anbouhi, Mahdi Habibi; Baraz, Aida Feiz; Bouzari, Saeid; Abolhassani, Mohsen; Khanahmad, Hossein; Golkar, Majid; Aghasadeghi, Mohammad Reza; Behdani, Mahdi; Najafabadi, Ali Jahanian; Shokrgozar, Mohammad Ali

    2012-01-01

    Background: Targeting of CD20 antigen with monoclonal antibodies has become the mainstay in the treatment of non-Hodgkin's lymphomas and immunotherapeutic depletion of malignant B cells. Accessibility of antigen is one of the crucial factors in development of monoclonal antibodies against this antigen. One major problem in expression of full length CD20 is aggregation and misfolding. Therefore, production of an alternative polypeptide is easer and favorable comparing to that of a full length ...

  5. A systematic approach for testing expression of human full-length proteins in cell-free expression systems

    OpenAIRE

    LaBaer Joshua; Ebert Lars; Scheuermann Tina; Wermke Nadja; Guilleaume Birgit; Langlais Claudia; Korn Bernhard

    2007-01-01

    Abstract Background The growing field of proteomics and systems biology is resulting in an ever increasing demand for purified recombinant proteins for structural and functional studies. Here, we show a systematic approach to successfully express a full-length protein of interest by using cell-free and cell-based expression systems. Results In a pre-screen, we evaluated the expression of 960 human full-length open reading frames in Escherichia coli (in vivo and in vitro). After analysing the ...

  6. Analysis of 4,664 high-quality sequence-finished poplar full-length

    Energy Technology Data Exchange (ETDEWEB)

    Ralph, S. [University of British Columbia, Vancouver; Gunter, Lee E [ORNL; Tuskan, Gerald A [ORNL; Douglas, Carl [University of British Columbia, Vancouver; Holt, Robert A. [Genome Sciences Centre, Vancouver, BC, Canada; Jones, Steven [Genome Sciences Centre, Vancouver, BC, Canada; Marra, Marco [Genome Sciences Centre, Vancouver, BC, Canada; Bohlmann, J. [University of British Columbia, Vancouver

    2008-01-01

    The genus Populus includes poplars, aspens and cottonwoods, which will be collectively referred to as poplars hereafter unless otherwise specified. Poplars are the dominant tree species in many forest ecosystems in the Northern Hemisphere and are of substantial economic value in plantation forestry. Poplar has been established as a model system for genomics studies of growth, development, and adaptation of woody perennial plants including secondary xylem formation, dormancy, adaptation to local environments, and biotic interactions. As part of the poplar genome sequencing project and the development of genomic resources for poplar, we have generated a full-length (FL)-cDNA collection using the biotinylated CAP trapper method. We constructed four FLcDNA libraries using RNA from xylem, phloem and cambium, and green shoot tips and leaves from the P. trichocarpa Nisqually-1 genotype, as well as insect-attacked leaves of the P. trichocarpa x P. deltoides hybrid. Following careful selection of candidate cDNA clones, we used a combined strategy of paired end reads and primer walking to generate a set of 4,664 high-accuracy, sequence-verified FLcDNAs, which clustered into 3,990 putative unique genes. Mapping FLcDNAs to the poplar genome sequence combined with BLAST comparisons to previously predicted protein coding sequences in the poplar genome identified 39 FLcDNAs that likely localize to gaps in the current genome sequence assembly. Another 173 FLcDNAs mapped to the genome sequence but were not included among the previously predicted genes in the poplar genome. Comparative sequence analysis against Arabidopsis thaliana and other species in the non-redundant database of GenBank revealed that 11.5% of the poplar FLcDNAs display no significant sequence similarity to other plant proteins. By mapping the poplar FLcDNAs against transcriptome data previously obtained with a 15.5 K cDNA microarray, we identified 153 FLcDNA clones for genes that were differentially expressed in

  7. IFPE/SPC-RE-GINNA, Full Length and Segmented Fuel Rodlet Irradiation in PWR

    International Nuclear Information System (INIS)

    The objective of this program was to develop a fuel design with increased margin to pellet-to-clad interaction (PCI) failure threshold and increased potential for higher burnup. The means by which this objective was to be attained was with annular pellets and zirconium barrier cladding. The annular pellets used have a void volume of approximately 10% higher than comparable solid pellets. Barrier cladding consists of Zircaloy-4 tubes with an integral inner layer of unalloyed zirconium comprising approximately 10% of the total wall thickness. The overall cladding dimensions are the same as the standard cladding. Four Siemens Power Corporation (SPC) 14x14 lead fuel assemblies (XT01, XT02, XT03 and XT04) were inserted into the R.E. Ginna reactor. The program included design and fabrication of the assemblies, irradiation in the R.E. Ginna reactor, pool-side examination and measurements, and post irradiation hot cell examination of selected segmented fuel rodlets in the CEA Laboratories in Grenoble. The aim of the program was to demonstrate and evaluate the in-reactor performance of the assemblies at high burnup, and the potential of annular pellets and zirconium barrier cladding for resisting fuel failures due to PCI. Each assembly contained 179 fuel rods, 16 guide tubes, and one instrument tube. XT03 and XT04 each contained 30 fully characterized fuel rods. 19 of the characterized fuel rods in each assembly were full length, annular pellet, zirconium barrier clad rods, and the remaining 11 rods were segmented fuel rods with different combinations of solid and annular pellets, standard and barrier cladding, and controlled variations in pellet-to-cladding gap. Three of the lead fuel assemblies were irradiated for four cycles to an average burnup of 42.5 MWd/kgU, while XT03 was irradiated for five cycles. This assembly reached an average burnup of 52.1 MWd/kgU, while the two central rodlets contained in the eleven segmented rods achieved a burnup of 52 MWd/kgU for solid

  8. Identification and Characterization of Full-Length cDNAs in Channel Catfish (Ictalurus punctatus) and Blue Catfish (Ictalurus furcatus)

    OpenAIRE

    Chen, Fei; Lee, Yoona; Jiang, Yanliang; Wang, Shaolin; Peatman, Eric; Abernathy, Jason; Liu, Hong; Liu, Shikai; Kucuktas, Huseyin; Ke, Caihuan; Liu, Zhanjiang

    2010-01-01

    Background Genome annotation projects, gene functional studies, and phylogenetic analyses for a given organism all greatly benefit from access to a validated full-length cDNA resource. While increasingly common in model species, full-length cDNA resources in aquaculture species are scarce. Methodology and Principal Findings Through in silico analysis of catfish (Ictalurus spp.) ESTs, a total of 10,037 channel catfish and 7,382 blue catfish cDNA clones were identified as potentially encoding f...

  9. Ensemble Structure of the Highly Flexible Complex Formed between Vesicular Stomatitis Virus Unassembled Nucleoprotein and its Phosphoprotein Chaperone.

    Science.gov (United States)

    Yabukarski, Filip; Leyrat, Cedric; Martinez, Nicolas; Communie, Guillaume; Ivanov, Ivan; Ribeiro, Euripedes A; Buisson, Marlyse; Gerard, Francine C; Bourhis, Jean-Marie; Jensen, Malene Ringkjøbing; Bernadó, Pau; Blackledge, Martin; Jamin, Marc

    2016-07-01

    Nucleocapsid assembly is an essential process in the replication of the non-segmented, negative-sense RNA viruses (NNVs). Unassembled nucleoprotein (N(0)) is maintained in an RNA-free and monomeric form by its viral chaperone, the phosphoprotein (P), forming the N(0)-P complex. Our earlier work solved the structure of vesicular stomatitis virus complex formed between an N-terminally truncated N (NΔ21) and a peptide of P (P60) encompassing the N(0)-binding site, but how the full-length P interacts with N(0) remained unknown. Here, we combine several experimental biophysical methods including size exclusion chromatography with detection by light scattering and refractometry, small-angle X-ray and neutron scattering and nuclear magnetic resonance spectroscopy with molecular dynamics simulation and computational modeling to characterize the NΔ21(0)-PFL complex formed with dimeric full-length P. We show that for multi-molecular complexes, simultaneous multiple-curve fitting using small-angle neutron scattering data collected at varying contrast levels provides additional information and can help refine structural ensembles. We demonstrate that (a) vesicular stomatitis virus PFL conserves its high flexibility within the NΔ21(0)-PFL complex and interacts with NΔ21(0) only through its N-terminal extremity; (b) each protomer of P can chaperone one N(0) client protein, leading to the formation of complexes with stoichiometries 1N:P2 and 2N:P2; and (c) phosphorylation of residues Ser60, Thr62 and Ser64 provides no additional interactions with N(0) but creates a metal binding site in PNTR. A comparison with the structures of Nipah virus and Ebola virus N(0)-P core complex suggests a mechanism for the control of nucleocapsid assembly that is common to all NNVs. PMID:27107640

  10. Characterization of near full-length genomes of HIV type 1 strains in Denmark: Basis for a universal therapeutic vaccine

    DEFF Research Database (Denmark)

    Andresen, Betina S.; Vinner, Lasse; Tang, Sheila Tuyet; Bragstad, Karoline; Kronborg, Gitte; Gerstoft, Jan; Corbet, Sylvie; Fomsgaard, Anders

    2007-01-01

    We report here the near full-length sequence characterization of 17 Danish clinical HIV-1 strains isolated from HLA-A02 patients not in need of ART, with relatively low viral loads and normal CD4 cell counts. Sequencing was performed directly on DNA extracted from short-term cocultures of PBMCs...

  11. Characterization of near full-length genomes of HIV type 1 strains in Denmark: basis for a universal therapeutic vaccine

    DEFF Research Database (Denmark)

    Andresen, Betina S; Vinner, Lasse; Tang, Sheila; Bragstad, Karoline; Kronborg, Gitte; Gerstoft, Jan; Corbet, Sylvie; Fomsgaard, Anders

    2007-01-01

    directly on DNA extracted from short-term cocultures of PBMCs. The near full-length genomes did not contain any major insertions, deletions, or rearrangements. Sixteen of the isolates were characterized as nonrecombinant subtype B and one isolate as nonrecombinant subtype C. Phylogenetic analysis...

  12. Transformation of Cowpea Vigna unguiculata with a Full-Length DNA Copy of Cowpea Mosaic Virus M-RNA

    NARCIS (Netherlands)

    Hille, Jacques; Goldbach, Rob

    1987-01-01

    A full-length DNA copy of the M-RNA of cowpea mosaic virus (CPMV), supplied with either the 35S promoter from cauliflower mosaic virus (CaMV) or the nopaline synthase promoter from Agrobacterium tumefaciens, was introduced into the T-DNA region of a Ti-plasmid-derived gene vector and transferred to

  13. The ability to form full-length intron RNA circles is a general property of nuclear group I introns

    DEFF Research Database (Denmark)

    Nielsen, Henrik; Fiskaa, Tonje; Birgisdottir, Asa Birna; Haugen, Peik; Einvik, Christer; Johansen, Steinar

    2003-01-01

    at the expense of the host. The circularization pathway has distinct structural requirements that differ from those of splicing and appears to be specifically suppressed in vivo. The ability to form full-length circles is found in all types of nuclear group I introns, including those from the...

  14. Analysis of expressed sequence tags generated from full-length enriched cDNA libraries of melon

    Directory of Open Access Journals (Sweden)

    Bendahmane Abdelhafid

    2011-05-01

    Full Text Available Abstract Background Melon (Cucumis melo, an economically important vegetable crop, belongs to the Cucurbitaceae family which includes several other important crops such as watermelon, cucumber, and pumpkin. It has served as a model system for sex determination and vascular biology studies. However, genomic resources currently available for melon are limited. Result We constructed eleven full-length enriched and four standard cDNA libraries from fruits, flowers, leaves, roots, cotyledons, and calluses of four different melon genotypes, and generated 71,577 and 22,179 ESTs from full-length enriched and standard cDNA libraries, respectively. These ESTs, together with ~35,000 ESTs available in public domains, were assembled into 24,444 unigenes, which were extensively annotated by comparing their sequences to different protein and functional domain databases, assigning them Gene Ontology (GO terms, and mapping them onto metabolic pathways. Comparative analysis of melon unigenes and other plant genomes revealed that 75% to 85% of melon unigenes had homologs in other dicot plants, while approximately 70% had homologs in monocot plants. The analysis also identified 6,972 gene families that were conserved across dicot and monocot plants, and 181, 1,192, and 220 gene families specific to fleshy fruit-bearing plants, the Cucurbitaceae family, and melon, respectively. Digital expression analysis identified a total of 175 tissue-specific genes, which provides a valuable gene sequence resource for future genomics and functional studies. Furthermore, we identified 4,068 simple sequence repeats (SSRs and 3,073 single nucleotide polymorphisms (SNPs in the melon EST collection. Finally, we obtained a total of 1,382 melon full-length transcripts through the analysis of full-length enriched cDNA clones that were sequenced from both ends. Analysis of these full-length transcripts indicated that sizes of melon 5' and 3' UTRs were similar to those of tomato, but

  15. Gymnastics of molecular chaperones.

    Science.gov (United States)

    Mayer, Matthias P

    2010-08-13

    Molecular chaperones assist folding processes and conformational changes in many proteins. In order to do so, they progress through complex conformational cycles themselves. In this review, I discuss the diverse conformational dynamics of the ATP-dependent chaperones of the Hsp60, Hsp70, Hsp90, and Hsp100 families. PMID:20705236

  16. Full-Length Structures of BenM and Two Variants Reveal Different Oligomerization Schemes for LysR-Type Transcriptional Regulators

    Energy Technology Data Exchange (ETDEWEB)

    Ruangprasert, Ajchareeya; Craven, Sarah H.; Neidle, Ellen L.; Momany, Cory (Georgia)

    2010-11-30

    BenM, a LysR-type transcriptional regulator (LTTR) from the bacterium Acinetobacter baylyi, responds synergistically to benzoate and cis,cis-muconate. With these effectors, BenM activates gene expression during benzoate consumption. Without effectors, BenM represses transcription. Here, X-ray crystallography was used to determine the full-length structures of BenM and two variants that activate transcription without benzoate or cis,cis-muconate: BenM(R156H) and BenM(E226K). Previous studies indicate that these regulators function as tetramers. Here, interconnections between subunits in the crystals prevented the formation of a closed oligomer and highlighted the inherent flexibility of this multidomain regulator. Nevertheless, analysis of subunit interfaces suggested the functional significance of key interactions. The structures of BenM and its variants were nearly identical, implying that transcriptional differences rely on factors beyond major conformational changes defined solely by sequence. Comparisons of BenM with other LTTRs, including unpublished structures in the Protein Data Bank, revealed extensive variation in the relative orientations of DNA-binding domains (DBDs) and effector-binding domains (EBDs). To form dimers, different LTTRs used similar interfaces between two EBDs, each containing two subdomains: EBD-I and EBD-II. Surprisingly, the dimers used three substantially different schemes to form higher-order oligomers. In one scheme used by BenM, oligomer assembly involved contacts between the EBD-II regions and the DBD regions of adjacent subunits. In another scheme, there were no contacts between the EBDs; only the DBDs were involved in tetramer formation. In the third scheme, the oligomer interface involved DBD and EBD-I/EBD-II contacts. These diverse schemes demonstrate novel variation in the oligomeric structures of individual LTTRs within this large and important family.

  17. [Full-length cDNA cloning of flavonol synthase genes of Carthamus tinctorius and construction plant expression vector].

    Science.gov (United States)

    Yang, Wen-ting; Liu, Xiu-ming; Wan, Qiu; Yao, Na; Wang, Nan; Zhang, Xue-meng; Jiao, Zhong-da; Li, Hai-yan; Li, Xiao-kun

    2015-02-01

    Flavonol synthase (FLS) is one of the key enzymes in flavonoids metabolic pathways. In this study, middle sequence was obtained from Carthamus tinctorius transcriptome sequencing results. Full-length cDNAs of FLS was cloned from petals of C. tinctorius to FLS by using RT-PCR and RACE technology. Its full-length cDNA was 1,201 bp, with an open reading frame of 1,101 bp and 336 encoded amino acids. The phylogenetic analysis showed that, FLS gene encoded amino acids in C. tinctorius were highly homologous with amino acids in congeneric Compositae species, especially Rudbeckia laciniata. The pBASTA-FLS plant expression vector was successfully built by the molecular biology method, which lays a foundation for further studying biology functions of the gene and biosynthesis mechanism of flavonoids. PMID:26137682

  18. Infective viruses produced from full-length complementary DNA of swine vesicular disease viruses HK/70 strain

    Institute of Scientific and Technical Information of China (English)

    ZHENG Haixue; FENG Xia; YIN Shuanghui; GUO Jianhong; CONG Guozheng; LIU Zaixin; CHANG Huiyun; MA Junwu; XIE Qingge; LIU Xiangtao; SHANG Youjun; WU Jinyan; BAI Xingwen; JIN Ye; SUN Shiqi; GUO Huichen; TIAN Hong

    2006-01-01

    The full-length cDNA clone of swine vesicular disease virus HK/70 strain named pSVOK12 was constructed in order to study the antigenicity, replication, maturation and pathogenicity of swine vesicular disease virus. In vitro transcription RNA from pSVOK12 transfected IBRS-2 cells and the recovered virus RNA were isolated and sequenced, then indirect hemagglutination test, indirect immunofluorescence assays, eleectron microscope test, 50% tissue culture infecting dose (TCID50) assays and mouse virulence studies were performed to study the antigenicity and virulence of the recovered virus. The result showed that the infectious clones we obtained and the virus derived from pSVOK12 had the same biological properties as the parental strain HK/70. The full-length infectious cDNA clone, pSVOK12, will be very useful in studies of the antigenicity, virulence, pathogenesis, maturation and replication of SVDV.

  19. Screening for plant transporter function by expressing a normalized Arabidopsis full-length cDNA library in Xenopus oocytes

    Directory of Open Access Journals (Sweden)

    Halkier Barbara A

    2006-10-01

    Full Text Available Abstract Background We have developed a functional genomics approach based on expression cloning in Xenopus oocytes to identify plant transporter function. We utilized the full-length cDNA databases to generate a normalized library consisting of 239 full-length Arabidopsis thaliana transporter cDNAs. The genes were arranged into a 96-well format and optimized for expression in Xenopus oocytes by cloning each coding sequence into a Xenopus expression vector. Results Injection of 96 in vitro transcribed cRNAs from the library in pools of columns and rows into oocytes and subsequent screening for glucose uptake activity identified three glucose transporters. One of these, AtSTP13, had not previously been experimentally characterized. Conclusion Expression of the library in Xenopus oocytes, combined with uptake assays, has great potential in assignment of plant transporter function and for identifying membrane transporters for the many plant metabolites where a transporter has not yet been identified.

  20. Massive Collection of Full-Length Complementary DNA Clones and Microarray Analyses:. Keys to Rice Transcriptome Analysis

    Science.gov (United States)

    Kikuchi, Shoshi

    2009-02-01

    Completion of the high-precision genome sequence analysis of rice led to the collection of about 35,000 full-length cDNA clones and the determination of their complete sequences. Mapping of these full-length cDNA sequences has given us information on (1) the number of genes expressed in the rice genome; (2) the start and end positions and exon-intron structures of rice genes; (3) alternative transcripts; (4) possible encoded proteins; (5) non-protein-coding (np) RNAs; (6) the density of gene localization on the chromosome; (7) setting the parameters of gene prediction programs; and (8) the construction of a microarray system that monitors global gene expression. Manual curation for rice gene annotation by using mapping information on full-length cDNA and EST assemblies has revealed about 32,000 expressed genes in the rice genome. Analysis of major gene families, such as those encoding membrane transport proteins (pumps, ion channels, and secondary transporters), along with the evolution from bacteria to higher animals and plants, reveals how gene numbers have increased through adaptation to circumstances. Family-based gene annotation also gives us a new way of comparing organisms. Massive amounts of data on gene expression under many kinds of physiological conditions are being accumulated in rice oligoarrays (22K and 44K) based on full-length cDNA sequences. Cluster analyses of genes that have the same promoter cis-elements, that have similar expression profiles, or that encode enzymes in the same metabolic pathways or signal transduction cascades give us clues to understanding the networks of gene expression in rice. As a tool for that purpose, we recently developed "RiCES", a tool for searching for cis-elements in the promoter regions of clustered genes.

  1. Full-length diaphyseal sequestrum as a consequence of segmental transverse fractures of the large metatarsal bone in a calf

    International Nuclear Information System (INIS)

    A 1-week-old calf was treated for fractured right metatarsus; however, at 3.5 months of age, the fracture site again became unstable. Radiography disclosed segmental transverse metatarsal fractures with an isolated, full-circumference diaphyseal sequestrum. Arteriography and nuclear scintigraphy confirmed the isolation and necrosis of the diaphyseal segment. Diaphyseal sequestra usually develop as cortical or partial circumferential necrotic fragments. The finding of a full-length diaphyseal sequestrum was unusual

  2. Stabilization of a Full-Length Infectious cDNA Clone of Transmissible Gastroenteritis Coronavirus by Insertion of an Intron

    OpenAIRE

    González, José M; Pénzes, Zoltan; Almazán, Fernando; Calvo, Enrique; Enjuanes, Luis

    2002-01-01

    The stable propagation of a full-length transmissible gastroenteritis coronavirus (TGEV) cDNA in Escherichia coli cells as a bacterial artificial chromosome has been considerably improved by the insertion of an intron to disrupt a toxic region identified in the viral genome. The viral RNA was expressed in the cell nucleus under the control of the cytomegalovirus promoter and the intron was efficiently removed during translocation of this RNA to the cytoplasm. The insertion in two different po...

  3. First full length sequences of the S gene of European isolates reveal further diversity among turkey coronaviruses.

    OpenAIRE

    MAUREL, Stéphan; Toquin, Didier; BRIAND, François-Xavier; QUEGUINER, Maryline; ALLEE, Chantal; BERTIN, Joel; RETAUX, Charlotte; TURBLIN, Vincent; Morvan, Hervé; Eterradossi, Nicolas

    2011-01-01

    Abstract An increasing incidence of enteric disorders clinically evocative of the poult enteritis complex has been observed in turkeys in France since 2003. Using a newly designed real-time RT-PCR assay specific for the nucleocapsid (N) gene of infectious bronchitis virus (IBV) and turkey coronaviruses (TCoV), coronaviruses were identified in 37 % of the intestinal samples collected from diseased turkey flocks. The full length Spike (S) gene of these viruses was amplified, cloned a...

  4. RT-PCR and sequence analysis of the full-length fusion protein of Canine Distemper Virus from domestic dogs.

    Science.gov (United States)

    Romanutti, Carina; Gallo Calderón, Marina; Keller, Leticia; Mattion, Nora; La Torre, José

    2016-02-01

    During 2007-2014, 84 out of 236 (35.6%) samples from domestic dogs submitted to our laboratory for diagnostic purposes were positive for Canine Distemper Virus (CDV), as analyzed by RT-PCR amplification of a fragment of the nucleoprotein gene. Fifty-nine of them (70.2%) were from dogs that had been vaccinated against CDV. The full-length gene encoding the Fusion (F) protein of fifteen isolates was sequenced and compared with that of those of other CDVs, including wild-type and vaccine strains. Phylogenetic analysis using the F gene full-length sequences grouped all the Argentinean CDV strains in the SA2 clade. Sequence identity with the Onderstepoort vaccine strain was 89.0-90.6%, and the highest divergence was found in the 135 amino acids corresponding to the F protein signal-peptide, Fsp (64.4-66.7% identity). In contrast, this region was highly conserved among the local strains (94.1-100% identity). One extra putative N-glycosylation site was identified in the F gene of CDV Argentinean strains with respect to the vaccine strain. The present report is the first to analyze full-length F protein sequences of CDV strains circulating in Argentina, and contributes to the knowledge of molecular epidemiology of CDV, which may help in understanding future disease outbreaks. PMID:26611227

  5. A novel full-length gene of human ribosomal protein L14.22 related to human glioma

    Institute of Scientific and Technical Information of China (English)

    QI Zhen-yu; HUI Guo-zhen; LI Yao; ZHOU Zong-xiang; GU Shao-hua; XIE Yi

    2006-01-01

    Background This study was undertaken to obtain differentially expressed genes related to human glioma by cDNA microarray and the characterization of a novel full-length gene. Methods Total RNA was extracted from human glioma and normal brain tissues, and mRNA was used as a probe. The results of hybridization procedure were scanned with the computer system. The gene named 507E08clone was subsequently analyzed by northern blot, bioinformatic approach, and protein expression.Results Fifteen differentially expressed genes were obtained from human glioma by hybridization and scanning for four times. Northern blot analysis confirmed that the 507E08 clone was low expressed in human brain tissue and over expressed in human glioma tissues. The analysis of BLASTn and BLASTx showed that the 507E08clone was a novel full-length gene, which codes 203 amino acid of protein and is called human ribosomal protein 14.22 gene. The nucleotide sequence had been submitted to the GenBankTM with the accession number of AF329277. After expression in E. Coli., protein yielded a major band of apparent molecular mass 22 kDa on an SDS-PAGE gel.Conclusions cDNA microarray technology can be successfully used to identify differentially expressed genes.The novel full-length gene of human ribosomal protein 14.22 may be correlated with the development of human glioma.

  6. A NEW METHOD TO CONSTRUCT A FULL-LENGTH cDNA LIBRARY OF HUMAN NORMAL BLADDER TISSUE

    Institute of Scientific and Technical Information of China (English)

    成瑜; 李旭; 陈葳; 杨玉琮; 赵乐

    2003-01-01

    Objective Using template-switch mechanism at the 5'-end of mRNA technique (SMART) to construct a full-length cDNA library of human normal bladder tissue. Methods The novel procedures used the template-switching activity of powerscript reverse transcriptase to synthesize and anchor first-strand cDNA in one step. Following reverse transcription, 5 cycles of PCR were performed using a modified oligo(dT) primer and an anchor primer to enrich the full-length cDNA population with 1.0 g human normal bladder poly(A)+RNA, then double-strand cDNA was synthesized. After digestion with sfiI and size-fractionation by CHROMA SPIN-400 columns, double-strand cDNA was ligated into λTripIEx2 vector and was packaged. We determined the titer of the primary library and the percentage of recombinant clones and finally amplified the library. Results The titer of the cDNA library constructed was 2.1×106 pfu*mL-1, and the amplified cDNA library was 6×1011 pfu*mL-1, the percentage of recombination clones was 99%. Conclusion Using SMART technique helps us to construct full-length cDNA library with high efficiency and high capacity which lays solid foundation for screening target genes of bladder diseases with probes and antibodies.

  7. Identification of 48 full-length MHC-DAB functional alleles in miiuy croaker and evidence for positive selection.

    Science.gov (United States)

    Liu, Jiang; Sun, Yueyan; Xu, Tianjun

    2016-07-01

    Major histocompatibility complex (MHC) molecules play a vital role in the immune response and are a highly polymorphic gene superfamily in vertebrates. As the molecular marker associated with polymorphism and disease susceptibility/resistance, the polymorphism of MHC genes has been investigated in many tetrapods and teleosts. Most studies were focused on the polymorphism of the second exon, which encodes the peptide-binding region (PBR) in the α1- or β1-domain, but few studies have examined the full-length coding region. To comprehensive investigate the polymorphism of MHC gene, we identified 48 full-length miiuy croaker (Miichthys miiuy) MHC class IIB (Mimi-DAB) functional alleles from 26 miiuy croaker individuals. All of the alleles encode 34 amino acid sequences, and a high level of polymorphism was detected in Mimi-DAB alleles. The rate of non-synonymous substitutions (dN) occurred at a significantly higher frequency than that of synonymous substitutions (dS) in the PBR, and this result suggests that balancing selection maintains polymorphisms at the Mimi-DAB locus. Phylogenetic analysis based on the full-length and exon 2 sequences of Mimi-DAB alleles both showed that the Mimi-DAB alleles were clustered into two major groups. A total of 19 positive selected sites were identified on the Mimi-DAB alleles after testing for positive selection, and 14 sites were predicted to be associated with antigen-binding sites, which suggests that most of selected sites are significant for disease resistance. The polymorphism of Mimi-DAB alleles provides an important resource for analyzing the association between the polymorphism of MHC gene and disease susceptibility/resistance, and for researching the molecular selective breeding of miiuy croaker with enhanced disease resistance. PMID:27164216

  8. Generation and Analysis of Expressed Sequence Tags (ESTs) from Muscle Full-Length cDNA Library of Wujin Pig

    Institute of Scientific and Technical Information of China (English)

    ZHAO Su-mei; LIU Yong-gang; PAN Hong-bing; ZHANG Xi; GE Chang-rong; JIA Jun-jing; GAO Shi-zheng

    2014-01-01

    Porcine skeletal muscle genes play a major role in determining muscle growth and meat quality. Construction of a full-length cDNA library is an effective way to understand the expression of functional genes in muscle tissues. In addition, novel genes for further research could be identiifed in the library. In this study, we constructed a full-length cDNA library from porcine muscle tissue. The estimated average size of the cDNA inserts was 1076 bp, and the cDNA fullness ratio was 86.2%. A total of 1058 unique sequences with 342 contigs (32.3%) and 716 singleton (67.7%) expressed sequence tags (EST) were obtained by clustering and assembling. Meanwhile, 826 (78.1%) ESTs were categorized as known genes, and 232 (21.9%) ESTs were categorized as unknown genes. 65 novel porcine genes that exhibit no identity in the TIGR gene index ofSus scrofa and 124 full-length sequences with unknown functions were deposited in the dbEST division of GenBank (accession numbers: EU650784-EU650788, GE843306, GH228978-GH229100). The abundantly expressed genes in porcine muscle tissue were related to muscle ifber development, energy metabolism and protein synthesis. Gene ontology analysis showed that sequences expressed in porcine muscle tissue contained a high percentage of binding activity, catalytic activity, structural molecule activity and motor activity, which involved mainly in metabolic, cellular and developmental process, distributed mainly in intracellular region. The sequence data generated in this study would provide valuable information for identifying porcine genes expressed in muscle tissue and help to advance the study on the structure and function of genes in pigs.

  9. Molecular Characterization of Full-length Genome of Japanese Encephalitis Virus Genotype V Isolated from Tibet, China

    Institute of Scientific and Technical Information of China (English)

    LI Ming Hua; FU Shi Hong; CHEN Wei Xin; WANG Huan Yu; CAO Yu Xi; LIANG Guo Dong

    2014-01-01

    Objective To determine the molecular characterization of full-length genome of Japanese encephalitis virus (JEV) genotype V. Methods The full-length nucleotide sequences of JEV strains isolated from different locations and sources were used in sequence and phylogenetic analysis. Results The full-length genome of genotypes V JEV, XZ0934, and Muar strain were composed of 10 983 and 10 988 nucleotides respectively and shared a lower level of identity with JEV genotypes I-IV, ranging from 78.4% (G I, KV1899) to 79.7% (G III, JaGAr01), for the nucleotide sequences, and from 90.0%(G I, KV1899) to 91.8%(G III, JaGAr01) for the amino acid sequences. The open reading frame (ORF) of JEV genotype V spanned nucleotides 96 to 10 397 and encoded 3 433 amino acids. Interestingly, a comparison with JEV genotype I-IV revealed that 3 nucleotides (encoded with a serine residue) were inserted in the NS4A gene of JEV genotype V, and the insertion of nucleotides was also found in downstream of the ORF stop codon in 3’-untranslated region. Moreover, numerous amino acid mutations were observed in 3 functional domains of the E gene of JEV genotype V. Conclusion The molecular characterization of JEV genotype V is significantly different from that of the known genotypes I-IV. The mutations located in the coding region and the non-coding region may be molecular markers of JEV genotype V and warrant further studies to determine their effects on biology and immunogenicity of genotype V strains.

  10. Isolation of a full-length mouse cDNA clone coding for an immunologically distinct p53 molecule.

    OpenAIRE

    Wolf, D; Harris, N; Goldfinger, N; Rotter, V

    1985-01-01

    Transfection of a cloned p53 gene into a p53 nonproducer Abelson murine leukemia virus-transformed cell line, L12, reconstituted p53 expression. The protein expressed in these cells was indistinguishable from that naturally expressed in p53 producer tumor cells. Conversely, p53 protein expressed in L12-derived clones that were established by transfection with a full-length p53 cDNA clone (pM8) exhibited a discrete immunological form. Immunoprecipitation of p53 with a panel of monoclonal anti-...

  11. THE RELATIONSHIPS AMONG SPATIOTEMPORAL COLLAGEN GENE EXPRESSION, HISTOLOGY, AND BIOMECHANICS FOLLOWING FULL-LENGTH INJURY IN THE MURINE PATELLAR TENDON

    OpenAIRE

    Dyment, Nathaniel A; Kazemi, Namdar; Lindsey E Aschbacher-Smith; Barthelery, Nicolas J.; Kenter, Keith; Gooch, Cynthia; Jason T Shearn; Wylie, Christopher; David L Butler

    2011-01-01

    Tendon injuries are major orthopaedic problems that worsen as the population ages. Type-I (Col1) and type-II (Col2) collagens play important roles in tendon midsubstance and tendon-to-bone insertion healing, respectively. Using double transgenic mice, this study aims to spatiotemporally monitor Col1 and Col2 gene expression, histology and biomechanics up to 8 weeks following a full-length patellar tendon injury. Gene expression and histology were analyzed weekly for up to 5 weeks while mechan...

  12. Full-length huntingtin levels modulate body weight by influencing insulin-like growth factor 1 expression

    OpenAIRE

    Pouladi, Mahmoud A.; Xie, Yuanyun; Skotte, Niels Henning; Ehrnhoefer, Dagmar E.; Graham, Rona K.; Kim, Jeong Eun; Bissada, Nagat; Yang, X. William; Paganetti, Paolo; Friedlander, Robert M.; Leavitt, Blair R.; Hayden, Michael R

    2010-01-01

    Levels of full-length huntingtin (FL htt) influence organ and body weight, independent of polyglutamine length. The growth hormone-insulin like growth factor-1 (GH-IGF-1) axis is well established as a regulator of organ growth and body weight. In this study, we investigate the involvement of the IGF-1 pathway in mediating the effect of htt on body weight. IGF-1 expression was examined in transgenic mouse lines expressing different levels of FL wild-type (WT) htt (YAC18 mice), FL mutant htt (Y...

  13. Recovery of infectious virus from full-length cowpox virus (CPXV) DNA cloned as a bacterial artificial chromosome (BAC)

    OpenAIRE

    Roth Swaantje J; Höper Dirk; Beer Martin; Feineis Silke; Tischer B Karsten; Osterrieder Nikolaus

    2011-01-01

    Abstract Transmission from pet rats and cats to humans as well as severe infection in felids and other animal species have recently drawn increasing attention to cowpox virus (CPXV). We report the cloning of the entire genome of cowpox virus strain Brighton Red (BR) as a bacterial artificial chromosome (BAC) in Escherichia coli and the recovery of infectious virus from cloned DNA. Generation of a full-length CPXV DNA clone was achieved by first introducing a mini-F vector, which allows mainte...

  14. The duplicated α7 subunits assemble and form functional nicotinic receptors with the full-length α7

    OpenAIRE

    Wang, Ying; Xiao, Cheng; Indersmitten, Tim; Freedman, Robert; Leonard, Sherry; Lester, Henry A.

    2014-01-01

    The α7 nicotinic acetylcholine receptor gene (CHRNA7) is linked to schizophrenia. A partial duplication of CHRNA7 (CHRFAM7A) is found in humans on 15q13-14. Exon 6 of CHRFAM7A harbors a 2 base pair deletion polymorphism, CHRFAM7AΔ2bp, which is also associated with schizophrenia. To understand the effects of the duplicated subunits on α7 receptors, we fused α7, dupα7, and dupΔα7 subunits with various fluorescent proteins. The duplicated subunits co-localized with full-length α7 subunits in mou...

  15. Cloning and Expression of Highly Pathogenic Avian Influenza Virus Full-Length Nonstructural Gene in Pichia pastoris

    OpenAIRE

    Abubakar, M. B.; I. Aini; Omar, A. R.; Hair-Bejo, M

    2011-01-01

    Avian influenza (AI) is a highly contagious and rapidly evolving pathogen of major concern to the poultry industry and human health. Rapid and accurate detection of avian influenza virus is a necessary tool for control of outbreaks and surveillance. The AI virus A/Chicken/Malaysia/5858/2004 (H5N1) was used as a template to produce DNA clones of the full-length NS1 genes via reverse transcriptase synthesis of cDNA by PCR amplification of the NS1 region. Products were cloned into pCR2.0 TOPO TA...

  16. [Rapid site-directed mutagenesis on full-length plasmid DNA by using designed restriction enzyme assisted mutagenesis].

    Science.gov (United States)

    Zhang, Baozhong; Ran, Duoliang; Zhang, Xin; An, Xiaoping; Shan, Yunzhu; Zhou, Yusen; Tong, Yigang

    2009-02-01

    To use the designed restriction enzyme assisted mutagenesis technique to perform rapid site-directed mutagenesis on double-stranded plasmid DNA. The target amino acid sequence was reversely translated into DNA sequences with degenerate codons, resulting in large amount of silently mutated sequences containing various restriction endonucleases (REs). Certain mutated sequence with an appropriate RE was selected as the target DNA sequence for designing mutation primers. The full-length plasmid DNA was amplified with high-fidelity Phusion DNA polymerase and the amplified product was 5' phosphorylated by T4 polynucleotide kinase and then self-ligated. After transformation into an E. coli host the transformants were rapidly screened by cutting with the designed RE. With this strategy we successfully performed the site-directed mutagenesis on an 8 kb plasmid pcDNA3.1-pIgR and recovered the wild-type amino acid sequence of human polymeric immunoglobulin receptor (pIgR). A novel site-directed mutagenesis strategy based on DREAM was developed which exploited RE as a rapid screening measure. The highly efficient, high-fidelity Phusion DNA polymerase was applied to ensure the efficient and faithful amplification of the full-length sequence of a plasmid of up to 8 kb. This rapid mutagenesis strategy avoids using any commercial site-directed mutagenesis kits, special host strains or isotopes. PMID:19459340

  17. A comparative phylogenetic analysis of full-length mariner elements isolated from the Indian tasar silkmoth, Antheraea mylitta (Lepidoptera: saturniidae)

    Indian Academy of Sciences (India)

    M Dharma Prasad; J Nagaraju

    2003-06-01

    Mariner like elements (MLEs) are widely distributed type II transposons with an open reading frame (ORF) for transposase. We studied comparative phylogenetic evolution and inverted terminal repeat (ITR) conservation of MLEs from Indian saturniid silkmoth, Antheraea mylitta with other full length MLEs submitted in the database. Full length elements from A. mylitta were inactive with multiple mutations. Many conserved amino acid blocks were identified after aligning transposase sequences. Mariner signature sequence, DD(34)D was almost invariable although a few new class of elements had different signatures. A. mylitta MLEs (Anmmar) get phylogenetically classified under cecropia subfamily and cluster closely with the elements from other Bombycoidea superfamily members implying vertical transmission from a common ancestor. ITR analysis showed a conserved sequence of AGGT(2-8N)ATAAGT for forward repeat and AGGT(2-8N)ATGAAAT for reverse repeat. These results and additional work may help us to understand the dynamics of MLE distribution in A. mylitta and construction of appropriate vectors for mariner mediated transgenics.

  18. Analysis of the mouse gut microbiome using full-length 16S rRNA amplicon sequencing.

    Science.gov (United States)

    Shin, Jongoh; Lee, Sooin; Go, Min-Jeong; Lee, Sang Yup; Kim, Sun Chang; Lee, Chul-Ho; Cho, Byung-Kwan

    2016-01-01

    Demands for faster and more accurate methods to analyze microbial communities from natural and clinical samples have been increasing in the medical and healthcare industry. Recent advances in next-generation sequencing technologies have facilitated the elucidation of the microbial community composition with higher accuracy and greater throughput than was previously achievable; however, the short sequencing reads often limit the microbial composition analysis at the species level due to the high similarity of 16S rRNA amplicon sequences. To overcome this limitation, we used the nanopore sequencing platform to sequence full-length 16S rRNA amplicon libraries prepared from the mouse gut microbiota. A comparison of the nanopore and short-read sequencing data showed that there were no significant differences in major taxonomic units (89%) except one phylotype and three taxonomic units. Moreover, both sequencing data were highly similar at all taxonomic resolutions except the species level. At the species level, nanopore sequencing allowed identification of more species than short-read sequencing, facilitating the accurate classification of the bacterial community composition. Therefore, this method of full-length 16S rRNA amplicon sequencing will be useful for rapid, accurate and efficient detection of microbial diversity in various biological and clinical samples. PMID:27411898

  19. Characterization of 40 full-length MHC class IIA functional alleles in miiuy croaker: Polymorphism and positive selection.

    Science.gov (United States)

    Xu, Tianjun; Liu, Jiang; Sun, Yueyan; Zhu, Zhihuang; Liu, Tianxing

    2016-02-01

    The major histocompatibility complex is a highly polymorphic gene superfamily in vertebrates that plays an important role in adaptive immune response. In the present study, we identified 40 full-length miiuy croaker MHC class IIA (Mimi-DAA) functional alleles from 26 miiuy croaker individuals and found that the alleles encode 30 amino acid sequences. A high level of polymorphism in Mimi-DAA was detected in miiuy croaker. The rate of non-synonymous substitutions (d(N)) occurred at a significantly higher frequency than that of synonymous substitutions (d(S)) in the peptide-binding region (PBR) and non-PBR. This result suggests that balancing selection maintains polymorphisms at the Mimi-DAA locus. Phylogenetic analysis based on the full-length sequences showed that the Mimi-DAA alleles clustered into three groups. However, the phylogenetic tree constructed using the exon 2 sequences indicated that the Mimi-DAA alleles clustered into two groups. A total of 22 positively selected sites were identified on the Mimi-DAA alleles after testing for positive selection, and five sites were predicted to be associated with the binding of peptide antigen, suggesting that a few selected residues may play a significant role in immune function. PMID:26598111

  20. Human uroporphyrinogen III synthase: Molecular cloning, nucleotide sequence, and expression of a full-length cDNA

    International Nuclear Information System (INIS)

    Uroporphyrinogen III synthase, the fourth enzyme in the heme biosynthetic pathway, is responsible for conversion of the linear tetrapyrrole, hydroxymethylbilane, to the cyclic tetrapyrrole, uroporphyrinogen III. The deficient activity of URO-synthase is the enzymatic defect in the autosomal recessive disorder congenital erythropoietic porphyria. To facilitate the isolation of a full-length cDNA for human URO-synthase, the human erythrocyte enzyme was purified to homogeneity and 81 nonoverlapping amino acids were determined by microsequencing the N terminus and four tryptic peptides. Two synthetic oligonucleotide mixtures were used to screen 1.2 x 106 recombinants from a human adult liver cDNA library. Eight clones were positive with both oligonucleotide mixtures. Of these, dideoxy sequencing of the 1.3 kilobase insert from clone pUROS-2 revealed 5' and 3' untranslated sequences of 196 and 284 base pairs, respectively, and an open reading frame of 798 base pairs encoding a protein of 265 amino acids with a predicted molecular mass of 28,607 Da. The isolation and expression of this full-length cDNA for human URO-synthase should facilitate studies of the structure, organization, and chromosomal localization of this heme biosynthetic gene as well as the characterization of the molecular lesions causing congenital erythropoietic porphyria

  1. Human uroporphyrinogen III synthase: Molecular cloning, nucleotide sequence, and expression of a full-length cDNA

    Energy Technology Data Exchange (ETDEWEB)

    Tsai, Shihfeng; Bishop, D.F.; Desnick, R.J. (Mount Sinai School of Medicine, New York, NY (USA))

    1988-10-01

    Uroporphyrinogen III synthase, the fourth enzyme in the heme biosynthetic pathway, is responsible for conversion of the linear tetrapyrrole, hydroxymethylbilane, to the cyclic tetrapyrrole, uroporphyrinogen III. The deficient activity of URO-synthase is the enzymatic defect in the autosomal recessive disorder congenital erythropoietic porphyria. To facilitate the isolation of a full-length cDNA for human URO-synthase, the human erythrocyte enzyme was purified to homogeneity and 81 nonoverlapping amino acids were determined by microsequencing the N terminus and four tryptic peptides. Two synthetic oligonucleotide mixtures were used to screen 1.2 {times} 10{sup 6} recombinants from a human adult liver cDNA library. Eight clones were positive with both oligonucleotide mixtures. Of these, dideoxy sequencing of the 1.3 kilobase insert from clone pUROS-2 revealed 5' and 3' untranslated sequences of 196 and 284 base pairs, respectively, and an open reading frame of 798 base pairs encoding a protein of 265 amino acids with a predicted molecular mass of 28,607 Da. The isolation and expression of this full-length cDNA for human URO-synthase should facilitate studies of the structure, organization, and chromosomal localization of this heme biosynthetic gene as well as the characterization of the molecular lesions causing congenital erythropoietic porphyria.

  2. Full length cDNA cloning and expression analysis of annexinA2 gene from deer antler tissue

    Institute of Scientific and Technical Information of China (English)

    Li Hao; Xianghong Xiao; Heping Li

    2014-01-01

    ANXA2(AnnexinA2), a calcium-dependent phospholipid bind-ing protein, is involved in various Ca2+-related biological activities. In the present study, full-length cDNA of ANXA2 was isolated from the velvet antler tip tissue of sika deer (Cervus nippon hortulorum);the amino acid sequence and gene expression was analyzed by using bioinformatics and real-time reverse transcriptase polymerase chain reaction (RT-PCR) techniques. Nucleotide sequence analysis reveals that the full-length cDNA of the ANXA2 gene was 1372 bp, of which 1020 bp was in the open-reading frame (ORF) encoding 339 amino acids; its relative mo-lecular weight was 38.3 kDa; and isoelectric point was 6.72. Sequence analysis indicates that the protein includes four conserved tan-dem-duplication ANX domains. The gene-accession nucleotide sequence number in GenBank is JX315571. Expression analysis by RT-PCR re-veals that ANXA2 gene expression has a significant positive correlation with the antler-tissue mineralization process, indicating that this gene may play an important role in the regulation of antler-tissue mineraliza-tion.

  3. Crystallization and preliminary X-ray crystallographic analysis of full-length yeast tropomyosin 2 from Saccharomyces cerevisiae

    International Nuclear Information System (INIS)

    Crystals of full-length yeast tropomyosin 2 from S. cerevisiae have been obtained. Tropomyosin is a highly conserved actin-binding protein that is found in most eukaryotic cells. It is critical for actin-filament stabilization and for cooperative regulation of many actin functions. Detailed structural information on tropomyosin is very important in order to understand the mechanisms of its action. Whereas structures of isolated tropomyosin fragments have been obtained at high resolution, the atomic structure of the entire tropomyosin molecule is still unknown. Here, the crystallization and preliminary crystallographic analysis of full-length yeast tropomyosin 2 (yTm2) from Saccharomyces cerevisiae are reported. Recombinant yTm2 expressed in Escherichia coli was crystallized using the hanging-drop vapour-diffusion method. The crystals belonged to space group C2, with unit-cell parameters a = 154.8, b = 49.9, c = 104.0 Å, α = γ = 90.0, β = 124.0° and two molecules in the asymmetric unit. A complete native X-ray diffraction data set was collected to 3.5 Å resolution using synchrotron radiation

  4. Calnexin and BiP act as sequential molecular chaperones during thyroglobulin folding in the endoplasmic reticulum

    OpenAIRE

    1995-01-01

    Before secretion, newly synthesized thyroglobulin (Tg) folds via a series of intermediates: disulfide-linked aggregates and unfolded monomers-->folded monomers-->dimers. Immediately after synthesis, very little Tg associated with calnexin (a membrane-bound molecular chaperone in the ER), while a larger fraction bound BiP (a lumenal ER chaperone); dissociation from these chaperones showed superficially similar kinetics. Calnexin might bind selectively to carbohydrates within glycoproteins, or ...

  5. Integrative annotation of 21,037 human genes validated by full-length cDNA clones.

    Directory of Open Access Journals (Sweden)

    Tadashi Imanishi

    2004-06-01

    Full Text Available The human genome sequence defines our inherent biological potential; the realization of the biology encoded therein requires knowledge of the function of each gene. Currently, our knowledge in this area is still limited. Several lines of investigation have been used to elucidate the structure and function of the genes in the human genome. Even so, gene prediction remains a difficult task, as the varieties of transcripts of a gene may vary to a great extent. We thus performed an exhaustive integrative characterization of 41,118 full-length cDNAs that capture the gene transcripts as complete functional cassettes, providing an unequivocal report of structural and functional diversity at the gene level. Our international collaboration has validated 21,037 human gene candidates by analysis of high-quality full-length cDNA clones through curation using unified criteria. This led to the identification of 5,155 new gene candidates. It also manifested the most reliable way to control the quality of the cDNA clones. We have developed a human gene database, called the H-Invitational Database (H-InvDB; http://www.h-invitational.jp/. It provides the following: integrative annotation of human genes, description of gene structures, details of novel alternative splicing isoforms, non-protein-coding RNAs, functional domains, subcellular localizations, metabolic pathways, predictions of protein three-dimensional structure, mapping of known single nucleotide polymorphisms (SNPs, identification of polymorphic microsatellite repeats within human genes, and comparative results with mouse full-length cDNAs. The H-InvDB analysis has shown that up to 4% of the human genome sequence (National Center for Biotechnology Information build 34 assembly may contain misassembled or missing regions. We found that 6.5% of the human gene candidates (1,377 loci did not have a good protein-coding open reading frame, of which 296 loci are strong candidates for non-protein-coding RNA

  6. Integrative Annotation of 21,037 Human Genes Validated by Full-Length cDNA Clones

    Directory of Open Access Journals (Sweden)

    Imanishi Tadashi

    2004-01-01

    Full Text Available The human genome sequence defines our inherent biological potential; the realization of the biology encoded therein requires knowledge of the function of each gene. Currently, our knowledge in this area is still limited. Several lines of investigation have been used to elucidate the structure and function of the genes in the human genome. Even so, gene prediction remains a difficult task, as the varieties of transcripts of a gene may vary to a great extent. We thus performed an exhaustive integrative characterization of 41,118 full-length cDNAs that capture the gene transcripts as complete functional cassettes, providing an unequivocal report of structural and functional diversity at the gene level. Our international collaboration has validated 21,037 human gene candidates by analysis of high-quality full-length cDNA clones through curation using unified criteria. This led to the identification of 5,155 new gene candidates. It also manifested the most reliable way to control the quality of the cDNA clones. We have developed a human gene database, called the H-Invitational Database (H-InvDB; http://www.h-invitational.jp/. It provides the following: integrative annotation of human genes, description of gene structures, details of novel alternative splicing isoforms, non-protein-coding RNAs, functional domains, subcellular localizations, metabolic pathways, predictions of protein three-dimensional structure, mapping of known single nucleotide polymorphisms (SNPs, identification of polymorphic microsatellite repeats within human genes, and comparative results with mouse full-length cDNAs. The H-InvDB analysis has shown that up to 4% of the human genome sequence (National Center for Biotechnology Information build 34 assembly may contain misassembled or missing regions. We found that 6.5% of the human gene candidates (1,377 loci did not have a good protein-coding open reading frame, of which 296 loci are strong candidates for non-protein-coding RNA

  7. Full-Length Fibronectin Drives Fibroblast Accumulation at the Surface of Collagen Microtissues during Cell-Induced Tissue Morphogenesis.

    Science.gov (United States)

    Foolen, Jasper; Shiu, Jau-Ye; Mitsi, Maria; Zhang, Yang; Chen, Christopher S; Vogel, Viola

    2016-01-01

    Generating and maintaining gradients of cell density and extracellular matrix (ECM) components is a prerequisite for the development of functionality of healthy tissue. Therefore, gaining insights into the drivers of spatial organization of cells and the role of ECM during tissue morphogenesis is vital. In a 3D model system of tissue morphogenesis, a fibronectin-FRET sensor recently revealed the existence of two separate fibronectin populations with different conformations in microtissues, i.e. 'compact and adsorbed to collagen' versus 'extended and fibrillar' fibronectin that does not colocalize with the collagen scaffold. Here we asked how the presence of fibronectin might drive this cell-induced tissue morphogenesis, more specifically the formation of gradients in cell density and ECM composition. Microtissues were engineered in a high-throughput model system containing rectangular microarrays of 12 posts, which constrained fibroblast-populated collagen gels, remodeled by the contractile cells into trampoline-shaped microtissues. Fibronectin's contribution during the tissue maturation process was assessed using fibronectin-knockout mouse embryonic fibroblasts (Fn-/- MEFs) and floxed equivalents (Fnf/f MEFs), in fibronectin-depleted growth medium with and without exogenously added plasma fibronectin (full-length, or various fragments). In the absence of full-length fibronectin, Fn-/- MEFs remained homogenously distributed throughout the cell-contracted collagen gels. In contrast, in the presence of full-length fibronectin, both cell types produced shell-like tissues with a predominantly cell-free compacted collagen core and a peripheral surface layer rich in cells. Single cell assays then revealed that Fn-/- MEFs applied lower total strain energy on nanopillar arrays coated with either fibronectin or vitronectin when compared to Fnf/f MEFs, but that the presence of exogenously added plasma fibronectin rescued their contractility. While collagen decoration of

  8. Effect of the electrostatic surface potential on the oligomerization of full-length human recombinant prion protein at single-molecule level

    Science.gov (United States)

    Wang, Bin; Lou, Zhichao; Zhang, Haiqian; Xu, Bingqian

    2016-03-01

    The electrostatic surface potential (ESP) of prion oligomers has critical influences on the aggregating processes of the prion molecules. The atomic force microscopy (AFM) and structural simulation were combined to investigate the molecular basis of the full-length human recombinant prion oligomerization on mica surfaces. The high resolution non-intrusive AFM images showed that the prion oligomers formed different patterns on mica surfaces at different buffer pH values. The basic binding units for the large oligomers were determined to be prion momoners (Ms), dimers (Ds), and trimers (Ts). The forming of the D and T units happened through the binding of hydrophobic β-sheets of the M units. In contrast, the α-helices of these M, D, and T units were the binding areas for the formation of large oligomers. At pH 4.5, the binding units M, D, and T showed clear polarized ESP distributions on the surface domains, while at pH 7.0, they showed more evenly distributed ESPs. Based on the conformations of oligomers observed from AFM images, the D and T units were more abundantly on mica surface at pH 4.5 because the ESP re-distribution of M units helped to stabilize these larger oligomers. The amino acid side chains involved in the binding interfaces were stabilized by hydrogen bonds and electrostatic interactions. The detailed analysis of the charged side chains at pH 4.5 indicated that the polarized ESPs induced the aggregations among M, D, and T to form larger oligomers. Therefore, the hydrogen bonds and electrostatic interactions worked together to form the stabilized prion oligomers.

  9. 5'-end sequences of budding yeast full-length cDNA clones - Budding yeast cDNA sequencing project | LSDB Archive [Life Science Database Archive metadata

    Lifescience Database Archive (English)

    Full Text Available Budding yeast cDNA sequencing project 5'-end sequences of budding yeast full-length cDNA clones Data detail Data name 5'-end sequence...s of budding yeast full-length cDNA clones Description of data contents cDNA sequence...e Update History of This Database Site Policy | Contact Us 5'-end sequences of budding yeast full-length cDNA clones - Budding yeast cDNA sequencing project | LSDB Archive ...

  10. Internalization of the Extracellular Full-Length Tau Inside Neuro2A and Cortical Cells Is Enhanced by Phosphorylation.

    Science.gov (United States)

    Wauters, Mathilde; Wattiez, Ruddy; Ris, Laurence

    2016-01-01

    Tau protein is mainly intracellular. However, several studies have demonstrated that full-length Tau can be released into the interstitial fluid of the brain. The physiological or pathological function of this extracellular Tau remains unknown. Moreover, as evidence suggests, extracellular Tau aggregates can be internalized by neurons, seeding Tau aggregation. However, much less is known about small species of Tau. In this study, we hypothesized that the status of phosphorylation could alter the internalization of recombinant Tau in Neuro2A and cortical cells. Our preliminary results revealed that the highly phosphorylated form of Tau entered the cells ten times more easily than a low phosphorylated one. This suggests that hyperphosphorylated Tau protein could spread between neurons in pathological conditions such as Alzheimer's disease. PMID:27548242

  11. High yield purification of full-length functional hERG K+ channels produced in Saccharomyces cerevisiae

    DEFF Research Database (Denmark)

    Molbaek, Karen; Scharff-Poulsen, Peter; Hélix-Nielsen, Claus;

    2015-01-01

    The hERG potassium channel is essential for repolarization of the cardiac action potential. Due to this vital function, absence of unintended and potentially life-threatening interactions with hERG is required for approval of new drugs. The structure of hERG is therefore one of the most sought......-after. To provide purified hERG for structural studies and new hERG biomimetic platforms for detection of undesirable interactions, we have developed a hERG expression platform generating unprecedented amounts of purified and functional hERG channels. Full-length hERG, with or without a C-terminally fused...... green fluorescent protein (GFP) His(8)-tag was produced from a codon-optimized hERG cDNA in Saccharomyces cerevisiae. Both constructs complemented the high potassium requirement of a knock-out Saccharomyces cerevisiae strain, indicating correct tetramer assembly in vivo. Functionality was further...

  12. Full-length huntingtin levels modulate body weight by influencing insulin-like growth factor 1 expression

    DEFF Research Database (Denmark)

    Pouladi, Mahmoud A; Xie, Yuanyun; Skotte, Niels Henning;

    2010-01-01

    Levels of full-length huntingtin (FL htt) influence organ and body weight, independent of polyglutamine length. The growth hormone-insulin like growth factor-1 (GH-IGF-1) axis is well established as a regulator of organ growth and body weight. In this study, we investigate the involvement...... of the IGF-1 pathway in mediating the effect of htt on body weight. IGF-1 expression was examined in transgenic mouse lines expressing different levels of FL wild-type (WT) htt (YAC18 mice), FL mutant htt (YAC128 and BACHD mice) and truncated mutant htt (shortstop mice). We demonstrate that htt influences...... body weight by modulating the IGF-1 pathway. Plasma IGF-1 levels correlate with body weight and htt levels in the transgenic YAC mice expressing human htt. The effect of htt on IGF-1 expression is independent of CAG size. No effect on body weight is observed in transgenic YAC mice expressing...

  13. Construction of Full-length cDNA Library of Non-diapause Pupae of the Onion Maggot, Delia antiqua

    Directory of Open Access Journals (Sweden)

    CHEN Bin

    2010-01-01

    Full Text Available The onion maggot, Delia antiqua has the characteristic of summer- and winter-diapause, and is close to Drosophila Melanogaster in phelogenetics. It is an ideal model species for the studies of the molecular mechanism of insect diapause and the comparison of winter- and summer-diapause-specific genes. The study aims to construct full-length cDNA library of summer-diapause pupae of the onion maggot, Delia antiqua, in order to play a base for further screening, cloning and expression analysis of diapause-specific genes. In this study, total RNA was extracted from non-diapause pupae of onion maggot, D. antiqua using RNAiso. Double-stranded cDNAs were synthesized with SMART technique and digested by SfiⅠ, and then the cDNAs were ligated into the vector pDNR-LIB. The ligation mixture was transformed into E. coli DH10B by eletroporation. According to the evaluation on quality, the titer of primary library was 2.3×107 cfu/mL. The results from random picking 15 clones showed that the inserted fragments ranged from 0.4 to 1.2 kb by PCR amplification, with an average size of 0.9 kb, and the recombination rate was 100 %. These results showed that a full-length cDNA library with high quality on Delia antiqua non-diapause pupae was well constructed. This indicates that the library is of high quality for cloning target genes and expressing target proteins.

  14. A Novel mouse model of enhanced proteostasis: Full-length human heat shock factor 1 transgenic mice

    Energy Technology Data Exchange (ETDEWEB)

    Pierce, Anson, E-mail: piercea2@uthscsa.edu [Department of Cellular and Structural Biology, The University of Texas Health Science Center at San Antonio, San Antonio, Texas, 78229 (United States); Barshop Institute for Longevity and Aging Studies, The University of Texas Health Science Center at San Antonio, San Antonio, Texas, 78229 (United States); The Department of Veteran' s Affairs, South Texas Veterans Health Care System, San Antonio, Texas, 78284 (United States); Wei, Rochelle; Halade, Dipti [Barshop Institute for Longevity and Aging Studies, The University of Texas Health Science Center at San Antonio, San Antonio, Texas, 78229 (United States); Yoo, Si-Eun [Department of Cellular and Structural Biology, The University of Texas Health Science Center at San Antonio, San Antonio, Texas, 78229 (United States); Barshop Institute for Longevity and Aging Studies, The University of Texas Health Science Center at San Antonio, San Antonio, Texas, 78229 (United States); Ran, Qitao; Richardson, Arlan [Department of Cellular and Structural Biology, The University of Texas Health Science Center at San Antonio, San Antonio, Texas, 78229 (United States); Barshop Institute for Longevity and Aging Studies, The University of Texas Health Science Center at San Antonio, San Antonio, Texas, 78229 (United States); The Department of Veteran' s Affairs, South Texas Veterans Health Care System, San Antonio, Texas, 78284 (United States)

    2010-11-05

    Research highlights: {yields} Development of mouse overexpressing native human HSF1 in all tissues including CNS. {yields} HSF1 overexpression enhances heat shock response at whole-animal and cellular level. {yields} HSF1 overexpression protects from polyglutamine toxicity and favors aggresomes. {yields} HSF1 overexpression enhances proteostasis at the whole-animal and cellular level. -- Abstract: The heat shock response (HSR) is controlled by the master transcriptional regulator heat shock factor 1 (HSF1). HSF1 maintains proteostasis and resistance to stress through production of heat shock proteins (HSPs). No transgenic model exists that overexpresses HSF1 in tissues of the central nervous system (CNS). We generated a transgenic mouse overexpressing full-length non-mutant HSF1 and observed a 2-4-fold increase in HSF1 mRNA and protein expression in all tissues studied of HSF1 transgenic (HSF1{sup +/0}) mice compared to wild type (WT) littermates, including several regions of the CNS. Basal expression of HSP70 and 90 showed only mild tissue-specific changes; however, in response to forced exercise, the skeletal muscle HSR was more elevated in HSF1{sup +/0} mice compared to WT littermates and in fibroblasts following heat shock, as indicated by levels of inducible HSP70 mRNA and protein. HSF1{sup +/0} cells elicited a significantly more robust HSR in response to expression of the 82 repeat polyglutamine-YFP fusion construct (Q82YFP) and maintained proteasome-dependent processing of Q82YFP compared to WT fibroblasts. Overexpression of HSF1 was associated with fewer, but larger Q82YFP aggregates resembling aggresomes in HSF1{sup +/0} cells, and increased viability. Therefore, our data demonstrate that tissues and cells from mice overexpressing full-length non-mutant HSF1 exhibit enhanced proteostasis.

  15. Recovery of infectious virus from full-length cowpox virus (CPXV DNA cloned as a bacterial artificial chromosome (BAC

    Directory of Open Access Journals (Sweden)

    Roth Swaantje J

    2011-01-01

    Full Text Available Abstract Transmission from pet rats and cats to humans as well as severe infection in felids and other animal species have recently drawn increasing attention to cowpox virus (CPXV. We report the cloning of the entire genome of cowpox virus strain Brighton Red (BR as a bacterial artificial chromosome (BAC in Escherichia coli and the recovery of infectious virus from cloned DNA. Generation of a full-length CPXV DNA clone was achieved by first introducing a mini-F vector, which allows maintenance of large circular DNA in E. coli, into the thymidine kinase locus of CPXV by homologous recombination. Circular replication intermediates were then electroporated into E. coli DH10B cells. Upon successful establishment of the infectious BR clone, we modified the full-length clone such that recombination-mediated excision of bacterial sequences can occur upon transfection in eukaryotic cells. This self-excision of the bacterial replicon is made possible by a sequence duplication within mini-F sequences and allows recovery of recombinant virus progeny without remaining marker or vector sequences. The in vitro growth properties of viruses derived from both BAC clones were determined and found to be virtually indistinguishable from those of parental, wild-type BR. Finally, the complete genomic sequence of the infectious clone was determined and the cloned viral genome was shown to be identical to that of the parental virus. In summary, the generated infectious clone will greatly facilitate studies on individual genes and pathogenesis of CPXV. Moreover, the vector potential of CPXV can now be more systematically explored using this newly generated tool.

  16. Characterization of full-length enterovirus 71 strains from severe and mild disease patients in northeastern China.

    Directory of Open Access Journals (Sweden)

    Xiaomei Wang

    Full Text Available Human enterovirus 71 (EV71-associated hand, foot, and mouth disease (HFMD has been a leading cause of childhood infection in China since 2008. Epidemic and molecular characteristics of HFMD have been examined in many areas of China, including the central and southern regions. However, clinical and genetic characterization of EV71 in the northeastern region of China is scarce. In this study, a series of analyses were performed on seven full-length EV71 sequences from HFMD patients who had either severe or mild disease. We have determined that these seven circulating EV71 viruses from Changchun, China are actually complex recombinant viruses involving multiple type A human enterovirus (HEV. Classified as EV71 subtype C4 (EV71 C4, these Changchun EV71 viruses contain genetic recombination events between the CA4, CA5, EV71B4 and EV71C1 strains. Most of the structural protein region (P1 of these viruses resembled that of the prototype EV71 C1 strains. The non-structural protein domains (P2 and P3 showed a high degree of similarity with CA4, CA5 and EV71 B4 in different regions. The 5'UTR had unclassified recombination,while partial 3D region of these viruses showed a high degree of similarity to CA16. Phylogenetic analysis of full-length or partial sequences of isolates from severe or mild disease patients in Changchun always formed a single cluster in various phylogenetic analyses of different genomic regions, suggesting that all seven strains originated from one single common ancestor. There was no correlation between viral genomic sequence and virulence. Thus, we found that circulating recombinant forms of EV71 are prevalent among HFMD patients in Northeastern China. The existence of a unique cluster of EV71 related viruses in Northeast China has important implications for vaccine development that would address the increasing prevalence of HFMD.

  17. Full-length cDNA cloning, molecular characterization and differential expression analysis of peroxiredoxin 6 from Ovis aries.

    Science.gov (United States)

    Liu, Nan-Nan; Liu, Zeng-Shan; Lu, Shi-Ying; Hu, Pan; Li, Yan-Song; Feng, Xiao-Li; Zhang, Shou-Yin; Wang, Nan; Meng, Qing-Feng; Yang, Yong-Jie; Tang, Feng; Xu, Yun-Ming; Zhang, Wen-Hui; Guo, Xing; Chen, Xiao-Feng; Zhou, Yu; Ren, Hong-Lin

    2015-04-15

    Peroxiredoxin 6 (Prdx6), an important antioxidant enzyme that can eliminate reactive oxygen species (ROS) to maintain homeostasis, is a bifunctional protein that possesses the activities of both glutathione peroxidase and phospholipase A2. In this study, a novel full-length Prdx6 cDNA (OaPrdx6) was cloned from Sheep (Ovis aries) using rapid amplification of cDNA ends (RACE). The full-length cDNA of OaPrdx6 was 1753bp containing a 5'-untranslated region (UTR) of 93bp, a 3'-UTR of 985bp with a poly(A) tail, and an open reading frame (ORF) of 675bp encoding a protein of 224 amino acid residues with a predicted molecular weight of 25.07kDa. The recombinant protein OaPrdx6 was expressed and purified, and its DNA protection activity was identified. In order to analyze the Prdx6 protein expression in tissues from O. aries, monoclonal antibodies against OaPrdx6 were prepared. Western blotting results indicated that OaPrdx6 protein could be detected in heart, liver, spleen, lung, kidney, stomach, intestine, muscle, lymph node and white blood cells, and the highest expression was found in lung while the lowest expression in muscle. Compared to the normal sheep group, the mRNA transcription level of Prdx6 in buffy coat was up-regulated in the group infected with a virulent field strain of Brucella melitensis, and down-regulated in the group inoculated with a vaccine strain S2 of brucellosis. The results indicated that Prdx6 was likely to be involved in the host immune responses against Brucella infection, and probably regarded as a molecular biomarker for distinguishing between animals infected with virulent Brucella infection and those inoculated with vaccine against brucellosis. PMID:25712755

  18. Full-Length cDNA Cloning, Molecular Characterization and Differential Expression Analysis of Lysophospholipase I from Ovis aries

    Directory of Open Access Journals (Sweden)

    Nan-Nan Liu

    2016-07-01

    Full Text Available Lysophospholipase I (LYPLA1 is an important protein with multiple functions. In this study, the full-length cDNA of the LYPLA1 gene from Ovis aries (OaLypla1 was cloned using primers and rapid amplification of cDNA ends (RACE technology. The full-length OaLypla1 was 2457 bp with a 5′-untranslated region (UTR of 24 bp, a 3′-UTR of 1740 bp with a poly (A tail, and an open reading frame (ORF of 693 bp encoding a protein of 230 amino acid residues with a predicted molecular weight of 24,625.78 Da. Phylogenetic analysis showed that the OaLypla1 protein shared a high amino acid identity with LYPLA1 of Bos taurus. The recombinant OaLypla1 protein was expressed and purified, and its phospholipase activity was identified. Monoclonal antibodies (mAb against OaLypla1 that bound native OaLypla1 were generated. Real-time PCR analysis revealed that OaLypla1 was constitutively expressed in the liver, spleen, lung, kidney, and white blood cells of sheep, with the highest level in the kidney. Additionally, the mRNA levels of OaLypla1 in the buffy coats of sheep challenged with virulent or avirulent Brucella strains were down-regulated compared to untreated sheep. The results suggest that OaLypla1 may have an important physiological role in the host response to bacteria. The function of OaLypla1 in the host response to bacterial infection requires further study in the future.

  19. Full-Length cDNA Cloning, Molecular Characterization and Differential Expression Analysis of Lysophospholipase I from Ovis aries.

    Science.gov (United States)

    Liu, Nan-Nan; Liu, Zeng-Shan; Hu, Pan; Zhang, Ying; Lu, Shi-Ying; Li, Yan-Song; Yang, Yong-Jie; Zhang, Dong-Song; Zhou, Yu; Ren, Hong-Lin

    2016-01-01

    Lysophospholipase I (LYPLA1) is an important protein with multiple functions. In this study, the full-length cDNA of the LYPLA1 gene from Ovis aries (OaLypla1) was cloned using primers and rapid amplification of cDNA ends (RACE) technology. The full-length OaLypla1 was 2457 bp with a 5'-untranslated region (UTR) of 24 bp, a 3'-UTR of 1740 bp with a poly (A) tail, and an open reading frame (ORF) of 693 bp encoding a protein of 230 amino acid residues with a predicted molecular weight of 24,625.78 Da. Phylogenetic analysis showed that the OaLypla1 protein shared a high amino acid identity with LYPLA1 of Bos taurus. The recombinant OaLypla1 protein was expressed and purified, and its phospholipase activity was identified. Monoclonal antibodies (mAb) against OaLypla1 that bound native OaLypla1 were generated. Real-time PCR analysis revealed that OaLypla1 was constitutively expressed in the liver, spleen, lung, kidney, and white blood cells of sheep, with the highest level in the kidney. Additionally, the mRNA levels of OaLypla1 in the buffy coats of sheep challenged with virulent or avirulent Brucella strains were down-regulated compared to untreated sheep. The results suggest that OaLypla1 may have an important physiological role in the host response to bacteria. The function of OaLypla1 in the host response to bacterial infection requires further study in the future. PMID:27483239

  20. Conditional expression of full-length humanized anti-prion protein antibodies in Chinese hamster ovary cells.

    Science.gov (United States)

    Mueller, Daniel A; Heinig, Lars; Ramljak, Sanja; Krueger, Astrid; Schulte, Reiner; Wrede, Arne; Stuke, Andreas W

    2010-12-01

    Because of their high antigen specificity and metabolic stability, genetically engineered human monoclonal antibodies are on the way to becoming one of the most promising medical diagnostics and therapeutics. In order to establish an in vitro system capable of producing such biosimilar antibodies, we used human constant chain sequences to design the novel human antibody expressing vector cassette pMAB-ABX. A bidirectional tetracycline (tet)-controllable promotor was used for harmonized expression of immunoglobulin type G (IgG) heavy and light chains. As an example we used anti-prion protein (anti-PrP) IgGs. Therefore, the variable heavy (V(H)) and light chain (V(L)) sequences of anti-PrP antibodies, previously generated in our laboratory by DNA immunization of prion protein knock-out mice, were isolated from murine hybridoma cell lines and inserted into pMAB-ABX vector. After transfection of Chinese hamster ovary (CHO) cells, a number of stable antibody producing cell clones were selected. One cell line (pMAB-ABX-13F10/3B5) stably expressing the recombinant humanized antibody (rechuAb) 13F10/3B5 was selected for detailed characterization by Western blot, immunofluorescence, and flow cytometric analyses. The full-length recombinant humanized IgG antibody showed a high level of expression in the cytoplasm. In conclusion, the new cell system described here is a suitable tool to produce functional intact full-length humanized IgG antibodies. PMID:21087094

  1. GST-His purification: a two-step affinity purification protocol yielding full-length purified proteins.

    Science.gov (United States)

    Maity, Ranjan; Pauty, Joris; Krietsch, Jana; Buisson, Rémi; Genois, Marie-Michelle; Masson, Jean-Yves

    2013-01-01

    Key assays in enzymology for the biochemical characterization of proteins in vitro necessitate high concentrations of the purified protein of interest. Protein purification protocols should combine efficiency, simplicity and cost effectiveness. Here, we describe the GST-His method as a new small-scale affinity purification system for recombinant proteins, based on a N-terminal Glutathione Sepharose Tag (GST) and a C-terminal 10xHis tag, which are both fused to the protein of interest. The latter construct is used to generate baculoviruses, for infection of Sf9 infected cells for protein expression. GST is a rather long tag (29 kDa) which serves to ensure purification efficiency. However, it might influence physiological properties of the protein. Hence, it is subsequently cleaved off the protein using the PreScission enzyme. In order to ensure maximum purity and to remove the cleaved GST, we added a second affinity purification step based on the comparatively small His-Tag. Importantly, our technique is based on two different tags flanking the two ends of the protein, which is an efficient tool to remove degraded proteins and, therefore, enriches full-length proteins. The method presented here does not require an expensive instrumental setup, such as FPLC. Additionally, we incorporated MgCl2 and ATP washes to remove heat shock protein impurities and nuclease treatment to abolish contaminating nucleic acids. In summary, the combination of two different tags flanking the N- and the C-terminal and the capability to cleave off one of the tags, guaranties the recovery of a highly purified and full-length protein of interest. PMID:24193370

  2. Short-read assembly of full-length 16S amplicons reveals bacterial diversity in subsurface sediments.

    Directory of Open Access Journals (Sweden)

    Christopher S Miller

    Full Text Available In microbial ecology, a fundamental question relates to how community diversity and composition change in response to perturbation. Most studies have had limited ability to deeply sample community structure (e.g. Sanger-sequenced 16S rRNA libraries, or have had limited taxonomic resolution (e.g. studies based on 16S rRNA hypervariable region sequencing. Here, we combine the higher taxonomic resolution of near-full-length 16S rRNA gene amplicons with the economics and sensitivity of short-read sequencing to assay the abundance and identity of organisms that represent as little as 0.01% of sediment bacterial communities. We used a new version of EMIRGE optimized for large data size to reconstruct near-full-length 16S rRNA genes from amplicons sheared and sequenced with Illumina technology. The approach allowed us to differentiate the community composition among samples acquired before perturbation, after acetate amendment shifted the predominant metabolism to iron reduction, and once sulfate reduction began. Results were highly reproducible across technical replicates, and identified specific taxa that responded to the perturbation. All samples contain very high alpha diversity and abundant organisms from phyla without cultivated representatives. Surprisingly, at the time points measured, there was no strong loss of evenness, despite the selective pressure of acetate amendment and change in the terminal electron accepting process. However, community membership was altered significantly. The method allows for sensitive, accurate profiling of the "long tail" of low abundance organisms that exist in many microbial communities, and can resolve population dynamics in response to environmental change.

  3. Full-length core sequence dependent complex-type glycosylation of hepatitis C virus E2 glycoprotein

    Institute of Scientific and Technical Information of China (English)

    Li-Xin Zhu; Jing Liu; Ying-Chun Li; Yu-Ying Kong; Caroline Staib; Gerd Sutter; Yuan Wang; Guang-Di Li

    2002-01-01

    AIM: To study HCV polyprotein processing is important forthe understanding of the natural history of HCV and thedesign of vaccines against HCV. The purpose of this studyis to investigate the affection of context sequences onhepatitis C virus (HCV) E2 processingMETHODS: HCV genes of different lengths were expressedand compared in vaccinia virus/T7 system with homologouspatient serum S94 and mouse anti-serum ME2116 raisedagainst E. coli-derived E2 peptide, respectively.Deglycosylation analysis and GNA (Galanthus nivalus )lectin binding assay were performed to study the post-translational processing of the expressed products.RESULTS: E2 glycoproteins with different molecular weights( ~ 75kDa end ~ 60kDa) were detected using S94 and ME2116,respectively. Deglycosylation analysis showed that thisdifference was mainly due to different glycosylation. Endo Hresistance and its failure to bind to GNA lectin demonstratedthat the higher molecular weight form (75kDa) of E2 wascomplex-type glycosylated, which was readily recognized byhomologous patient serum S94. Expression of complex-typeglycosylated E2 could not be detected in all of the core-truncated constructs tested, but readily detected inconstructs encoding full-length core sequences.CONCLUSION: The upstream conserved full-length corecoding sequence was required for the production of E2glycoproteins carrying complex-type N-glycans whichreacted strongly with homologous patient serum andtherefore possibly represented more mature forms of E2. Ascomplex-type N-glycans indicated modification by Golgienzymes, the results suggest that the presence of full-lengthcore might be critical for E1/E2 complex to leave ER. Ourdata may contribute to a better understanding of theprocessing of HCV structural proteins as well as HCVmorphogenesis.

  4. A Novel mouse model of enhanced proteostasis: Full-length human heat shock factor 1 transgenic mice

    International Nuclear Information System (INIS)

    Research highlights: → Development of mouse overexpressing native human HSF1 in all tissues including CNS. → HSF1 overexpression enhances heat shock response at whole-animal and cellular level. → HSF1 overexpression protects from polyglutamine toxicity and favors aggresomes. → HSF1 overexpression enhances proteostasis at the whole-animal and cellular level. -- Abstract: The heat shock response (HSR) is controlled by the master transcriptional regulator heat shock factor 1 (HSF1). HSF1 maintains proteostasis and resistance to stress through production of heat shock proteins (HSPs). No transgenic model exists that overexpresses HSF1 in tissues of the central nervous system (CNS). We generated a transgenic mouse overexpressing full-length non-mutant HSF1 and observed a 2-4-fold increase in HSF1 mRNA and protein expression in all tissues studied of HSF1 transgenic (HSF1+/0) mice compared to wild type (WT) littermates, including several regions of the CNS. Basal expression of HSP70 and 90 showed only mild tissue-specific changes; however, in response to forced exercise, the skeletal muscle HSR was more elevated in HSF1+/0 mice compared to WT littermates and in fibroblasts following heat shock, as indicated by levels of inducible HSP70 mRNA and protein. HSF1+/0 cells elicited a significantly more robust HSR in response to expression of the 82 repeat polyglutamine-YFP fusion construct (Q82YFP) and maintained proteasome-dependent processing of Q82YFP compared to WT fibroblasts. Overexpression of HSF1 was associated with fewer, but larger Q82YFP aggregates resembling aggresomes in HSF1+/0 cells, and increased viability. Therefore, our data demonstrate that tissues and cells from mice overexpressing full-length non-mutant HSF1 exhibit enhanced proteostasis.

  5. Chaperoning ribosome assembly

    OpenAIRE

    Karbstein, Katrin

    2010-01-01

    Chaperones help proteins fold in all cellular compartments, and many associate directly with ribosomes, capturing nascent chains to assist their folding and prevent aggregation. In this issue, new data from Koplin et al. (2010. J. Cell Biol. doi: 10.1083/jcb.200910074) and Albanèse et al. (2010. J. Cell Biol. doi: 10.1083/jcb.201001054) suggest that in addition to promoting protein folding, the chaperones ribosome-associated complex (RAC), nascent chain–associated complex (NAC), and Jjj1 also...

  6. A potent malaria transmission blocking vaccine based on codon harmonized full length Pfs48/45 expressed in Escherichia coli.

    Directory of Open Access Journals (Sweden)

    Debabani Roy Chowdhury

    Full Text Available Malaria caused by Plasmodium falciparum is responsible for nearly 1 million deaths annually. Although much progress has been made in the recent past, the development of a safe, effective and affordable malaria vaccine has remained a challenge. A vaccine targeting sexual stages of the parasite will not only reduce malaria transmission by female Anopheles mosquitoes, but also reduce the spread of parasites able to evade immunity elicited by vaccines targeting pre-erythrocytic and erythrocytic asexual stages. We focused our studies on Pfs48/45, a protein expressed in the sexual stages developing within an infected person and one of the most promising transmission-blocking vaccine targets. Functional immunogenicity of Pfs48/45 protein requires proper disulfide bond formation, consequently evaluation of the immunogenicity of recombinant full-length Pfs48/45 has been hampered by difficulties in expressing properly folded protein to date. Here we present a strategy involving harmonization of codons for successful recombinant expression of full length Pfs48/45 in Escherichia coli. The purified protein, designated CH-rPfs48/45, was recognized by monoclonal antibodies directed against reduction-sensitive conformational epitopes in the native protein. Immunogenicity evaluation in mice revealed potent transmission blocking activity in membrane feeding assays of antisera elicited by CH-rPfs48/45 formulated in three different adjuvants, i.e. Alum, Montanide ISA-51 and complete Freund's adjuvant. More importantly, CH-rPfs48/45 formulated with Montanide ISA-51 when administered to nonhuman primates (Olive baboons, Papio anubis resulted in uniformly high antibody responses (ELISA titers >2 million in all five animals. Sera from these animals displayed greater than 93% blocking activity in membrane feeding assays after a single immunization, reaching nearly complete blocking after a booster dose of the vaccine. The relative ease of expression and induction of

  7. A full-length Plasmodium falciparum recombinant circumsporozoite protein expressed by Pseudomonas fluorescens platform as a malaria vaccine candidate.

    Directory of Open Access Journals (Sweden)

    Amy R Noe

    Full Text Available The circumsporozoite protein (CSP of Plasmodium falciparum is a major surface protein, which forms a dense coat on the sporozoite's surface. Preclinical research on CSP and clinical evaluation of a CSP fragment-based RTS, S/AS01 vaccine have demonstrated a modest degree of protection against P. falciparum, mediated in part by humoral immunity and in part by cell-mediated immunity. Given the partial protective efficacy of the RTS, S/AS01 vaccine in a recent Phase 3 trial, further improvement of CSP-based vaccines is crucial. In this report, we describe the preclinical development of a full-length, recombinant CSP (rCSP-based vaccine candidate against P. falciparum malaria suitable for current Good Manufacturing Practice (cGMP production. Utilizing a novel high-throughput Pseudomonas fluorescens expression platform, we demonstrated greater efficacy of full-length rCSP as compared to N-terminally truncated versions, rapidly down-selected a promising lead vaccine candidate, and developed a high-yield purification process to express immunologically active, intact antigen for clinical trial material production. The rCSP, when formulated with various adjuvants, induced antigen-specific antibody responses as measured by enzyme-linked immunosorbent assay (ELISA and immunofluorescence assay (IFA, as well as CD4+ T-cell responses as determined by ELISpot. The adjuvanted rCSP vaccine conferred protection in mice when challenged with transgenic P. berghei sporozoites containing the P. falciparum repeat region of CSP. Furthermore, heterologous prime/boost regimens with adjuvanted rCSP and an adenovirus type 35-vectored CSP (Ad35CS showed modest improvements in eliciting CSP-specific T-cell responses and anti-malarial protection, depending on the order of vaccine delivery. Collectively, these data support the importance of further clinical development of adjuvanted rCSP, either as a stand-alone product or as one of the components in a heterologous prime

  8. A Glutamine/Asparagine-Rich Fragment of Gln3, but not the Full-Length Protein, Aggregates in Saccharomyces cerevisiae.

    Science.gov (United States)

    Antonets, K S; Sargsyan, H M; Nizhnikov, A A

    2016-04-01

    The amino acid sequence of protein Gln3 in yeast Saccharomyces cerevisiae has a region enriched with Gln (Q) and Asn (N) residues. In this study, we analyzed the effects of overexpression of Gln3 and its Q/N-rich fragment fused with yellow fluorescent protein (YFP). Being overexpressed, full-length Gln3-YFP does not form aggregates, inhibits vegetative growth, and demonstrates nuclear localization, while the Q/N-rich fragment (Gln3QN) fused with YFP forms aggregates that do not colocalize with the nucleus and do not affect growth of the cells. Although detergent-resistant aggregates of Gln3QN are formed in the absence of yeast prions, the aggregation of Gln3QN significantly increases in the presence of [PIN(+)] prion, while in the presence of two prions, [PSI(+)] and [PIN(+)], the percentage of cells with Gln3QN aggregates is significantly lower than in the strain bearing only [PIN(+)]. Data on colocalization demonstrate that this effect is mediated by interaction between Gln3QN aggregates and [PSI(+)] and [PIN(+)] prions. PMID:27293098

  9. THE RELATIONSHIPS AMONG SPATIOTEMPORAL COLLAGEN GENE EXPRESSION, HISTOLOGY, AND BIOMECHANICS FOLLOWING FULL-LENGTH INJURY IN THE MURINE PATELLAR TENDON

    Science.gov (United States)

    Dyment, Nathaniel A; Kazemi, Namdar; Aschbacher-Smith, Lindsey E; Barthelery, Nicolas J.; Kenter, Keith; Gooch, Cynthia; Shearn, Jason T; Wylie, Christopher; Butler, David L

    2011-01-01

    Tendon injuries are major orthopaedic problems that worsen as the population ages. Type-I (Col1) and type-II (Col2) collagens play important roles in tendon midsubstance and tendon-to-bone insertion healing, respectively. Using double transgenic mice, this study aims to spatiotemporally monitor Col1 and Col2 gene expression, histology and biomechanics up to 8 weeks following a full-length patellar tendon injury. Gene expression and histology were analyzed weekly for up to 5 weeks while mechanical properties were measured at 1, 2, 5, and 8 weeks. At week 1, the healing region displayed loose granulation tissue with little Col1 expression. Col1 expression peaked at 2 weeks, but the ECM was highly disorganized and hypercellular. By 3 weeks, Col1 expression had reduced and by 5 weeks, the ECM was generally aligned along the tendon axis. Col2 expression was not seen in the healing midsubstance or insertion at any time point. The biomechanics of the healing tissue was inadequate at all time points, achieving ultimate loads and stiffnesses of 48% and 63% of normal values by 8 weeks. Future studies will further characterize the cells within the healing midsubstance and insertion using tenogenic markers and compare these results to those of tendon cells during normal development. PMID:21698662

  10. Development of a full-length cDNA-derived enterovirus A71 vaccine candidate using reverse genetics technology.

    Science.gov (United States)

    Yang, Ya-Ting; Chow, Yen-Hung; Hsiao, Kuang-Nan; Hu, Kai-Chieh; Chiang, Jen-Ron; Wu, Suh-Chin; Chong, Pele; Liu, Chia-Chyi

    2016-08-01

    Enterovirus A71 (EV-A71) is responsible for epidemics of hand, foot and mouth disease (HFMD) in young children. To circumvent difficulties in obtaining clinical enterovirus isolates that might be contaminated with other viruses, a platform technology was developed to quickly generate vaccine virus strains based on the published enterovirus genomic sequences. A recombinant plasmid containing the full-length infectious cDNA clone of EV-A71 vaccine strain E59 was directly generated after transfecting the recombinant plasmid into Vero, RD or HEK293A cells, and phenotypic characteristics similar to the parental strain were observed. The cDNA-derived infectious EV-A71 virus grown in Vero cells produced relatively stable virus titers in both T-flasks and microcarrier culture systems. To evaluate the genetic stability of the cDNA-derived EV-A71 viruses, the immunodominant structural proteins, VP1 and VP2, of the recombinant EV-A71 viruses were sequenced and analyzed. The cDNA-derived EV-A71 virus showed weak pathogenicity in a human SCARB2 mouse model. These results show the successful generation of a recombinant virus derived from a published viral genomic sequence that demonstrated good genetic stability and viral yields, which could represent an efficient and safe vaccine strain for cGMP-grade manufacturing. PMID:27387826

  11. Association of murine lupus and thymic full-length endogenous retroviral expression maps to a bone marrow stem cell

    Energy Technology Data Exchange (ETDEWEB)

    Krieg, A.M.; Gourley, M.F.; Steinberg, A.D. (National Institutes of Health, Bethesda, MD (USA))

    1991-05-01

    Recent studies of thymic gene expression in murine lupus have demonstrated 8.4-kb (full-length size) modified polytropic (Mpmv) endogenous retroviral RNA. In contrast, normal control mouse strains do not produce detectable amounts of such RNA in their thymuses. Prior studies have attributed a defect in experimental tolerance in murine lupus to a bone marrow stem cell rather than to the thymic epithelium; in contrast, infectious retroviral expression has been associated with the thymic epithelium, rather than with the bone marrow stem cell. The present study was designed to determine whether the abnormal Mpmv expression associated with murine lupus mapped to thymic epithelium or to a marrow precursor. Lethally irradiated control and lupus-prone mice were reconstituted with T cell depleted bone marrow; one month later their thymuses were studied for endogenous retroviral RNA and protein expression. Recipients of bone marrow from nonautoimmune donors expressed neither 8.4-kb Mpmv RNA nor surface MCF gp70 in their thymuses. In contrast, recipients of bone marrow from autoimmune NZB or BXSB donors expressed thymic 8.4-kb Mpmv RNA and mink cell focus-forming gp70. These studies demonstrate that lupus-associated 8.4-kb Mpmv endogenous retroviral expression is determined by bone marrow stem cells.

  12. Improved yields of full-length functional human FGF1 can be achieved using the methylotrophic yeast Pichia pastoris.

    Science.gov (United States)

    Fantoni, Adele; Bill, Roslyn M; Gustafsson, Lena; Hedfalk, Kristina

    2007-03-01

    We have produced human fibroblast growth factor 1 (hFGF1) in the methylotrophic yeast Pichia pastoris in order to obtain the large amounts of active protein required for subsequent functional and structural characterization. Four constructs were made to examine both intracellular and secreted expression, with variations in the location of the His6 tag at either end of the peptide. hFGF1 could be produced from all four constructs in shake flasks, but production was optimized by growing only the highest-yielding of these strains, which produced hFGF1 intracellularly, under tightly controlled conditions in a 3 L fermentor. One hundred and eight milligrams of pure protein was achieved per liter culture (corresponding to 0.68 mg of protein per gram of wet cells), the function of which was verified using NIH 3T3 cell cultures. This is a 30-fold improvement over previously reported yields of full-length hFGF1. PMID:17134911

  13. Cloning and Expression of Highly Pathogenic Avian Influenza Virus Full-Length Nonstructural Gene in Pichia pastoris

    Directory of Open Access Journals (Sweden)

    M. B. Abubakar

    2011-01-01

    Full Text Available Avian influenza (AI is a highly contagious and rapidly evolving pathogen of major concern to the poultry industry and human health. Rapid and accurate detection of avian influenza virus is a necessary tool for control of outbreaks and surveillance. The AI virus A/Chicken/Malaysia/5858/2004 (H5N1 was used as a template to produce DNA clones of the full-length NS1 genes via reverse transcriptase synthesis of cDNA by PCR amplification of the NS1 region. Products were cloned into pCR2.0 TOPO TA plasmid and subsequently subcloned into pPICZαA vector to construct a recombinant plasmid. Recombinant plasmid designated as pPICZαA-NS1 gene was confirmed by PCR colony screening, restriction enzyme digestion, and nucleotide sequence analysis. The recombinant plasmid was transformed into Pichia pastoris GS115 strain by electroporation, and expressed protein was identified by SDS-PAGE and western blotting. A recombinant protein of approximately ~28 kDa was produced. The expressed protein was able to bind a rabbit polyclonal antibody of nonstructural protein (NS1 avian influenza virus H5N1. The result of the western blotting and solid-phase ELISA assay using H5N1 antibody indicated that the recombinant protein produced retained its antigenicity. This further indicates that Pichia pastoris could be an efficient expression system for a avian influenza virus nonstructural (NS1.

  14. Association of murine lupus and thymic full-length endogenous retroviral expression maps to a bone marrow stem cell

    International Nuclear Information System (INIS)

    Recent studies of thymic gene expression in murine lupus have demonstrated 8.4-kb (full-length size) modified polytropic (Mpmv) endogenous retroviral RNA. In contrast, normal control mouse strains do not produce detectable amounts of such RNA in their thymuses. Prior studies have attributed a defect in experimental tolerance in murine lupus to a bone marrow stem cell rather than to the thymic epithelium; in contrast, infectious retroviral expression has been associated with the thymic epithelium, rather than with the bone marrow stem cell. The present study was designed to determine whether the abnormal Mpmv expression associated with murine lupus mapped to thymic epithelium or to a marrow precursor. Lethally irradiated control and lupus-prone mice were reconstituted with T cell depleted bone marrow; one month later their thymuses were studied for endogenous retroviral RNA and protein expression. Recipients of bone marrow from nonautoimmune donors expressed neither 8.4-kb Mpmv RNA nor surface MCF gp70 in their thymuses. In contrast, recipients of bone marrow from autoimmune NZB or BXSB donors expressed thymic 8.4-kb Mpmv RNA and mink cell focus-forming gp70. These studies demonstrate that lupus-associated 8.4-kb Mpmv endogenous retroviral expression is determined by bone marrow stem cells

  15. Accumulation of human full-length tau induces degradation of nicotinic acetylcholine receptor α4 via activating calpain-2

    Science.gov (United States)

    Yin, Yaling; Wang, Yali; Gao, Di; Ye, Jinwang; Wang, Xin; Fang, Lin; Wu, Dongqin; Pi, Guilin; Lu, Chengbiao; Zhou, Xin-Wen; Yang, Ying; Wang, Jian-Zhi

    2016-01-01

    Cholinergic impairments and tau accumulation are hallmark pathologies in sporadic Alzheimer’s disease (AD), however, the intrinsic link between tau accumulation and cholinergic deficits is missing. Here, we found that overexpression of human wild-type full-length tau (termed hTau) induced a significant reduction of α4 subunit of nicotinic acetylcholine receptors (nAChRs) with an increased cleavage of the receptor producing a ~55kDa fragment in primary hippocampal neurons and in the rat brains, meanwhile, the α4 nAChR currents decreased. Further studies demonstrated that calpains, including calpain-1 and calpain-2, were remarkably activated with no change of caspase-3, while simultaneous suppression of calpain-2 by selective calpain-2 inhibitor but not calpain-1 attenuated the hTau-induced degradation of α4 nAChR. Finally, we demonstrated that hTau accumulation increased the basal intracellular calcium level in primary hippocampal neurons. We conclude that the hTau accumulation inhibits nAChRs α4 by activating calpain-2. To our best knowledge, this is the first evidence showing that the intracellular accumulation of tau causes cholinergic impairments. PMID:27277673

  16. Sequencing and analysis of 10967 full-length cDNA clones from Xenopus laevis and Xenopus tropicalis

    Energy Technology Data Exchange (ETDEWEB)

    Morin, R D; Chang, E; Petrescu, A; Liao, N; Kirkpatrick, R; Griffith, M; Butterfield, Y; Stott, J; Barber, S; Babakaiff, R; Matsuo, C; Wong, D; Yang, G; Smailus, D; Brown-John, M; Mayo, M; Beland, J; Gibson, S; Olson, T; Tsai, M; Featherstone, R; Chand, S; Siddiqui, A; Jang, W; Lee, E; Klein, S; Prange, C; Myers, R M; Green, E D; Wagner, L; Gerhard, D; Marra, M; Jones, S M; Holt, R

    2005-10-31

    Sequencing of full-insert clones from full-length cDNA libraries from both Xenopus laevis and Xenopus tropicalis has been ongoing as part of the Xenopus Gene Collection initiative. Here we present an analysis of 10967 clones (8049 from X. laevis and 2918 from X. tropicalis). The clone set contains 2013 orthologs between X. laevis and X. tropicalis as well as 1795 paralog pairs within X. laevis. 1199 are in-paralogs, believed to have resulted from an allotetraploidization event approximately 30 million years ago, and the remaining 546 are likely out-paralogs that have resulted from more ancient gene duplications, prior to the divergence between the two species. We do not detect any evidence for positive selection by the Yang and Nielsen maximum likelihood method of approximating d{sub N}/d{sub S}. However, d{sub N}/d{sub S} for X. laevis in-paralogs is elevated relative to X. tropicalis orthologs. This difference is highly significant, and indicates an overall relaxation of selective pressures on duplicated gene pairs. Within both groups of paralogs, we found evidence of subfunctionalization, manifested as differential expression of paralogous genes among tissues, as measured by EST information from public resources. We have observed, as expected, a higher instance of subfunctionalization in out-paralogs relative to in-paralogs.

  17. Cloning and Analysis of Full-Length cDNA of PumNPR1 Gene from Pyrus ussuriensis Maxim

    Institute of Scientific and Technical Information of China (English)

    CHE Daidi; FAN Jinping; WANG Jingang; XU Ping; YANG Tao; LIU Shenkui

    2008-01-01

    The purpose of this study is to find a new gene resource for the researches of molecular breeding of Rosaceae plants disease-resistance. Pyrus ussuriensis Maxim is used as a starting material to clone the full-length cDNA of NPR1(nonexpressor of pathogenesis- related genes 1) which is a key regulator in SA (salicylic acid)-mediated systemic acquired resistance (SAR) by homologous cloning and RACE techniques. The length of the cDNA sequence was 1 767 bp, the ORF was 1 761 bp, it coded 586 amino acids, pI=5.58, the relative molecular weight was 65.009 ku, contained 19 kinds of amino acids, and had full BTB/POZ and ANK domains. Compared the homology of NPR1 gene in GenBank database, the homology with Pyrus pyrifolia, Arabidopsis thaliana, Nicotiana tabacum, Lycopersicon esculentum, Oryza sativa, Helianthus annuus were 98%, 62%, 68%, 65%, 57%, 63%. The homology of functional area were 99%, 78%, 82%, 79%, 74%, 77%. This NPR1 gene was considered as homologic gene of Pyrus ussuriensis Maxim and named PumNPR1.

  18. Computational Analysis of Full-length cDNAs Reveals Frequent Coupling Between Transcriptional and Splicing Programs

    Science.gov (United States)

    Chern, Tzu-Ming; Paul, Nicodeme; van Nimwegen, Erik; Zavolan, Mihaela

    2008-01-01

    High-throughput sequencing studies revealed that the majority of human and mouse multi-exon genes have multiple splice forms. High-density oligonucleotide array-based measurements have further established that many exons are expressed in a tissue-specific manner. The mechanisms underlying the tissue-dependent expression of most alternative exons remain, however, to be understood. In this study, we focus on one possible mechanism, namely the coupling of (tissue specific) transcription regulation with alternative splicing. We analyzed the FANTOM3 and H-Invitational datasets of full-length mouse and human cDNAs, respectively, and found that in transcription units with multiple start sites, the inclusion of at least 15% and possibly up to 30% of the ‘cassette’ exons correlates with the use of specific transcription start sites (TSS). The vast majority of TSS-associated exons are conserved between human and mouse, yet the conservation is weaker when compared with TSS-independent exons. Additionally, the currently available data only support a weak correlation between the probabilities of TSS association of orthologous exons. Our analysis thus suggests frequent coupling of transcriptional and splicing programs, and provides a large dataset of exons on which the molecular basis of this coupling can be further studied. PMID:18276623

  19. REAL-Select: full-length antibody display and library screening by surface capture on yeast cells.

    Directory of Open Access Journals (Sweden)

    Laura Rhiel

    Full Text Available We describe a novel approach named REAL-Select for the non-covalent display of IgG-molecules on the surface of yeast cells for the purpose of antibody engineering and selection. It relies on the capture of secreted native full-length antibodies on the cell surface via binding to an externally immobilized ZZ domain, which tightly binds antibody Fc. It is beneficial for high-throughput screening of yeast-displayed IgG-libraries during antibody discovery and development. In a model experiment, antibody-displaying yeast cells were isolated from a 1:1,000,000 mixture with control cells confirming the maintenance of genotype-phenotype linkage. Antibodies with improved binding characteristics were obtained by affinity maturation using REAL-Select, demonstrating the ability of this system to display antibodies in their native form and to detect subtle changes in affinity by flow cytometry. The biotinylation of the cell surface followed by functionalization with a streptavidin-ZZ fusion protein is an approach that is independent of the genetic background of the antibody-producing host and therefore can be expected to be compatible with other eukaryotic expression hosts such as P. pastoris or mammalian cells.

  20. X-Ray Crystal Structure of the Full Length Human Chitotriosidase (CHIT1) Reveals Features of Its Chitin Binding Domain

    Science.gov (United States)

    Fadel, Firas; Zhao, Yuguang; Cousido-Siah, Alexandra; Ruiz, Francesc X.; Mitschler, André; Podjarny, Alberto

    2016-01-01

    Chitinases are enzymes that catalyze the hydrolysis of chitin. Human chitotriosidase (CHIT1) is one of the two active human chitinases, involved in the innate immune response and highly expressed in a variety of diseases. CHIT1 is composed of a catalytic domain linked by a hinge to its chitin binding domain (ChBD). This latter domain belongs to the carbohydrate-binding module family 14 (CBM14 family) and facilitates binding to chitin. So far, the available crystal structures of the human chitinase CHIT1 and the Acidic Mammalian Chitinase (AMCase) comprise only their catalytic domain. Here, we report a crystallization strategy combining cross-seeding and micro-seeding cycles which allowed us to obtain the first crystal structure of the full length CHIT1 (CHIT1-FL) at 1.95 Å resolution. The CHIT1 chitin binding domain (ChBDCHIT1) structure shows a distorted β-sandwich 3D fold, typical of CBM14 family members. Accordingly, ChBDCHIT1 presents six conserved cysteine residues forming three disulfide bridges and several exposed aromatic residues that probably are involved in chitin binding, including the highly conserved Trp465 in a surface- exposed conformation. Furthermore, ChBDCHIT1 presents a positively charged surface which may be involved in electrostatic interactions. Our data highlight the strong structural conservation of CBM14 family members and uncover the structural similarity between the human ChBDCHIT1, tachycitin and house mite dust allergens. Overall, our new CHIT1-FL structure, determined with an adapted crystallization approach, is one of the few complete bi-modular chitinase structures available and reveals the structural features of a human CBM14 domain. PMID:27111557

  1. Improved strategy for phylogenetic analysis of classical swine fever virus based on full-length E2 encoding sequences

    Directory of Open Access Journals (Sweden)

    Postel Alexander

    2012-06-01

    Full Text Available Abstract Molecular epidemiology has proven to be an essential tool in the control of classical swine fever (CSF and its use has significantly increased during the past two decades. Phylogenetic analysis is a prerequisite for virus tracing and thus allows implementing more effective control measures. So far, fragments of the 5´NTR (150 nucleotides, nt and the E2 gene (190 nt have frequently been used for phylogenetic analyses. The short sequence lengths represent a limiting factor for differentiation of closely related isolates and also for confidence levels of proposed CSFV groups and subgroups. In this study, we used a set of 33 CSFV isolates in order to determine the nucleotide sequences of a 3508–3510 nt region within the 5´ terminal third of the viral genome. Including 22 additional sequences from GenBank database different regions of the genome, comprising the formerly used short 5´NTR and E2 fragments as well as the genomic regions encoding the individual viral proteins Npro, C, Erns, E1, and E2, were compared with respect to variability and suitability for phylogenetic analysis. Full-length E2 encoding sequences (1119 nt proved to be most suitable for reliable and statistically significant phylogeny and analyses revealed results as good as obtained with the much longer entire 5´NTR-E2 sequences. This strategy is therefore recommended by the EU and OIE Reference Laboratory for CSF as it provides a solid and improved basis for CSFV molecular epidemiology. Finally, the power of this method is illustrated by the phylogenetic analysis of closely related CSFV isolates from a recent outbreak in Lithuania.

  2. Isolation and characterization of a full length cDNA for dentatorubral-pallidoluysian atrophy (DRPLA) gene

    Energy Technology Data Exchange (ETDEWEB)

    Oyake, M.; Onodera, O.; Ikeuchi, T. [Niigata Univ. (Japan)] [and others

    1994-09-01

    Hereditary dentatorubral-pallidoluysian atrophy (DRPLA) is an autosomal dominant spinocerebellar degeneration characterized by anticipation and variable combination of symptoms including myoclonus, epilepsy, cerebellar ataxia, choleoathetosis, and dementia. Recently, we discovered that DRPLA is caused by unstable expansion of a CAG repeat of a B37 gene on chromosome 12. To characterize functions of the DRPLA gene product, we isolated several cDNA clones for the DRPLA gene from human adult and fetus brain cDNA libraries, using an oligonucleotide flanking the CAG repeat. The cDNA spans 4247 bp in length and there is only an open reading frame coding for 986 amino acids. The CAG repeat, which is expanded in DRPLA, is located 291 bp downstream from the initiation methionine and encodes a polyglutamine tract. The deduced amino acid sequence from amino acids residues 582 to 707 has a high homology to published human hippocampus derived expressed sequence (M78755) located at chromosome 1p (63.8% identity), and 3{prime}-untranslated region of the DRPLA cDNA revealed homology to the mouse small nuclear RNA U7 gene (X54165). Northern blot analysis revealed a 4.7 knt transcript which is widely expressed in various tissues including heart, lung, kidney, placenta, skeletal muscle, and brain. In human adult brain, the transcript was broadly expressed including amygdala, caudate nucleus, corpus callosum, hippocampus, hypothalamus, substantia nigra, subthalamic nucleus and thalamus, and was not specific to the dentatorubral-pallidoluysian system. The availability of a full length cDNA will be highly useful for analyzing the pathogenesis of this unique neurodegenerative disease as well as for analyzing other CAG repeat related neurodegenerative diseases.

  3. Potency of Full- Length MGF to Induce Maximal Activation of the IGF-I R Is Similar to Recombinant Human IGF-I at High Equimolar Concentrations

    OpenAIRE

    Janssen, Joseph A. M. J. L.; Hofland, Leo J.; Strasburger, Christian J.; Elisabeth S R van den Dungen; Mario Thevis

    2016-01-01

    textabstractAims To compare full-length mechano growth factor (full-length MGF) with human recombinant insulin-like growth factor-I (IGF-I) and human recombinant insulin (HI) in their ability to activate the human IGF-I receptor (IGF-IR), the human insulin receptor (IR-A) and the human insulin receptor-B (IR-B), respectively. In addition, we tested the stimulatory activity of human MGF and its stabilized analog Goldspink-MGF on the IGF-IR. Methods The effects of full-length MGF, IGF-I, human ...

  4. Transcripts from a single full-length cDNA clone of hepatitis C virus are infectious when directly transfected into the liver of a chimpanzee

    OpenAIRE

    Yanagi, Masayuki; Purcell, Robert H.; Emerson, Suzanne U.; Bukh, Jens

    1997-01-01

    We have succeeded in constructing a stable full-length cDNA clone of strain H77 (genotype 1a) of hepatitis C virus (HCV). We devised a cassette vector with fixed 5′ and 3′ termini and constructed multiple full-length cDNA clones of H77 in a single step by cloning of the entire ORF, which was amplified by long reverse transcriptase–PCR, directly into this vector. The infectivity of two complete full-length cDNA clones was tested by the direct intrahepatic injection of a chimpanzee with RNA tra...

  5. Potency of Full- Length MGF to Induce Maximal Activation of the IGF-I R Is Similar to Recombinant Human IGF-I at High Equimolar Concentrations.

    Directory of Open Access Journals (Sweden)

    Joseph A M J L Janssen

    Full Text Available To compare full-length mechano growth factor (full-length MGF with human recombinant insulin-like growth factor-I (IGF-I and human recombinant insulin (HI in their ability to activate the human IGF-I receptor (IGF-IR, the human insulin receptor (IR-A and the human insulin receptor-B (IR-B, respectively. In addition, we tested the stimulatory activity of human MGF and its stabilized analog Goldspink-MGF on the IGF-IR.The effects of full-length MGF, IGF-I, human mechano growth factor (MGF, Goldspink-MGF and HI were compared using kinase specific receptor activation (KIRA bioassays specific for IGF-I, IR-A or IR-B, respectively. These assays quantify activity by measuring auto-phosphorylation of the receptor upon ligand binding.IGF-IR: At high equimolar concentrations maximal IGF-IR stimulating effects generated by full-length MGF were similar to that of IGF-I (89-fold vs. 77-fold, respectively. However, EC50 values of IGF-I and full-length MGF for the IGF-I receptor were 0.86 nmol/L (95% CI 0.69-1.07 and 7.83 nmol/L (95% CI: 4.87-12.58, respectively. No IGF-IR activation was observed by human MGF and Goldspink-MGF, respectively. IR-A/IR-B: At high equimolar concentrations similar maximal IR-A stimulating effects were observed for full -length MGF and HI, but maximal IR-B stimulation achieved by full -length MGF was stronger than that by HI (292-fold vs. 98-fold. EC50 values of HI and full-length MGF for the IR-A were 1.13 nmol/L (95% CI 0.69-1.84 and 73.11 nmol/L (42.87-124.69, respectively; for IR-B these values were 1.28 nmol/L (95% CI 0.64-2.57 and 35.10 nmol/L (95% 17.52-70.33, respectively.Full-length MGF directly stimulates the IGF-IR. Despite a higher EC50 concentration, at high equimolar concentrations full-length MGF showed a similar maximal potency to activate the IGF-IR as compared to IGF-I. Further research is needed to understand the actions of full-length MGF in vivo and to define the physiological relevance of our in vitro findings.

  6. Potency of Full- Length MGF to Induce Maximal Activation of the IGF-I R Is Similar to Recombinant Human IGF-I at High Equimolar Concentrations

    Science.gov (United States)

    Janssen, Joseph A. M. J. L.; Hofland, Leo J.; Strasburger, Christian J.; van den Dungen, Elisabeth S. R.; Thevis, Mario

    2016-01-01

    Aims To compare full-length mechano growth factor (full-length MGF) with human recombinant insulin-like growth factor-I (IGF-I) and human recombinant insulin (HI) in their ability to activate the human IGF-I receptor (IGF-IR), the human insulin receptor (IR-A) and the human insulin receptor-B (IR-B), respectively. In addition, we tested the stimulatory activity of human MGF and its stabilized analog Goldspink-MGF on the IGF-IR. Methods The effects of full-length MGF, IGF-I, human mechano growth factor (MGF), Goldspink-MGF and HI were compared using kinase specific receptor activation (KIRA) bioassays specific for IGF-I, IR-A or IR-B, respectively. These assays quantify activity by measuring auto-phosphorylation of the receptor upon ligand binding. Results IGF-IR: At high equimolar concentrations maximal IGF-IR stimulating effects generated by full-length MGF were similar to that of IGF-I (89-fold vs. 77-fold, respectively). However, EC50 values of IGF-I and full-length MGF for the IGF-I receptor were 0.86 nmol/L (95% CI 0.69–1.07) and 7.83 nmol/L (95% CI: 4.87–12.58), respectively. No IGF-IR activation was observed by human MGF and Goldspink-MGF, respectively. IR-A/IR-B: At high equimolar concentrations similar maximal IR-A stimulating effects were observed for full -length MGF and HI, but maximal IR-B stimulation achieved by full -length MGF was stronger than that by HI (292-fold vs. 98-fold). EC50 values of HI and full-length MGF for the IR-A were 1.13 nmol/L (95% CI 0.69–1.84) and 73.11 nmol/L (42.87–124.69), respectively; for IR-B these values were 1.28 nmol/L (95% CI 0.64–2.57) and 35.10 nmol/L (95% 17.52–70.33), respectively. Conclusions Full-length MGF directly stimulates the IGF-IR. Despite a higher EC50 concentration, at high equimolar concentrations full-length MGF showed a similar maximal potency to activate the IGF-IR as compared to IGF-I. Further research is needed to understand the actions of full-length MGF in vivo and to define the

  7. Identification and isolation of full-length cDNA sequences by sequencing and analysis of expressed sequence tags from guarana (Paullinia cupana).

    Science.gov (United States)

    Figueirêdo, L C; Faria-Campos, A C; Astolfi-Filho, S; Azevedo, J L

    2011-01-01

    The current intense production of biological data, generated by sequencing techniques, has created an ever-growing volume of unanalyzed data. We reevaluated data produced by the guarana (Paullinia cupana) transcriptome sequencing project to identify cDNA clones with complete coding sequences (full-length clones) and complete sequences of genes of biotechnological interest, contributing to the knowledge of biological characteristics of this organism. We analyzed 15,490 ESTs of guarana in search of clones with complete coding regions. A total of 12,402 sequences were analyzed using BLAST, and 4697 full-length clones were identified, responsible for the production of 2297 different proteins. Eighty-four clones were identified as full-length for N-methyltransferase and 18 were sequenced in both directions to obtain the complete genome sequence, and confirm the search made in silico for full-length clones. Phylogenetic analyses were made with the complete genome sequences of three clones, which showed only 0.017% dissimilarity; these are phylogenetically close to the caffeine synthase of Theobroma cacao. The search for full-length clones allowed the identification of numerous clones that had the complete coding region, demonstrating this to be an efficient and useful tool in the process of biological data mining. The sequencing of the complete coding region of identified full-length clones corroborated the data from the in silico search, strengthening its efficiency and utility. PMID:21732283

  8. Identification and expression analysis of a full-length cDNA encoding a Kandelia candel tonoplast intrinsic protein.

    Science.gov (United States)

    Huang, Wei; Fang, Xiao-Dong; Lin, Qi-Fen; Li, Guan-Yi; Zhao, Wen-Ming

    2003-03-01

    corresponding to the 5' end of this gene was obtained using the GSP2 primer. Two primers that flank the putative open reading frame (ORF) were designed to obtain the cDNA containing the complete ORF by RACE PCR reaction. The full-length cDNA of KCTIP1, containing a 756 bp open reading frame (ORF), was approximately 1.1 kb; the start codon was located at the nucleotides of 99-101 and stop codon at the nucleotides of 855-857 followed by a poly (A) tail. The KCTIP1 cDNA sequence in this research was released in GenBank with accession number AF521135. Using ExPASy Proteomics tools provided by EMBL, the isoelectric point and MWt of KCTIP1 are estimated as 5.77 and 26.3 kD respectively. Transmembrane prediction analysis revealed the deduced KCTIP1 protein sequence contains six transmembrane regions at amino acid residues of 20 - 42, 57 - 79, 86 - 108, 113 - 135, 142 - 164 and 217 - 239. Two highly conserved asparagine-proline-alanine (NPA) motifs were located at 85 - 87 and 199 - 201 amino acid residues respectively. KCTIP1 is also predicted to contain the Cys residue (Cys 118) that are shown to confer Hg-sensitivity in Arabidopsis gamma-TIP and delta-TIP. Similarity analysis showed that KCTIP1 shared 77% - 79% amino acid sequence identity with the TIPs from Vitis berlandieri, Brassica oleracea and Arabidopsis thaliana. Expression analyses indicated that KCTIP1 had different expression among species of Mangroves. Expressions of KCTIP1 in Kandelia candel, Rhizophora apoculata and Ceriops tagal were suppressed by salt, and were insensitive to salt stress in unknown species of Mangroves. Previous studied showed that salt conditions might result in large and rapid changes in extracellular water potential and serious disturbance to the cytoplasm. In order to compensate for this imbalance, the relative contribution of water channels to flow across the root could thus vary. K. candel is a species that is native to intertial zone of tropical and subtropical coast and is well-adapted to salt

  9. Large-scale collection and analysis of full-length cDNAs from Brachypodium distachyon and integration with Pooideae sequence resources.

    Directory of Open Access Journals (Sweden)

    Keiichi Mochida

    Full Text Available A comprehensive collection of full-length cDNAs is essential for correct structural gene annotation and functional analyses of genes. We constructed a mixed full-length cDNA library from 21 different tissues of Brachypodium distachyon Bd21, and obtained 78,163 high quality expressed sequence tags (ESTs from both ends of ca. 40,000 clones (including 16,079 contigs. We updated gene structure annotations of Brachypodium genes based on full-length cDNA sequences in comparison with the latest publicly available annotations. About 10,000 non-redundant gene models were supported by full-length cDNAs; ca. 6,000 showed some transcription unit modifications. We also found ca. 580 novel gene models, including 362 newly identified in Bd21. Using the updated transcription start sites, we searched a total of 580 plant cis-motifs in the -3 kb promoter regions and determined a genome-wide Brachypodium promoter architecture. Furthermore, we integrated the Brachypodium full-length cDNAs and updated gene structures with available sequence resources in wheat and barley in a web-accessible database, the RIKEN Brachypodium FL cDNA database. The database represents a "one-stop" information resource for all genomic information in the Pooideae, facilitating functional analysis of genes in this model grass plant and seamless knowledge transfer to the Triticeae crops.

  10. Engineering of recombinant crystallization chaperones

    OpenAIRE

    Koide, Shohei

    2009-01-01

    The preparation of diffraction quality crystals remains the major bottleneck in macromolecular x-ray crystallography. A crystallization chaperone is an auxiliary protein, such as fragments of monoclonal antibodies, that binds to and increases the crystallization probability of a target molecule of interest. Such chaperones reduce conformational heterogeneity, mask counterproductive surfaces while extending surfaces predisposed to forming crystal contacts, and provide phasing information. Crys...

  11. Molecular chaperones and neurodegenerative diseases

    Institute of Scientific and Technical Information of China (English)

    2006-01-01

    Neurodegenerative diseases are characterized by the accumulation of intracellular or extracellular protein aggregates that result from conformational changes in proteins. These diseases may result from an imbalance between the production of misfolded proteins and normal chaperone capacity. Molecular chaperones provide a first line of defence against misfolded, aggregation-prone proteins and are, therefore, promising therapeutic targets for neurodegenerative diseases.

  12. Robust full-length hepatitis C virus genotype 2a and 2b infectious cultures using mutations identified by a systematic approach applicable to patient strains

    DEFF Research Database (Denmark)

    Li, Yi-Ping; Ramirez, Santseharay; Gottwein, Judith M;

    2012-01-01

    replicates spontaneously in hepatoma cells and releases infectious virus. A JFH1 chimera with the 5' end through NS2 from another genotype 2a strain, J6, had enhanced infectivity. However, the full-length J6 clone (J6CF), which we previously found to be fully functional in vivo, was replication incompetent...... in vitro. Through a systematic approach of culturing J6 with minimal JFH1 sequences, we identified three mutations in NS3, NS4A, and NS5B that permitted full-length J6 propagation and adaptation with infectivity titers comparable to JFH1-based systems. The most efficient recombinant, J6cc, had six...... adaptive mutations and did not accumulate additional changes following viral passage. We demonstrated that HCV NS3/NS4A protease-, NS5A- and NS5B polymerase-directed drugs respectively inhibited full-length J6 infection dose dependently. Importantly, the three J6-derived mutations enabled culture...

  13. Purification and characterization of recombinant full-length and protease domain of murine MMP-9 expressed in Drosophila S2 cells

    DEFF Research Database (Denmark)

    Rasch, Morten G; Lund, Ida K.; Illemann, Martin; Høyer-Hansen, Gunilla; Gårdsvoll, Henrik

    2010-01-01

    MMP-9. Constructs encoding zymogens of full-length murine MMP-9 and a version lacking the O-glycosylated linker region and hemopexin domains were therefore generated and expressed in stably transfected Drosophila S2 insect cells. After 7 days of induction the expression levels of the full-length and...... truncated versions were 5 mg/l and 2 mg/l, respectively. The products were >95% pure after gelatin Sepharose chromatography and possessed proteolytic activity when analyzed by gelatin zymography. Using the purified full-length murine MMP-9 we raised polyclonal antibodies by immunizations of rabbits. These......Matrix metalloproteinase-9 (MMP-9) is a 92-kDa soluble pro-enzyme implicated in pathological events including cancer invasion. It is therefore an attractive target for therapeutic intervention studies in mouse models. Development of inhibitors requires sufficient amounts of correctly folded murine...

  14. Full-length cloning and phylogenetic analyses of translationally controlled tumour protein and ferritin genes from the Indian white prawn, Fenneropenaeus indicus (H. Milne Edwards)

    Digital Repository Service at National Institute of Oceanography (India)

    Nayak, S.; Ramaiah, N.; Meena, R.M.; Sreepada, R.A.

    in this study clearly shows that the crustacean members distributed in four different groups (Fig 3). F. indicus TCTP along with other penaeid family representatives clustered to a separate clade and the protein sequences, taken for the tree construction... of the four groups. A high similarity shared by TCTP from F. indicus with other penaeid species suggests its conserved nature within the penaeid family. Full length cloning of ferritin gene After 3′ and 5′ ends RACE PCR, the full-length cDNA fragment...

  15. Evidence for alternative quaternary structure in a bacterial Type III secretion system chaperone

    Energy Technology Data Exchange (ETDEWEB)

    Barta, Michael L.; Zhang, Lingling; Picking, Wendy L.; Geisbrecht, Brian V. (UMKC); (OKLU)

    2010-10-05

    Type III secretion systems are a common virulence mechanism in many Gram-negative bacterial pathogens. These systems use a nanomachine resembling a molecular needle and syringe to provide an energized conduit for the translocation of effector proteins from the bacterial cytoplasm to the host cell cytoplasm for the benefit of the pathogen. Prior to translocation specialized chaperones maintain proper effector protein conformation. The class II chaperone, Invasion plasmid gene (Ipg) C, stabilizes two pore forming translocator proteins. IpgC exists as a functional dimer to facilitate the mutually exclusive binding of both translocators. In this study, we present the 3.3 {angstrom} crystal structure of an amino-terminally truncated form (residues 10-155, denoted IpgC10-155) of the class II chaperone IpgC from Shigella flexneri. Our structure demonstrates an alternative quaternary arrangement to that previously described for a carboxy-terminally truncated variant of IpgC (IpgC{sup 1-151}). Specifically, we observe a rotationally-symmetric 'head-to-head' dimerization interface that is far more similar to that previously described for SycD from Yersinia enterocolitica than to IpgC1-151. The IpgC structure presented here displays major differences in the amino terminal region, where extended coil-like structures are seen, as opposed to the short, ordered alpha helices and asymmetric dimerization interface seen within IpgC{sup 1-151}. Despite these differences, however, both modes of dimerization support chaperone activity, as judged by a copurification assay with a recombinant form of the translocator protein, IpaB. Conclusions: From primary to quaternary structure, these results presented here suggest that a symmetric dimerization interface is conserved across bacterial class II chaperones. In light of previous data which have described the structure and function of asymmetric dimerization, our results raise the possibility that class II chaperones may

  16. Evidence for alternative quaternary structure in a bacterial Type III secretion system chaperone

    Directory of Open Access Journals (Sweden)

    Picking Wendy L

    2010-07-01

    Full Text Available Abstract Background Type III secretion systems are a common virulence mechanism in many Gram-negative bacterial pathogens. These systems use a nanomachine resembling a molecular needle and syringe to provide an energized conduit for the translocation of effector proteins from the bacterial cytoplasm to the host cell cytoplasm for the benefit of the pathogen. Prior to translocation specialized chaperones maintain proper effector protein conformation. The class II chaperone, Invasion plasmid gene (Ipg C, stabilizes two pore forming translocator proteins. IpgC exists as a functional dimer to facilitate the mutually exclusive binding of both translocators. Results In this study, we present the 3.3 Å crystal structure of an amino-terminally truncated form (residues 10-155, denoted IpgC10-155 of the class II chaperone IpgC from Shigella flexneri. Our structure demonstrates an alternative quaternary arrangement to that previously described for a carboxy-terminally truncated variant of IpgC (IpgC1-151. Specifically, we observe a rotationally-symmetric "head-to- head" dimerization interface that is far more similar to that previously described for SycD from Yersinia enterocolitica than to IpgC1-151. The IpgC structure presented here displays major differences in the amino terminal region, where extended coil-like structures are seen, as opposed to the short, ordered alpha helices and asymmetric dimerization interface seen within IpgC1-151. Despite these differences, however, both modes of dimerization support chaperone activity, as judged by a copurification assay with a recombinant form of the translocator protein, IpaB. Conclusions From primary to quaternary structure, these results presented here suggest that a symmetric dimerization interface is conserved across bacterial class II chaperones. In light of previous data which have described the structure and function of asymmetric dimerization, our results raise the possibility that class II

  17. Genome-wide comparisons of phylogenetic similarities between partial genomic regions and the full-length genome in Hepatitis E virus genotyping.

    Directory of Open Access Journals (Sweden)

    Shuai Wang

    Full Text Available Besides the complete genome, different partial genomic sequences of Hepatitis E virus (HEV have been used in genotyping studies, making it difficult to compare the results based on them. No commonly agreed partial region for HEV genotyping has been determined. In this study, we used a statistical method to evaluate the phylogenetic performance of each partial genomic sequence from a genome wide, by comparisons of evolutionary distances between genomic regions and the full-length genomes of 101 HEV isolates to identify short genomic regions that can reproduce HEV genotype assignments based on full-length genomes. Several genomic regions, especially one genomic region at the 3'-terminal of the papain-like cysteine protease domain, were detected to have relatively high phylogenetic correlations with the full-length genome. Phylogenetic analyses confirmed the identical performances between these regions and the full-length genome in genotyping, in which the HEV isolates involved could be divided into reasonable genotypes. This analysis may be of value in developing a partial sequence-based consensus classification of HEV species.

  18. Association of the exon 3 deleted/full-length GHR polymorphism with recombinant growth hormone dose in growth hormone-deficient adults

    NARCIS (Netherlands)

    S. Meyer; W.J. Weise (Wolfgang); A. Pfützner (Andreas); P. Kann (P.); S. Schaefer (Stephan); L. Stolk (Lisette); P.P. Arp (Pascal); A.G. Uitterlinden (André); U. Plöckinger (Ursula); G.K. Stalla (Günter); U. Tuschy (Ulrich); M.M. Weber (Matthias)

    2009-01-01

    textabstractAims: Contradictory reports exist regarding the influence of the exon 3 deleted (d3)/full-length (fl) growth hormone receptor (GHR) polymorphism on responsiveness to recombinant human growth-hormone therapy in idiopathic short stature, small for gestational age and GH-deficient children,

  19. Full-Length Coding Sequences for 12 Bovine Viral Diarrhea Virus Isolates from Persistently Infected Cattle in a Feedyard in Kansas

    OpenAIRE

    Workman, Aspen M.; Harhay, Gregory P.; Heaton, Michael P; Grotelueschen, Dale M.; Sjeklocha, David; Smith, Timothy P. L.

    2015-01-01

    We report here the full-length coding sequences of 12 bovine viral diarrhea virus (BVDV) isolates from persistently infected cattle in a feedyard in southwest KS. These 12 genomes represent the three major subtypes of BVDV (BVDV-1a, 1b, and 2a) currently circulating in the United States.

  20. Particle infectivity of HIV-1 full-length genome infectious molecular clones in a subtype C heterosexual transmission pair following high fidelity amplification and unbiased cloning

    International Nuclear Information System (INIS)

    The high genetic diversity of HIV-1 impedes high throughput, large-scale sequencing and full-length genome cloning by common restriction enzyme based methods. Applying novel methods that employ a high-fidelity polymerase for amplification and an unbiased fusion-based cloning strategy, we have generated several HIV-1 full-length genome infectious molecular clones from an epidemiologically linked transmission pair. These clones represent the transmitted/founder virus and phylogenetically diverse non-transmitted variants from the chronically infected individual's diverse quasispecies near the time of transmission. We demonstrate that, using this approach, PCR-induced mutations in full-length clones derived from their cognate single genome amplicons are rare. Furthermore, all eight non-transmitted genomes tested produced functional virus with a range of infectivities, belying the previous assumption that a majority of circulating viruses in chronic HIV-1 infection are defective. Thus, these methods provide important tools to update protocols in molecular biology that can be universally applied to the study of human viral pathogens. - Highlights: • Our novel methodology demonstrates accurate amplification and cloning of full-length HIV-1 genomes. • A majority of plasma derived HIV variants from a chronically infected individual are infectious. • The transmitted/founder was more infectious than the majority of the variants from the chronically infected donor

  1. Establishment of Stably Transfected Cells Constitutively Expressing the Full-Length and Truncated Antigenic Proteins of Two Genetically Distinct Mink Astroviruses

    DEFF Research Database (Denmark)

    Bidokhti, Mehdi R. M.; Ullman, Karin; Jensen, Trine Hammer;

    2013-01-01

    as evaluated by in-situ PLA and IFA, and confirmed by Western blotting. The recombinant full-length and truncated proteins induced a high level of antibodies in mink, evaluated by ELISA, demonstrating their immunogenicity. In a challenge experiment in mink, a reduction in presentation clinical signs and virus...

  2. Sequencing and comparative genomics analysis in Senecio scandens Buch.-Ham. Ex D. Don, based on full-length cDNA library

    Science.gov (United States)

    Qian, Gang; Ping, Junjiao; Zhang, Zhen; Xu, Delin

    2014-01-01

    Senecio scandens Buch.-Ham. ex D. Don, an important antibacterial source of Chinese traditional medicine, has a widespread distribution in a few ecological habitats of China. We generated a full-length complementary DNA (cDNA) library from a sample of elite individuals with superior antibacterial properties, with satisfactory parameters such as library storage (4.30 × 106 CFU), efficiency of titre (1.30 × 106 CFU/mL), transformation efficiency (96.35%), full-length ratio (64.00%) and redundancy ratio (3.28%). The BLASTN search revealed the facile formation of counterparts between the experimental sample and Arabidopsis thaliana in view of high-homology cDNA sequence (90.79%) with e-values <1e – 50. Sequence similarities to known proteins indicate that the entire sequences of the full-length cDNA clones consist of the major of functional genes identified by a large set of microarray data from the present experimental material. For other Compositae species, a large set of full-length cDNA clones reported in the present article will serve as a useful resource to facilitate further research on the transferability of expressed sequence tag-derived simple sequence repeats (EST-SSR) development, comparative genomics and novel transcript profiles. PMID:26740776

  3. Salmo salar and Esox lucius full-length cDNA sequences reveal changes in evolutionary pressures on a post-tetraploidization genome

    Directory of Open Access Journals (Sweden)

    Holt Robert A

    2010-04-01

    Full Text Available Abstract Background Salmonids are one of the most intensely studied fish, in part due to their economic and environmental importance, and in part due to a recent whole genome duplication in the common ancestor of salmonids. This duplication greatly impacts species diversification, functional specialization, and adaptation. Extensive new genomic resources have recently become available for Atlantic salmon (Salmo salar, but documentation of allelic versus duplicate reference genes remains a major uncertainty in the complete characterization of its genome and its evolution. Results From existing expressed sequence tag (EST resources and three new full-length cDNA libraries, 9,057 reference quality full-length gene insert clones were identified for Atlantic salmon. A further 1,365 reference full-length clones were annotated from 29,221 northern pike (Esox lucius ESTs. Pairwise dN/dS comparisons within each of 408 sets of duplicated salmon genes using northern pike as a diploid out-group show asymmetric relaxation of selection on salmon duplicates. Conclusions 9,057 full-length reference genes were characterized in S. salar and can be used to identify alleles and gene family members. Comparisons of duplicated genes show that while purifying selection is the predominant force acting on both duplicates, consistent with retention of functionality in both copies, some relaxation of pressure on gene duplicates can be identified. In addition, there is evidence that evolution has acted asymmetrically on paralogs, allowing one of the pair to diverge at a faster rate.

  4. Particle infectivity of HIV-1 full-length genome infectious molecular clones in a subtype C heterosexual transmission pair following high fidelity amplification and unbiased cloning

    Energy Technology Data Exchange (ETDEWEB)

    Deymier, Martin J., E-mail: mdeymie@emory.edu [Emory Vaccine Center, Yerkes National Primate Research Center, 954 Gatewood Road NE, Atlanta, GA 30329 (United States); Claiborne, Daniel T., E-mail: dclaibo@emory.edu [Emory Vaccine Center, Yerkes National Primate Research Center, 954 Gatewood Road NE, Atlanta, GA 30329 (United States); Ende, Zachary, E-mail: zende@emory.edu [Emory Vaccine Center, Yerkes National Primate Research Center, 954 Gatewood Road NE, Atlanta, GA 30329 (United States); Ratner, Hannah K., E-mail: hannah.ratner@emory.edu [Emory Vaccine Center, Yerkes National Primate Research Center, 954 Gatewood Road NE, Atlanta, GA 30329 (United States); Kilembe, William, E-mail: wkilembe@rzhrg-mail.org [Zambia-Emory HIV Research Project (ZEHRP), B22/737 Mwembelelo, Emmasdale Post Net 412, P/BagE891, Lusaka (Zambia); Allen, Susan, E-mail: sallen5@emory.edu [Zambia-Emory HIV Research Project (ZEHRP), B22/737 Mwembelelo, Emmasdale Post Net 412, P/BagE891, Lusaka (Zambia); Department of Pathology and Laboratory Medicine, Emory University, Atlanta, GA (United States); Hunter, Eric, E-mail: eric.hunter2@emory.edu [Emory Vaccine Center, Yerkes National Primate Research Center, 954 Gatewood Road NE, Atlanta, GA 30329 (United States); Department of Pathology and Laboratory Medicine, Emory University, Atlanta, GA (United States)

    2014-11-15

    The high genetic diversity of HIV-1 impedes high throughput, large-scale sequencing and full-length genome cloning by common restriction enzyme based methods. Applying novel methods that employ a high-fidelity polymerase for amplification and an unbiased fusion-based cloning strategy, we have generated several HIV-1 full-length genome infectious molecular clones from an epidemiologically linked transmission pair. These clones represent the transmitted/founder virus and phylogenetically diverse non-transmitted variants from the chronically infected individual's diverse quasispecies near the time of transmission. We demonstrate that, using this approach, PCR-induced mutations in full-length clones derived from their cognate single genome amplicons are rare. Furthermore, all eight non-transmitted genomes tested produced functional virus with a range of infectivities, belying the previous assumption that a majority of circulating viruses in chronic HIV-1 infection are defective. Thus, these methods provide important tools to update protocols in molecular biology that can be universally applied to the study of human viral pathogens. - Highlights: • Our novel methodology demonstrates accurate amplification and cloning of full-length HIV-1 genomes. • A majority of plasma derived HIV variants from a chronically infected individual are infectious. • The transmitted/founder was more infectious than the majority of the variants from the chronically infected donor.

  5. Functional characterization of a full length pregnane X receptor, expression in vivo, and identification of PXR alleles, in Zebrafish (Danio rerio)

    Energy Technology Data Exchange (ETDEWEB)

    Bainy, Afonso C.D. [Biology Department, Woods Hole Oceanographic Institution, Woods Hole, MA 02543 (United States); Departamento de Bioquímica, CCB, Universidade Federal de Santa Catarina, Florianópolis, SC 88040-900 (Brazil); Kubota, Akira; Goldstone, Jared V. [Biology Department, Woods Hole Oceanographic Institution, Woods Hole, MA 02543 (United States); Lille-Langøy, Roger [Department of Biology, University of Bergen, N-5020 Bergen (Norway); Karchner, Sibel I. [Biology Department, Woods Hole Oceanographic Institution, Woods Hole, MA 02543 (United States); Celander, Malin C. [Department of Biological and Environmental Sciences, University of Gothenburg, SE 405 30 Göteborg (Sweden); Hahn, Mark E. [Biology Department, Woods Hole Oceanographic Institution, Woods Hole, MA 02543 (United States); Goksøyr, Anders [Department of Biology, University of Bergen, N-5020 Bergen (Norway); Stegeman, John J., E-mail: jstegeman@whoi.edu [Biology Department, Woods Hole Oceanographic Institution, Woods Hole, MA 02543 (United States)

    2013-10-15

    Highlights: •Full-length pxr has been cloned from zebrafish. •Alleles of pxr were identified in zebrafish. •Full length Pxr was activated less strongly than ligand binding domain in cell-based reporter assays. •High levels of pxr expression were found in eye and brain as well as in liver. •TCPOBOP and PB did not significantly alter expression of pxr in liver. -- Abstract: The pregnane X receptor (PXR) (nuclear receptor NR1I2) is a ligand activated transcription factor, mediating responses to diverse xenobiotic and endogenous chemicals. The properties of PXR in fish are not fully understood. Here we report on cloning and characterization of full-length PXR of zebrafish, Danio rerio, and pxr expression in vivo. Initial efforts gave a cDNA encoding a 430 amino acid protein identified as zebrafish pxr by phylogenetic and synteny analysis. The sequence of the cloned Pxr DNA binding domain (DBD) was highly conserved, with 74% identity to human PXR-DBD, while the ligand-binding domain (LBD) of the cloned sequence was only 44% identical to human PXR-LBD. Sequence variation among clones in the initial effort prompted sequencing of multiple clones from a single fish. There were two prominent variants, one sequence with S183, Y218 and H383 and the other with I183, C218 and N383, which we designate as alleles pxr*1 (nr1i2*1) and pxr*2 (nr1i2*2), respectively. In COS-7 cells co-transfected with a PXR-responsive reporter gene, the full-length Pxr*1 (the more common variant) was activated by known PXR agonists clotrimazole and pregnenolone 16α-carbonitrile but to a lesser extent than the full-length human PXR. Activation of full-length Pxr*1 was only 10% of that with the Pxr*1 LBD. Quantitative real time PCR analysis showed prominent expression of pxr in liver and eye, as well as brain and intestine of adult zebrafish. The pxr was expressed in heart and kidney at levels similar to that in intestine. The expression of pxr in liver was weakly induced by ligands for

  6. Chaperoning prions: the story unfolds

    OpenAIRE

    O'Connor, David; Jones, Gary

    2006-01-01

    Prions are infectious proteins that are responsible for a number of mammalian degenerative diseases. The discovery of prions in yeast has allowed detailed genetic analysis to be carried out to identify cellular factors involved in prion propagation. It is now clear that a complex relationship exists between molecular chaperones and prion propagation. Prions may actually have evolved to exploit the cell's chaperone machinery to ensure their own propaga...

  7. Amplification of the Full-Length PAMP Gene and Difference of the mRNA Expression Among Three Lean Pig Breeds

    Institute of Scientific and Technical Information of China (English)

    2008-01-01

    To understand the function of porcine adipocyte-special membrane protein (PAMP) gene and the difference of fat deposition ability among various lean pig breeds, a full-length porcine adipocyte-special membrane protein (PAMP) gene was successfully amplified using reverse transcription polymerase chain reaction (RT-PCR) and 5'-rapid amplification of cDNA end (5'-RACE). The open reading frame was 1 587 bp encoding 529 amino acids. The nucleotide sequence of the full-length PAMP gene was deposited in the GenBank under the accession number EF433431. The PAMP gene mRNA expression was analyzed on three lean pig breeds by quantitative reverse transcription polymerase chain reaction (QRT-PCR). The PAMP gene mRNA levels in YHM (Yorkshire × Hampshire × Meishan) pig and DLY (Duroc×Landrance× Yorkshire) pig were about 0.82 and 0.38 times of that in SW (Shanxi-White) pig, respectively.

  8. Gene expression of a truncated and the full-length growth hormone (GH) receptor in subcutaneous fat and skeletal muscle in GH-deficient adults

    DEFF Research Database (Denmark)

    Fisker, Sidse; Kristensen, K; Rosenfalck, A M;

    2001-01-01

    In humans at least two GH receptors are significantly expressed. One is the full-length receptor (GHR); the other is a truncated form (GHRtr), that lacks most of the intracellular domain. This receptor may inhibit the action of the full-length receptor. Circulating GH-binding protein (GHBP) is a...... proteolytically cleaved product from both of these receptors. The clinical relevance of the different receptor types is unknown. We examined the gene expression of GHR and GHRtr in human adipose tissue and skeletal muscle and the influence of GH treatment on this expression. Furthermore, we studied the...... relationship of circulating GHBP and body composition to GHR and GHRtr gene expression. Eleven adult GH-deficient patients were studied before and after 4 months of GH substitution therapy. Abdominal fat obtained by liposuction and femoral muscle biopsies were taken at baseline and after 4 months. Gene...

  9. Infectious foot-and-mouth disease virus derived from a cloned full-length cDNA of OH/CHA/99

    Institute of Scientific and Technical Information of China (English)

    LIU Guangqing; LIU Zaixin; XIE Qingge; CHEN Yingli; BAO Huifang; LIU Xiangtao

    2004-01-01

    The China foot-and-mouth virus (FMDV) isolate OH/CHA/99 was isolated from swine, which was unable to infect bovine thyroid cells in vitro or to cause typical disease in bovines following intradermal inoculation in the tongue. To enhance antigenicity, replication, maturation and pathogenicity studies of OH/CHA/99, an infectious full- length cDNA clone, designated pBlFMDV, was prepared. The in vitro and in vivo biological properties of the virus derived from pBlFMDV were studied by analyzing antigenicity, plaque morphology and virulence in pigs. The results showed that the virus derived from pBlFMDV had the same biological properties as the parent strain OH/CHA/99; the full- length infectious cDNA clone, pBlFMDV, will be very useful in studies of the antigenicity, virulence, pathogenesis, maturation and replication of FMDV.

  10. A new set of ESTs and cDNA clones from full-length and normalized libraries for gene discovery and functional characterization in citrus

    OpenAIRE

    Alamar Santiago; Arribas Raquel; Forment Javier; Alonso-Cantabrana Hugo; Marques M Carmen; Conejero Vicente; Perez-Amador Miguel A

    2009-01-01

    Abstract Background Interpretation of ever-increasing raw sequence information generated by modern genome sequencing technologies faces multiple challenges, such as gene function analysis and genome annotation. Indeed, nearly 40% of genes in plants encode proteins of unknown function. Functional characterization of these genes is one of the main challenges in modern biology. In this regard, the availability of full-length cDNA clones may fill in the gap created between sequence information an...

  11. Sequencing and analysis of full-length cDNAs, 5'-ESTs and 3'-ESTs from a cartilaginous fish, the elephant shark (Callorhinchus milii).

    KAUST Repository

    Brenner, Sydney

    2012-10-08

    Cartilaginous fishes are the most ancient group of living jawed vertebrates (gnathostomes) and are, therefore, an important reference group for understanding the evolution of vertebrates. The elephant shark (Callorhinchus milii), a holocephalan cartilaginous fish, has been identified as a model cartilaginous fish genome because of its compact genome (∼910 Mb) and a genome project has been initiated to obtain its whole genome sequence. In this study, we have generated and sequenced full-length enriched cDNA libraries of the elephant shark using the \\'oligo-capping\\' method and Sanger sequencing. A total of 6,778 full-length protein-coding cDNA and 10,701 full-length noncoding cDNA were sequenced from six tissues (gills, intestine, kidney, liver, spleen, and testis) of the elephant shark. Analysis of their polyadenylation signals showed that polyadenylation usage in elephant shark is similar to that in mammals. Furthermore, both coding and noncoding transcripts of the elephant shark use the same proportion of canonical polyadenylation sites. Besides BLASTX searches, protein-coding transcripts were annotated by Gene Ontology, InterPro domain, and KEGG pathway analyses. By comparing elephant shark genes to bony vertebrate genes, we identified several ancient genes present in elephant shark but differentially lost in tetrapods or teleosts. Only ∼6% of elephant shark noncoding cDNA showed similarity to known noncoding RNAs (ncRNAs). The rest are either highly divergent ncRNAs or novel ncRNAs. In addition to full-length transcripts, 30,375 5\\'-ESTs and 41,317 3\\'-ESTs were sequenced and annotated. The clones and transcripts generated in this study are valuable resources for annotating transcription start sites, exon-intron boundaries, and UTRs of genes in the elephant shark genome, and for the functional characterization of protein sequences. These resources will also be useful for annotating genes in other cartilaginous fishes whose genomes have been targeted for

  12. Cloning of the Full-Length Rhesus Cytomegalovirus Genome as an Infectious and Self-Excisable Bacterial Artificial Chromosome for Analysis of Viral Pathogenesis

    OpenAIRE

    Chang, W. L. William; Peter A Barry

    2003-01-01

    Rigorous investigation of many functions encoded by cytomegaloviruses (CMVs) requires analysis in the context of virus-host interactions. To facilitate the construction of rhesus CMV (RhCMV) mutants for in vivo studies, a bacterial artificial chromosome (BAC) containing an enhanced green fluorescent protein (EGFP) cassette was engineered into the intergenic region between unique short 1 (US1) and US2 of the full-length viral genome by Cre/lox-mediated recombination. Infectious virions were re...

  13. Construction of occluded recombinant baculoviruses containing the full-length cry1Ab and cry1Ac genes from Bacillus thuringiensis

    Directory of Open Access Journals (Sweden)

    B.M. Ribeiro

    1998-06-01

    Full Text Available The administration of baculoviruses to insects for bioassay purposes is carried out, in most cases, by contamination of food surfaces with a known amount of occlusion bodies (OBs. Since per os infection is the natural route of infection, occluded recombinant viruses containing crystal protein genes (cry1Ab and cry1Ac from Bacillus thuringiensis were constructed for comparison with the baculovirus prototype Autographa californica nucleopolyhedrovirus (AcNPV. The transfer vector pAcUW2B was used for construction of occluded recombinant viruses. The transfer vector containing the crystal protein genes was cotransfected with linearized DNA from a non-occluded recombinant virus. The isolation of recombinant viruses was greatly facilitated by the reduction of background "wild type" virus and the increased proportion of recombinant viruses. Since the recombinant viruses containing full-length and truncated forms of the crystal protein genes did not seem to improve the pathogenicity of the recombinant viruses when compared with the wild type AcNPV, and in order to compare expression levels of the full-length crystal proteins produced by non-occluded and occluded recombinant viruses the full-length cry1Ab and cry1Ac genes were chosen for construction of occluded recombinant viruses. The recombinant viruses containing full-length and truncated forms of the crystal protein genes did not seem to improve its pathogenicity but the size of the larvae infected with the recombinant viruses was significantly smaller than that of larvae infected with the wild type virus.

  14. Update on the efficacy, safety, and adherence to treatment of full length parathyroid hormone, PTH (1-84), in the treatment of postmenopausal osteoporosis

    OpenAIRE

    Luca Pietrogrande

    2009-01-01

    Luca PietrograndeDipartimento di Medicina Chirurgia e Odontoiatria Polo San Paolo, Università degli Studi di Milano, Milan, ItalyAbstract: Full length (1-84) parathyroid hormone (PTH) was introduced in Europe as a treatment for postmenopausal osteoporosis in 2006. The efficacy of PTH (1-84) in the prevention of vertebral fractures is very high, and is similar to that of teriparatide. Its action in the prevention of femoral fractures has yet to be fully demonstrated, but the inciden...

  15. Modification of the full-length cDNA clone of Newcastle disease virus Isolated from an outbreak In the goose

    Institute of Scientific and Technical Information of China (English)

    LIU Yuliang; HU Shunli; ZHANG Yanmei; WU Yantao; LIU Xiufan; R(o)emer-Oberdoerfer Angela; Veits Jutta; Lange Martina

    2006-01-01

    A 6.5-kb specific fragment containing the T7 promoter and the transcription vector was excised from the full-length eDNA clone of the Newcastle disease virus (NDV) strain ZJI of goose origin,and thereafter it was self-ligated to form a high quality plasmid for mutagenesis.Site-directed mutagenesis was used for inserting three additional G nucleotides (nts) into the region between the T7 promoter and the leader sequence of the NDV genome.RT-PCR was employed to amplify the F/HN gene fragments,and then they were ligated by the shared restriction enzyme BsmBI.Finally,the corresponding fragment in the mutant full-length eDNA was substituted with the new one.The sequencing results showed that the three additional Gnts were successfully inserted and the mutant nts in the full-length eDNA were corrected.This study lays a good foundation for research on the reverse genetics of NDV strain ZJI.

  16. Enhanced expression of full-length human cytomegalovirus fusion protein in non-swelling baculovirus-infected cells with a minimal fed-batch strategy.

    Directory of Open Access Journals (Sweden)

    Marco Patrone

    Full Text Available Human cytomegalovirus congenital infection represents an unmet medical issue and attempts are ongoing to develop an effective vaccine. The virion fusion players of this enveloped virus are the natural targets to achieve this goal and to develop novel anti-viral therapies. The secreted ectodomain of the viral fusion factor glycoprotein B (gB has been exploited so far as an alternative to the cumbersome expression of the wild type trans-membrane protein. In the soluble form, gB showed encouraging but limited potential as antigen candidate calling for further efforts. Here, the exhaustive evaluation of the Baculovirus/insect cell expression system has been coupled to an orthogonal screening for expression additives to produce full-length gB. In detail, rapamycin was found to prolong gB intracellular accumulation while inhibiting the infection-induced cell swelling. Not obvious to predict, this inhibition did not affect Baculovirus growth, revealing that the virus-induced cell size increase is a dispensable side phenotype. In parallel, a feeding strategy for the limiting nutrient cysteine has been set up which improved gB stability. This multi-modal scheme allowed the production of full-length, mutation-free gB in the milligram scale. The recombinant full-length gB obtained was embedded into a stable mono-dispersed particle substantially larger than the protein trimer itself, according to the reported association of this protein with detergent-resistant lipid domains.

  17. Construction and characterization of a full-length cDNA library for the wheat stripe rust pathogen (Puccinia striiformis f. sp. tritici

    Directory of Open Access Journals (Sweden)

    Chen Xianming

    2007-06-01

    Full Text Available Abstract Background Puccinia striiformis is a plant pathogenic fungus causing stripe rust, one of the most important diseases on cereal crops and grasses worldwide. However, little is know about its genome and genes involved in the biology and pathogenicity of the pathogen. We initiated the functional genomic research of the fungus by constructing a full-length cDNA and determined functions of the first group of genes by sequence comparison of cDNA clones to genes reported in other fungi. Results A full-length cDNA library, consisting of 42,240 clones with an average cDNA insert of 1.9 kb, was constructed using urediniospores of race PST-78 of P. striiformis f. sp. tritici. From 196 sequenced cDNA clones, we determined functions of 73 clones (37.2%. In addition, 36 clones (18.4% had significant homology to hypothetical proteins, 37 clones (18.9% had some homology to genes in other fungi, and the remaining 50 clones (25.5% did not produce any hits. From the 73 clones with functions, we identified 51 different genes encoding protein products that are involved in amino acid metabolism, cell defense, cell cycle, cell signaling, cell structure and growth, energy cycle, lipid and nucleotide metabolism, protein modification, ribosomal protein complex, sugar metabolism, transcription factor, transport metabolism, and virulence/infection. Conclusion The full-length cDNA library is useful in identifying functional genes of P. striiformis.

  18. Delivery of full-length factor VIII using a piggyBac transposon vector to correct a mouse model of hemophilia A.

    Directory of Open Access Journals (Sweden)

    Hideto Matsui

    Full Text Available Viral vectors have been used for hemophilia A gene therapy. However, due to its large size, full-length Factor VIII (FVIII cDNA has not been successfully delivered using conventional viral vectors. Moreover, viral vectors may pose safety risks, e.g., adverse immunological reactions or virus-mediated cytotoxicity. Here, we took advantages of the non-viral vector gene delivery system based on piggyBac DNA transposon to transfer the full-length FVIII cDNA, for the purpose of treating hemophilia A. We tested the efficiency of this new vector system in human 293T cells and iPS cells, and confirmed the expression of the full-length FVIII in culture media using activity-sensitive coagulation assays. Hydrodynamic injection of the piggyBac vectors into hemophilia A mice temporally treated with an immunosuppressant resulted in stable production of circulating FVIII for over 300 days without development of anti-FVIII antibodies. Furthermore, tail-clip assay revealed significant improvement of blood coagulation time in the treated mice. piggyBac transposon vectors can facilitate the long-term expression of therapeutic transgenes in vitro and in vivo. This novel gene transfer strategy should provide safe and efficient delivery of FVIII.

  19. Assessment of adaptive evolution between wheat and rice as deduced from full-length common wheat cDNA sequence data and expression patterns

    Directory of Open Access Journals (Sweden)

    Hayashizaki Yoshihide

    2009-06-01

    Full Text Available Abstract Background Wheat is an allopolyploid plant that harbors a huge, complex genome. Therefore, accumulation of expressed sequence tags (ESTs for wheat is becoming particularly important for functional genomics and molecular breeding. We prepared a comprehensive collection of ESTs from the various tissues that develop during the wheat life cycle and from tissues subjected to stress. We also examined their expression profiles in silico. As full-length cDNAs are indispensable to certify the collected ESTs and annotate the genes in the wheat genome, we performed a systematic survey and sequencing of the full-length cDNA clones. This sequence information is a valuable genetic resource for functional genomics and will enable carrying out comparative genomics in cereals. Results As part of the functional genomics and development of genomic wheat resources, we have generated a collection of full-length cDNAs from common wheat. By grouping the ESTs of recombinant clones randomly selected from the full-length cDNA library, we were able to sequence 6,162 independent clones with high accuracy. About 10% of the clones were wheat-unique genes, without any counterparts within the DNA database. Wheat clones that showed high homology to those of rice were selected in order to investigate their expression patterns in various tissues throughout the wheat life cycle and in response to abiotic-stress treatments. To assess the variability of genes that have evolved differently in wheat and rice, we calculated the substitution rate (Ka/Ks of the counterparts in wheat and rice. Genes that were preferentially expressed in certain tissues or treatments had higher Ka/Ks values than those in other tissues and treatments, which suggests that the genes with the higher variability expressed in these tissues is under adaptive selection. Conclusion We have generated a high-quality full-length cDNA resource for common wheat, which is essential for continuation of the

  20. Correlation of the level of full-length CFTR transcript with pulmonary phenotype in patients carrying R117H and 1342-1,-2delAG mutations

    Energy Technology Data Exchange (ETDEWEB)

    Hamosh, A.; Cutting, G.R. [Johns Hopkins Univ. School of Medicine, Balitmore, MD (United States); Oates, R.; Amos, J. [Boston Univ. School of Medicine, Boston, MA (United States)

    1994-09-01

    The R117H mutation occurs on two chromosome backgrounds, one associated with a 7 thymidine tract (7T-R11H) in the splice-acceptor site of intron 8, the other with a 5 thymidine tract (5T-R117H). We examined exon 9 splicing efficiency in 5 patients of genotype R117H/{delta}F508 and one carrying 1342-1,-2delAG{delta}F508, an obligate exon 9 slice site mutation. Four patients carried R117H on a 7T background -- three adult men with congenital bilateral absence of the vas deferens and one adolescent female with pancreatitis and borderline sweat chloride concentration. The patient with R117H on a 5T background had pancreatic sufficient CF (PS-CF). The 1342-1,-2delAG patient has classic pancreatic insufficient CF (PI-CF). cDNA was synthesized from total RNA extracted from nasal epithlial cells and analyzed for CFTR splicing by 35 cycle PCR using primers in exon 7 and 11. The quantity of full length transcript derived from the R117H or {delta}F508 alleles was assessed by allele-specific oligonucleotide hybridization. While 91.4% of transcript from the 5T-R117H allele was full-length, only 42.2% of CFTR transcript from the 5T-R117H allele was full length. Since CBAVD patients have no lung disease and PS-CF patients do, this indicates that the threshold of developing CF lung disease is crossed when the amount of CFTR transcript bearing R117H is reduced by half. Interestingly, 17.1% of transcript derived from the 1342-1,-2delAG allele (or 8.6% of total CFTR transcript) was normal and full length. This suggests that up to 9% of full length wild-type CFTR transcript may be inadequate to escape the lung disease of CF and that a 9 thymidine tract followed by AAC (the result of the AG deletion) can be used as a splice donor with 2-9% efficiency.

  1. A new set of ESTs and cDNA clones from full-length and normalized libraries for gene discovery and functional characterization in citrus

    Directory of Open Access Journals (Sweden)

    Alamar Santiago

    2009-09-01

    Full Text Available Abstract Background Interpretation of ever-increasing raw sequence information generated by modern genome sequencing technologies faces multiple challenges, such as gene function analysis and genome annotation. Indeed, nearly 40% of genes in plants encode proteins of unknown function. Functional characterization of these genes is one of the main challenges in modern biology. In this regard, the availability of full-length cDNA clones may fill in the gap created between sequence information and biological knowledge. Full-length cDNA clones facilitate functional analysis of the corresponding genes enabling manipulation of their expression in heterologous systems and the generation of a variety of tagged versions of the native protein. In addition, the development of full-length cDNA sequences has the power to improve the quality of genome annotation. Results We developed an integrated method to generate a new normalized EST collection enriched in full-length and rare transcripts of different citrus species from multiple tissues and developmental stages. We constructed a total of 15 cDNA libraries, from which we isolated 10,898 high-quality ESTs representing 6142 different genes. Percentages of redundancy and proportion of full-length clones range from 8 to 33, and 67 to 85, respectively, indicating good efficiency of the approach employed. The new EST collection adds 2113 new citrus ESTs, representing 1831 unigenes, to the collection of citrus genes available in the public databases. To facilitate functional analysis, cDNAs were introduced in a Gateway-based cloning vector for high-throughput functional analysis of genes in planta. Herein, we describe the technical methods used in the library construction, sequence analysis of clones and the overexpression of CitrSEP, a citrus homolog to the Arabidopsis SEP3 gene, in Arabidopsis as an example of a practical application of the engineered Gateway vector for functional analysis. Conclusion The new

  2. Do nucleic acids moonlight as molecular chaperones?

    Science.gov (United States)

    Docter, Brianne E.; Horowitz, Scott; Gray, Michael J.; Jakob, Ursula; Bardwell, James C.A.

    2016-01-01

    Organisms use molecular chaperones to combat the unfolding and aggregation of proteins. While protein chaperones have been widely studied, here we demonstrate that DNA and RNA exhibit potent chaperone activity in vitro. Nucleic acids suppress the aggregation of classic chaperone substrates up to 300-fold more effectively than the protein chaperone GroEL. Additionally, RNA cooperates with the DnaK chaperone system to refold purified luciferase. Our findings reveal a possible new role for nucleic acids within the cell: that nucleic acids directly participate in maintaining proteostasis by preventing protein aggregation. PMID:27105849

  3. Construction of a full-length cDNA library for Senecio scandens%千里光全长cDNA文库的构建及分析

    Institute of Scientific and Technical Information of China (English)

    平军娇; 张珍; 蔡振锋; 汤贤春; 钱刚

    2012-01-01

    目的 构建千里光全长cDNA文库,以期研究千里光的功能基因组学信息,为克隆药理学性状相关的功能基因提供数据资源.方法 Trizol法提取千里光叶片总RNA,通过SMART(switching mechanism at 5’end of RNA transcript)构建全长cDNA文库,随机挑取600个单克隆测序分析文库滴度、全长率及冗余率,得到的EST序列进行Blast分析(NR、NT、Swiss-Prot、KEGG)及COG功能分类.结果 文库的库容为4.3×106 cfu/mL,插入片段大小平均1.7 kb,文库重组率96.35%,全长率58.24%,冗余率10.88%;获得524条全长EST序列,含有467条独立基因(unigenes),其中5条序列与千里光次生代谢产物的合成、运输与代谢有关.结论 经检测,SMART技术成功构建了千里光全长cDNA文库,该文库可用于千里光功能基因组鉴定、新基因筛选及次生代谢产物生物合成的表达调控研究.%Objective In the present study, our information from Senecio scandens full-length cDNA clones will serve as a useful resource for elucidating functional genes and will also aid a precise annotation of genomics in Compositae plants. Methods The total RNA was extracted from S. Scandens using Trizol method. SMART (switching mechanism at 5' end of RNA transcript) was applied to constructing the full-length cDNA library. Titer of the library, full-length ratio, and redundancy rate for 600 monoclone randomly selected sequencing library were evaluated by PCR amplification. NCBI and COG database was used to compare those sequences. Results Parameters of the the quality of cDNA library were as follows: the capacity of the library (4.3* 106 cfu/mL), the average size of the inserted fragment (1.7 kb), the recombination rate (96.35%), the full-length rate (58.24%), and the redundancy rate (10.88%). EST sequences for 524 full-length were obtained in this study, involving 467 unigenes, among which five sequences associated with synthesis, transport, and metabolism of S. Scandens secondary

  4. Distinct roles for histone chaperones in the deposition of Htz1 in chromatin

    Science.gov (United States)

    Liu, Hongde; Zhu, Min; Mu, Yawen; Liu, Lingjie; Li, Guanghui; Wan, Yakun

    2014-01-01

    Histone variant Htz1 substitution for H2A plays important roles in diverse DNA transactions. Histone chaperones Chz1 and Nap1 (nucleosome assembly protein 1) are important for the deposition Htz1 into nucleosomes. In literatures, it was suggested that Chz1 is a Htz1–H2B-specific chaperone, and it is relatively unstructured in solution but it becomes structured in complex with the Htz1–H2B histone dimer. Nap1 (nucleosome assembly protein 1) can bind (H3–H4)2 tetramers, H2A–H2B dimers and Htz1–H2B dimers. Nap1 can bind H2A–H2B dimer in the cytoplasm and shuttles the dimer into the nucleus. Moreover, Nap1 functions in nucleosome assembly by competitively interacting with non-nucleosomal histone–DNA. However, the exact roles of these chaperones in assembling Htz1-containing nucleosome remain largely unknown. In this paper, we revealed that Chz1 does not show a physical interaction with chromatin. In contrast, Nap1 binds exactly at the genomic DNA that contains Htz1. Nap1 and Htz1 show a preferential interaction with AG-rich DNA sequences. Deletion of chz1 results in a significantly decreased binding of Htz1 in chromatin, whereas deletion of nap1 dramatically increases the association of Htz1 with chromatin. Furthermore, genome-wide nucleosome-mapping analysis revealed that nucleosome occupancy for Htz1p-bound genes decreases upon deleting htz1 or chz1, suggesting that Htz1 is required for nucleosome structure at the specific genome loci. All together, these results define the distinct roles for histone chaperones Chz1 and Nap1 to regulate Htz1 incorporation into chromatin. PMID:25338502

  5. Direct demonstration of NCAM cis-dimerization and inhibitory effect of palmitoylation using the BRET2 technique

    DEFF Research Database (Denmark)

    Kulahin, Nikolaj; Grunnet, Lars Groth; Lundh, Morten;

    2011-01-01

    , cis-dimerization in living cells has not been shown directly and the role of the cytoplasmic part in NCAM dimerization is poorly understood. Here, we used the bioluminescence resonance energy transfer (BRET(2)) technique to directly demonstrate that full-length NCAM cis-homodimerizes in living cells...

  6. Rapid Construction of Stable Infectious Full-Length cDNA Clone of Papaya Leaf Distortion Mosaic Virus Using In-Fusion Cloning.

    Science.gov (United States)

    Tuo, Decai; Shen, Wentao; Yan, Pu; Li, Xiaoying; Zhou, Peng

    2015-12-01

    Papaya leaf distortion mosaic virus (PLDMV) is becoming a threat to papaya and transgenic papaya resistant to the related pathogen, papaya ringspot virus (PRSV). The generation of infectious viral clones is an essential step for reverse-genetics studies of viral gene function and cross-protection. In this study, a sequence- and ligation-independent cloning system, the In-Fusion(®) Cloning Kit (Clontech, Mountain View, CA, USA), was used to construct intron-less or intron-containing full-length cDNA clones of the isolate PLDMV-DF, with the simultaneous scarless assembly of multiple viral and intron fragments into a plasmid vector in a single reaction. The intron-containing full-length cDNA clone of PLDMV-DF was stably propagated in Escherichia coli. In vitro intron-containing transcripts were processed and spliced into biologically active intron-less transcripts following mechanical inoculation and then initiated systemic infections in Carica papaya L. seedlings, which developed similar symptoms to those caused by the wild-type virus. However, no infectivity was detected when the plants were inoculated with RNA transcripts from the intron-less construct because the instability of the viral cDNA clone in bacterial cells caused a non-sense or deletion mutation of the genomic sequence of PLDMV-DF. To our knowledge, this is the first report of the construction of an infectious full-length cDNA clone of PLDMV and the splicing of intron-containing transcripts following mechanical inoculation. In-Fusion cloning shortens the construction time from months to days. Therefore, it is a faster, more flexible, and more efficient method than the traditional multistep restriction enzyme-mediated subcloning procedure. PMID:26633465

  7. Cloning and Sequence Analysis of Interleukin 10 (IL-10) Full-length cDNA from Cyprinus carpio L.

    Institute of Scientific and Technical Information of China (English)

    Xiangru FENG; Yilong CHEN; Xiao ZHAO; Wendong WANG; Junhui ZHANG; Zhenguo YANG SUN; Shengmei JIA; Qiang LU

    2012-01-01

    Abstract [Objective] This study aimed to obtain IL-IO (interleukin 10) full-length cD- NA of common carp (Cyprinus carpio L.) and conduct the sequence analysis. []~lethod] The differentially expressed cDNA fragment was obtained by DD-RTPCR (differential display RT-PCR). The cDNA library of peripheral blood leukocytes which were separated from common carp and stimulated by mitogen was screened with a probe labeled with DIG (digoxigenin). The IL-IO full-length cDNA was cloned from 0.8x104 pfu of recombinant phages, and the sequence analysis and homology com- parison were carried out. [Result] Sequence analysis indicated that the IL-IO full- length cDNA of common carp was 1 117 bp long, containing a.55 bp 5'-UTR, a 522 bp 3"-UTR, and a 540 bp open reading frame(ORF) encoding 179 amino acids. In addition, there were three mRNA instability motifs (ATTTA) in the 3"-untranslated region. The deduced protein sequence shared typical sequence features of the IL-IO family. Homology comparison indicated that the obtained sequence shared 89.1% homology with the carp IL-IO gene from GenBank. [Conclusion] This study laid foun- dation for further study of the expression manner, functional characteristic and regu- lation mechanism of IL-IO in vivo and the interaction mechanism in the inflammatory reaction and immune response.

  8. Sequencing and rescuing a highly virulent classical swine fever virus: Chinese strain cF114 from a full-length cDNA clone

    Institute of Scientific and Technical Information of China (English)

    2003-01-01

    The complete nucleotide sequence of classical swine fever virus (CSFV) strain cF114 (F114 strain propa- gated on PK-15 cells) was cloned by RT-PCR. The analyses of nucleotide and amino acids identity between cF114 and F114, Brescia, Alfort or C strain were 99.41%, 96.80%, 86.03%, 95.70% and 99.28%, 98.54%, 93.33%, 97.41% re- spectively. The cDNA fragments with correct sequence were ligated into a full-length cDNA and inserted into pMC18 plasmid (pMC12297). A full-length infectious viral RNA was synthesized by runoff transcription and transfected to PK15 cells. Viruses were recovered from transfected cells which wese titrated on PK-15 cells by endpoint dilution and indirect immunofluorescence with a CSFV-specific monoclonal antibody. The antigenicity and replication kinetics of the plasmid-derived virus (vM12297) were similar to the parental virus in vitro. The E01 or E2 gene was replaced with the genes from strain C and the pM/CE01 and pM/CE2 with chimeric full-length cDNA of cF114 were generated. The infectious viruses were obtained from pM/CE01 and pM/CE2. Both of the chimeric viruses can infect PK-15, SK- 6 and primary testicle cell of swine. The chimeric viruses can grow to a titer of 8×105 F-PFU/mL. These results are very important for understanding the genes related to the CSFV propagation and pathogenesis.

  9. Analysis of the full-length genome sequence of papaya lethal yellowing virus (PLYV), determined by deep sequencing, confirms its classification in the genus Sobemovirus.

    Science.gov (United States)

    Pereira, Alvaro J; Alfenas-Zerbini, Poliane; Cascardo, Renan S; Andrade, Eduardo C; Murilo Zerbini, F

    2012-10-01

    Papaya lethal yellowing virus (PLYV) causes an economically important disease in papayas in northeastern Brazil. Based on biological and molecular properties, PLYV has been tentatively assigned to the genus Sobemovirus. We report the sequence of the full-length genome of a PLYV isolate from Brazil, determined by deep sequencing. The PLYV genome is 4,145 nt long and contains four ORFs, with an arrangement identical to that of sobemoviruses. The polyprotein and CP display significant sequence identity with the corresponding proteins of other sobemoviruses. Pairwise comparisons and phylogenetic analysis based on complete nucleotide sequences confirm the classification of PLYV in the genus Sobemovirus. PMID:22743825

  10. A novel full-length isoform of murine pregnancy-specific glycoprotein 16 (psg16) is expressed in the brain but does not mediate murine coronavirus (MHV) entry

    OpenAIRE

    Phillips, Judith M.; Kuo, I-Ting; Richardson, Chelsea; Weiss, Susan R.

    2012-01-01

    The mouse pregnancy-specific glycoprotein 16 (PSG16) has been reported to be an alternative receptor for mouse hepatitis virus (MHV), some strains of which cause encephalitis in mice lacking the canonical receptor CEACAM1a. The known isoforms of PSG16 are N-terminally truncated relative to other PSG family proteins and are expressed in neurons as well as in the placenta. We have cloned a novel full-length isoform of psg16 that is also expressed in the brain, placenta, and retina but, like the...

  11. Molecular cloning of the genome of a cardiotropic Coxsackie B3 virus: full-length reverse-transcribed recombinant cDNA generates infectious virus in mammalian cells.

    OpenAIRE

    Kandolf, R; Hofschneider, P H

    1985-01-01

    The molecular cloning of double-stranded cDNA synthesized from the single-stranded RNA genome of the cardiotropic Coxsackie B3 virus (Nancy strain) is reported. Full-length reverse-transcribed cloned viral cDNA of approximately equal to 7500 nucleotides generated infectious antigenically identical Coxsackie B3 virus upon transfection of recombinant plasmid DNA into mammalian cells, demonstrating the molecular cloning of a biologically active viral cDNA copy. Furthermore, the cloned cDNA is ch...

  12. Cloning and expression of full-length human insulin-like growth factor binding protein 3 (IGFBP3 in the Escherichia coli

    Directory of Open Access Journals (Sweden)

    Emad Khodadadi

    2015-01-01

    Conclusion: DNA fragment encoding the full-length IGFBP3 protein was accurately cloned in the pET-11a expression vector and the recombinant plasmid transformed to E. coli BL21 (DE3 expression host. Results of the SDS-PAGE analysis verified that recombinant IGFBP3 (31.6 kDa are successfully expressed under the control of T7 promoter. As we shown pET-11a can be successfully used for expression of the IGFBP3 protein.

  13. Transfer of the Full-Length Dystrophin-Coding Sequence into Muscle Cells by a Dual High-Capacity Hybrid Viral Vector with Site-Specific Integration Ability

    OpenAIRE

    Gonçalves, Manuel A. F. V.; van Nierop, Gijsbert P.; Tijssen, Marloes R.; Lefesvre, Pierre; Knaän-Shanzer, Shoshan; van der Velde, Ietje; van Bekkum, Dirk W; Valerio, Dinko; de Vries, Antoine A. F.

    2005-01-01

    Duchenne muscular dystrophy (DMD) is caused by mutations in the DMD gene, making it a potential target for gene therapy. There is, however, a scarcity of vectors that can accommodate the 14-kb DMD cDNA and permanently genetically correct muscle tissue in vivo or proliferating myogenic progenitors in vitro for use in autologous transplantation. Here, a dual high-capacity adenovirus-adeno-associated virus (hcAd/AAV) vector with two full-length human dystrophin-coding sequences flanked by AAV in...

  14. Expression of a full-length cDNA for the human "MDR1" gene confers resistance to colchicine, doxorubicin, and vinblastine.

    OpenAIRE

    Ueda, K; Cardarelli, C; Gottesman, M M; Pastan, I

    1987-01-01

    Intrinsic and acquired multidrug resistance (MDR) is an important problem in cancer therapy. MDR in human KB carcinoma cells selected for resistance to colchicine, vinblastine, or doxorubicin (former generic name adriamycin) is associated with overexpression of the "MDR1" gene, which encodes P-glycoprotein. We previously have isolated an overlapping set of cDNA clones for the human MDR1 gene from multidrug-resistant KB cells. Here we report the construction of a full-length cDNA for the human...

  15. Inconsistencies of genome annotations in apicomplexan parasites revealed by 5'-end-one-pass and full-length sequences of oligo-capped cDNAs

    Directory of Open Access Journals (Sweden)

    Sugano Sumio

    2009-07-01

    Full Text Available Abstract Background Apicomplexan parasites are causative agents of various diseases including malaria and have been targets of extensive genomic sequencing. We generated 5'-EST collections for six apicomplexa parasites using our full-length oligo-capping cDNA library method. To improve upon the current genome annotations, as well as to validate the importance for physical cDNA clone resources, we generated a large-scale collection of full-length cDNAs for several apicomplexa parasites. Results In this study, we used a total of 61,056 5'-end-single-pass cDNA sequences from Plasmodium falciparum, P. vivax, P. yoelii, P. berghei, Cryptosporidium parvum, and Toxoplasma gondii. We compared these partially sequenced cDNA sequences with the currently annotated gene models and observed significant inconsistencies between the two datasets. In particular, we found that on average 14% of the exons in the current gene models were not supported by any cDNA evidence, and that 16% of the current gene models may contain at least one mis-annotation and should be re-evaluated. We also identified a large number of transcripts that had been previously unidentified. For 732 cDNAs in T. gondii, the entire sequences were determined in order to evaluate the annotated gene models at the complete full-length transcript level. We found that 41% of the T. gondii gene models contained at least one inconsistency. We also identified and confirmed by RT-PCR 140 previously unidentified transcripts found in the intergenic regions of the current gene annotations. We show that the majority of these discrepancies are due to questionable predictions of one or two extra exons in the upstream or downstream regions of the genes. Conclusion Our data indicates that the current gene models are likely to still be incomplete and have much room for improvement. Our unique full-length cDNA information is especially useful for further refinement of the annotations for the genomes of

  16. Cloning, expression, purification, crystallization and preliminary X-ray characterization of the full-length single-stranded DNA-binding protein from the hyperthermophilic bacterium Aquifex aeolicus

    OpenAIRE

    Clarke, David J.; Northey, Christopher G; Mack, Lynsey A.; McNae, Iain W.; Alexeev, Dmitriy; Sawyer, Lindsay; Campopiano, Dominic J.

    2004-01-01

    Single-stranded DNA-binding (SSB) proteins stabilize single-stranded DNA, which is exposed by separation of the duplex during DNA replication, recombination and repair. The SSB protein from the hyperthermophile Aquifex aeolicus has been overexpressed in Escherichia coli, purified and characterized and crystals of the full-length protein (147 amino acids; M(r) 17 131.20) have been grown by vapour diffusion from ammonium sulfate pH 7.5 in both the absence and presence of ssDNA [dT(pT)(68)]. All...

  17. Nucleotide sequence of classical swine fever virus strain Alfort/187 and transcription of infectious RNA from stably cloned full-length cDNA.

    OpenAIRE

    Ruggli, N; Tratschin, J D; Mittelholzer, C.; Hofmann, M A

    1996-01-01

    The complete nucleotide sequence of the genome of classical swine fever virus (CSFV) strain Alfort/187 was determined from three cDNA libraries constructed by cloning of DNA fragments obtained from independent sets of reverse transcription and PCR. The cDNA fragments were then assembled and inserted downstream of a T7 promoter in a P15A-derived plasmid vector to obtain the full-length cDNA clone pA187-1. The first nucleotide of the CSFV genome was positioned at the transcription start site of...

  18. Cloning and expression of full-length human insulin-like growth factor binding protein 3 (IGFBP3) in the Escherichia coli

    OpenAIRE

    Emad Khodadadi; Mojtaba Panjepour; Mahdi Abbasian; Zahra Khalili Broujeni; Mohammad Reza Mofid

    2015-01-01

    Background: The effect of the growth hormone on target cells is mediated by the insulin-like growth factor 1 (IGF-1). IGF-1 binds to the insulin-like growth factor binding proteins (IGFBPs) in blood and biological fluids. Considering the important application of IGBP3 as a drug component, in this research we cloned and expressed the full-length IGFBP3 in the pET-11a vector and BL21 (DE3) expression host. Materials and Methods: First the sequence encoding of IGFBP3 was designed based on th...

  19. Novel insights into structure–function mechanism and tissue-specific expression profiling of full-length dxr gene from Cymbopogon winterianus

    OpenAIRE

    Devi, Kamalakshi; Dehury, Budheswar; Phukon, Munmi; Modi, Mahendra Kumar; Sen, Priyabrata

    2015-01-01

    The 1-deoxy-d-xylulose-5-phosphate reductoisomerase (DXR; EC1.1.1.267), an NADPH-dependent reductase, plays a pivotal role in the methylerythritol 4-phosphate pathway (MEP), in the conversion of 1-deoxy-d-xylulose-5-phosphate (DXP) into MEP. The sheath and leaf of citronella (Cymbopogon winterianus) accumulates large amount of terpenes and sesquiterpenes with proven medicinal value and economic uses. Thus, sequencing of full length dxr gene and its characterization seems to be a valuable reso...

  20. Full-length insulin analogues and the effect of N-methylation of peptide bonds at positions B24, B25, B26

    Czech Academy of Sciences Publication Activity Database

    Antolíková, Emília; Žáková, Lenka; Cooper, A.; Kraus, Tomáš; Jiráček, Jiří

    2009-01-01

    Roč. 92, č. 4 (2009), s. 328-328. ISSN 0006-3525. [American Peptide Symposium /21./. 07.06.2009-12.06.2009, Bloomington] Institutional research plan: CEZ:AV0Z40550506 Keywords : ITC * N- methylation * dimerization Subject RIV: CC - Organic Chemistry

  1. Production of full-length soluble Plasmodium falciparum RH5 protein vaccine using a Drosophila melanogaster Schneider 2 stable cell line system

    Science.gov (United States)

    Hjerrild, Kathryn A.; Jin, Jing; Wright, Katherine E.; Brown, Rebecca E.; Marshall, Jennifer M.; Labbé, Geneviève M.; Silk, Sarah E.; Cherry, Catherine J.; Clemmensen, Stine B.; Jørgensen, Thomas; Illingworth, Joseph J.; Alanine, Daniel G. W.; Milne, Kathryn H.; Ashfield, Rebecca; de Jongh, Willem A.; Douglas, Alexander D.; Higgins, Matthew K.; Draper, Simon J.

    2016-01-01

    The Plasmodium falciparum reticulocyte-binding protein homolog 5 (PfRH5) has recently emerged as a leading candidate antigen against the blood-stage human malaria parasite. However it has proved challenging to identify a heterologous expression platform that can produce a soluble protein-based vaccine in a manner compliant with current Good Manufacturing Practice (cGMP). Here we report the production of full-length PfRH5 protein using a cGMP-compliant platform called ExpreS2, based on a Drosophila melanogaster Schneider 2 (S2) stable cell line system. Five sequence variants of PfRH5 were expressed that differed in terms of mutagenesis strategies to remove potential N-linked glycans. All variants bound the PfRH5 receptor basigin and were recognized by a panel of monoclonal antibodies. Analysis following immunization of rabbits identified quantitative and qualitative differences in terms of the functional IgG antibody response against the P. falciparum parasite. The antibodies induced by one protein variant were shown to be qualitatively similar to responses induced by other vaccine platforms. This work identifies Drosophila S2 cells as a clinically-relevant platform suited for the production of ‘difficult-to-make’ proteins from Plasmodium parasites, and identifies a PfRH5 sequence variant that can be used for clinical production of a non-glycosylated, soluble full-length protein vaccine immunogen. PMID:27457156

  2. Construction of a full-length cDNA library of Solen grandis dunker and identification of defense- and immune-related genes

    Science.gov (United States)

    Sun, Guohua; Liu, Xiangquan; Ren, Lihua; Yang, Jianmin; Wei, Xiumei; Yang, Jialong

    2013-11-01

    The basic genetic characteristics, important functional genes, and entire transcriptome of Solen grandis Dunker were investigated by constructing a full-length cDNA library with the `switching mechanism at the 5'-end of the RNA transcript' (SMART) technique. Total RNA was isolated from the immune-relevant tissues, gills and hemocytes, using the Trizol reagent, and cDNA fragments were digested with Sfi I before being ligated to the pBluescript II SK* vector. The cDNA library had a titer of 1048 cfu μL-1 and a storage capacity of 1.05×106 cfu. Approximately 98% of the clones in the library were recombinants, and the fragment lengths of insert cDNA ranged from 0.8 kb to 3.0 kb. A total of 2038 expressed sequence tags were successfully sequenced and clustered into 965 unigenes. BLASTN analysis showed that 240 sequences were highly similar to the known genes (E-value 80%), accounting for 25% of the total unigenes. According to the Gene Ontology, these unigenes were related to several biological processes, including cell structure, signal transport, protein synthesis, transcription, energy metabolism, and immunity. Fifteen of the identified sequences were related to defense and immunity. The full-length cDNA sequence of HSC70 was obtained. The cDNA library of S. grandis provided a useful resource for future researches of functional genomics related to stress tolerance, immunity, and other physiological activities.

  3. Sequencing analysis of 20,000 full-length cDNA clones from cassava reveals lineage specific expansions in gene families related to stress response

    Directory of Open Access Journals (Sweden)

    Sakaki Yoshiyuki

    2007-12-01

    Full Text Available Abstract Background Cassava, an allotetraploid known for its remarkable tolerance to abiotic stresses is an important source of energy for humans and animals and a raw material for many industrial processes. A full-length cDNA library of cassava plants under normal, heat, drought, aluminum and post harvest physiological deterioration conditions was built; 19968 clones were sequence-characterized using expressed sequence tags (ESTs. Results The ESTs were assembled into 6355 contigs and 9026 singletons that were further grouped into 10577 scaffolds; we found 4621 new cassava sequences and 1521 sequences with no significant similarity to plant protein databases. Transcripts of 7796 distinct genes were captured and we were able to assign a functional classification to 78% of them while finding more than half of the enzymes annotated in metabolic pathways in Arabidopsis. The annotation of sequences that were not paired to transcripts of other species included many stress-related functional categories showing that our library is enriched with stress-induced genes. Finally, we detected 230 putative gene duplications that include key enzymes in reactive oxygen species signaling pathways and could play a role in cassava stress response features. Conclusion The cassava full-length cDNA library here presented contains transcripts of genes involved in stress response as well as genes important for different areas of cassava research. This library will be an important resource for gene discovery, characterization and cloning; in the near future it will aid the annotation of the cassava genome.

  4. Sequencing analysis of 20,000 full-length cDNA clones from cassava reveals lineage specific expansions in gene families related to stress response

    Science.gov (United States)

    Sakurai, Tetsuya; Plata, Germán; Rodríguez-Zapata, Fausto; Seki, Motoaki; Salcedo, Andrés; Toyoda, Atsushi; Ishiwata, Atsushi; Tohme, Joe; Sakaki, Yoshiyuki; Shinozaki, Kazuo; Ishitani, Manabu

    2007-01-01

    Background Cassava, an allotetraploid known for its remarkable tolerance to abiotic stresses is an important source of energy for humans and animals and a raw material for many industrial processes. A full-length cDNA library of cassava plants under normal, heat, drought, aluminum and post harvest physiological deterioration conditions was built; 19968 clones were sequence-characterized using expressed sequence tags (ESTs). Results The ESTs were assembled into 6355 contigs and 9026 singletons that were further grouped into 10577 scaffolds; we found 4621 new cassava sequences and 1521 sequences with no significant similarity to plant protein databases. Transcripts of 7796 distinct genes were captured and we were able to assign a functional classification to 78% of them while finding more than half of the enzymes annotated in metabolic pathways in Arabidopsis. The annotation of sequences that were not paired to transcripts of other species included many stress-related functional categories showing that our library is enriched with stress-induced genes. Finally, we detected 230 putative gene duplications that include key enzymes in reactive oxygen species signaling pathways and could play a role in cassava stress response features. Conclusion The cassava full-length cDNA library here presented contains transcripts of genes involved in stress response as well as genes important for different areas of cassava research. This library will be an important resource for gene discovery, characterization and cloning; in the near future it will aid the annotation of the cassava genome. PMID:18096061

  5. cDNA microarray in isolation of novel differentially expressed genes related to human glioma and clone of a novel full-length gene

    Institute of Scientific and Technical Information of China (English)

    QI Zhen-yu; HUI Guo-zhen; LI Yao; ZHOU Zong-xiang; GU Shao-hua; YING Kang; XIE Yi

    2005-01-01

    Background This investigation was undertaken to obtain differentially expressed genes related to human glioma using cDNA microarray and the characterization of one novel full-length gene. Methods Total RNA was extracted from human glioma tissues and normal brain tissues, and mRNA was used to make probes. After hybridization and washing, the results were scanned using a computer system. The gene named 681F05 clone was an expressed gene to human glioma through four-time hybridization and scanning. Subsequently northern blot analysis was performed by northern blot, 5'RACE and bioinformatics. Results Fifteen differentially expressed genes to human glioma were obtained through four-time hybridization and scanning. Northern blot analysis confirmed that 681F05 clone was low-expressed in human brain tissues and over-expressed in human glioma tissues. The analysis of BLASTn and BLASTx showed that 681F05 clone is two cDNA clones encoding two novel proteins that are highly identified to the cyclophilin isoform 10 of C. Elgans, respectively. Sequence analysis revealed the two cDNA clones are two different splicing variants of a novel cycophilin-like gene (PPIL3a and PPIL3b).Conclusions cDNA microarray technology can be successfully used to identify differentially expressed genes. The novel full-length gene of human PPIL3 may be correlated with the formation of human glioma.

  6. Update on the efficacy, safety, and adherence to treatment of full length parathyroid hormone, PTH (1-84, in the treatment of postmenopausal osteoporosis

    Directory of Open Access Journals (Sweden)

    Luca Pietrogrande

    2009-11-01

    Full Text Available Luca PietrograndeDipartimento di Medicina Chirurgia e Odontoiatria Polo San Paolo, Università degli Studi di Milano, Milan, ItalyAbstract: Full length (1-84 parathyroid hormone (PTH was introduced in Europe as a treatment for postmenopausal osteoporosis in 2006. The efficacy of PTH (1-84 in the prevention of vertebral fractures is very high, and is similar to that of teriparatide. Its action in the prevention of femoral fractures has yet to be fully demonstrated, but the incidence of such fractures in trials was very low, and a decrease in nonvertebral fractures was seen in high-risk patients. The effect on bone mineral density (BMD was clearly demonstrated in the spine and also in the hip. The effects on BMD were evident and increased progressively with treatment until 36 months. After its discontinuation there was a clear decrease in BMD if no antiresorptive treatment was initiated. Increases in bone volumetric density and bone volume in trabecular sites were also reported. Moreover, a bone volume increase was detected in cortical sites. Hypercalcemia and hypercalciuria are frequent consequences of PTH treatment, but rarely have clinical effects and are usually well controlled by reducing calcium and vitamin D supplementation.Keywords: PTH (1-84, full-length parathyroid hormone, osteoporosis treatment

  7. Rational Design of High-Number dsDNA Fragments Based on Thermodynamics for the Construction of Full-Length Genes in a Single Reaction.

    Directory of Open Access Journals (Sweden)

    Bhagyashree S Birla

    Full Text Available Gene synthesis is frequently used in modern molecular biology research either to create novel genes or to obtain natural genes when the synthesis approach is more flexible and reliable than cloning. DNA chemical synthesis has limits on both its length and yield, thus full-length genes have to be hierarchically constructed from synthesized DNA fragments. Gibson Assembly and its derivatives are the simplest methods to assemble multiple double-stranded DNA fragments. Currently, up to 12 dsDNA fragments can be assembled at once with Gibson Assembly according to its vendor. In practice, the number of dsDNA fragments that can be assembled in a single reaction are much lower. We have developed a rational design method for gene construction that allows high-number dsDNA fragments to be assembled into full-length genes in a single reaction. Using this new design method and a modified version of the Gibson Assembly protocol, we have assembled 3 different genes from up to 45 dsDNA fragments at once. Our design method uses the thermodynamic analysis software Picky that identifies all unique junctions in a gene where consecutive DNA fragments are specifically made to connect to each other. Our novel method is generally applicable to most gene sequences, and can improve both the efficiency and cost of gene assembly.

  8. Efficient generation of recombinant RNA viruses using targeted recombination-mediated mutagenesis of bacterial artificial chromosomes containing full-length cDNA

    DEFF Research Database (Denmark)

    Rasmussen, Thomas Bruun; Risager, Peter Christian; Fahnøe, Ulrik;

    2013-01-01

    Background Infectious cDNA clones are a prerequisite for directed genetic manipulation of RNA viruses. Here, a strategy to facilitate manipulation and rescue of classical swine fever viruses (CSFVs) from full-length cDNAs present within bacterial artificial chromosomes (BACs) is described. This...... strategy allows manipulation of viral cDNA by targeted recombination-mediated mutagenesis within bacteria. Results A new CSFV-BAC (pBeloR26) derived from the Riems vaccine strain has been constructed and subsequently modified in the E2 coding sequence, using the targeted recombination strategy to enable...... rescue of chimeric pestiviruses (vR26_E2gif and vR26_TAV) with potential as new marker vaccine candidates. Sequencing of the BACs revealed a high genetic stability during passages within bacteria. The complete genome sequences of rescued viruses, after extensive passages in mammalian cells showed that...

  9. Isolation of full-length putative rat lysophospholipase cDNA using improved methods for mRNA isolation and cDNA cloning

    International Nuclear Information System (INIS)

    The authors have cloned a full-length putative rat pancreatic lysophospholipase cDNA by an improved mRNA isolation method and cDNA cloning strategy using [32P]-labelled nucleotides. These new methods allow the construction of a cDNA library from the adult rat pancreas in which the majority of recombinant clones contained complete sequences for the corresponding mRNAs. A previously recognized but unidentified long and relatively rare cDNA clone containing the entire sequence from the cap site at the 5' end to the poly(A) tail at the 3' end of the mRNA was isolated by single-step screening of the library. The size, amino acid composition, and the activity of the protein expressed in heterologous cells strongly suggest this mRNA codes for lysophospholipase

  10. Purification and characterization of recombinant full-length and protease domain of murine MMP-9 expressed in Drosophila S2 cells

    DEFF Research Database (Denmark)

    Rasch, Morten G; Lund, Ida K; Illemann, Martin;

    2010-01-01

    truncated versions were 5 mg/l and 2 mg/l, respectively. The products were >95% pure after gelatin Sepharose chromatography and possessed proteolytic activity when analyzed by gelatin zymography. Using the purified full-length murine MMP-9 we raised polyclonal antibodies by immunizations of rabbits. These....... No immunoreactivity was observed when the antibody was probed against skin wound material from MMP-9 deficient mice. In conclusion, we have generated and purified two proteolytically active recombinant murine MMP-9 protein constructs, which are critical reagents for future cancer drug discovery...... antibodies specifically identified pro-MMP-9 in incisional skin wound extracts from mice when used for Western blotting. Immunohistochemical analysis of paraffin embedded skin wounds from mice showed that MMP-9 protein was localized at the leading-edge keratinocytes in front of the migrating epidermal layer...

  11. Cloning a Full-length cDNA Encoding UDP-glucose Pyrophosphorylase from Amorpha fruticosa by PCR-based Methods

    Institute of Scientific and Technical Information of China (English)

    2002-01-01

    A method based on degenerate Oligo-primed polymerase chain reaction (PCR) and random amplification of cDNA end (RACE) PCR for cloning a full-length cDNA is described. An Amorpha fruticosa cDNA clone encoding UDP-glucose pyrophosphorylase (UGP), a key enzyme producing UDP-glucose in the synthesis of sucrose and cell ulose, is cloned by using this method. We design 5' RACE primers based on UGP A1 fragment, which obtains from degenerate PCR. Inverse PCR and nested PCR enable cloning of the remainder 5' and 3' end fragments of the gene. The deduced amino acid sequence exhibits significant homology with the other UGP genes cloned. This method is more simple and inexpensive than screening cDNA library, and can be easily adapted to clone other genes.

  12. From selective full-length genes isolation by TAR cloning in yeast to their expression from HAC vectors in human cells.

    Science.gov (United States)

    Kouprina, Natalay; Lee, Nicholas C O; Kononenko, Artem V; Samoshkin, Alexander; Larionov, Vladimir

    2015-01-01

    Transformation-associated recombination (TAR) cloning allows selective isolation of full-length genes and genomic loci as large circular Yeast Artificial Chromosomes (YACs) in yeast. The method has a broad application for structural and functional genomics, long-range haplotyping, characterization of chromosomal rearrangements, and evolutionary studies. In this paper, we describe a basic protocol for gene isolation by TAR as well as a method to convert TAR isolates into Bacterial Artificial Chromosomes (BACs) using a retrofitting vector. The retrofitting vector contains a 3' HPRT-loxP cassette to allow subsequent gene loading into a unique loxP site of the HAC-based (Human Artificial Chromosome) gene delivery vector. The benefit of combining the TAR gene cloning technology with the HAC gene delivery system for gene expression studies is discussed. PMID:25239739

  13. Molecular and biological characterization of highly infectious transcripts from full-length cDNA clones of broad bean wilt virus 1.

    Science.gov (United States)

    Ferriol, Inmaculada; Ambrós, Silvia; da Silva, Dorivaldo M; Falk, Bryce W; Rubio, Luis

    2016-06-01

    Broad bean wilt virus 1 (BBWV-1), genus Fabavirus, has a genome composed of two single-stranded positive-sense RNAs of ∼5.8 (RNA1) and 3.4kb (RNA2). Full-length cDNA clones of both genomic RNAs (pBenR1 and pBenR2) from BBWV-1 isolate Ben were constructed under the control of the T7 promoter. In vitro derived capped transcripts were infectious in Nicotiana benthamiana, Chenopodium quinoa and Vicia faba plants. The biological activity of viral transcripts was not affected by extra bases at the 5'-terminus introduced during in vitro transcription. Virions derived from the infectious cDNA clones displayed similar viral infectivity and accumulation, as well as symptom induction as the wild-type BBWV-1 isolate. PMID:26951858

  14. Full-length characterization of A1/D intersubtype recombinant genomes from a therapy-induced HIV type 1 controller during acute infection and his noncontrolling partner

    DEFF Research Database (Denmark)

    Fomsgaard, A.; Vinner, L.; Therrien, D.;

    2008-01-01

    To increase the understanding of mechanisms of HIV control we have genetically and immunologically characterized a full-length HIV-1 isolated from an acute infection in a rare case of undetectable viremia. The subject, a 43-year-old Danish white male (DK1), was diagnosed with acute HIV-1 infection...... after 1 year in Uganda. Following transient antiretroviral therapy DK1 maintained undetectable viral load for more than 10 years. His Ugandan wife (UG1) developed high viral load. HIV-1 sequences from both individuals were compared by bootscanning for recombination break points. Diversity plots and...... homology in shared regions. Four of seven crossover points were identical; however, the env gene from UG1 was subtype D, but A1 in DK1. Both viruses encoded proteins of the expected length and replicated equally well in vitro. DK1 and UG1 shared the HLA-A02 tissue type. HLA-A02-restricted CD8(+) T cell IFN...

  15. Copper and Zinc Interactions with Cellular Prion Proteins Change Solubility of Full-Length Glycosylated Isoforms and Induce the Occurrence of Heterogeneous Phenotypes

    Science.gov (United States)

    Brim, Svetlana; Groschup, Martin H.; Kuczius, Thorsten

    2016-01-01

    Prion diseases are characterized biochemically by protein aggregation of infectious prion isoforms (PrPSc), which result from the conformational conversion of physiological prion proteins (PrPC). PrPC are variable post-translationally modified glycoproteins, which exist as full length and as aminoterminally truncated glycosylated proteins and which exhibit differential detergent solubility. This implicates the presence of heterogeneous phenotypes, which overlap as protein complexes at the same molecular masses. Although the biological function of PrPC is still enigmatic, evidence reveals that PrPC exhibits metal-binding properties, which result in structural changes and decreased solubility. In this study, we analyzed the yield of PrPC metal binding affiliated with low solubility and changes in protein banding patterns. By implementing a high-speed centrifugation step, the interaction of zinc ions with PrPC was shown to generate large quantities of proteins with low solubility, consisting mainly of full-length glycosylated PrPC; whereas unglycosylated PrPC remained in the supernatants as well as truncated glycosylated proteins which lack of octarepeat sequence necessary for metal binding. This effect was considerably lower when PrPC interacted with copper ions; the presence of other metals tested exhibited no effect under these conditions. The binding of zinc and copper to PrPC demonstrated differentially soluble protein yields within distinct PrPC subtypes. PrPC–Zn2+-interaction may provide a means to differentiate glycosylated and unglycosylated subtypes and offers detailed analysis of metal-bound and metal-free protein conversion assays. PMID:27093554

  16. Development of three full-length infectious cDNA clones of distinct brassica yellows virus genotypes for agrobacterium-mediated inoculation.

    Science.gov (United States)

    Zhang, Xiao-Yan; Dong, Shu-Wei; Xiang, Hai-Ying; Chen, Xiang-Ru; Li, Da-Wei; Yu, Jia-Lin; Han, Cheng-Gui

    2015-02-01

    Brassica yellows virus is a newly identified species in the genus of Polerovirus within the family Luteoviridae. Brassica yellows virus (BrYV) is prevalently distributed throughout Mainland China and South Korea, is an important virus infecting cruciferous crops. Based on six BrYV genomic sequences of isolates from oilseed rape, rutabaga, radish, and cabbage, three genotypes, BrYV-A, BrYV-B, and BrYV-C, exist, which mainly differ in the 5' terminal half of the genome. BrYV is an aphid-transmitted and phloem-limited virus. The use of infectious cDNA clones is an alternative means of infecting plants that allows reverse genetic studies to be performed. In this study, full-length cDNA clones of BrYV-A, recombinant BrYV5B3A, and BrYV-C were constructed under control of the cauliflower mosaic virus 35S promoter. An agrobacterium-mediated inoculation system of Nicotiana benthamiana was developed using these cDNA clones. Three days after infiltration with full-length BrYV cDNA clones, necrotic symptoms were observed in the inoculated leaves of N. benthamiana; however, no obvious symptoms appeared in the upper leaves. Reverse transcription-PCR (RT-PCR) and western blot detection of samples from the upper leaves showed that the maximum infection efficiency of BrYVs could reach 100%. The infectivity of the BrYV-A, BrYV-5B3A, and BrYV-C cDNA clones was further confirmed by northern hybridization. The system developed here will be useful for further studies of BrYV, such as host range, pathogenicity, viral gene functions, and plant-virus-vector interactions, and especially for discerning the differences among the three genotypes. PMID:25499296

  17. Upright MRI measurement of mechanical axis and frontal plane alignment as a new technique: a comparative study with weight bearing full length radiographs

    Energy Technology Data Exchange (ETDEWEB)

    Liodakis, Emmanouil; Kenawey, Mohamed; Doxastaki, Iosifina; Krettek, Christian; Haasper, Carl; Hankemeier, Stefan [Medical School Hannover, Department of Trauma Surgery, Hannover (Germany)

    2011-07-15

    The purpose of this prospective study was to investigate the practicality, accuracy, and reliability of upright MR imaging as a new radiation-free technique for the measurement of mechanical axis. We used upright MRI in 15 consecutive patients (30 limbs, 44.7 {+-} 20.6 years old) to measure mechanical axis deviation (MAD), hip-knee-ankle (HKA) angle, leg length, and all remaining angles of the frontal plane alignment according to Paley (mLPFA, mLDTA, mMPTA, mLDTA, JLCA). The measurements were compared to weight bearing full length radiographs, which are considered to be the standard of reference for planning corrective surgery. FDA-approved medical planning software (MediCAD) was used for the above measurements. Intra- and inter-observer reproducibility using mean absolute differences was also calculated for both methods. The correlation coefficient between angles determined with upright MRI and weight bearing full length radiographs was high for mLPFA, mLDTA, mMPTA, mLDTA, and the HKA angle (r > 0.70). Mean interobserver and intraobserver agreements for upright MRI were also very high (r > 0.89). The leg length and the MAD were significantly underestimated by MRI (-3.2 {+-} 2.2 cm, p < 0.001 and -6.2 {+-} 4.4 mm, p = 0.006, respectively). With the exception of underestimation of leg length and MAD, upright MR imaging measurements of the frontal plane angles are precise and produce reliable, reproducible results. (orig.)

  18. Characterization of a full-length infectious cDNA clone and a GFP reporter derivative of the oncolytic picornavirus SVV-001.

    Science.gov (United States)

    Poirier, John T; Reddy, P Seshidhar; Idamakanti, Neeraja; Li, Shawn S; Stump, Kristine L; Burroughs, Kevin D; Hallenbeck, Paul L; Rudin, Charles M

    2012-12-01

    Seneca Valley virus (SVV-001) is an oncolytic picornavirus with selective tropism for a subset of human cancers with neuroendocrine differentiation. To characterize further the specificity of SVV-001 and its patterns and kinetics of intratumoral spread, bacterial plasmids encoding a cDNA clone of the full-length wild-type virus and a derivative virus expressing GFP were generated. The full-length cDNA of the SVV-001 RNA genome was cloned into a bacterial plasmid under the control of the T7 core promoter sequence to create an infectious cDNA clone, pNTX-09. A GFP reporter virus cDNA clone, pNTX-11, was then generated by cloning a fusion protein of GFP and the 2A protein from foot-and-mouth disease virus immediately following the native SVV-001 2A sequence. Recombinant GFP-expressing reporter virus, SVV-GFP, was rescued from cells transfected with in vitro RNA transcripts from pNTX-11 and propagated in cell culture. The proliferation kinetics of SVV-001 and SVV-GFP were indistinguishable. The SVV-GFP reporter virus was used to determine that a subpopulation of permissive cells is present in small-cell lung cancer cell lines previously thought to lack permissivity to SVV-001. Finally, it was shown that SVV-GFP administered to tumour-bearing animals homes in to and infects tumours whilst having no detectable tropism for normal mouse tissues at 1×10(11) viral particles kg(-1), a dose equivalent to that administered in ongoing clinical trials. These infectious clones will be of substantial value in further characterizing the biology of this virus and as a backbone for the generation of additional oncolytic derivatives. PMID:22971818

  19. Transcriptome and full-length cDNA resources for the mountain pine beetle, Dendroctonus ponderosae Hopkins, a major insect pest of pine forests.

    Science.gov (United States)

    Keeling, Christopher I; Henderson, Hannah; Li, Maria; Yuen, Mack; Clark, Erin L; Fraser, Jordie D; Huber, Dezene P W; Liao, Nancy Y; Docking, T Roderick; Birol, Inanc; Chan, Simon K; Taylor, Greg A; Palmquist, Diana; Jones, Steven J M; Bohlmann, Joerg

    2012-08-01

    Bark beetles (Coleoptera: Curculionidae: Scolytinae) are major insect pests of many woody plants around the world. The mountain pine beetle (MPB), Dendroctonus ponderosae Hopkins, is a significant historical pest of western North American pine forests. It is currently devastating pine forests in western North America--particularly in British Columbia, Canada--and is beginning to expand its host range eastward into the Canadian boreal forest, which extends to the Atlantic coast of North America. Limited genomic resources are available for this and other bark beetle pests, restricting the use of genomics-based information to help monitor, predict, and manage the spread of these insects. To overcome these limitations, we generated comprehensive transcriptome resources from fourteen full-length enriched cDNA libraries through paired-end Sanger sequencing of 100,000 cDNA clones, and single-end Roche 454 pyrosequencing of three of these cDNA libraries. Hybrid de novo assembly of the 3.4 million sequences resulted in 20,571 isotigs in 14,410 isogroups and 246,848 singletons. In addition, over 2300 non-redundant full-length cDNA clones putatively containing complete open reading frames, including 47 cytochrome P450s, were sequenced fully to high quality. This first large-scale genomics resource for bark beetles provides the relevant sequence information for gene discovery; functional and population genomics; comparative analyses; and for future efforts to annotate the MPB genome. These resources permit the study of this beetle at the molecular level and will inform research in other Dendroctonus spp. and more generally in the Curculionidae and other Coleoptera. PMID:22516182

  20. A Quantitative Characterization of Nucleoplasmin/Histone Complexes Reveals Chaperone Versatility.

    Science.gov (United States)

    Fernández-Rivero, Noelia; Franco, Aitor; Velázquez-Campoy, Adrian; Alonso, Edurne; Muga, Arturo; Prado, Adelina

    2016-01-01

    Nucleoplasmin (NP) is an abundant histone chaperone in vertebrate oocytes and embryos involved in storing and releasing maternal histones to establish and maintain the zygotic epigenome. NP has been considered a H2A-H2B histone chaperone, and recently it has been shown that it can also interact with H3-H4. However, its interaction with different types of histones has not been quantitatively studied so far. We show here that NP binds H2A-H2B, H3-H4 and linker histones with Kd values in the subnanomolar range, forming different complexes. Post-translational modifications of NP regulate exposure of the polyGlu tract at the disordered distal face of the protein and induce an increase in chaperone affinity for all histones. The relative affinity of NP for H2A-H2B and linker histones and the fact that they interact with the distal face of the chaperone could explain their competition for chaperone binding, a relevant process in NP-mediated sperm chromatin remodelling during fertilization. Our data show that NP binds H3-H4 tetramers in a nucleosomal conformation and dimers, transferring them to DNA to form disomes and tetrasomes. This finding might be relevant to elucidate the role of NP in chromatin disassembly and assembly during replication and transcription. PMID:27558753

  1. Measuring the Energetics of Membrane Protein Dimerization in Mammalian Membranes

    OpenAIRE

    Chen, Lirong; Novicky, Lawrence; Merzlyakov, Mikhail; Hristov, Tihomir; Hristova, Kalina

    2010-01-01

    Thus far, methods that give quantitative information about lateral interactions in membranes have been restricted peptides or simplified protein constructs studied in detergents, lipid vesicles or bacterial membranes. None of the available methods have been extended to complex or full length membrane proteins. Here we show how free energies of membrane protein dimerization can be measured in mammalian plasma membrane-derived vesicles. The measurements, performed in single vesicles, utilize th...

  2. Gene discovery from Jatropha curcas by sequencing of ESTs from normalized and full-length enriched cDNA library from developing seeds

    Directory of Open Access Journals (Sweden)

    Sugantham Priyanka Annabel

    2010-10-01

    Full Text Available Abstract Background Jatropha curcas L. is promoted as an important non-edible biodiesel crop worldwide. Jatropha oil, which is a triacylglycerol, can be directly blended with petro-diesel or transesterified with methanol and used as biodiesel. Genetic improvement in jatropha is needed to increase the seed yield, oil content, drought and pest resistance, and to modify oil composition so that it becomes a technically and economically preferred source for biodiesel production. However, genetic improvement efforts in jatropha could not take advantage of genetic engineering methods due to lack of cloned genes from this species. To overcome this hurdle, the current gene discovery project was initiated with an objective of isolating as many functional genes as possible from J. curcas by large scale sequencing of expressed sequence tags (ESTs. Results A normalized and full-length enriched cDNA library was constructed from developing seeds of J. curcas. The cDNA library contained about 1 × 106 clones and average insert size of the clones was 2.1 kb. Totally 12,084 ESTs were sequenced to average high quality read length of 576 bp. Contig analysis revealed 2258 contigs and 4751 singletons. Contig size ranged from 2-23 and there were 7333 ESTs in the contigs. This resulted in 7009 unigenes which were annotated by BLASTX. It showed 3982 unigenes with significant similarity to known genes and 2836 unigenes with significant similarity to genes of unknown, hypothetical and putative proteins. The remaining 191 unigenes which did not show similarity with any genes in the public database may encode for unique genes. Functional classification revealed unigenes related to broad range of cellular, molecular and biological functions. Among the 7009 unigenes, 6233 unigenes were identified to be potential full-length genes. Conclusions The high quality normalized cDNA library was constructed from developing seeds of J. curcas for the first time and 7009 unigenes coding

  3. Lysine-Specific Demethylase 1A (KDM1A/LSD1): Product Recognition and Kinetic Analysis of Full-Length Histones.

    Science.gov (United States)

    Burg, Jonathan M; Gonzalez, Julie J; Maksimchuk, Kenneth R; McCafferty, Dewey G

    2016-03-22

    Lysine-specific demethylase 1A (KDM1A/LSD1) is a FAD-dependent enzyme that catalyzes the oxidative demethylation of histone H3K4me1/2 and H3K9me1/2 repressing and activating transcription, respectively. Although the active site is expanded compared to that of members of the greater amine oxidase superfamily, it is too sterically restricted to encompass the minimal 21-mer peptide substrate footprint. The remainder of the substrate/product is therefore expected to extend along the surface of KDM1A. We show that full-length histone H3, which lacks any posttranslational modifications, is a tight-binding, competitive inhibitor of KDM1A demethylation activity with a Ki of 18.9 ± 1.2 nM, a value that is approximately 100-fold higher than that of the 21-mer peptide product. The relative H3 affinity is independent of preincubation time, suggesting that H3 rapidly reaches equilibrium with KDM1A. Jump dilution experiments confirmed the increased binding affinity of full-length H3 was at least partially due to a slow off rate (koff) of 1.2 × 10(-3) s(-1), corresponding to a half-life (t1/2) of 9.63 min, and a residence time (τ) of 13.9 min. Independent affinity capture surface plasmon resonance experiments confirmed the tight-binding nature of the H3/KDM1A interaction, revealing a Kd of 9.02 ± 2.3 nM, a kon of (9.3 ± 1.5) × 10(4) M(-1) s(-1), and a koff of (8.4 ± 0.3) × 10(-4) s(-1). Additionally, no other core histones exhibited inhibition of KDM1A demethylation activity, which is consistent with H3 being the preferred histone substrate of KDM1A versus H2A, H2B, and H4. Together, these data suggest that KDM1A likely contains a histone H3 secondary specificity element on the enzyme surface that contributes significantly to its recognition of substrates and products. PMID:26673564

  4. Neutron scattering in dimers

    DEFF Research Database (Denmark)

    Gudel, H. U.; Furrer, A.; Kjems, Jørgen

    1986-01-01

    Insulating compounds containing dimers of transition metal and rare earth ions have been studied by inelastic neutron scattering (INS). Energy splittings can be directly determined, and the corresponding parameters are easily extracted from the experimental data. The intensities of dimer...

  5. 5'-end sequences of budding yeast full-length cDNA clones and quality scores - Budding yeast cDNA sequencing project | LSDB Archive [Life Science Database Archive metadata

    Lifescience Database Archive (English)

    Full Text Available Budding yeast cDNA sequencing project 5'-end sequences of budding yeast full-length cDNA clones and quality ...scores Data detail Data name 5'-end sequences of budding yeast full-length cDNA clones and quality scores De...from the budding yeast full-length cDNA library by the vector-capping method, the sequence quality score gen...s accession only. Sequence 5'-end sequence data of budding yeast full-length cDNA clones. FASTA format. Quality Phred's quality... Update History of This Database Site Policy | Contact Us 5'-end sequences of budding yeast full-length cDNA clones and quality

  6. Neutron scattering in dimers

    DEFF Research Database (Denmark)

    Gudel, H. U.; Furrer, A.; Kjems, Jørgen

    Insulating compounds containing dimers of transition metal and rare earth ions have been studied by inelastic neutron scattering (INS). Energy splittings can be directly determined, and the corresponding parameters are easily extracted from the experimental data. The intensities of dimer excitati......Insulating compounds containing dimers of transition metal and rare earth ions have been studied by inelastic neutron scattering (INS). Energy splittings can be directly determined, and the corresponding parameters are easily extracted from the experimental data. The intensities of dimer...

  7. Poplar defense against insects: genome analysis, full-length cDNA cloning, and transcriptome and protein analysis of the poplar Kunitz-type protease inhibitor family.

    Science.gov (United States)

    Philippe, Ryan N; Ralph, Steven G; Külheim, Carsten; Jancsik, Sharon I; Bohlmann, Jörg

    2009-12-01

    *Kunitz protease inhibitors (KPIs) feature prominently in poplar defense responses against insects. The increasing availability of genomics resources enabled a comprehensive analysis of the poplar (p)KPI family. *Using genome analysis, expressed sequence tag (EST) mining and full-length (FL)cDNA cloning we established an inventory and phylogeny of pKPIs. Microarray and real-time PCR analyses were used to profile pKPI gene expression following real or simulated insect attack. Proteomics of insect midgut content was used to monitor stability of pKPI protein. *We identified 31 pKPIs in the genome and validated gene models by EST mining and cloning of 41 unique FLcDNAs. Genome organization of the pKPI family, with six poplar-specific subfamilies, suggests that tandem duplications have played a major role in its expansion. pKPIs are expressed throughout the plant and many are strongly induced by insect attack, although insect-specific signals seem initially to suppress the tree pKPI response. We found substantial peptide coverage for a potentially intact pKPI protein in insect midgut after eating poplar leaves. *These results highlight the complexity of an important defense gene family in poplar with regard to gene family size, differential constitutive and insect-induced gene expression, and resilience of at least one pKPI protein to digestion by herbivores. PMID:19780988

  8. Phylogenetic Analysis of Ethiopian HIV-1 Subtype C Near Full-Length Genomes Reveals High Intrasubtype Diversity and a Strong Geographical Cluster.

    Science.gov (United States)

    Amogne, Wondwossen; Bontell, Irene; Grossmann, Sebastian; Aderaye, Getachew; Lindquist, Lars; Sönnerborg, Anders; Neogi, Ujjwal

    2016-05-01

    In this study, we characterize HIV-1 subtype C (HIV-1C) strains at the near full-length genome (NFLG) level and perform genotypic drug resistance testing (GRT) and genotypic tropism testing (GTT) from Ethiopia (HIV-1CET). Plasma samples (n = 150) were obtained from therapy-naive individuals residing in Addis Ababa, Ethiopia in 2008. HIV-NFLG was performed in a subset of patients (n = 30). GRT (pol) and GTT (V3 env) were performed using in-house methods. GTT was analyzed by PhenoSeq-C. The phylogenetic analysis of the NLFG identified two separate clusters of HIV-1CET, although all strains formed one large overarching cluster together. At NFLG, greater diversity was found among HIV-1CET strains compared to HIV-1C strains from other geographical locations. The geographic clustering was weak in the small subgenomic (pol and env) regions. The primary drug-resistant mutations were identified at a low level (<5%). GTT identified that 12% (12/102) of the patients were predicted to be harboring X4-tropic or both R5/X4-tropic viruses. PMID:26881451

  9. Cloning of the Full-length cDNA of the Wheat Involved in Salt Stress: Root Hair Defective 3 Gene (RHD3)

    Institute of Scientific and Technical Information of China (English)

    Lei SHAN; Shuang-Yi ZHAO; Guang-Min XIA

    2005-01-01

    The full-length cDNA of the wheat (Triticum aestivum L.) root hair defective 3 gene (RHD3) has been cloned from the salt-tolerant hybrid wheat variety Shanrong No. 3 (Za3) using the mRNA differential display and 5′ rapid amplification of cDNA ends (RACE) methods. Analysis of the amino acid sequence deduced from the wheat RHD3 gene shows that two conservative GTP-binding motifs, namely GXXXXGKS and DXXG, in eukaryotes also exist at the N-terminal of wheat RHD3. In addition, an 18 amino acid residue transmembrane domain, namely FYLAVMFVVFLVGKAIWV, exists at positions 701-718 of the C-terminal of the deduced protein of wheat RHD3 obtained, but this domain is absent in another three proteins aligned,including rice RHD3, Arabidopsis RHD3, and yeast homologue SEY1. Northern blot revealed that transcription of the wheat RHD3 gene is down-regulated in both the salt-tolerant line and in JN177 under saline stress. A possible stress-responsive mechanism for this gene is discussed.

  10. Characterization of the cloned full-length and a truncated human target of rapamycin: Activity, specificity, and enzyme inhibition as studied by a high capacity assay

    International Nuclear Information System (INIS)

    The mammalian target of rapamycin (mTOR/TOR) is implicated in cancer and other human disorders and thus an important target for therapeutic intervention. To study human TOR in vitro, we have produced in large scale both the full-length TOR (289 kDa) and a truncated TOR (132 kDa) from HEK293 cells. Both enzymes demonstrated a robust and specific catalytic activity towards the physiological substrate proteins, p70 S6 ribosomal protein kinase 1 (p70S6K1) and eIF4E binding protein 1 (4EBP1), as measured by phosphor-specific antibodies in Western blotting. We developed a high capacity dissociation-enhanced lanthanide fluorescence immunoassay (DELFIA) for analysis of kinetic parameters. The Michaelis constant (K m) values of TOR for ATP and the His6-S6K substrate were shown to be 50 and 0.8 μM, respectively. Dose-response and inhibition mechanisms of several known inhibitors, the rapamycin-FKBP12 complex, wortmannin and LY294002, were also studied in DELFIA. Our data indicate that TOR exhibits kinetic features of those shared by traditional serine/threonine kinases and demonstrate the feasibility for TOR enzyme screen in searching for new inhibitors

  11. A truncated fragment of Ov-ASP-1 consisting of the core pathogenesis-related-1 (PR-1) domain maintains adjuvanticity as the full-length protein.

    Science.gov (United States)

    Guo, Jingjing; Yang, Yi; Xiao, Wenjun; Sun, Weilai; Yu, Hong; Du, Lanying; Lustigman, Sara; Jiang, Shibo; Kou, Zhihua; Zhou, Yusen

    2015-04-15

    The Onchocerca volvulus activation-associated secreted protein-1 (Ov-ASP-1) has good adjuvanticity for a variety of antigens and vaccines, probably due to its ability activate antigen-processing cells (APCs). However, the functional domain of Ov-ASP-1 as an adjuvant is not clearly defined. Based on the structural prediction of this protein family, we constructed a 16-kDa recombinant protein of Ov-ASP-1 that contains only the core pathogenesis-related-1 (PR-1) domain (residues 10-153), designated ASPPR. We found that ASPPR exhibits adjuvanticity similar to that of the full-length Ov-ASP-1 (residues 10-220) for various antigens, including ovalbumin (OVA), HBsAg protein antigen, and the HIV peptide 5 (Pep5) antigen, but it is more suitable for vaccine design in ASPPR-antigen fusion proteins, and more stable in PBS than Ov-ASP-1 stored at -70 °C. These results suggest that ASPPR might be the functional region of Ov-ASP-1 as an adjuvant, and therefore could be developed as an adjuvant for human use. PMID:25736195

  12. Strategies to facilitate the development of uncloned or cloned infectious full-length viral cDNAs: Apple chlorotic leaf spot virus as a case study

    Directory of Open Access Journals (Sweden)

    Marais Armelle

    2011-10-01

    Full Text Available Abstract Background Approaches to simplify and streamline the construction of full-length infectious cDNA clones (FL-cDNAs are needed. Among desirable improvements are the ability to use total nucleic acids (TNA extracts from infected hosts (to bypass viral purification limitations for the direct one-step amplification of large FL-cDNAs, the possibility to inoculate plants with uncloned FL-cDNAs and the simplified cloning of these large molecules. Results Using the 7.55 kb genome of Apple chlorotic leaf spot trichovirus (ACLSV approaches allowing the rapid generation from TNA extracts of FL-cDNAs under the control of the T7 promoter and the successful inoculation of plants using in vitro transcripts obtained from these uncloned amplification products have been developed. We also show that the yeast homologous recombination system permits efficient cloning of FL-cDNAs and the simultaneous one-step tailoring of a ternary Yeast-Escherichia coli-Agrobacterium tumefaciens shuttle vector allowing efficient inoculation of both herbaceous and woody host plants by agroinfiltration. Conclusions The fast and efficient strategies described here should have broad applications, in particular for the study of "difficult" plant viruses, such as those infecting woody hosts, and potentially for other, non plant-infecting viral agents.

  13. Crystallization and preliminary X-ray crystallographic analysis of a full-length active form of the Cry4Ba toxin from Bacillus thuringiensis

    International Nuclear Information System (INIS)

    The crystallization of the Cry4Ba toxin from B. thuringiensis is described. To obtain a complete structure of the Bacillus thuringiensis Cry4Ba mosquito-larvicidal protein, a 65 kDa functional form of the Cry4Ba-R203Q mutant toxin was generated for crystallization by eliminating the tryptic cleavage site at Arg203. The 65 kDa trypsin-resistant fragment was purified and crystallized using the sitting-drop vapour-diffusion method. The crystals belonged to the rhombohedral space group R32, with unit-cell parameters a = b = 184.62, c = 187.36 Å. Diffraction data were collected to at least 2.07 Å resolution using synchrotron radiation and gave a data set with an overall Rmerge of 9.1% and a completeness of 99.9%. Preliminary analysis indicated that the asymmetric unit contained one molecule of the active full-length mutant, with a VM coefficient and solvent content of 4.33 Å3 Da−1 and 71%, respectively

  14. RNase HI overproduction is required for efficient full-length RNA synthesis in the absence of topoisomerase I in Escherichia coli.

    Science.gov (United States)

    Baaklini, Imad; Hraiky, Chadi; Rallu, Fabien; Tse-Dinh, Yuk-Ching; Drolet, Marc

    2004-10-01

    It has long been known that Escherichia coli cells deprived of topoisomerase I (topA null mutants) do not grow. Because mutations reducing DNA gyrase activity and, as a consequence, negative supercoiling, occur to compensate for the loss of topA function, it has been assumed that excessive negative supercoiling is somehow involved in the growth inhibition of topA null mutants. However, how excess negative supercoiling inhibits growth is still unknown. We have previously shown that the overproduction of RNase HI, an enzyme that degrades the RNA portion of an R-loop, can partially compensate for the growth defects because of the absence of topoisomerase I. In this article, we have studied the effects of gyrase reactivation on the physiology of actively growing topA null cells. We found that growth immediately and almost completely ceases upon gyrase reactivation, unless RNase HI is overproduced. Northern blot analysis shows that the cells have a significantly reduced ability to accumulate full-length mRNAs when RNase HI is not overproduced. Interestingly, similar phenotypes, although less severe, are also seen when bacterial cells lacking RNase HI activity are grown and treated in the same way. All together, our results suggest that excess negative supercoiling promotes the formation of R-loops, which, in turn, inhibit RNA synthesis. PMID:15458416

  15. Expression of a full-length cDNA for the human MDR1 gene confers resistance to colchicine, doxorubicin, and vinblastine

    International Nuclear Information System (INIS)

    Intrinsic and acquired multidrug resistance (MDR) is an important problem in cancer therapy. MDR in human KB carcinoma cells selected for resistance to colchicine, vinblastine, or doxorubicin (former generic name adriamycin) is associated with overexpression of the MDR1 gene, which encodes P-glycoprotein. The authors previously have isolated an overlapping set of cDNA clones for the human MDR1 gene from multidrug-resistant KB cells. Here they report the construction of a full-length cDNA for the human MDR1 gene and show that this reconstructed cDNA, when inserted into a retroviral expression vector containing the long terminal repeats of Moloney leukemia virus or Harvey sarcoma virus, functions in mouse NIH 3T3 and human KB cells to confer the complete multidrug-resistance phenotype. These results suggest that the human MDR1 gene may be used as a positive selectable marker to introduce genes into human cells and to transform human cells to multidrug resistance without introducing nonhuman antigens

  16. Short Communication: Phylogenetic and Molecular Characterization of Six Full-Length HIV-1 Genomes from India Reveals a Monophyletic Lineage of Indian Sub-Subtype A1.

    Science.gov (United States)

    Pandey, Sudhanshu Shekhar; Cherian, Sarah; Thakar, Madhuri; Paranjape, Ramesh S

    2016-05-01

    Although HIV-1 epidemic in India is mainly driven by subtype C, subtype A has been reported for over two decades. This is the first comprehensive analysis of sequences of HIV-1 subtype A from India, based on the near full-length genome sequences of six different HIV-1 subtype A Indian isolates along with available partial gene sequences from India and global sequences. The phylogenetic analyses revealed the convergence of all Indian whole-genome sequences and majority of the partial gene sequences to a single node with the sequences most closely related to African sub-subtype A1. The presence of the signature motifs consistent with those observed in subtype A and CTL epitopes characterized specifically for subtype A1 were observed among the study sequences. Deletion of LY amino acid of LYPXnL motif of p6gag and one amino acid in V3 loop have been observed among the study isolates, which have also been observed in a few sequences from East Africa. Overall, the results are indicative of a monophyletic lineage or founder effect of the Indian epidemic due to sub-subtype A1 and supportive of a possible migration of subtype A1 into India from East Africa. PMID:26756665

  17. Identification of cDNAs encoding viper venom hyaluronidases: cross-generic sequence conservation of full-length and unusually short variant transcripts.

    Science.gov (United States)

    Harrison, Robert A; Ibison, Frances; Wilbraham, Davina; Wagstaff, Simon C

    2007-05-01

    The immobilisation of prey by snakes is most efficiently achieved by the rapid dissemination of venom from its site of injection into the blood stream. Hyaluronidase is a common component of snake venoms and has been termed the "venom spreading factor". In the absence of nucleotide or protein sequence data to confirm the functional identity of this venom component, we interrogated a venom gland EST database for the saw-scaled viper, Echis ocellatus (Nigeria), using the gene ontology (GO) term "carbohydrate metabolism". A single hyalurononglucosaminadase-activity matching sequence (EOC00242) was found and used to design PCR primers to acquire the full-length cDNA sequence. Although very different from the bee venom and mammalian hyaluronidase sequences, the E. ocellatus sequence retained all the catalytic, positional and structural residues that characterise this class of carbohydrate metabolising hydrolases. An extraordinarily high level of sequence identity (>95%) was observed in analogous venom gland cDNA sequences isolated (by PCR) from another saw-scaled viper species, E. pyramidum leakeyi (Kenya), and from the sahara horned viper, Cerastes cerastes cerastes (Egypt) and the puff adder, Bitis arietans (Nigeria). Smaller amplicons, lacking hyaluronidase catalytic residues because of 768 bp or 855 bp central deletions, appear to encode either truncated peptides without hyaluronidase activity, or are non-translated transcripts because they lack consensus translation initiating motifs. PMID:17210232

  18. Construction and characterisation of a full-length infectious molecular clone from a fast replicating, X4-tropic HIV-1 CRF02.AG primary isolate

    International Nuclear Information System (INIS)

    Based on our previous analysis of HIV-1 isolates from Cameroon, we constructed a full-length infectious molecular clone from a primary isolate belonging to the CRF02.AG group of recombinant viruses which dominate the HIV-epidemic in West and Central Africa. The virus derived by transfection of the proviral clone pBD6-15 replicated with similar efficiency compared to its parental isolate and used CXCR4 as coreceptor as well. Furthermore, HIV-1 BD6-15 exhibited similar replication properties and virus yield as the reference B-type HIV-1 strain NL4-3. Sequence analysis revealed open reading frames for all structural and accessory genes apart from vpr. Phylogenetic and bootscanning analyses confirmed that BD6-15 clusters with CRF02.AG recombinant strains from West and Central Africa with similar cross-over points as described for the CRF02.AG prototype strain lbNG. Thus, pBD6-15 represents the first non-subtype B infectious molecular clone of a fast replicating, high producer, X4-tropic primary HIV-1 isolate, which had only been briefly passaged in primary cells

  19. Complete genome sequence and construction of infectious full-length cDNA clones of tobacco ringspot Nepovirus, a viral pathogen causing bud blight in soybean.

    Science.gov (United States)

    Zhao, Fumei; Hwang, Un Sun; Lim, Seungmo; Yoo, Ran Hee; Igori, Davaajargal; Lee, Su-Heon; Lim, Hyoun-Sub; Moon, Jae Sun

    2015-08-01

    Tobacco ringspot virus (TRSV, genus Nepovirus), causes severe diseases in soybean and tobacco plants. TRSV-induced bud blight disease significantly reduced both the yield and quality of soybeans. The function of the encoded viral gene product involved in TRSV infection was unclear due to the limitation of reverse genetics studies on the viral genome. Here, we represent the successful construction of infectious full-length cDNA clones of TRSV genome (RNA1 and RNA2). The cDNAs of TRSV RNA1 and RNA2 were cloned into the binary vector pPZP211 immediately downstream of a double cauliflower mosaic virus 35S promoter and upstream of the nopaline synthase terminator. Seven days after agrobacterium-mediated co-inoculation of these two constructs, Nicotiana benthamiana plants developed a systemic infection with necrotic ringspot symptoms and weak stunting of the leaves, similar to that induced by natural TRSV. The systemic infection was confirmed by transmission electron microscopy and Western blot analysis. Simultaneously, soybean, tomato, and Arabidopsis ecotype Estland were mechanically inoculated with sap prepared from TRSV-agroinfiltrated N. benthamiana leaves, showing typical symptoms of bud blight, necrotic spots, and lethal systemic necrosis, respectively. The system developed herein will be an appealing way to determine TRSV viral gene functions and study host-TRSV interactions. PMID:26159876

  20. Characterization of the role of full-length CRMP3 and its calpain-cleaved product in inhibiting microtubule polymerization and neurite outgrowth

    International Nuclear Information System (INIS)

    Collapsin response mediator proteins (CRMPs) are key modulators of cytoskeletons during neurite outgrowth in response to chemorepulsive guidance molecules. However, their roles in adult injured neurons are not well understood. We previously demonstrated that CRMP3 underwent calcium-dependent N-terminal protein cleavage during excitotoxicity-induced neurite retraction and neuronal death. Here, we report findings that the full-length CRMP3 inhibits tubulin polymerization and neurite outgrowth in cultured mature cerebellar granule neurons, while the N-terminal truncated CRMP3 underwent nuclear translocation and caused a significant nuclear condensation. The N-terminal truncated CRMP3 underwent nuclear translocation through nuclear pores. Nuclear protein pull-down assay and mass spectrometry analysis showed that the N-terminal truncated CRMP3 was associated with nuclear vimentin. In fact, nuclear-localized CRMP3 co-localized with vimentin during glutamate-induced excitotoxicity. However, the association between the truncated CRMP3 and vimentin was not critical for nuclear condensation and neurite outgrowth since over-expression of truncated CRMP3 in vimentin null neurons did not alleviate nuclear condensation and neurite outgrowth inhibition. Together, these studies showed CRMP3's role in attenuating neurite outgrowth possibility through inhibiting microtubule polymerization, and also revealed its novel association with vimentin during nuclear condensation prior to neuronal death.

  1. Characterization of the role of full-length CRMP3 and its calpain-cleaved product in inhibiting microtubule polymerization and neurite outgrowth

    Energy Technology Data Exchange (ETDEWEB)

    Aylsworth, Amy [Experimental NeuroTherapeutics Laboratory, Institute for Biological Sciences, National Research Council Canada, 1200 Montreal Road, Bldg M54, Ottawa, Ontario, Canada K1A 0R6 (Canada); Department of Biochemistry, Microbiology and Immunology, University of Ottawa, Ottawa, Ontario (Canada); Jiang, Susan X.; Desbois, Angele [Experimental NeuroTherapeutics Laboratory, Institute for Biological Sciences, National Research Council Canada, 1200 Montreal Road, Bldg M54, Ottawa, Ontario, Canada K1A 0R6 (Canada); Hou, Sheng T., E-mail: Sheng.hou@nrc-cnrc.gc.ca [Experimental NeuroTherapeutics Laboratory, Institute for Biological Sciences, National Research Council Canada, 1200 Montreal Road, Bldg M54, Ottawa, Ontario, Canada K1A 0R6 (Canada); Department of Biochemistry, Microbiology and Immunology, University of Ottawa, Ottawa, Ontario (Canada)

    2009-10-01

    Collapsin response mediator proteins (CRMPs) are key modulators of cytoskeletons during neurite outgrowth in response to chemorepulsive guidance molecules. However, their roles in adult injured neurons are not well understood. We previously demonstrated that CRMP3 underwent calcium-dependent N-terminal protein cleavage during excitotoxicity-induced neurite retraction and neuronal death. Here, we report findings that the full-length CRMP3 inhibits tubulin polymerization and neurite outgrowth in cultured mature cerebellar granule neurons, while the N-terminal truncated CRMP3 underwent nuclear translocation and caused a significant nuclear condensation. The N-terminal truncated CRMP3 underwent nuclear translocation through nuclear pores. Nuclear protein pull-down assay and mass spectrometry analysis showed that the N-terminal truncated CRMP3 was associated with nuclear vimentin. In fact, nuclear-localized CRMP3 co-localized with vimentin during glutamate-induced excitotoxicity. However, the association between the truncated CRMP3 and vimentin was not critical for nuclear condensation and neurite outgrowth since over-expression of truncated CRMP3 in vimentin null neurons did not alleviate nuclear condensation and neurite outgrowth inhibition. Together, these studies showed CRMP3's role in attenuating neurite outgrowth possibility through inhibiting microtubule polymerization, and also revealed its novel association with vimentin during nuclear condensation prior to neuronal death.

  2. [Rescue of bovine Asia 1 serotype foot-and-mouth disease virus from a full-length cDNA clone].

    Science.gov (United States)

    Li, Shuang; Zhang, Runxiang; Song, Ge; Gao, Mingchun; Liu, Xiangtao; Wang, Junwei

    2009-11-01

    After sequencing the Asia 1 foot-and-mouth disease virus (FMDV) (As01 strain), we amplified the two fragments covering the whole genome by overlapping PCR and long PCR. The 5' fragment was 1.8 kb in length including 15Cs, and the 3' fragment was 6.7 kb in length. The two fragments were cloned into the pBluescript SK vector to construct recombinant plasmid pBSAs carrying the full-length cDNA of FMDV As01 strain. The RNA transcript was synthesized in vitro using T7 polymerase and transfected into BHK-21 cells. We observed the typical CPE caused by rescued FMDV. The harvested virus was confirmed to be Asia 1 FMDV by RT-PCR, indirect immunofluorescence assay (IFA) and electron microscope observation. The rescued virus showed a similar pathogenicity in suckling mouse (LD50) compared to its wild-type virus. The infectious cDNA clone of the FMDV As01 strain laid a new ground for further investigation of FMDV virulence determinants and development of novel vaccines against FMD. PMID:20222458

  3. Genome-wide characterization of the biggest grass, bamboo, based on 10,608 putative full-length cDNA sequences

    Directory of Open Access Journals (Sweden)

    Zhu Chuanrang

    2010-06-01

    Full Text Available Abstract Background With the availability of rice and sorghum genome sequences and ongoing efforts to sequence genomes of other cereal and energy crops, the grass family (Poaceae has become a model system for comparative genomics and for better understanding gene and genome evolution that underlies phenotypic and ecological divergence of plants. While the genomic resources have accumulated rapidly for almost all major lineages of grasses, bamboo remains the only large subfamily of Poaceae with little genomic information available in databases, which seriously hampers our ability to take a full advantage of the wealth of grass genomic data for effective comparative studies. Results Here we report the cloning and sequencing of 10,608 putative full length cDNAs (FL-cDNAs primarily from Moso bamboo, Phyllostachys heterocycla cv. pubescens, a large woody bamboo with the highest ecological and economic values of all bamboos. This represents the third largest FL-cDNA collection to date of all plant species, and provides the first insight into the gene and genome structures of bamboos. We developed a Moso bamboo genomic resource database that so far contained the sequences of 10,608 putative FL-cDNAs and nearly 38,000 expressed sequence tags (ESTs generated in this study. Conclusion Analysis of FL-cDNA sequences show that bamboo diverged from its close relatives such as rice, wheat, and barley through an adaptive radiation. A comparative analysis of the lignin biosynthesis pathway between bamboo and rice suggested that genes encoding caffeoyl-CoA O-methyltransferase may serve as targets for genetic manipulation of lignin content to reduce pollutants generated from bamboo pulping.

  4. The E1 copper binding domain of full-length amyloid precursor protein mitigates copper-induced growth inhibition in brain metastatic prostate cancer DU145 cells

    Energy Technology Data Exchange (ETDEWEB)

    Gough, Mallory, E-mail: m.gough1@lancaster.ac.uk; Blanthorn-Hazell, Sophee, E-mail: s.blanthorn-hazell@lancaster.ac.uk; Delury, Craig, E-mail: c.delury@lancaster.ac.uk; Parkin, Edward, E-mail: e.parkin@lancaster.ac.uk

    2014-10-31

    Highlights: • Copper levels are elevated in the tumour microenvironment. • APP mitigates copper-induced growth inhibition of DU145 prostate cancer (PCa) cells. • The APP intracellular domain is a prerequisite; soluble forms have no effect. • The E1 CuBD of APP is also a prerequisite. • APP copper binding potentially mitigates copper-induced PCa cell growth inhibition. - Abstract: Copper plays an important role in the aetiology and growth of tumours and levels of the metal are increased in the serum and tumour tissue of patients affected by a range of cancers including prostate cancer (PCa). The molecular mechanisms that enable cancer cells to proliferate in the presence of elevated copper levels are, therefore, of key importance in our understanding of tumour growth progression. In the current study, we have examined the role played by the amyloid precursor protein (APP) in mitigating copper-induced growth inhibition of the PCa cell line, DU145. A range of APP molecular constructs were stably over-expressed in DU145 cells and their effects on cell proliferation in the presence of copper were monitored. Our results show that endogenous APP expression was induced by sub-toxic copper concentrations in DU145 cells and over-expression of the wild-type protein was able to mitigate copper-induced growth inhibition via a mechanism involving the cytosolic and E1 copper binding domains of the full-length protein. APP likely represents one of a range of copper binding proteins that PCa cells employ in order to ensure efficient proliferation despite elevated concentrations of the metal within the tumour microenvironment. Targeting the expression of such proteins may contribute to therapeutic strategies for the treatment of cancers.

  5. Generation and analysis of a large-scale expressed sequence tags from a full-length enriched cDNA library of Siberian tiger (Panthera tigris altaica).

    Science.gov (United States)

    Guo, Yu; Liu, Changqing; Lu, Taofeng; Liu, Dan; Bai, Chunyu; Li, Xiangchen; Ma, Yuehui; Guan, Weijun

    2014-05-15

    In this study, a full-length enriched cDNA library was successfully constructed from Siberian tiger, the world's most endangered species. The titers of primary and amplified libraries were 1.28×10(6)pfu/mL and 1.59×10(10)pfu/mL respectively. The proportion of recombinants from unamplified library was 91.3% and the average length of exogenous inserts was 1.06kb. A total of 279 individual ESTs with sizes ranging from 316 to 1258bps were then analyzed. Furthermore, 204 unigenes were successfully annotated and involved in 49 functions of the GO classification, cell (175, 85.5%), cellular process (165, 80.9%), and binding (152, 74.5%) are the dominant terms. 198 unigenes were assigned to 156 KEGG pathways, and the pathways with the most representation are metabolic pathways (18, 9.1%). The proportion pattern of each COG subcategory was similar among Panthera tigris altaica, P. tigris tigris and Homo sapiens, and general function prediction only cluster (44, 15.8%) represents the largest group, followed by translation, ribosomal structure and biogenesis (33, 11.8%), replication, recombination and repair (24, 8.6%), and only 7.2% ESTs classified as novel genes. Moreover, the recombinant plasmid pET32a-TAT-COL6A2 was constructed, coded for the Trx-TAT-COL6A2 fusion protein with two 6× His-tags in N and C-terminal. After BCA assay, the concentration of soluble Trx-TAT-COL6A2 recombinant protein was 2.64±0.18mg/mL. This library will provide a useful platform for the functional genome and transcriptome research of for the P. tigris and other felid animals in the future. PMID:24630959

  6. The E1 copper binding domain of full-length amyloid precursor protein mitigates copper-induced growth inhibition in brain metastatic prostate cancer DU145 cells

    International Nuclear Information System (INIS)

    Highlights: • Copper levels are elevated in the tumour microenvironment. • APP mitigates copper-induced growth inhibition of DU145 prostate cancer (PCa) cells. • The APP intracellular domain is a prerequisite; soluble forms have no effect. • The E1 CuBD of APP is also a prerequisite. • APP copper binding potentially mitigates copper-induced PCa cell growth inhibition. - Abstract: Copper plays an important role in the aetiology and growth of tumours and levels of the metal are increased in the serum and tumour tissue of patients affected by a range of cancers including prostate cancer (PCa). The molecular mechanisms that enable cancer cells to proliferate in the presence of elevated copper levels are, therefore, of key importance in our understanding of tumour growth progression. In the current study, we have examined the role played by the amyloid precursor protein (APP) in mitigating copper-induced growth inhibition of the PCa cell line, DU145. A range of APP molecular constructs were stably over-expressed in DU145 cells and their effects on cell proliferation in the presence of copper were monitored. Our results show that endogenous APP expression was induced by sub-toxic copper concentrations in DU145 cells and over-expression of the wild-type protein was able to mitigate copper-induced growth inhibition via a mechanism involving the cytosolic and E1 copper binding domains of the full-length protein. APP likely represents one of a range of copper binding proteins that PCa cells employ in order to ensure efficient proliferation despite elevated concentrations of the metal within the tumour microenvironment. Targeting the expression of such proteins may contribute to therapeutic strategies for the treatment of cancers

  7. Characterization of a new full length TMPRSS3 isoform and identification of mutant alleles responsible for nonsyndromic recessive deafness in Newfoundland and Pakistan

    Directory of Open Access Journals (Sweden)

    Shotland Lawrence I

    2004-09-01

    Full Text Available Abstract Background Mutant alleles of TMPRSS3 are associated with nonsyndromic recessive deafness (DFNB8/B10. TMPRSS3 encodes a predicted secreted serine protease, although the deduced amino acid sequence has no signal peptide. In this study, we searched for mutant alleles of TMPRSS3 in families from Pakistan and Newfoundland with recessive deafness co-segregating with DFNB8/B10 linked haplotypes and also more thoroughly characterized the genomic structure of TMPRSS3. Methods We enrolled families segregating recessive hearing loss from Pakistan and Newfoundland. Microsatellite markers flanking the TMPRSS3 locus were used for linkage analysis. DNA samples from participating individuals were sequenced for TMPRSS3. The structure of TMPRSS3 was characterized bioinformatically and experimentally by sequencing novel cDNA clones of TMPRSS3. Results We identified mutations in TMPRSS3 in four Pakistani families with recessive, nonsyndromic congenital deafness. We also identified two recessive mutations, one of which is novel, of TMPRSS3 segregating in a six-generation extended family from Newfoundland. The spectrum of TMPRSS3 mutations is reviewed in the context of a genotype-phenotype correlation. Our study also revealed a longer isoform of TMPRSS3 with a hitherto unidentified exon encoding a signal peptide, which is expressed in several tissues. Conclusion Mutations of TMPRSS3 contribute to hearing loss in many communities worldwide and account for 1.8% (8 of 449 of Pakistani families segregating congenital deafness as an autosomal recessive trait. The newly identified TMPRSS3 isoform e will be helpful in the functional characterization of the full length protein.

  8. Cloning and sequence analysis of a full-length cDNA of SmPP1cb encoding turbot protein phosphatase 1 beta catalytic subunit

    Science.gov (United States)

    Qi, Fei; Guo, Huarong; Wang, Jian

    2008-02-01

    Reversible protein phosphorylation, catalyzed by protein kinases and phosphatases, is an important and versatile mechanism by which eukaryotic cells regulate almost all the signaling processes. Protein phosphatase 1 (PP1) is the first and well-characterized member of the protein serine/threonine phosphatase family. In the present study, a full-length cDNA encoding the beta isoform of the catalytic subunit of protein phosphatase 1(PP1cb), was for the first time isolated and sequenced from the skin tissue of flatfish turbot Scophthalmus maximus, designated SmPP1cb, by the rapid amplification of cDNA ends (RACE) technique. The cDNA sequence of SmPP1cb we obtained contains a 984 bp open reading frame (ORF), flanked by a complete 39 bp 5' untranslated region and 462 bp 3' untranslated region. The ORF encodes a putative 327 amino acid protein, and the N-terminal section of this protein is highly acidic, Met-Ala-Glu-Gly-Glu-Leu-Asp-Val-Asp, a common feature for PP1 catalytic subunit but absent in protein phosphatase 2B (PP2B). And its calculated molecular mass is 37 193 Da and pI 5.8. Sequence analysis indicated that, SmPP1cb is extremely conserved in both amino acid and nucleotide acid levels compared with the PP1cb of other vertebrates and invertebrates, and its Kozak motif contained in the 5'UTR around ATG start codon is GXXAXXGXX ATGG, which is different from mammalian in two positions A-6 and G-3, indicating the possibility of different initiation of translation in turbot, and also the 3'UTR of SmPP1cb is highly diverse in the sequence similarity and length compared with other animals, especially zebrafish. The cloning and sequencing of SmPP1cb gene lays a good foundation for the future work on the biological functions of PP1 in the flatfish turbot.

  9. Cloning, expression and protective immunity evaluation of the full-length cDNA encoding succinate dehydrogenase iron-sulfur protein of Schistosoma japonicum

    Institute of Scientific and Technical Information of China (English)

    YU JunLong; WANG ShiPing; LI WenKai; DAI Gan; XU ShaoRui; HE Zhuo; PENG XianChu; ZHOU SongHua; LIU XueQin

    2007-01-01

    1071-bp fragment was obtained from the Schistosoma japonicum (Chinese strain) adult cDNA library after the 3' and 5' ends of the incomplete expression sequence tag (EST) of succinate dehydrogenase iron-sulfur protein of Schistosoma japonicum (SjSDISP) were amplified by the anchored PCR with 2pairs of primers designed according to the EST of SjSDISP and the sequence of multiclone sites of the library vector. Sequence analysis indicated that the fragment was a full-length cDNA with a complete open reading frame (ORF), encoding 278 amino acid residues. The fragment was cloned into prokaryotic expression vector pQE30, and subsequently sequenced and expressed in Escherichia coll.SDS-PAGE and Western-blot analyses showed that the recombinant protein was about 32 kD and could be recognized by the polyclonal antisera from rabbits immunized with Schistosoma japonicum adult worm antigen. Compared with the FCA controls, mice vaccinated with rSjSDISP (test) or rSjGST (positive control) all revealed high levels of specific antibody and significant reduction in worm burden, liver eggs per gram (LEPG), fecal eggs per gram (FEPG) and intrauterine eggs. These results suggest that SjSDISP may be a novel and partially protective vaccine candidate against schistosomiasis. In contrast to the worm burden reduction rate, the higher degree of egg reduction rate in the test group also suggested that SjSDISP vaccine may primarily play a role in anti-embryonation or anti-fecundity immunity.

  10. Engineering infectious foot-and-mouth disease virus in vivo from a full-length genomic cDNA clone of the A/AKT/58 strain

    Institute of Scientific and Technical Information of China (English)

    2009-01-01

    Two full-length genomic cDNA clones, pTA/FMDV and pCA/FMDV, were constructed that contained three point-mutants [A174G and A308G (not present in pTA/FMDV); T1029G] in the genome compared with the wild type A/AKT/58 strain of foot-and-mouth disease virus. These two viruses were rescued by co-transfection of pCA/FMDV with pCT7RNAP, which can express T7 RNA polymerase in BHK-21 cell-lines, or by transfection of the in vitro transcribed RNA. Their biological properties were analyzed for their antigenicity, virulence in suckling-mice (LD50) and growth kinetics in BHK-21 cells. The in vivo rescued viruses showed high pathogenicity for 3-day-old unweaned mice (LD50=10?7.5). However, the in vitro transcribed RNA derived from pTA/FMDV had lower pathogenicity for suckling-mice (LD50=10?6), and the in vivo transcribed RNA recovered from pCA/FMDV co-transfected with pCT7RNAP showed no significant differences from the wild type virus. These data showed that recovery of the infectious foot-and-mouth disease virus directly from the use of in vivo techniques was better than from in vitro methods. Furthermore, the reverse genetic procedure technique was simplified to a faster one-step procedure based on co-transfection with pCT7RNAP. These results suggest that in vivo RNA tran- scripts may be more valuable for engineering recombinant foot-and-mouth disease virus than in vitro RNA transcripts, and may contribute to further understanding of the biological properties, such as replication, maturation and quasispecies, of the foot-and-mouth disease virus.

  11. Recurrence of occult hepatitis B virus infection in a recipient of a liver transplant for HCV-related cirrhosis: full length genome, mutations analysis and literature review

    Directory of Open Access Journals (Sweden)

    Tahar Bajjou

    2014-08-01

    Full Text Available The outcome of liver transplant recipients in HCV chronic carriers with Anti-HBc only concerning occult HBV infection is unknown. We report here the case of a patient who underwent liver transplantation (LT for cirrhosis post chronic hepatitis C who received an allograft from a donor with no marker of hepatitis B infection. After LT, HBV DNA was detected in the serum in the absence of HBsAg while HCV RNA remained negative. To determine the origin of this occult HBV infection, we retrospectively examined stored serum and liver tissue, pre and post-transplantation, for HBV DNA by PCR. A stored liver biopsy of the donor before transplantation was also tested. HBV DNA was detected in the pre-transplant liver but not in the donor liver. HBV viral load quantified by real time PCR after LT ranged from about 102 to 5x103 HBV DNA copies/mg of liver, while in sera, concentrations ranged from 102 to 3x103 HBV DNA copies/ml. All PCR products in the S gene from liver and sera were sequenced. Analysis of sequences showed the presence of an HBV strain genotype D. The nucleotide homology between the patient's HBV strains before and after LT was 96 % across the analyzed regions. Full length HBV genomes were amplified from the sera using Rolling Circle Amplification and then sequenced. Analysis of sequences confirmed the genotype D, but did not show obvious mutations that could contribute to HBsAg seronegativity and low HBV viral replication. Factors leading to occult HBV infection are still unclear, but it is well establish that occult HBV infection is frequent in HCV patients. This underlines the role of extra hepatic sites for HBV replication, potentially lymphocytes acting as and ldquo;reservoirs and rdquo;. [Int J Res Med Sci 2014; 2(4.000: 1294-1301

  12. BAY 81-8973, a full-length recombinant factor VIII: Human heat shock protein 70 improves the manufacturing process without affecting clinical safety.

    Science.gov (United States)

    Maas Enriquez, Monika; Thrift, John; Garger, Stephen; Katterle, Yvonne

    2016-11-01

    BAY 81-8973 is a full-length, unmodified recombinant human factor VIII (FVIII) approved for the treatment of hemophilia A. BAY 81-8973 has the same amino acid sequence as the currently marketed sucrose-formulated recombinant FVIII (rFVIII-FS) product and is produced using additional advanced manufacturing technologies. One of the key manufacturing advances for BAY 81-8973 is introduction of the gene for human heat shock protein 70 (HSP70) into the rFVIII-FS cell line. HSP70 facilitates proper folding of proteins, enhances cell survival by inhibiting apoptosis, and potentially impacts rFVIII glycosylation. HSP70 expression in the BAY 81-8973 cell line along with other manufacturing advances resulted in a higher-producing cell line and improvements in the pharmacokinetics of the final product as determined in clinical studies. HSP70 protein is not detected in the harvest or in the final BAY 81-8973 product. However, because this is a new process, clinical trial safety assessments included monitoring for anti-HSP70 antibodies. Most patients, across all age groups, had low levels of anti-HSP70 antibodies before exposure to the investigational product. During BAY 81-8973 treatment, 5% of patients had sporadic increases in anti-HSP70 antibody levels above a predefined threshold (cutoff value, 239 ng/mL). No clinical symptoms related to anti-HSP70 antibody development occurred. In conclusion, addition of HSP70 to the BAY 81-8973 cell line is an innovative technology for manufacturing rFVIII aimed at improving protein folding and expression. Improved pharmacokinetics and no effect on safety of BAY 81-8973 were observed in clinical trials in patients with hemophilia A. PMID:27436242

  13. ASC-J9 Suppresses Castration-Resistant Prostate Cancer Growth through Degradation of Full-length and Splice Variant Androgen Receptors

    Directory of Open Access Journals (Sweden)

    Shinichi Yamashita

    2012-01-01

    Full Text Available Early studies suggested androgen receptor (AR splice variants might contribute to the progression of prostate cancer (PCa into castration resistance. However, the therapeutic strategy to target these AR splice variants still remains unresolved. Through tissue survey of tumors from the same patients before and after castration resistance, we found that the expression of AR3, a major AR splice variant that lacks the AR ligand-binding domain, was substantially increased after castration resistance development. The currently used antiandrogen, Casodex, showed little growth suppression in CWR22Rv1 cells. Importantly, we found that AR degradation enhancer ASC-J9 could degrade both full-length (fAR and AR3 in CWR22Rv1 cells as well as in C4-2 and C81 cells with addition of AR3. The consequences of such degradation of both fAR and AR3 might then result in the inhibition of AR transcriptional activity and cell growth in vitro. More importantly, suppression of AR3 specifically by short-hairpin AR3 or degradation of AR3 by ASC-J9 resulted in suppression of AR transcriptional activity and cell growth in CWR22Rv1-fARKD (fAR knockdown cells in which DHT failed to induce, suggesting the importance of targeting AR3. Finally, we demonstrated the in vivo therapeutic effects of ASC-J9 by showing the inhibition of PCa growth using the xenografted model of CWR22Rv1 cells orthotopically implanted into castrated nude mice with undetectable serum testosterone. These results suggested that targeting both fAR- and AR3-mediated PCa growth by ASC-J9 may represent the novel therapeutic approach to suppress castration-resistant PCa. Successful clinical trials targeting both fAR and AR3 may help us to battle castration-resistant PCa in the future.

  14. Crystal Structure of Full-length Mycobacterium tuberculosis H37Rv Glycogen Branching Enzyme; Insights of N-Terminal [beta]-Sandwich in Sustrate Specifity and Enzymatic Activity

    Energy Technology Data Exchange (ETDEWEB)

    Pal, Kuntal; Kumar, Shiva; Sharma, Shikha; Garg, Saurabh Kumar; Alam, Mohammad Suhail; Xu, H. Eric; Agrawal, Pushpa; Swaminathan, Kunchithapadam (NU Sinapore); (Van Andel); (IMT-India)

    2010-07-13

    The open reading frame Rv1326c of Mycobacterium tuberculosis (Mtb) H37Rv encodes for an {alpha}-1,4-glucan branching enzyme (MtbGlgB, EC 2.4.1.18, Uniprot entry Q10625). This enzyme belongs to glycoside hydrolase (GH) family 13 and catalyzes the branching of a linear glucose chain during glycogenesis by cleaving a 1 {yields} 4 bond and making a new 1 {yields} 6 bond. Here, we show the crystal structure of full-length MtbGlgB (MtbGlgBWT) at 2.33-{angstrom} resolution. MtbGlgBWT contains four domains: N1 {beta}-sandwich, N2 {beta}-sandwich, a central ({beta}/{alpha}){sub 8} domain that houses the catalytic site, and a C-terminal {beta}-sandwich. We have assayed the amylase activity with amylose and starch as substrates and the glycogen branching activity using amylose as a substrate for MtbGlgBWT and the N1 domain-deleted (the first 108 residues deleted) Mtb{Delta}108GlgB protein. The N1 {beta}-sandwich, which is formed by the first 105 amino acids and superimposes well with the N2 {beta}-sandwich, is shown to have an influence in substrate binding in the amylase assay. Also, we have checked and shown that several GH13 family inhibitors are ineffective against MtbGlgBWT and Mtb{Delta}108GlgB. We propose a two-step reaction mechanism, for the amylase activity (1 {yields} 4 bond breakage) and isomerization (1 {yields} 6 bond formation), which occurs in the same catalytic pocket. The structural and functional properties of MtbGlgB and Mtb{Delta}108GlgB are compared with those of the N-terminal 112-amino acid-deleted Escherichia coli GlgB (EC{Delta}112GlgB).

  15. Uroporphyrinogen-III synthase: Molecular cloning, nucleotide sequence, expression of a mouse full-length cDNA, and its localization on mouse chromosome 7

    Energy Technology Data Exchange (ETDEWEB)

    Xu, W.; Desnick, R.J. [Mount Sinai School of Medicine, New York, NY (United States); Kozak, C.A. [National Institute of Health, Bethesda, MD (United States)

    1995-04-10

    Uroporphyrinogen-III synthase, the fourth enzyme in the heme biosynthetic pathway, is responsible for the conversion of hydroxymethylbilane to the cyclic tetrapyrrole, uroporphyrinogen III. The deficient activity of URO-S is the enzymatic defect in congenital erythropoietic porphyria (CEP), an autosomal recessive disorder. For the generation of a mouse model of CEP, the human URO-S cDNA was used to screen 2 X 10{sup 6} recombinants from a mouse adult liver cDNA library. Ten positive clones were isolated, and dideoxy sequencing of the entire 1.6-kb insert of clone pmUROS-1 revealed 5{prime} and 3{prime} untranslated sequences of 144 and 623 bp, respectively, and an open reading frame of 798 bp encoding a 265-amino-acid polypeptide with a predicted molecular mass of 28,501 Da. The mouse and human coding sequences had 80.5 and 77.8% nucleotide and amino acid identity, respectively. The authenticity of the mouse cDNA was established by expression of the active monomeric enzyme in Escherichia coli. In addition, the analysis of two multilocus genetic crosses localized the mouse gene on chromosome 7, consistent with the mapping of the human gene to a position of conserved synteny on chromosome 10. The isolation, expression, and chromosomal mapping of this full-length cDNA should facilitate studies of the structure and organization of the mouse genomic sequence and the development of a mouse model of CEP for characterization of the disease pathogenesis and evaluation of gene therapy. 38 refs., 1 tab.

  16. Characterization of a Full-Length Endogenous Beta-Retrovirus, EqERV-Beta1, in the Genome of the Horse (Equus caballus

    Directory of Open Access Journals (Sweden)

    Antoinette C. van der Kuyl

    2011-06-01

    Full Text Available Information on endogenous retroviruses fixed in the horse (Equus caballus genome is scarce. The recent availability of a draft sequence of the horse genome enables the detection of such integrated viruses by similarity search. Using translated nucleotide fragments from gamma-, beta-, and delta-retroviral genera for initial searches, a full-length beta-retrovirus genome was retrieved from a horse chromosome 5 contig. The provirus, tentatively named EqERV-beta1 (for the first equine endogenous beta-retrovirus, was 10434 nucleotide (nt in length with the usual retroviral genome structure of 5’LTR-gag-pro-pol-env-3’LTR. The LTRs were 1361 nt long, and differed approximately 1% from each other, suggestive of a relatively recent integration. Coding sequences for gag, pro and pol were present in three different reading-frames, as common for beta-retroviruses, and the reading frames were completely open, except that the env gene was interrupted by a single stopcodon. No reading frame was apparent downstream of the env gene, suggesting that EqERV-beta1 does not encode a superantigen like mouse mammary tumor virus (MMTV. A second proviral genome of EqERV-beta1, with no stopcodon in env, is additionally integrated on chromosome 5 downstream of the first virus. Single EqERV-beta1 LTRs were abundantly present on all chromosomes except chromosome 24. Phylogenetically, EqERV-beta1 most closely resembles an unclassified retroviral sequence from cattle (Bos taurus, and the murine beta-retrovirus MMTV.

  17. trans activation by the full-length E2 proteins of human papillomavirus type 16 and bovine papillomavirus type 1 in vitro and in vivo: cooperation with activation domains of cellular transcription factors.

    OpenAIRE

    Ushikai, M; Lace, M J; Yamakawa, Y.; Kono, M; Anson, J; Ishiji, T; Parkkinen, S; Wicker, N.; Valentine, M E; Davidson, I

    1994-01-01

    Papillomaviral E2 genes encode proteins that regulate viral transcription. While the full-length bovine papillomavirus type 1 (BPV-1) E2 peptide is a strong trans activator, the homologous full-length E2 product of human papillomavirus type 16 (HPV-16) appeared to vary in function in previous studies. Here we show that when expressed from comparable constructs, the full-length E2 products of HPV-16 and BPV-1 trans activate a simple E2- and Sp1-dependent promoter up to approximately 100-fold i...

  18. Insight into the assembly of chaperones

    Energy Technology Data Exchange (ETDEWEB)

    May, R.P. [Institut Max von Laue - Paul Langevin (ILL), 38 - Grenoble (France); Stegmann, R.; Manakova, E.; Roessle, M.; Hermann, T.; Heumann, H. [Max-Planck-Institut fuer Biochemie, Martinsried (Germany); Axmann, S.; Plueckthun, A. [Zurich Univ. (Switzerland); Wiedenmann, A. [HMI, Berlin (Germany)

    1997-04-01

    Chaperones are proteins that help other proteins (substrate proteins) to acquire a `good` conformation. The folding is a dynamic process and involves repetitive binding and release of the chaperone components and of the substrate protein. Small-angle neutron scattering is used to investigate the structural changes that appear to happen during the folding process. (author). 2 refs.

  19. The F4 fimbrial chaperone FaeE is stable as a monomer that does not require self-capping of its pilin-interactive surfaces.

    Science.gov (United States)

    Van Molle, Inge; Moonens, Kristof; Buts, Lieven; Garcia-Pino, Abel; Panjikar, Santosh; Wyns, Lode; De Greve, Henri; Bouckaert, Julie

    2009-05-01

    Many Gram-negative bacteria use the chaperone-usher pathway to express adhesive surface structures, such as fimbriae, in order to mediate attachment to host cells. Periplasmic chaperones are required to shuttle fimbrial subunits or pilins through the periplasmic space in an assembly-competent form. The chaperones cap the hydrophobic surface of the pilins through a donor-strand complementation mechanism. FaeE is the periplasmic chaperone required for the assembly of the F4 fimbriae of enterotoxigenic Escherichia coli. The FaeE crystal structure shows a dimer formed by interaction between the pilin-binding interfaces of the two monomers. Dimerization and tetramerization have been observed previously in crystal structures of fimbrial chaperones and have been suggested to serve as a self-capping mechanism that protects the pilin-interactive surfaces in solution in the absence of the pilins. However, thermodynamic and biochemical data show that FaeE occurs as a stable monomer in solution. Other lines of evidence indicate that self-capping of the pilin-interactive interfaces is not a mechanism that is conservedly applied by all periplasmic chaperones, but is rather a case-specific solution to cap aggregation-prone surfaces. PMID:19390146

  20. Generation and analysis of large-scale expressed sequence tags (ESTs from a full-length enriched cDNA library of porcine backfat tissue

    Directory of Open Access Journals (Sweden)

    Lee Hae-Young

    2006-02-01

    Full Text Available Abstract Background Genome research in farm animals will expand our basic knowledge of the genetic control of complex traits, and the results will be applied in the livestock industry to improve meat quality and productivity, as well as to reduce the incidence of disease. A combination of quantitative trait locus mapping and microarray analysis is a useful approach to reduce the overall effort needed to identify genes associated with quantitative traits of interest. Results We constructed a full-length enriched cDNA library from porcine backfat tissue. The estimated average size of the cDNA inserts was 1.7 kb, and the cDNA fullness ratio was 70%. In total, we deposited 16,110 high-quality sequences in the dbEST division of GenBank (accession numbers: DT319652-DT335761. For all the expressed sequence tags (ESTs, approximately 10.9 Mb of porcine sequence were generated with an average length of 674 bp per EST (range: 200–952 bp. Clustering and assembly of these ESTs resulted in a total of 5,008 unique sequences with 1,776 contigs (35.46% and 3,232 singleton (65.54% ESTs. From a total of 5,008 unique sequences, 3,154 (62.98% were similar to other sequences, and 1,854 (37.02% were identified as having no hit or low identity (Sus scrofa. Gene ontology (GO annotation of unique sequences showed that approximately 31.7, 32.3, and 30.8% were assigned molecular function, biological process, and cellular component GO terms, respectively. A total of 1,854 putative novel transcripts resulted after comparison and filtering with the TIGR SsGI; these included a large percentage of singletons (80.64% and a small proportion of contigs (13.36%. Conclusion The sequence data generated in this study will provide valuable information for studying expression profiles using EST-based microarrays and assist in the condensation of current pig TCs into clusters representing longer stretches of cDNA sequences. The isolation of genes expressed in backfat tissue is the

  1. The shortened disabilities of the arm, shoulder and hand questionnaire (QuickDASH: validity and reliability based on responses within the full-length DASH

    Directory of Open Access Journals (Sweden)

    Ward Michael M

    2006-05-01

    Full Text Available Abstract Background The 30-item disabilities of the arm, shoulder and hand (DASH questionnaire is increasingly used in clinical research involving upper extremity musculoskeletal disorders. From the original DASH a shorter version, the 11-item QuickDASH, has been developed. Little is known about the discriminant ability of score changes for the QuickDASH compared to the DASH. The aim of this study was to assess the performance of the QuickDASH and its cross-sectional and longitudinal validity and reliability. Methods The study was based on extracting QuickDASH item responses from the responses to the full-length DASH questionnaire completed by 105 patients with a variety of upper extremity disorders before surgery and at follow-up 6 to 21 months after surgery. The DASH and QuickDASH scores were compared for the whole population and for different diagnostic groups. For longitudinal construct validity the effect size and standardized response mean were calculated. Analyses with ROC curves were performed to compare the ability of the DASH and QuickDASH to discriminate among patients classified according to the magnitude of self-rated improvement. Cross-sectional and test-retest reliability was assessed. Results The mean DASH score was 34 (SD 22 and the mean QuickDASH score was 39 (SD 24 at baseline. For the different diagnostic groups the mean and median QuickDASH scores were higher than the corresponding DASH scores. For the whole population, the mean difference between the QuickDASH and DASH baseline scores was 4.2 (95% CI 3.2–5.3, follow-up scores was 2.6 (1.7–3.4, and change scores was 1.7 (0.6–2.8. The overall effect size and standardized response mean measured with the DASH and the QuickDASH were similar. In the ROC analysis of change scores among patients who rated their arm status as somewhat or much better and those who rated it as unchanged the difference in the area under the ROC curve for the DASH and QuickDASH was 0.01 (95% CI -0

  2. The imprinted retrotransposon-like gene PEG11 (RTL1 is expressed as a full-length protein in skeletal muscle from Callipyge sheep.

    Directory of Open Access Journals (Sweden)

    Keren Byrne

    Full Text Available Members of the Ty3-Gypsy retrotransposon family are rare in mammalian genomes despite their abundance in invertebrates and some vertebrates. These elements contain a gag-pol-like structure characteristic of retroviruses but have lost their ability to retrotranspose into the mammalian genome and are thought to be inactive relics of ancient retrotransposition events. One of these retrotransposon-like elements, PEG11 (also called RTL1 is located at the distal end of ovine chromosome 18 within an imprinted gene cluster that is highly conserved in placental mammals. The region contains several conserved imprinted genes including BEGAIN, DLK1, DAT, GTL2 (MEG3, PEG11 (RTL1, PEG11as, MEG8, MIRG and DIO3. An intergenic point mutation between DLK1 and GTL2 causes muscle hypertrophy in callipyge sheep and is associated with large changes in expression of the genes linked in cis between DLK1 and MEG8. It has been suggested that over-expression of DLK1 is the effector of the callipyge phenotype; however, PEG11 gene expression is also strongly correlated with the emergence of the muscling phenotype as a function of genotype, muscle type and developmental stage. To date, there has been no direct evidence that PEG11 encodes a protein, especially as its anti-sense transcript (PEG11as contains six miRNA that cause cleavage of the PEG11 transcript. Using immunological and mass spectrometry approaches we have directly identified the full-length PEG11 protein from postnatal nuclear preparations of callipyge skeletal muscle and conclude that its over-expression may be involved in inducing muscle hypertrophy. The developmental expression pattern of the PEG11 gene is consistent with the callipyge mutation causing recapitulation of the normal fetal-like gene expression program during postnatal development. Analysis of the PEG11 sequence indicates strong conservation of the regions encoding the antisense microRNA and in at least two cases these correspond with structural

  3. Chemical chaperones improve protein secretion and rescue mutant factor VIII in mice with hemophilia A.

    Directory of Open Access Journals (Sweden)

    Stefanie D Roth

    Full Text Available Inefficient intracellular protein trafficking is a critical issue in the pathogenesis of a variety of diseases and in recombinant protein production. Here we investigated the trafficking of factor VIII (FVIII, which is affected in the coagulation disorder hemophilia A. We hypothesized that chemical chaperones may be useful to enhance folding and processing of FVIII in recombinant protein production, and as a therapeutic approach in patients with impaired FVIII secretion. A tagged B-domain-deleted version of human FVIII was expressed in cultured Chinese Hamster Ovary cells to mimic the industrial production of this important protein. Of several chemical chaperones tested, the addition of betaine resulted in increased secretion of FVIII, by increasing solubility of intracellular FVIII aggregates and improving transport from endoplasmic reticulum to Golgi. Similar results were obtained in experiments monitoring recombinant full-length FVIII. Oral betaine administration also increased FVIII and factor IX (FIX plasma levels in FVIII or FIX knockout mice following gene transfer. Moreover, in vitro and in vivo applications of betaine were also able to rescue a trafficking-defective FVIII mutant (FVIIIQ305P. We conclude that chemical chaperones such as betaine might represent a useful treatment concept for hemophilia and other diseases caused by deficient intracellular protein trafficking.

  4. Characterization of RNA binding and chaperoning activities of HIV-1 Vif protein

    Science.gov (United States)

    Sleiman, Dona; Bernacchi, Serena; Xavier Guerrero, Santiago; Brachet, Franck; Larue, Valéry; Paillart, Jean-Christophe; Tisné, Carine

    2014-01-01

    The viral infectivity factor (Vif) is essential for the productive infection and dissemination of HIV-1 in non-permissive cells, containing the cellular anti-HIV defense cytosine deaminases APOBEC3 (A3G and A3F). Vif neutralizes the antiviral activities of the APOBEC3G/F by diverse mechanisms including their degradation through the ubiquitin/proteasome pathway and their translational inhibition. In addition, Vif appears to be an active partner of the late steps of viral replication by interacting with Pr55Gag, reverse transcriptase and genomic RNA. Here, we expressed and purified full-length and truncated Vif proteins, and analyzed their RNA binding and chaperone properties. First, we showed by CD and NMR spectroscopies that the N-terminal domain of Vif is highly structured in solution, whereas the C-terminal domain remains mainly unfolded. Both domains exhibited substantial RNA binding capacities with dissociation constants in the nanomolar range, whereas the basic unfolded C-terminal domain of Vif was responsible in part for its RNA chaperone activity. Second, we showed by NMR chemical shift mapping that Vif and NCp7 share the same binding sites on tRNALys3, the primer of HIV-1 reverse transcriptase. Finally, our results indicate that Vif has potent RNA chaperone activity and provide direct evidence for an important role of the unstructured C-terminal domain of Vif in this capacity. PMID:25144404

  5. Lipid Chaperones and Metabolic Inflammation

    Directory of Open Access Journals (Sweden)

    Masato Furuhashi

    2011-01-01

    Full Text Available Over the past decade, a large body of evidence has emerged demonstrating an integration of metabolic and immune response pathways. It is now clear that obesity and associated disorders such as insulin resistance and type 2 diabetes are associated with a metabolically driven, low-grade, chronic inflammatory state, referred to as “metaflammation.” Several inflammatory cytokines as well as lipids and metabolic stress pathways can activate metaflammation, which targets metabolically critical organs and tissues including adipocytes and macrophages to adversely affect systemic homeostasis. On the other hand, inside the cell, fatty acid-binding proteins (FABPs, a family of lipid chaperones, as well as endoplasmic reticulum (ER stress, and reactive oxygen species derived from mitochondria play significant roles in promotion of metabolically triggered inflammation. Here, we discuss the molecular and cellular basis of the roles of FABPs, especially FABP4 and FABP5, in metaflammation and related diseases including obesity, diabetes, and atherosclerosis.

  6. Peptide binding specificity of the chaperone calreticulin

    DEFF Research Database (Denmark)

    Sandhu, N.; Duus, K.; Jorgensen, C.S.;

    2007-01-01

    Calreticulin is a molecular chaperone with specificity for polypeptides and N-linked monoglucosylated glycans. In order to determine the specificity of polypeptide binding, the interaction of calreticulin with polypeptides was investigated using synthetic peptides of different length and composit...

  7. Polypeptide binding properties of the chaperone calreticulin

    DEFF Research Database (Denmark)

    Jørgensen, C S; Heegaard, N H; Holm, A; Højrup, P; Houen, G

    2000-01-01

    Calreticulin is a highly conserved eukaryotic ubiquitious protein located mainly in the endoplasmic reticulum. Two major characteristics of calreticulin are its chaperone activity and its lectin properties, but its precise function in intracellular protein and peptide processing remains to be...

  8. Aging cellular networks: chaperones as major participants

    OpenAIRE

    Soti, Csaba; Csermely, Peter

    2006-01-01

    We increasingly rely on the network approach to understand the complexity of cellular functions. Chaperones (heat shock proteins) are key "networkers", which have among their functions to sequester and repair damaged protein. In order to link the network approach and chaperones with the aging process, we first summarize the properties of aging networks suggesting a "weak link theory of aging". This theory suggests that age-related random damage primarily affects the overwhelming majority of t...

  9. Revised genomic consensus for the hypermethylated CpG island region of the human L1 transposon and integration sites of full length L1 elements from recombinant clones made using methylation-tolerant host strains

    DEFF Research Database (Denmark)

    Crowther, P J; Doherty, J P; Linsenmeyer, M E; Williamson, M R; Woodcock, D M

    1991-01-01

    Efficient recovery of clones from the 5' end of the human L1 dispersed repetitive elements necessitates the use of deletion mcr- host strains since this region contains a CpG island which is hypermethylated in vivo. Clones recovered with conventional mcr+ hosts seem to have been derived...... preferentially from L1 members which have accumulated mutations that have removed sites of methylation. We present a revised consensus from the 5' presumptive control region of these elements. This revised consensus contains a consensus RNA polymerase III promoter which would permit the synthesis of transcripts...... from the 5' end of full length L1 elements. Such potential transcripts are likely to exhibit a high degree of secondary structure. In addition, we have determined the flanking sequences for 6 full length L1 elements. The majority of full length L1 clones show no convincing evidence for target site...

  10. Structural and functional conversion of molecular chaperone ClpB from the gram-positive halophilic lactic acid bacterium Tetragenococcus halophilus mediated by ATP and stress.

    Science.gov (United States)

    Sugimoto, Shinya; Yoshida, Hiroyuki; Mizunoe, Yoshimitsu; Tsuruno, Keigo; Nakayama, Jiro; Sonomoto, Kenji

    2006-12-01

    In this study, we report the purification, initial structural characterization, and functional analysis of the molecular chaperone ClpB from the gram-positive, halophilic lactic acid bacterium Tetragenococcus halophilus. A recombinant T. halophilus ClpB (ClpB(Tha)) was overexpressed in Escherichia coli and purified by affinity chromatography, hydroxyapatite chromatography, and gel filtration chromatography. As demonstrated by gel filtration chromatography, chemical cross-linking with glutaraldehyde, and electron microscopy, ClpB(Tha) forms a homohexameric single-ring structure in the presence of ATP under nonstress conditions. However, under stress conditions, such as high-temperature (>45 degrees C) and high-salt concentrations (>1 M KCl), it dissociated into dimers and monomers, regardless of the presence of ATP. The hexameric ClpB(Tha) reactivated heat-aggregated proteins dependent upon the DnaK system from T. halophilus (KJE(Tha)) and ATP. Interestingly, the mixture of dimer and monomer ClpB(Tha), which was formed under stress conditions, protected substrate proteins from thermal inactivation and aggregation in a manner similar to those of general molecular chaperones. From these results, we hypothesize that ClpB(Tha) forms dimers and monomers to function as a holding chaperone under stress conditions, whereas it forms a hexamer ring to function as a disaggregating chaperone in cooperation with KJE(Tha) and ATP under poststress conditions. PMID:16997952

  11. Structural and Functional Conversion of Molecular Chaperone ClpB from the Gram-Positive Halophilic Lactic Acid Bacterium Tetragenococcus halophilus Mediated by ATP and Stress▿

    Science.gov (United States)

    Sugimoto, Shinya; Yoshida, Hiroyuki; Mizunoe, Yoshimitsu; Tsuruno, Keigo; Nakayama, Jiro; Sonomoto, Kenji

    2006-01-01

    In this study, we report the purification, initial structural characterization, and functional analysis of the molecular chaperone ClpB from the gram-positive, halophilic lactic acid bacterium Tetragenococcus halophilus. A recombinant T. halophilus ClpB (ClpBTha) was overexpressed in Escherichia coli and purified by affinity chromatography, hydroxyapatite chromatography, and gel filtration chromatography. As demonstrated by gel filtration chromatography, chemical cross-linking with glutaraldehyde, and electron microscopy, ClpBTha forms a homohexameric single-ring structure in the presence of ATP under nonstress conditions. However, under stress conditions, such as high-temperature (>45°C) and high-salt concentrations (>1 M KCl), it dissociated into dimers and monomers, regardless of the presence of ATP. The hexameric ClpBTha reactivated heat-aggregated proteins dependent upon the DnaK system from T. halophilus (KJETha) and ATP. Interestingly, the mixture of dimer and monomer ClpBTha, which was formed under stress conditions, protected substrate proteins from thermal inactivation and aggregation in a manner similar to those of general molecular chaperones. From these results, we hypothesize that ClpBTha forms dimers and monomers to function as a holding chaperone under stress conditions, whereas it forms a hexamer ring to function as a disaggregating chaperone in cooperation with KJETha and ATP under poststress conditions. PMID:16997952

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

    Directory of Open Access Journals (Sweden)

    Miyoung Kim

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

  13. Modulation and elimination of yeast prions by protein chaperones and co-chaperones

    OpenAIRE

    Reidy, Michael; Masison, Daniel C.

    2011-01-01

    The yeast system has provided considerable insight into the biology of amyloid and prions. Here we focus on how alterations in abundance or function of protein chaperones and co-chaperones affect propagation of yeast prions. In spite of a considerable amount of information, a clear understanding of the molecular mechanisms underlying these effects remains wanting.

  14. Evaluation of Neisseria gonorrhoeae Multiple-Locus Variable-Number Tandem-Repeat Analysis, N. gonorrhoeae Multiantigen Sequence Typing, and Full-Length porB Gene Sequence Analysis for Molecular Epidemiological Typing

    OpenAIRE

    Heymans, Raymond; Golparian, Daniel; Bruisten, Sylvia M; Schouls, Leo M.; Unemo, Magnus

    2012-01-01

    The performance characteristics of Neisseria gonorrhoeae multilocus variable-number tandem-repeat analysis were evaluated, by comparison with N. gonorrhoeae multiantigen sequence typing and full-length porB sequence typing. Assessment of the relatedness of intra- and interpatient isolates showed that all three genotyping techniques display a high resolution and typeability.

  15. Production of enzymatically active recombinant full-length barley high pI alpha-glucosidase of glycoside family 31 by high cell-density fermentation of Pichia pastoris and affinity purification

    DEFF Research Database (Denmark)

    Næsted, Henrik; Kramhøft, Birte; Lok, F.; Bojsen, K.; Yu, S.; Svensson, Birte

    2006-01-01

    Recombinant barley high pI alpha-glucosidase was produced by high cell-density fermentation of Pichia pastoris expressing the cloned full-length gene. The gene was amplified from a genomic clone and exons (coding regions) were assembled by overlap PCR. The resulting cDNA was expressed under control...

  16. Chaperone-like activities of different molecular forms of beta-casein. Importance of polarity of N-terminal hydrophilic domain.

    Science.gov (United States)

    Yousefi, Reza; Shchutskaya, Yulia Y; Zimny, Jaroslaw; Gaudin, Jean-Charles; Moosavi-Movahedi, Ali A; Muronetz, Vladimir I; Zuev, Yuriy F; Chobert, Jean-Marc; Haertlé, Thomas

    2009-08-01

    As a member of intrinsically unstructured protein family, beta-casein (beta-CN) contains relatively high amount of prolyl residues, adopts noncompact and flexible structure and exhibits chaperone-like activity in vitro. Like many chaperones, native beta-CN does not contain cysteinyl residues and exhibits strong tendencies for self-association. The chaperone-like activities of three recombinant beta-CNs wild type (WT) beta-CN, C4 beta-CN (with cysteinyl residue in position 4) and C208 beta-CN (with cysteinyl residue in position 208), expressed and purified from E. coli, which, consequently, lack the phosphorylated residues, were examined and compared with that of native beta-CN using insulin and alcohol dehydrogenase as target/substrate proteins. The dimers (beta-CND) of C4-beta-CN and C208 beta-CN were also studied and their chaperone-like activities were compared with those of their monomeric forms. Lacking phosphorylation, WT beta-CN, C208 beta-CN, C4 beta-CN and C4 beta-CND exhibited significantly lower chaperone-like activities than native beta-CN. Dimerization of C208 beta-CN with two distal hydrophilic domains considerably improved its chaperone-like activity in comparison with its monomeric form. The obtained results demonstrate the significant role played by the polar contributions of phosphorylated residues and N-terminal hydrophilic domain as important functional elements in enhancing the chaperone-like activity of native beta-CN. (c) 2009 Wiley Periodicals, Inc. Biopolymers 91: 623-632, 2009.This article was originally published online as an accepted preprint. The "Published Online" date corresponds to the preprint version. You can request a copy of the preprint by emailing the Biopolymers editorial office at biopolymers@wiley.com. PMID:19322774

  17. The FNIP co-chaperones decelerate the Hsp90 chaperone cycle and enhance drug binding

    Science.gov (United States)

    Woodford, Mark R.; Dunn, Diana M.; Blanden, Adam R.; Capriotti, Dante; Loiselle, David; Prodromou, Chrisostomos; Panaretou, Barry; Hughes, Philip F.; Smith, Aaron; Ackerman, Wendi; Haystead, Timothy A.; Loh, Stewart N.; Bourboulia, Dimitra; Schmidt, Laura S.; Marston Linehan, W.; Bratslavsky, Gennady; Mollapour, Mehdi

    2016-01-01

    Heat shock protein-90 (Hsp90) is an essential molecular chaperone in eukaryotes involved in maintaining the stability and activity of numerous signalling proteins, also known as clients. Hsp90 ATPase activity is essential for its chaperone function and it is regulated by co-chaperones. Here we show that the tumour suppressor FLCN is an Hsp90 client protein and its binding partners FNIP1/FNIP2 function as co-chaperones. FNIPs decelerate the chaperone cycle, facilitating FLCN interaction with Hsp90, consequently ensuring FLCN stability. FNIPs compete with the activating co-chaperone Aha1 for binding to Hsp90, thereby providing a reciprocal regulatory mechanism for chaperoning of client proteins. Lastly, downregulation of FNIPs desensitizes cancer cells to Hsp90 inhibitors, whereas FNIPs overexpression in renal tumours compared with adjacent normal tissues correlates with enhanced binding of Hsp90 to its inhibitors. Our findings suggest that FNIPs expression can potentially serve as a predictive indicator of tumour response to Hsp90 inhibitors. PMID:27353360

  18. The DNAJA2 Substrate Release Mechanism Is Essential for Chaperone-mediated Folding*

    Science.gov (United States)

    Baaklini, Imad; Wong, Michael J. H.; Hantouche, Christine; Patel, Yogita; Shrier, Alvin; Young, Jason C.

    2012-01-01

    DNAJA1 (DJA1/Hdj2) and DNAJA2 (DJA2) are the major J domain partners of human Hsp70/Hsc70 chaperones. Although they have overall similarity with the well characterized type I co-chaperones from yeast and bacteria, they are biologically distinct, and their functional mechanisms are poorly characterized. We identified DJA2-specific activities in luciferase folding and repression of human ether-a-go-go-related gene (HERG) trafficking that depended on its expression levels in cells. Mutations in different internal domains of DJA2 abolished these effects. Using purified proteins, we addressed the mechanistic defects. A mutant lacking the region between the zinc finger motifs (DJA2-Δm2) was able to bind substrate similar to wild type but was incapable of releasing substrate during its transfer to Hsc70. The equivalent mutation in DJA1 also abolished its substrate release. A DJA2 mutant (DJA-221), which had its C-terminal dimerization region replaced by that of DJA1, was inactive but retained its ability to release substrate. The release mechanism required the J domain and ATP hydrolysis by Hsc70, although the nucleotide dependence diverged between DJA2 and DJA1. Limited proteolysis suggested further conformational differences between the two wild-type co-chaperones and the mutants. Our results demonstrate an essential role of specific DJA domains in the folding mechanism of Hsc70. PMID:23091061

  19. The DNAJA2 substrate release mechanism is essential for chaperone-mediated folding.

    Science.gov (United States)

    Baaklini, Imad; Wong, Michael J H; Hantouche, Christine; Patel, Yogita; Shrier, Alvin; Young, Jason C

    2012-12-01

    DNAJA1 (DJA1/Hdj2) and DNAJA2 (DJA2) are the major J domain partners of human Hsp70/Hsc70 chaperones. Although they have overall similarity with the well characterized type I co-chaperones from yeast and bacteria, they are biologically distinct, and their functional mechanisms are poorly characterized. We identified DJA2-specific activities in luciferase folding and repression of human ether-a-go-go-related gene (HERG) trafficking that depended on its expression levels in cells. Mutations in different internal domains of DJA2 abolished these effects. Using purified proteins, we addressed the mechanistic defects. A mutant lacking the region between the zinc finger motifs (DJA2-Δm2) was able to bind substrate similar to wild type but was incapable of releasing substrate during its transfer to Hsc70. The equivalent mutation in DJA1 also abolished its substrate release. A DJA2 mutant (DJA-221), which had its C-terminal dimerization region replaced by that of DJA1, was inactive but retained its ability to release substrate. The release mechanism required the J domain and ATP hydrolysis by Hsc70, although the nucleotide dependence diverged between DJA2 and DJA1. Limited proteolysis suggested further conformational differences between the two wild-type co-chaperones and the mutants. Our results demonstrate an essential role of specific DJA domains in the folding mechanism of Hsc70. PMID:23091061

  20. Photoionization of helium dimers

    International Nuclear Information System (INIS)

    The helium dimer is one of the most weakly bound systems in the universe. This makes it an interesting quantum mechanical object for investigation. These Van der Waals Clusters can be produced in an expansion of a cryogenic gas jet through a small nozzle into vacuum. In the present experiment we examine the interaction of He dimers with synchrotron radiation at an energy range from 64 to 78 eV. We observed different pathways leading to single ionization of both He atoms of the dimer compound. This two close standing ions begin now to dissociate in cause of their coulomb potential. All charged fragments were detected in coincidence with a COLTRIMS system. Especially Interatomic Coulombic Decay (ICD) and the two step process (TS1) were clearly identified. Furthermore a distribution of the internuclear distance was obtained from the measured Kinetic Energy Release (KER). (orig.)

  1. Analysis of ORF5 and Full-Length Genome Sequences of Porcine Reproductive and Respiratory Syndrome Virus Isolates of Genotypes 1 and 2 Retrieved Worldwide Provides Evidence that Recombination Is a Common Phenomenon and May Produce Mosaic Isolates

    OpenAIRE

    Martin-Valls, G. E.; Kvisgaard, L. K.; Tello, M.; Darwich, L.; Cortey, M.; Burgara-Estrella, A. J.; Hernandez, J; Larsen, L. E.; MATEU, E.

    2014-01-01

    Recombination is currently recognized as a factor for high genetic diversity, but the frequency of such recombination events and the genome segments involved are not well known. In the present study, we initially focused on the detection of recombinant porcine reproductive and respiratory syndrome virus (PRRSV) isolates by examining previously published data sets of ORF5 sequences (genotypes 1 and 2) obtained worldwide. We then examined full-length genome sequences in order to determine poten...

  2. Isolation and characterization of the full-length cDNA encoding a member of a novel cytochrome p450 family (CYP320A1) from the tropical freshwater snail, Biomphalaria glabrata, intermediate host for Schistosoma mansoni

    OpenAIRE

    Lockyer, Anne E.; Noble, Leslie R.; David Rollinson; Jones, Catherine S.

    2005-01-01

    Cytochrome p450s (cyp450s) are a family of structurally related proteins, with diverse functions, including steroid synthesis and breakdown of toxins. This paper reports the full-length sequence of a novel cyp450 gene, the first to be isolated from the tropical freshwater snail Biomphalaria glabrata, an important intermediate host of Schistosoma mansoni. The nucleotide sequence is 2291 bp with a predicted amino acid sequence of 584aa. The sequence demonstrates conserved cyp450 structural moti...

  3. Chaperone binding at the ribosomal exit tunnel

    DEFF Research Database (Denmark)

    Kristensen, Ole; Gajhede, Michael

    2003-01-01

    The exit tunnel region of the ribosome is well established as a focal point for interaction between the components that guide the fate of nascent polypeptides. One of these, the chaperone trigger factor (TF), associates with the 50S ribosomal subunit through its N-terminal domain. Targeting of TF...

  4. Alkane dimers interaction

    DEFF Research Database (Denmark)

    Ferrighi, Lara; Madsen, Georg Kent Hellerup; Hammer, Bjørk

    The interaction energies of a series of n-alkane dimers, from methane to decane, have been investigated with Density Functional Theory (DFT), using the MGGA-M06-L density functional. The results are compared both to the available wavefunction-based values as well as to dispersion corrected DFT...

  5. Characterization of RNA binding and chaperoning activities of HIV-1 Vif protein. Importance of the C-terminal unstructured tail.

    Science.gov (United States)

    Sleiman, Dona; Bernacchi, Serena; Xavier Guerrero, Santiago; Brachet, Franck; Larue, Valéry; Paillart, Jean-Christophe; Tisne, Carine

    2014-01-01

    The viral infectivity factor (Vif) is essential for the productive infection and dissemination of HIV-1 in non-permissive cells, containing the cellular anti-HIV defense cytosine deaminases APOBEC3 (A3G and A3F). Vif neutralizes the antiviral activities of the APOBEC3G/F by diverse mechanisms including their degradation through the ubiquitin/proteasome pathway and their translational inhibition. In addition, Vif appears to be an active partner of the late steps of viral replication by interacting with Pr55(Gag), reverse transcriptase and genomic RNA. Here, we expressed and purified full-length and truncated Vif proteins, and analyzed their RNA binding and chaperone properties. First, we showed by CD and NMR spectroscopies that the N-terminal domain of Vif is highly structured in solution, whereas the C-terminal domain remains mainly unfolded. Both domains exhibited substantial RNA binding capacities with dissociation constants in the nanomolar range, whereas the basic unfolded C-terminal domain of Vif was responsible in part for its RNA chaperone activity. Second, we showed by NMR chemical shift mapping that Vif and NCp7 share the same binding sites on tRNA(Lys) 3, the primer of HIV-1 reverse transcriptase. Finally, our results indicate that Vif has potent RNA chaperone activity and provide direct evidence for an important role of the unstructured C-terminal domain of Vif in this capacity. PMID:25144404

  6. Using SANS to monitor the interaction of misfolding alcohol dehydrogenase with the molecular chaperone protein 14-3-3ζ

    International Nuclear Information System (INIS)

    14-3-3 is a family of acidic, dimeric proteins which are highly conserved across many species. Each monomer is approximately 30kDa in mass and contains 9 α-helices. Dimer formation is initiated at the N-terminal region of the protein as a result of the interaction between several buried polar and hydrophobic residues in this region. 14-3-3 proteins interact with a wide range of proteins to regulate many cellular processes, e.g. apoptosis and mitosis, as well as protein misfolding associated with conformational diseases such as Alzheimer’s and Parkinson’s Disease. A potential role of 14-3-3 in these diseases was discovered with the observation that 14-3-3ζ can act as a molecular chaperone, whereby it stabilises intermediately folded proteins to prevent their aggregation. The binding site and mechanism of the chaperone action of 14-3-3ζ are not known, despite being narrowed down in our NMR study. We produced deuterated 14-3-3ζ and used it in SANS experiments with a model misfolding protein, alcohol dehydrogenase (ADH). Contrast variation allowed us to monitor changes in each component separately after the initiation of ADH misfolding. The Rg and Dmax values of ADH under stress show an increase in size with time, consistent with unfolding and aggregation. In the presence of 14-3-3ζ, the unfolding of ADH is reduced and the protein maintains a globular expanded conformation consistent with an adoption of an intermediately folded (molten globule) state. 14-3-3ζ whilst chaperoning showed a reduction in size, possibly due to dissociation. Ab initio models were also obtained. This is the first instance where conformational changes during chaperoning of either a partly folded target protein, or 14-3-3ζ, have been observed.

  7. Bacteriochlorophyll dimers in photosynthesis

    International Nuclear Information System (INIS)

    The X-ray crysallagraphic study of reaction center (RC) single crystals of the photosynthetic bacteria Rps.Viridis and Rb. sphaeroides confirms the existence of bacteriochlorophyll (BChl) dimers which were postulated earlier from EPR and ENDOR studies at low temperature to be the primary electron donors P960 and P870. Apart from the spatial structure of these dimers a knowlegde of the electron density distribution in various electronic stated in indispensable for an understanding of their functional properties. For P870+ and P960+ under physiological ocnditions the electron spin density distrubutions were obtained by ENDOR-in-solution via the hyperifne couplings. The comparison between the EPR/ENDOR data of P870+ and P960+ in RC's and of onomeric BChl a. and BChl b. shows that the primary donors are pie conjugated supermolecules with more or less asymmetric spin dnesity distrubutions over the dimer halves. Theoretical spian and charge densities were calculated by an all-valence electron SCF MO method, RHF-INDO/SP, using coordinates from refined X-ray data. These calculations yield asymmetry ratios similar to those observed.Consequences of the asymmetries in the charge distribution with respect to the observed unidirectionality of the electron transfer are discussed. (author). 29 refs.; 4 figs

  8. Structural and biochemical characterization of SrcA, a multi-cargo type III secretion chaperone in Salmonella required for pathogenic association with a host.

    Directory of Open Access Journals (Sweden)

    Colin A Cooper

    2010-02-01

    Full Text Available Many Gram-negative bacteria colonize and exploit host niches using a protein apparatus called a type III secretion system (T3SS that translocates bacterial effector proteins into host cells where their functions are essential for pathogenesis. A suite of T3SS-associated chaperone proteins bind cargo in the bacterial cytosol, establishing protein interaction networks needed for effector translocation into host cells. In Salmonella enterica serovar Typhimurium, a T3SS encoded in a large genomic island (SPI-2 is required for intracellular infection, but the chaperone complement required for effector translocation by this system is not known. Using a reverse genetics approach, we identified a multi-cargo secretion chaperone that is functionally integrated with the SPI-2-encoded T3SS and required for systemic infection in mice. Crystallographic analysis of SrcA at a resolution of 2.5 A revealed a dimer similar to the CesT chaperone from enteropathogenic E. coli but lacking a 17-amino acid extension at the carboxyl terminus. Further biochemical and quantitative proteomics data revealed three protein interactions with SrcA, including two effector cargos (SseL and PipB2 and the type III-associated ATPase, SsaN, that increases the efficiency of effector translocation. Using competitive infections in mice we show that SrcA increases bacterial fitness during host infection, highlighting the in vivo importance of effector chaperones for the SPI-2 T3SS.

  9. Chaperones in hepatitis C virus infection

    Institute of Scientific and Technical Information of China (English)

    2016-01-01

    The hepatitis C virus (HCV) infects approximately 3% ofthe world population or more than 185 million peopleworldwide. Each year, an estimated 350000-500000deaths occur worldwide due to HCV-associated diseasesincluding cirrhosis and hepatocellular carcinoma. HCV isthe most common indication for liver transplantation inpatients with cirrhosis worldwide. HCV is an envelopedRNA virus classified in the genus Hepacivirus in theFlaviviridae family. The HCV viral life cycle in a cellcan be divided into six phases (1) binding and internalization;(2) cytoplasmic release and uncoating; (3)viral polyprotein translation and processing; (4) RNAgenome replication; (5) encapsidation (packaging) andassembly; and (6) virus morphogenesis (maturation)and secretion. Many host factors are involved in theHCV life cycle. Chaperones are an important group ofhost cytoprotective molecules that coordinate numerouscellular processes including protein folding, multimericprotein assembly, protein trafficking, and proteindegradation. All phases of the viral life cycle requirechaperone activity and the interaction of viral proteinswith chaperones. This review will present our currentknowledge and understanding of the role of chaperonesin the HCV life cycle. Analysis of chaperones in HCVinfection will provide further insights into viral/hostinteractions and potential therapeutic targets for bothHCV and other viruses.

  10. ISOLATION AND IDENTIFICATION OF cDNA FRAGMENTS AND FULL-LENGTH cDNA DIFFERENTIALLY EXPRESSEDIN HUMAN GLIOBLASTOMA CELL LINE BT-325 VERSUS ALL-TRANS RETINOIC ACID INDUCTION

    Institute of Scientific and Technical Information of China (English)

    金虎林; 胡松年; 李光涛; 涂纯; 袁建刚; 强伯勤

    2000-01-01

    Objective. To investigate the differentiation process of the human glioblastoma cells. Methods. Differential display reverse transcribed-PCR(DDRT-PCR) was used to isolate the genes differentially expressed in control and all-trans retinoic acid treated human glioblastoma cell line BT-325. Routine method of cDNA library screening was performed to clone full-length cDNA. Results. Thirty-six RT-PCR reactions were performed and 64 differentially expressed fragments were recovered, amplified and cloned. Of them,46 ESTs were sequenced and delivered into the GenBank. The homology comparison us-ing BLAST algorithm revealed that 22ESFs are highly homologous with the known genes and many of them play impor-tant roles in the cell differentiation progress. A dot-blot hybridization was conducted to certify the differemiation expres-sion. The result showed that 27 EST clones are expressed at different level in control and all-traus retinoic acid treated BT-325 cells. A full-length cDNA was cloned using the EST-HGBB098.Conclusion. DDRT-PCR was a simple and effective method to segally analyze the differentially expressed genes.

  11. ISOLATION AND IDENTIFICATION OF cDNA FRAGMENTS AND FULL-LENGTH cDNA DIFFERENTIALLY EXPRESSED IN HUMAN GLIOBLASTOMA CELL LINE BT-325 VERSUS ALL-TRANS RETINOIC ACID INDUCTION

    Institute of Scientific and Technical Information of China (English)

    金虎林; 胡松年; 李光涛; 涂纯; 袁建刚; 强伯勤

    2000-01-01

    Objective. To investigate the differentiation process of the human glioblastoma cells. Methods. Differential display reverse transcribed-PCR(DDRT-PCR) was used to isolate the genes differentially expressed in control and all-trans retinoic acid treated human glioblastoma cell line BT-325. Routine method of cDNA library screening was performed to clone full-length cDNA. Results. Thirty-six RT-PCR reactions were performed and 64 differentially expressed fragments were recovered, amplified and cloned. Of them,46 ESTs were sequenced and delivered into the GenBank. The homology comparison us ing BLAST algorithm revealed that 22ESTs are highly homologous with the known genes and many of them play impor tant roles in the cell differentiation progress. A dot-blot hybridization was conducted to certify the differentiation expres sion. The result showed that 27 EST clones are expressed at different level in control and all-trans retinoi c acid treated BT-325 cells. A full-length cDNA was cloned using the ES T-HGBB098. Conclusion. DDRT-PCR was a simple and effective method to serially analyze the differentially expressed genes.

  12. Model for growth hormone receptor activation based on subunit rotation within a receptor dimer

    Energy Technology Data Exchange (ETDEWEB)

    Brown, Richard J.; Adams, Julian J.; Pelekanos, Rebecca A.; Wan, Yu; McKinstry, William J.; Palethorpe, Kathryn; Seeber, Ruth M.; Monks, Thea A.; Eidne, Karin A.; Parker, Michael W.; Waters, Michael J. (UWA); (St. Vincent); (Queensland)

    2010-07-13

    Growth hormone is believed to activate the growth hormone receptor (GHR) by dimerizing two identical receptor subunits, leading to activation of JAK2 kinase associated with the cytoplasmic domain. However, we have reported previously that dimerization alone is insufficient to activate full-length GHR. By comparing the crystal structure of the liganded and unliganded human GHR extracellular domain, we show here that there is no substantial change in its conformation on ligand binding. However, the receptor can be activated by rotation without ligand by inserting a defined number of alanine residues within the transmembrane domain. Fluorescence resonance energy transfer (FRET), bioluminescence resonance energy transfer (BRET) and coimmunoprecipitation studies suggest that receptor subunits undergo specific transmembrane interactions independent of hormone binding. We propose an activation mechanism involving a relative rotation of subunits within a dimeric receptor as a result of asymmetric placement of the receptor-binding sites on the ligand.

  13. Antiviral activity of α-helical stapled peptides designed from the HIV-1 capsid dimerization domain

    Directory of Open Access Journals (Sweden)

    Cowburn David

    2011-05-01

    Full Text Available Abstract Background The C-terminal domain (CTD of HIV-1 capsid (CA, like full-length CA, forms dimers in solution and CTD dimerization is a major driving force in Gag assembly and maturation. Mutations of the residues at the CTD dimer interface impair virus assembly and render the virus non-infectious. Therefore, the CTD represents a potential target for designing anti-HIV-1 drugs. Results Due to the pivotal role of the dimer interface, we reasoned that peptides from the α-helical region of the dimer interface might be effective as decoys to prevent CTD dimer formation. However, these small peptides do not have any structure in solution and they do not penetrate cells. Therefore, we used the hydrocarbon stapling technique to stabilize the α-helical structure and confirmed by confocal microscopy that this modification also made these peptides cell-penetrating. We also confirmed by using isothermal titration calorimetry (ITC, sedimentation equilibrium and NMR that these peptides indeed disrupt dimer formation. In in vitro assembly assays, the peptides inhibited mature-like virus particle formation and specifically inhibited HIV-1 production in cell-based assays. These peptides also showed potent antiviral activity against a large panel of laboratory-adapted and primary isolates, including viral strains resistant to inhibitors of reverse transcriptase and protease. Conclusions These preliminary data serve as the foundation for designing small, stable, α-helical peptides and small-molecule inhibitors targeted against the CTD dimer interface. The observation that relatively weak CA binders, such as NYAD-201 and NYAD-202, showed specificity and are able to disrupt the CTD dimer is encouraging for further exploration of a much broader class of antiviral compounds targeting CA. We cannot exclude the possibility that the CA-based peptides described here could elicit additional effects on virus replication not directly linked to their ability to bind

  14. Impact of a deletion of the full-length and short isoform of p75NTR on cholinergic innervation and the population of postmitotic doublecortin positive cells in the dentate gyrus

    Science.gov (United States)

    Poser, Robert; Dokter, Martin; von Bohlen und Halbach, Viola; Berger, Stefan M.; Busch, Ruben; Baldus, Marian; Unsicker, Klaus; von Bohlen und Halbach, Oliver

    2015-01-01

    Analyses of mice carrying a deletion of the pan-neurotrophin receptor p75NTR have allowed identifying p75NTR as an important structural regulator of the hippocampus. Most of the previous analyses were done using p75NTRExIII knockout mice which still express the short isoform of p75NTR. To scrutinize the role of p75NTR in the hippocampus, we analyzed adult and aged p75NTRExIV knockout mice, in which both, the short and the full-length isoform are deleted. Deletion of these isoforms induced morphological alterations in the adult dentate gyrus (DG), leading to an increase in the thickness of the molecular and granular layer. Based on these observations, we next determined the morphological substrates that might contribute to this phenotype. The cholinergic innervation of the molecular and granular layer of the DG was found to be significantly increased in the knockout mice. Furthermore, adult neurogenesis in the DG was found to be significantly altered with increased numbers of doublecortin (DCX) positive cells and reduced numbers of apoptotic cells in p75NTRExIV knockout mice. However, cell proliferation as measured by phosphohiston H3 (PH3) positive cell numbers was not affected. These morphological alterations (number of DCX-positive cells and increased cholinergic fiber densities) as well as reduced cell death in the DG are likely to contribute to the observed thickening of the granular layer in p75NTRExIV knockout mice. In addition, Sholl-analysis of DCX-positive neurons revealed a higher dendritic complexity and could thus be a possible morphological correlate for the increased thickness of the molecular layer in p75NTR deficient animals. Our data clearly demonstrate that deletion of both, the short and the full-length isoform of p75NTR affects DG morphology, due to alterations of the cholinergic system and an imbalance between neurogenesis and programmed cell death within the subgranular zone. PMID:26074780

  15. Impact of a deletion of the full-length and short isoform of p75NTR on cholinergic innervation and the population of postmitotic doublecortin positive cells in the dentate gyrus

    Directory of Open Access Journals (Sweden)

    Robert ePoser

    2015-05-01

    Full Text Available Analyses of mice carrying a deletion of the pan-neurotrophin receptor p75NTR have allowed identifying p75NTR as an important structural regulator of the hippocampus. Most of the previous analyses were done using p75NTRExIII knockout mice which still express the short isoform of p75NTR. To scrutinize the role of p75NTR in the hippocampus, we analyzed adult and aged p75NTRExIV knockout mice, in which both, the short and the full-length isoform are deleted. Deletion of these isoforms induced morphological alterations in the adult dentate gyrus (DG, leading to an increase in the thickness of the molecular and granular layer. Based on these observations, we next determined the morphological substrates that might contribute to this phenotype. The cholinergic innervation of the molecular and granular layer of the DG was found to be significantly increased in the knockout mice. Furthermore, adult neurogenesis in the DG was found to be significantly altered with increased numbers of doublecortin (DCX positive cells and reduced numbers of apoptotic cells in p75NTRExIV knockout mice. However, cell proliferation as measured by phosphohiston H3 (PH3 positive cell numbers was not affected. These morphological alterations (number of DCX-positive cells and increased cholinergic fiber densities as well as reduced cell death in the DG are likely to contribute to the observed thickening of the granular layer in p75NTRExIV knockout mice. In addition, Sholl-analysis of DCX-positive neurons revealed a higher dendritic complexity and could thus be a possible morphological correlate for the increased thickness of the molecular layer in p75NTR deficient animals. Our data clearly demonstrate that deletion of both, the short and the full-length isoform of p75NTR affects DG morphology, due to alterations of the cholinergic system and an imbalance between neurogenesis and programmed cell death within the subgranular zone.

  16. The extracellular chaperone clusterin sequesters oligomeric forms of the Aβ1–40 peptide

    Science.gov (United States)

    Narayan, Priyanka; Orte, Angel; Clarke, Richard W.; Bolognesi, Benedetta; Hook, Sharon; Ganzinger, Kristina A.; Meehan, Sarah; Wilson, Mark R.; Dobson, Christopher M.; Klenerman, David

    2016-01-01

    In recent genome-wide association studies, the extracellular chaperone protein, clusterin, has been identified as a novel risk factor in Alzheimer’s disease (AD). We have examined the interactions between clusterin and the AD-associated amyloid-β1–40 peptide (Aβ1–40) which is prone to aggregate into an ensemble of oligomeric intermediates implicated in both the proliferation of amyloid fibrils and in neuronal toxicity. Using highly sensitive single molecule fluorescence methods, we have found that Aβ1–40 forms a heterogeneous distribution of small oligomers (from dimers to 50mers), all of which interact with clusterin to form long-lived, stable complexes. Consequently, clusterin is able to influence strongly both the aggregation and disaggregation of Aβ1–40 by sequestration of the Aβ oligomers. These results not only elucidate the protective role of clusterin but also provide a molecular basis for the genetic link between clusterin and AD. PMID:22179788

  17. Pharmacological chaperone reshapes the energy landscape for folding and aggregation of the prion protein.

    Science.gov (United States)

    Gupta, Amar Nath; Neupane, Krishna; Rezajooei, Negar; Cortez, Leonardo M; Sim, Valerie L; Woodside, Michael T

    2016-01-01

    The development of small-molecule pharmacological chaperones as therapeutics for protein misfolding diseases has proven challenging, partly because their mechanism of action remains unclear. Here we study Fe-TMPyP, a tetrapyrrole that binds to the prion protein PrP and inhibits misfolding, examining its effects on PrP folding at the single-molecule level with force spectroscopy. Single PrP molecules are unfolded with and without Fe-TMPyP present using optical tweezers. Ligand binding to the native structure increases the unfolding force significantly and alters the transition state for unfolding, making it more brittle and raising the barrier height. Fe-TMPyP also binds the unfolded state, delaying native refolding. Furthermore, Fe-TMPyP binding blocks the formation of a stable misfolded dimer by interfering with intermolecular interactions, acting in a similar manner to some molecular chaperones. The ligand thus promotes native folding by stabilizing the native state while also suppressing interactions driving aggregation. PMID:27346148

  18. The role of Vif oligomerization and RNA chaperone activity in HIV-1 replication.

    Science.gov (United States)

    Batisse, Julien; Guerrero, Santiago; Bernacchi, Serena; Sleiman, Dona; Gabus, Caroline; Darlix, Jean-Luc; Marquet, Roland; Tisné, Carine; Paillart, Jean-Christophe

    2012-11-01

    The viral infectivity factor (Vif) is essential for the productive infection and dissemination of HIV-1 in non-permissive cells that involve most natural HIV-1 target cells. Vif counteracts the packaging of two cellular cytidine deaminases named APOBEC3G (A3G) and A3F by diverse mechanisms including the recruitment of an E3 ubiquitin ligase complex and the proteasomal degradation of A3G/A3F, the inhibition of A3G mRNA translation or by a direct competition mechanism. In addition, Vif appears to be an active partner of the late steps of viral replication by participating in virus assembly and Gag processing, thus regulating the final stage of virion formation notably genomic RNA dimerization and by inhibiting the initiation of reverse transcription. Vif is a small pleiotropic protein with multiple domains, and recent studies highlighted the importance of Vif conformation and flexibility in counteracting A3G and in binding RNA. In this review, we will focus on the oligomerization and RNA chaperone properties of Vif and show that the intrinsic disordered nature of some Vif domains could play an important role in virus assembly and replication. Experimental evidence demonstrating the RNA chaperone activity of Vif will be presented. PMID:22728817

  19. Pharmacological chaperone reshapes the energy landscape for folding and aggregation of the prion protein

    Science.gov (United States)

    Gupta, Amar Nath; Neupane, Krishna; Rezajooei, Negar; Cortez, Leonardo M.; Sim, Valerie L.; Woodside, Michael T.

    2016-01-01

    The development of small-molecule pharmacological chaperones as therapeutics for protein misfolding diseases has proven challenging, partly because their mechanism of action remains unclear. Here we study Fe-TMPyP, a tetrapyrrole that binds to the prion protein PrP and inhibits misfolding, examining its effects on PrP folding at the single-molecule level with force spectroscopy. Single PrP molecules are unfolded with and without Fe-TMPyP present using optical tweezers. Ligand binding to the native structure increases the unfolding force significantly and alters the transition state for unfolding, making it more brittle and raising the barrier height. Fe-TMPyP also binds the unfolded state, delaying native refolding. Furthermore, Fe-TMPyP binding blocks the formation of a stable misfolded dimer by interfering with intermolecular interactions, acting in a similar manner to some molecular chaperones. The ligand thus promotes native folding by stabilizing the native state while also suppressing interactions driving aggregation. PMID:27346148

  20. Screening for Pharmacological Chaperones in Fabry Disease

    OpenAIRE

    Shin, Sang-Hoon; Murray, Gary J.; Kluepfel-Stahl, Stefanie; Cooney, Adele M.; Quirk, Jane M.; Schiffmann, Raphael; Brady, Roscoe O.; Kaneski, Christine R.

    2007-01-01

    As a prerequisite for full clinical trials of pharmacological chaperone therapy (PCT) for Fabry disease we developed a rapid screening assay for enhancement of endogenous α-galactosidase A (α-Gal A) in patient-derived cells. We used a T-cell based system to screen 11 mutations causing Fabry disease for enhanceability using 1- deoxygalactonojirimycin (DGJ). When patient derived T-cells were grown in the presence of DGJ α-Gal A activity increased to more than 50% of normal in several mutations ...

  1. Structure of the RuBisCO chaperone RbcX from the thermophilic cyanobacterium Thermosynechococcus elongatus

    International Nuclear Information System (INIS)

    The crystal structure of the RuBisCO assembly chaperone RbcX from a thermophilic cyanobacterium has been determined at 1.7 Å resolution. The dimeric structure is capable of a hinge movement (probably connected with binding of the RuBisCO large subunit) pivoted on a kink in two long antiparallel α-helices. The crystal structure of TeRbcX, a RuBisCO assembly chaperone from the cyanobacterium Thermosynechococcus elongatus, a thermophilic organism, has been determined at 1.7 Å resolution. TeRbcX has an unusual cysteine residue at position 103 that is not found in RbcX proteins from mesophilic organisms. Unlike wild-type TeRbcX, a mutant protein with Cys103 replaced by Ala (TeRbcX-C103A) could be readily crystallized. The structure revealed that the overall fold of the TeRbcX homodimer is similar to those of previously crystallized RbcX proteins. Normal-mode analysis suggested that TeRbcX might adopt an open or closed conformation through a hinge movement pivoted on a kink in two long α4 helices. This type of conformational transition is presumably connected to RbcL (the large RuBisCO subunit) binding during the chaperone function of the RuBisCO assembly

  2. Molecular chaperones: The modular evolution of cellular networks

    Indian Academy of Sciences (India)

    Tamás Korcsmáros; István A Kovács; Máté S Szalay; Péter Csermely

    2007-04-01

    Molecular chaperones play a prominent role in signaling and transcriptional regulatory networks of the cell. Recent advances uncovered that chaperones act as genetic buffers stabilizing the phenotype of various cells and organisms and may serve as potential regulators of evolvability. Chaperones have weak links, connect hubs, are in the overlaps of network modules and may uncouple these modules during stress, which gives an additional protection for the cell at the network-level. Moreover, after stress chaperones are essential to re-build inter-modular contacts by their low affinity sampling of the potential interaction partners in different modules. This opens the way to the chaperone-regulated modular evolution of cellular networks, and helps us to design novel therapeutic and anti-aging strategies.

  3. In Vivo Dopamine Efflux is Decreased in Striatum of both Fragment (R6/2 and Full-length (YAC128 Transgenic Mouse Models of Huntington’s Disease

    Directory of Open Access Journals (Sweden)

    Joshua W Callahan

    2011-07-01

    Full Text Available Huntington’s disease (HD is characterized by alterations within the corticostriatal circuitry. The striatum is innervated by a dense array of dopaminergic (DA terminals and these DA synapses are critical to the proper execution of motor functions. As motor disturbances are prevalent in HD we examined DA neurotransmission in the striatum in transgenic (tg murine models of HD. We used in vivo microdialysis to compare extracellular concentrations of striatal DA in both a fragment (R6/2 model, which displays a rapid and severe phenotype, and a full-length (YAC128 model that expresses a more progressive phenotype. Extracellular striatal DA concentrations were significantly reduced in R6/2 mice and decreased concomitantly with age-dependent increasing motor impairments on the rotarod task (7, 9, and 11 weeks. In a sample of 11-week-old R6/2 mice, we also measured tissue concentrations of striatal DA and found that total levels of DA were significantly depleted. However, the loss of total DA content (<50% was insufficient to account for the full extent of DA depletion in the extracellular fluid (ECF (~75%. We also observed a significant reduction in extracellular DA concentrations in the striatum of 7-month-old YAC128 mice. In a separate set of experiments, we applied d-amphetamine (AMPH (10 μm locally into the striatum to stimulate the release of intracellular DA into the ECF. The AMPH-induced increase in extracellular DA levels was significantly blunted in 9-week-old R6/2 mice. There also was a decrease in AMPH-stimulated DA efflux in 7-month-old YAC128 mice in comparison to WT controls, although the effect was milder. In the same cohort of 7-month-old YAC128 mice we observed a significant reduction in the total locomotor activity in response to systemic AMPH (2 mg/kg. Our data demonstrate that extracellular DA release is attenuated in both a fragment and full-length tg mouse model of HD and support the concept of DA involvement in aspects of the

  4. Final Report on TREAT Tests R4 and R5; Seven-pin, Loss-of-flow Tests with Full-length, Unirradiated FFTF-type Fuel Pins -- Part 1

    Energy Technology Data Exchange (ETDEWEB)

    Spencer, B. W.

    1979-04-01

    Two seven-pin loss-of-flow simulation tests have been performed in-pile in support of LMFBR safety analyses. These integral-type tests were executed in the R-series test apparatus at TREAT, designed to provide a prototypic thermal hydraulic flowing sodium system. The test fuel pins were full-length FFTF-type, containing unirradiated UO{sub 2} fuel. In LOF test R4, the sequence was run at constant fuel power well beyond the inception of molten fuel motion; in R5, the sequence was terminated prior to fuel melting to preserve evidence of early molten cladding motion. The tests were consistent with one another, showing the anticipated sequence of sodium boiling, channel voiding, cladding dryout, cladding failures, molten cladding motion and, for R4, fuel melting and motion. Thin planar upper cladding blockages were formed as were thick lower blockages, effectively plugging the bundle at the extremities of the active fuel region. There was no sodium reentry, and there were no significant pressurization events. Comparison of test results with SAS code calculations revealed differences associated with early voiding behavior, release of noncondensible fuel pin plenum gas, and the thickness and location of the upper blockage. In general, however, the overall sequence of significant events observed in R4 and R5 is in quite good agreement with SAS code analysis of the test conditions. Features of SAS related to overall voiding, cladding motion, and to some extent fuel motion have been substantiated in these results.

  5. Expression Analysis and Nuclear Import Study of Full-length Isoforms Importin α as 6x Histidin-tagged Fusion Protein on the Intracellular Localization of Recombinant HBV Core Protein

    Directory of Open Access Journals (Sweden)

    Aris Haryanto

    2015-10-01

    Full Text Available Isoform importin α molecules play a central role in the classical nuclear import pathway, that occurs throughthe nuclear pore complex (NPC and typically requires a specific nuclear localization signal (NLS. In this study,it was investigated the role of isoforms importin α in the nuclear import of wild type recombinant hepatitis B viruscore protein (WT rHBc, phosphorylated recombinant HBV core (rHBc and recombinant HBV core without NLSby co-immunoprecipitation. Four recombinant full-length isoforms importin α as 6x histidin-tagged fusion proteinwere expressed and analysed from expression plasmid vectors Rch1, pHM 1969, pHM 1967 and pHM 1965. Theresults indicated that importin α-1, importin α-3, importin α-4 and importin α-5 can be expressed and isolatedfrom E. coli transformed recombinant DNA plasmid as protein in size around 58-60 kDa. By the nuclear transportstudy shown that isoforms importin α are involved in the nuclear import of WT rHBc, phosphorylated rHBc andrHBc without NLS. It also indicated that they have an important role for nuclear transport of from cytoplasm intothe nucleus.Keywords: NPC, NLS, importin α, importin β, isoforms importin α as 6x histidin-tagged fusion protein, WTrHBc, SV40 Tag, co-immunoprecipitation, westernblotting.

  6. Using RNA-Seq to assemble a rose transcriptome with more than 13,000 full-length expressed genes and to develop the WagRhSNP 68k Axiom SNP array for rose (Rosa L.).

    Science.gov (United States)

    Koning-Boucoiran, Carole F S; Esselink, G Danny; Vukosavljev, Mirjana; van 't Westende, Wendy P C; Gitonga, Virginia W; Krens, Frans A; Voorrips, Roeland E; van de Weg, W Eric; Schulz, Dietmar; Debener, Thomas; Maliepaard, Chris; Arens, Paul; Smulders, Marinus J M

    2015-01-01

    In order to develop a versatile and large SNP array for rose, we set out to mine ESTs from diverse sets of rose germplasm. For this RNA-Seq libraries containing about 700 million reads were generated from tetraploid cut and garden roses using Illumina paired-end sequencing, and from diploid Rosa multiflora using 454 sequencing. Separate de novo assemblies were performed in order to identify single nucleotide polymorphisms (SNPs) within and between rose varieties. SNPs among tetraploid roses were selected for constructing a genotyping array that can be employed for genetic mapping and marker-trait association discovery in breeding programs based on tetraploid germplasm, both from cut roses and from garden roses. In total 68,893 SNPs were included on the WagRhSNP Axiom array. Next, an orthology-guided assembly was performed for the construction of a non-redundant rose transcriptome database. A total of 21,740 transcripts had significant hits with orthologous genes in the strawberry (Fragaria vesca L.) genome. Of these 13,390 appeared to contain the full-length coding regions. This newly established transcriptome resource adds considerably to the currently available sequence resources for the Rosaceae family in general and the genus Rosa in particular. PMID:25954285

  7. Construction of a full-length cDNA Library from Chinese oak silkworm pupa and identification of a KK-42-binding protein gene in relation to pupa-diapause termination

    Directory of Open Access Journals (Sweden)

    Yu-Ping Li, Run-Xi Xia, Huan Wang, Xi-Sheng Li, Yan-Qun Liu, Zhao-Jun Wei, Cheng Lu, Zhong-Huai Xiang

    2009-01-01

    Full Text Available In this study we successfully constructed a full-length cDNA library from Chinese oak silkworm, Antheraea pernyi, the most well-known wild silkworm used for silk production and insect food. Total RNA was extracted from a single fresh female pupa at the diapause stage. The titer of the library was 5 × 105 cfu/ml and the proportion of recombinant clones was approximately 95%. Expressed sequence tag (EST analysis was used to characterize the library. A total of 175 clustered ESTs consisting of 24 contigs and 151 singlets were generated from 250 effective sequences. Of the 175 unigenes, 97 (55.4% were known genes but only five from A. pernyi, 37 (21.2% were known ESTs without function annotation, and 41 (23.4% were novel ESTs. By EST sequencing, a gene coding KK-42-binding protein in A. pernyi (named as ApKK42-BP; GenBank accession no. FJ744151 was identified and characterized. Protein sequence analysis showed that ApKK42-BP was not a membrane protein but an extracellular protein with a signal peptide at position 1-18, and contained two putative conserved domains, abhydro_lipase and abhydrolase_1, suggesting it may be a member of lipase superfamily. Expression analysis based on number of ESTs showed that ApKK42-BP was an abundant gene in the period of diapause stage, suggesting it may also be involved in pupa-diapause termination.

  8. Full-length genome analyses of two new simian immunodeficiency virus (SIV) strains from mustached monkeys (C. Cephus) in Gabon illustrate a complex evolutionary history among the SIVmus/mon/gsn lineage.

    Science.gov (United States)

    Liégeois, Florian; Schmidt, Fabian; Boué, Vanina; Butel, Christelle; Mouacha, Fatima; Ngari, Paul; Ondo, Bertrand Mve; Leroy, Eric; Heeney, Jonathan L; Delaporte, Eric; Peeters, Martine; Rouet, François

    2014-07-01

    The Simian Immunodeficiency Virus (SIV) mus/mon/gsn lineage is a descendant of one of the precursor viruses to the HIV-1/SIVcpz/gor viral lineage. SIVmus and SIVgsn were sequenced from mustached and greater spot nosed monkeys in Cameroon and SIVmon from mona monkeys in Cameroon and Nigeria. In order to further document the genetic diversity of SIVmus, we analyzed two full-length genomes of new strains identified in Gabon. The whole genomes obtained showed the expected reading frames for gag, pol, vif, vpr, tat, rev, env, nef, and also for a vpu gene. Analyses showed that the Gabonese SIVmus strains were closely related and formed a monophyletic clade within the SIVmus/mon/gsn lineage. Nonetheless, within this lineage, the position of both new SIVmus differed according to the gene analyzed. In pol and nef gene, phylogenetic topologies suggested different evolutions for each of the two new SIVmus strains whereas in the other nucleic fragments studied, their positions fluctuated between SIVmon, SIVmus-1, and SIVgsn. In addition, in C1 domain of env, we identified an insertion of seven amino acids characteristic for the SIVmus/mon/gsn and HIV‑1/SIVcpz/SIVgor lineages. Our results show a high genetic diversity of SIVmus in mustached monkeys and suggest cross-species transmission events and recombination within SIVmus/mon/gsn lineage. Additionally, in Central Africa, hunters continue to be exposed to these simian viruses, and this represents a potential threat to humans. PMID:25054885

  9. Full-Length Genome Analyses of Two New Simian Immunodeficiency Virus (SIV Strains from Mustached Monkeys (C. Cephus in Gabon Illustrate a Complex Evolutionary History among the SIVmus/mon/gsn Lineage

    Directory of Open Access Journals (Sweden)

    Florian Liégeois

    2014-07-01

    Full Text Available The Simian Immunodeficiency Virus (SIV mus/mon/gsn lineage is a descendant of one of the precursor viruses to the HIV-1/SIVcpz/gor viral lineage. SIVmus and SIVgsn were sequenced from mustached and greater spot nosed monkeys in Cameroon and SIVmon from mona monkeys in Cameroon and Nigeria. In order to further document the genetic diversity of SIVmus, we analyzed two full-length genomes of new strains identified in Gabon. The whole genomes obtained showed the expected reading frames for gag, pol, vif, vpr, tat, rev, env, nef, and also for a vpu gene. Analyses showed that the Gabonese SIVmus strains were closely related and formed a monophyletic clade within the SIVmus/mon/gsn lineage. Nonetheless, within this lineage, the position of both new SIVmus differed according to the gene analyzed. In pol and nef gene, phylogenetic topologies suggested different evolutions for each of the two new SIVmus strains whereas in the other nucleic fragments studied, their positions fluctuated between SIVmon, SIVmus-1, and SIVgsn. In addition, in C1 domain of env, we identified an insertion of seven amino acids characteristic for the SIVmus/mon/gsn and HIV‑1/SIVcpz/SIVgor lineages. Our results show a high genetic diversity of SIVmus in mustached monkeys and suggest cross-species transmission events and recombination within SIVmus/mon/gsn lineage. Additionally, in Central Africa, hunters continue to be exposed to these simian viruses, and this represents a potential threat to humans.

  10. Construction and characterization of a full-length infectious cDNA clone of foot-and-mouth disease virus strain O/JPN/2010 isolated in Japan in 2010.

    Science.gov (United States)

    Nishi, Tatsuya; Onozato, Hiroyuki; Ohashi, Seiichi; Fukai, Katsuhiko; Yamada, Manabu; Morioka, Kazuki; Kanno, Toru

    2016-06-01

    A full-length infectious cDNA clone of the genome of a foot-and-mouth disease virus isolated from the 2010 epidemic in Japan was constructed and designated pSVL-f02. Transfection of Cos-7 or IBRS-2 cells with this clone allowed the recovery of infectious virus. The recovered virus had the same in vitro characterization as the parental virus with regard to antigenicity in neutralization and indirect immunofluorescence tests, plaque size and one-step growth. Pigs were experimentally infected with the parental virus or the recombinant virus recovered from pSVL-f02 transfected cells. There were no significant differences in clinical signs or antibody responses between the two groups, and virus isolation and viral RNA detection from clinical samples were similar. Virus recovered from transfected cells therefore retained the in vitro characteristics and the in vivo pathogenicity of their parental strain. This cDNA clone should be a valuable tool to analyze determinants of pathogenicity and mechanisms of virus replication, and to develop genetically engineered vaccines against foot-and-mouth disease virus. PMID:27234555

  11. In vitro binding of anthrax protective antigen on bacteriophage T4 capsid surface through Hoc-capsid interactions: A strategy for efficient display of large full-length proteins

    International Nuclear Information System (INIS)

    An in vitro binding system is described to display large full-length proteins on bacteriophage T4 capsid surface at high density. The phage T4 icosahedral capsid features 155 copies of a nonessential highly antigenic outer capsid protein, Hoc, at the center of each major capsid protein hexon. Gene fusions were engineered to express the 83-kDa protective antigen (PA) from Bacillus anthracis fused to the N-terminus of Hoc and the 130-kDa PA-Hoc protein was expressed in Escherichia coli and purified. The purified PA-Hoc was assembled in vitro on hoc - phage particles. Binding was specific, stable, and of high affinity. This defined in vitro system allowed manipulation of the copy number of displayed PA and imposed no significant limitation on the size of the displayed antigen. In contrast to in vivo display systems, the in vitro approach allows all the capsid binding sites to be occupied by the 130-kDa PA-Hoc fusion protein. The PA-T4 particles were immunogenic in mice in the absence of an adjuvant, eliciting strong PA-specific antibodies and anthrax lethal toxin neutralizing antibodies. The in vitro display on phage T4 offers a novel platform for potential construction of customized vaccines against anthrax and other infectious diseases

  12. Isolation and characterization of the full-length cDNA encoding a member of a novel cytochrome p450 family (CYP320A1 from the tropical freshwater snail, Biomphalaria glabrata, intermediate host for Schistosoma mansoni

    Directory of Open Access Journals (Sweden)

    Anne E Lockyer

    2005-05-01

    Full Text Available Cytochrome p450s (cyp450s are a family of structurally related proteins, with diverse functions, including steroid synthesis and breakdown of toxins. This paper reports the full-length sequence of a novel cyp450 gene, the first to be isolated from the tropical freshwater snail Biomphalaria glabrata, an important intermediate host of Schistosoma mansoni. The nucleotide sequence is 2291 bp with a predicted amino acid sequence of 584aa. The sequence demonstrates conserved cyp450 structural motifs, but is sufficiently different from previously reported cyp450 sequences to be given a new classification, CYP320A1. Initially identified as down-regulated in partially resistant snails in response to S. mansoni infection, amplification of this gene using RT-PCR in both totally resistant or susceptible snail lines when exposed to infection, and all tissues examined, suggests ubiquitous expression. Characterization of the first cyp450 from B. glabrata is significant in understanding the evolution of these metabolically important proteins.

  13. Molecular characterization of two novel molecular chaperones in bacterial-challenged Apostichopus japonicus.

    Science.gov (United States)

    Wang, Haihong; Shao, Yina; Zhang, Weiwei; Li, Chenghua; Lv, Zhimeng; Jin, Chunhua

    2015-10-01

    Molecular chaperones of 78 kDa glucose-regulated protein (GRP78) and protein disulfide isomerase (PDI) are involved in protein folding and assembly in the endoplasmic reticulum (ER). Increasing evidences also suggest that these two molecules play an important role in immune response. In the present study, we cloned and characterized GRP78 and PDI genes from Apostichopus japonicus by RNA-seq and RACE approaches (designated as AjGRP78 and AjPDI, respectively). The AjGRP78 cDNA was of 2355bp including an open reading frame (ORF) of 2013 bp encoding a protein of 670 amino acids with three heat shock protein 70 (HSP70) family signatures. AjGRP78 contained a 23-amino acid signal peptide at the N-terminus and a HDEL motif at the C-terminus, which supported the location of the protein in the ER. The full length cDNA of AjPDI was of 1893 bp with a 5' untranslated region (UTR) of 153 bp, a 3' UTR of 228 bp and an ORF of 1512 bp encoding a protein of 503 amino acids. A 17-amino acid signal peptide, two thioredoxin domains with two active sites of CGHC, and KDEL retention signal were totally conserved in the deduced amino acid of AjPDI. Phylogenic analysis and multiple alignments have shown that both genes shared remarkably higher degree of structural conservation and sequence identities with other counterparts from invertebrates and vertebrates, further supporting that the two proteins were novel members of molecular chaperone family. Spatial expression analysis revealed that AjGRP78 mRNA transcripts were dominantly expressed in the tentacle, while AjPDI mRNA levels were abundant in the muscle, intestine and respiratory trees. For Vibrio splendidus challenged sea cucumber, the peak expression of AjGRP78 and AjPDI mRNAs in coelomocytes were detected at 24h with 1.73-fold increase and at 6h with 1.83-fold increase compared with the control group, respectively. Similarly, a significant increase in the relative mRNA levels of AjGRP78 and AjPDI was also identified in 1 μg mL(-1

  14. Expression and variability of molecular chaperones in the sugarcane expressome.

    Science.gov (United States)

    Borges, Júlio C; Cagliari, Thiago C; Ramos, Carlos H I

    2007-04-01

    Molecular chaperones perform folding assistance in newly synthesized polypeptides preventing aggregation processes, recovering proteins from aggregates, among other important cellular functions. Thus their study presents great biotechnological importance. The present work discusses the mining for chaperone-related sequences within the sugarcane EST genome project database, which resulted in approximately 300 different sequences. Since molecular chaperones are highly conserved in most organisms studied so far, the number of sequences related to these proteins in sugarcane was very similar to the number found in the Arabidopsis thaliana genome. The Hsp70 family was the main molecular chaperone system present in the sugarcane expressome. However, many other relevant molecular chaperones systems were also present. A digital RNA blot analysis showed that 5'ESTs from all molecular chaperones were found in every sugarcane library, despite their heterogeneous expression profiles. The results presented here suggest the importance of molecular chaperones to polypeptide metabolism in sugarcane cells, based on their abundance and variability. Finally, these data have being used to guide more in deep analysis, permitting the choice of specific targets to study. PMID:16687190

  15. Maintenance of structure and function of mitochondrial Hsp70 chaperones requires the chaperone Hep1

    Science.gov (United States)

    Sichting, Martin; Mokranjac, Dejana; Azem, Abdussalam; Neupert, Walter; Hell, Kai

    2005-01-01

    Hsp70 chaperones mediate folding of proteins and prevent their misfolding and aggregation. We report here on a new kind of Hsp70 interacting protein in mitochondria, Hep1. Hep1 is a highly conserved protein present in virtually all eukaryotes. Deletion of HEP1 results in a severe growth defect. Cells lacking Hep1 are deficient in processes that need the function of mitochondrial Hsp70s, such as preprotein import and biogenesis of proteins containing FeS clusters. In the mitochondria of these cells, Hsp70s, Ssc1 and Ssq1 accumulate as insoluble aggregates. We show that it is the nucleotide-free form of mtHsp70 that has a high tendency to self-aggregate. This process is efficiently counteracted by Hep1. We conclude that Hep1 acts as a chaperone that is necessary and sufficient to prevent self-aggregation and to thereby maintain the function of the mitochondrial Hsp70 chaperones. PMID:15719019

  16. Principles of Quantitative Estimation of the Chaperone-Like Activity

    Institute of Scientific and Technical Information of China (English)

    2002-01-01

    Molecular chaperones are able to interact with unfolded states of the protein molecule preventing their aggregation and facilitating folding of the polypeptide chain into the native structure. An understanding of the mechanism of protein aggregation is required to estimate the efficiency of action of chaperones in the test-systems based on the suppression of aggregation of protein substrates. The kinetic regimes of aggregation of proteins are discussed. The analysis of the aggregation kinetics of proteins shows that after passing the lag phase, aggregation follows, as a rule, first order kinetics. The quantitative characterization methods of the ability of chaperones to prevent aggregation of protein substrates have been elaborated.

  17. Low risk of inhibitor formation in haemophilia A patients following en masse switch in treatment to a third generation full length plasma and albumin-free recombinant factor VIII product (ADVATE®).

    LENUS (Irish Health Repository)

    Bacon, C L

    2011-05-01

    Previous studies have suggested that development of inhibitors in previously treated patients (PTPs) may be attributable to a switch in factor VIII (FVIII) therapeutic product. Consequently, it is widely recognized that inhibitor development must be assessed in PTPs following the introduction of any new FVIII product. Following a national tender process in 2006, all patients with haemophilia A in Ireland changed their FVIII treatment product en masse to a plasma and albumin-free recombinant full-length FVIII product (ADVATE(®)). In this study, we retrospectively reviewed the case records of Irish PTPs to evaluate risk of inhibitor formation following this treatment switch. One hundred and thirteen patients participated in the study. Most patients (89%) had severe haemophilia. Only one of 96 patients with no inhibitor history developed an inhibitor. Prior to the switch in his recombinant FVIII (rFVIII) treatment of choice, this child had only experienced three exposure days (EDs). Consequently, in total he had only received 6 EDs when his inhibitor was first diagnosed. In keeping with this lack of de novo inhibitor development, we observed no evidence of any recurrent inhibitor formation in any of 16 patients with previously documented inhibitors. Similarly, following a previous en masse switch, we have previously reported that changing from a Chinese hamster ovary cell-produced to a baby hamster kidney cell-produced rFVIII was also associated with a low risk of inhibitor formation in PTPs. Our cumulative findings from these two studies clearly emphasizes that the risk of inhibitor development for PTPs following changes in commercial rFVIII product is low, at least in the Irish population.

  18. Multiple different defense mechanisms are activated in the young transgenic tobacco plants which express the full length genome of the Tobacco mosaic virus, and are resistant against this virus.

    Science.gov (United States)

    Jada, Balaji; Soitamo, Arto J; Siddiqui, Shahid Aslam; Murukesan, Gayatri; Aro, Eva-Mari; Salakoski, Tapio; Lehto, Kirsi

    2014-01-01

    Previously described transgenic tobacco lines express the full length infectious Tobacco mosaic virus (TMV) genome under the 35S promoter (Siddiqui et al., 2007. Mol Plant Microbe Interact, 20: 1489-1494). Through their young stages these plants exhibit strong resistance against both the endogenously expressed and exogenously inoculated TMV, but at the age of about 7-8 weeks they break into TMV infection, with typical severe virus symptoms. Infections with some other viruses (Potato viruses Y, A, and X) induce the breaking of the TMV resistance and lead to synergistic proliferation of both viruses. To deduce the gene functions related to this early resistance, we have performed microarray analysis of the transgenic plants during the early resistant stage, and after the resistance break, and also of TMV-infected wild type tobacco plants. Comparison of these transcriptomes to those of corresponding wild type healthy plants indicated that 1362, 1150 and 550 transcripts were up-regulated in the transgenic plants before and after the resistance break, and in the TMV-infected wild type tobacco plants, respectively, and 1422, 1200 and 480 transcripts were down-regulated in these plants, respectively. These transcriptome alterations were distinctly different between the three types of plants, and it appears that several different mechanisms, such as the enhanced expression of the defense, hormone signaling and protein degradation pathways contributed to the TMV-resistance in the young transgenic plants. In addition to these alterations, we also observed a distinct and unique gene expression alteration in these plants, which was the strong suppression of the translational machinery. This may also contribute to the resistance by slowing down the synthesis of viral proteins. Viral replication potential may also be suppressed, to some extent, by the reduction of the translation initiation and elongation factors eIF-3 and eEF1A and B, which are required for the TMV replication

  19. Comparison of radiation dose, workflow, patient comfort and financial break-even of standard digital radiography and a novel biplanar low-dose X-ray system for upright full-length lower limb and whole spine radiography

    International Nuclear Information System (INIS)

    To compare the radiation dose, workflow, patient comfort, and financial break-even of a standard digital radiography and a biplanar low-dose X-ray system. A standard digital radiography system (Ysio, Siemens Healthcare, Erlangen, Germany) was compared with a biplanar X-ray unit (EOS, EOS imaging, Paris, France) consisting of two X-ray tubes and slot-scanning detectors, arranged at an angle of 90 allowing simultaneous vertical biplanar linear scanning in the upright patient position. We compared data of standing full-length lower limb radiographs and whole spine radiographs of both X-ray systems. Dose-area product was significantly lower for radiographs of the biplanar X-ray system than for the standard digital radiography system (e.g. whole spine radiographs; standard digital radiography system: 392.2 ± 231.7 cGy*cm2 versus biplanar X-ray system: 158.4 ± 103.8 cGy*cm2). The mean examination time was significantly shorter for biplanar radiographs compared with standard digital radiographs (e.g. whole spine radiographs: 449 s vs 248 s). Patients' comfort regarding noise was significantly higher for the standard digital radiography system. The financial break-even point was 2,602 radiographs/year for the standard digital radiography system compared with 4,077 radiographs/year for the biplanar X-ray unit. The biplanar X-ray unit reduces radiation exposure and increases subjective noise exposure to patients. The biplanar X-ray unit demands a higher number of examinations per year for the financial break-even point, despite the lower labour cost per examination due to the shorter examination time. (orig.)

  20. N-terminal arm of orchardgrass Hsp17.2 (DgHsp17.2) is essential for both in vitro chaperone activity and in vivo thermotolerance in yeast.

    Science.gov (United States)

    Cha, Joon-Yung; Lee, Sang-Hoon; Seo, Kyung Hye; Choi, Young Jin; Cheong, Mi Sun; Son, Daeyoung

    2016-02-01

    Small heat shock proteins are well-known to function as chaperone in the protection of proteins and subcellular structures against stress-induced denaturation in many cell compartments. Irrespective of such general functional assignment, a proof of function in a living organism is missing. Here, we used heat-induced orchardgrass small Hsp17.2 (DgHsp17.2). Its function in in vitro chaperone properties has shown in protecting the model substrate, malate dehydrogenase (MDH) and citrate synthase (CS). Overexpression of DgHsp17.2 triggering strong chaperone activity enhanced in vivo thermotolerance of yeast cells. To identify the functional domain on DgHsp17.2 and correlationship between in vitro chaperone property and in vivo thermotolerance, we generated truncation mutants of DgHsp17.2 and showed essentiality of the N-terminal arm of DgHsp17.2 for the chaperone function. In addition, beyond for acquisition of thermotolerance irrespective of sequences are diverse among the small Hsps. However, any truncation mutants of DgHsp17.2 did not exhibit strong interaction with orchardgrass heat shock protein 70 (DgHsp70) different from mature DgHsp17.2, indicating that full-length DgHsp17.2 is necessary for cooperating with Hsp70 protein. Our study indicates that the N-terminal arm of DgHsp17.2 is an important region for chaperone activity and thermotolerance. PMID:26724757

  1. DAXX envelops a histone H3.3-H4 dimer for H3.3-specific recognition

    Energy Technology Data Exchange (ETDEWEB)

    Elsässer, Simon J; Huang, Hongda; Lewis, Peter W; Chin, Jason W; Allis, C David; Patel, Dinshaw J [MSKCC; (Rockefeller); (MRC)

    2013-01-24

    Histone chaperones represent a structurally and functionally diverse family of histone-binding proteins that prevent promiscuous interactions of histones before their assembly into chromatin. DAXX is a metazoan histone chaperone specific to the evolutionarily conserved histone variant H3.3. Here we report the crystal structures of the DAXX histone-binding domain with a histone H3.3–H4 dimer, including mutants within DAXX and H3.3, together with in vitro and in vivo functional studies that elucidate the principles underlying H3.3 recognition specificity. Occupying 40% of the histone surface-accessible area, DAXX wraps around the H3.3–H4 dimer, with complex formation accompanied by structural transitions in the H3.3–H4 histone fold. DAXX uses an extended α-helical conformation to compete with major inter-histone, DNA and ASF1 interaction sites. Our structural studies identify recognition elements that read out H3.3-specific residues, and functional studies address the contributions of Gly90 in H3.3 and Glu225 in DAXX to chaperone-mediated H3.3 variant recognition specificity.

  2. Structural features and interactions of substrates complexed with molecular chaperones

    OpenAIRE

    Ungelenk, Sophia Maria

    2015-01-01

    Protein misfolding and aggregation perturbs cellular functions and is involved in aging and numerous medical disorders. In cells, the first line of defense is the association of deleterious aggregating proteins with small Heat shock proteins (sHsp). These oligomeric, ATP-independent chaperones sequester misfolded proteins into complexes and facilitate subsequent substrate solubilization and refolding by ATP-dependent chaperones. The cytosol of S. cerevisiae contains two sHsps: Hsp42 is consti...

  3. The use of a chaperone in obstetrical and gynaecological practice.

    LENUS (Irish Health Repository)

    Afaneh, I

    2010-05-01

    The aim of this study was to assess the use of a chaperone in obstetrical and gynaecological practice in Ireland and to explore patients\\' opinions. Two questionnaires were designed; one for patients and the other one was sent to 145 gynaecologists in Ireland. One hundred and fifty two women took part in this survey of whom 74 were gynaecological and 78 were obstetric patients. Ninety five (65%) patients felt no need for a chaperone during a vaginal examination (VE) by a male doctor. On the other hand 34 (23%) participating women would request a chaperone if being examined by a female doctor. Among clinicians 116 (80%) responded by returning the questionnaire. Overall 60 (52%) always used a chaperone in public practice, in contrast to 24 (27%) in private practice. The study demonstrated that most patients do not wish to have a chaperone during a VE but a small proportion would still request one regardless of the examiner\\'s gender. Patients should be offered the choice of having a chaperone and their opinion should be respected and documented.

  4. The use of a chaperone in obstetrical and gynaecological practice.

    LENUS (Irish Health Repository)

    Afaneh, I

    2012-02-01

    The aim of this study was to assess the use of a chaperone in obstetrical and gynaecological practice in Ireland and to explore patients\\' opinions. Two questionnaires were designed; one for patients and the other one was sent to 145 gynaecologists in Ireland. One hundred and fifty two women took part in this survey of whom 74 were gynaecological and 78 were obstetric patients. Ninety five (65%) patients felt no need for a chaperone during a vaginal examination (VE) by a male doctor. On the other hand 34 (23%) participating women would request a chaperone if being examined by a female doctor. Among clinicians 116 (80%) responded by returning the questionnaire. Overall 60 (52%) always used a chaperone in public practice, in contrast to 24 (27%) in private practice. The study demonstrated that most patients do not wish to have a chaperone during a VE but a small proportion would still request one regardless of the examiner\\'s gender. Patients should be offered the choice of having a chaperone and their opinion should be respected and documented.

  5. Chaperoning Roles of Macromolecules Interacting with Proteins in Vivo

    Directory of Open Access Journals (Sweden)

    Baik L. Seong

    2011-03-01

    Full Text Available The principles obtained from studies on molecular chaperones have provided explanations for the assisted protein folding in vivo. However, the majority of proteins can fold without the assistance of the known molecular chaperones, and little attention has been paid to the potential chaperoning roles of other macromolecules. During protein biogenesis and folding, newly synthesized polypeptide chains interact with a variety of macromolecules, including ribosomes, RNAs, cytoskeleton, lipid bilayer, proteolytic system, etc. In general, the hydrophobic interactions between molecular chaperones and their substrates have been widely believed to be mainly responsible for the substrate stabilization against aggregation. Emerging evidence now indicates that other features of macromolecules such as their surface charges, probably resulting in electrostatic repulsions, and steric hindrance, could play a key role in the stabilization of their linked proteins against aggregation. Such stabilizing mechanisms are expected to give new insights into our understanding of the chaperoning functions for de novo protein folding. In this review, we will discuss the possible chaperoning roles of these macromolecules in de novo folding, based on their charge and steric features.

  6. 虹鳟 Ndufb2基因全长 cDNA 序列的克隆与分析%Cloning and sequence analysis of Ndufb2 full-length cDNA derived from Oncorhynchus mykiss

    Institute of Scientific and Technical Information of China (English)

    王家庆; 边佳; 李代宗; 马爽; 王亮; 那广宁

    2013-01-01

    Summary Rainbow trout belongs to Salmonidae aerobic fish,and it is necessary for high dissolved oxygen content of living water environment.If the dissolved oxygen content of living water is less than 5 mg/L,it will cause the increase of respiratory rate,which is the so-called“aquaculture floating head”phenomenon.Because the fish lives in hypoxia environment and the 90% oxygen consumption is in the mitochondria,the transmission mechanism in composition and electronic respiratory chain may be different from the terrestrial animal.At the mitochondrial inner membrane,electrons from NADH and succinate pass through the electron transport chain to oxygen,which is reduced to water.Complex I is one of the main sites at which premature electron leakage to oxygen occurs,thus being one of the main sites of production of harmful superoxide.The first isolation of mitochondrial complex I since 1 961,its composition and structure have had a primary understanding,but the specific mechanism of its participation in respiration,especially the function of each subunit is not clear.The protein encoded by Ndufb2 gene is a subunit of the multisubunit NADH:ubiquinone oxidoreductase(complex I). Mammalian complex I is composed of 45 different subunits.This protein has NADH dehydrogenase activity and oxidoreductase activity.It plays an important role in transferring electrons from NADH to the respiratory chain. Reverse transcription PCR(RT-PCR) and rapid amplification of cDNA ends(RACE)methods were used for the isolation of the whole cDNA of Ndufb2 gene from brain of Oncorhynchus mykiss .The assembly taskes of 3' and 5'-RACE sequence were completed by DNAman program.A pair of gene specific primers were designed to amplify the full-length cDNA sequence.ClustalX 1.81 and MEGA 3.0 software were used to calculate the amino acid sequence differences,and then the phylogenetic relationships of rainbow trout Ndufb2 gene sequence with other species were analyzed.Protein phosphorylation sites and

  7. Adventures in Holographic Dimer Models

    Energy Technology Data Exchange (ETDEWEB)

    Kachru, Shamit; /Stanford U., Phys. Dept. /SLAC; Karch, Andreas; /Washington U., Seattle; Yaida, Sho; /Stanford U., Phys. Dept.

    2011-08-12

    We abstract the essential features of holographic dimer models, and develop several new applications of these models. Firstly, semi-holographically coupling free band fermions to holographic dimers, we uncover novel phase transitions between conventional Fermi liquids and non-Fermi liquids, accompanied by a change in the structure of the Fermi surface. Secondly, we make dimer vibrations propagate through the whole crystal by way of double trace deformations, obtaining nontrivial band structure. In a simple toy model, the topology of the band structure experiences an interesting reorganization as we vary the strength of the double trace deformations. Finally, we develop tools that would allow one to build, in a bottom-up fashion, a holographic avatar of the Hubbard model.

  8. Heat shock protein 90: the cancer chaperone

    Indian Academy of Sciences (India)

    Len Neckers

    2007-04-01

    Heat shock protein 90 (Hsp90) is a molecular chaperone required for the stability and function of a number of conditionally activated and/or expressed signalling proteins, as well as multiple mutated, chimeric, and/or over-expressed signalling proteins, that promote cancer cell growth and/or survival. Hsp90 inhibitors are unique in that, although they are directed towards a specific molecular target, they simultaneously inhibit multiple cellular signalling pathways. By inhibiting nodal points in multiple overlapping survival pathways utilized by cancer cells, combination of an Hsp90 inhibitor with standard chemotherapeutic agents may dramatically increase the in vivo efficacy of the standard agent. Hsp90 inhibitors may circumvent the characteristic genetic plasticity that has allowed cancer cells to eventually evade the toxic effects of most molecularly targeted agents. The mechanism-based use of Hsp90 inhibitors, both alone and in combination with other drugs, should be effective toward multiple forms of cancer. Further, because Hsp90 inhibitors also induce Hsf-1-dependent expression of Hsp70, and because certain mutated Hsp90 client proteins are neurotoxic, these drugs display ameliorative properties in several neurodegenerative disease models, suggesting a novel role for Hsp90 inhibitors in treating multiple pathologies involving neurodegeneration.

  9. Analysis of 4,664 high-quality sequence-finished poplar full-length cDNA clones and their utility for the discovery of genes responding to insect feeding

    Directory of Open Access Journals (Sweden)

    Douglas Carl J

    2008-01-01

    Full Text Available Abstract Background The genus Populus includes poplars, aspens and cottonwoods, which will be collectively referred to as poplars hereafter unless otherwise specified. Poplars are the dominant tree species in many forest ecosystems in the Northern Hemisphere and are of substantial economic value in plantation forestry. Poplar has been established as a model system for genomics studies of growth, development, and adaptation of woody perennial plants including secondary xylem formation, dormancy, adaptation to local environments, and biotic interactions. Results As part of the poplar genome sequencing project and the development of genomic resources for poplar, we have generated a full-length (FL-cDNA collection using the biotinylated CAP trapper method. We constructed four FLcDNA libraries using RNA from xylem, phloem and cambium, and green shoot tips and leaves from the P. trichocarpa Nisqually-1 genotype, as well as insect-attacked leaves of the P. trichocarpa × P. deltoides hybrid. Following careful selection of candidate cDNA clones, we used a combined strategy of paired end reads and primer walking to generate a set of 4,664 high-accuracy, sequence-verified FLcDNAs, which clustered into 3,990 putative unique genes. Mapping FLcDNAs to the poplar genome sequence combined with BLAST comparisons to previously predicted protein coding sequences in the poplar genome identified 39 FLcDNAs that likely localize to gaps in the current genome sequence assembly. Another 173 FLcDNAs mapped to the genome sequence but were not included among the previously predicted genes in the poplar genome. Comparative sequence analysis against Arabidopsis thaliana and other species in the non-redundant database of GenBank revealed that 11.5% of the poplar FLcDNAs display no significant sequence similarity to other plant proteins. By mapping the poplar FLcDNAs against transcriptome data previously obtained with a 15.5 K cDNA microarray, we identified 153 FLcDNA clones

  10. Cloning and Bioimformatic Analysis of Full Length cDNA of LHY Gene from Cicer arietinum L.%鹰嘴豆 LHY 基因 cDNA 克隆及生物信息学分析

    Institute of Scientific and Technical Information of China (English)

    韩慧; 王希东; 姚正培; 夏木斯亚·卡坎

    2014-01-01

    从鹰嘴豆中克隆生物节律钟 LHY 基因的 cDNA 全长序列,进行序列信息学分析.通过同源克隆策略,利用 RT-PCR 技术获得核心片段,结合5′-RACE 和3′-RACE 技术,克隆得到鹰嘴豆生物节律钟基因 LHY 的 cD-NA 全长序列,其核苷酸序列长度为3061 bp,包括2220 bp 的完整开放阅读框(ORF),编码739个氨基酸.验证后命名为 CarLHY 基因,获得基因登录号为 KJ558378.生物信息学研究表明 CarLHY 基因 cDNA 序列与其他植物 LHY 基因具有较高的相似性;预测 CarLHY 蛋白不具有跨膜结构;为转录因子,定位于细胞核中;不具备信号肽.对 CarLHY 蛋白功能结构域预测表明,蛋白质核心结构存在符合转录因子与 DNA 结合的常见功能域 HTH.蛋白系统进化树显示,与大豆分子进化距离最近,其次是黑杨、拟南芥.%The sequence analysis was carried out to investigate the full-length cDNA sequence of biothythm clock gene LHY cloned from Cicer arietinum Linn with 5′-RACE and 3′-RACE.RT-PCR tech-nology was used to obtain core fragment with the homologous cloning strategy.The cDNA sequence was 3061 bp,and the open reading frame (ORF)was 2 220 bp,encoded 739 amino acids.It was named CarLHY after checked.The registration number was KJ558378 in Genebank datebase.The research of biological in-formation science indicated that cDNA sequence of CarLHY gene and other plant LHY gene have higher similarity.It was predicted that CarLHY protein have no transmembrane structure.It was speculated that CarLHY protein have no transmembrane spiral;It was a transcriptional factors and located in the nucleus. It has no signal peptide.CarLHY protein functional domains forecast indicated that there were transcription factor and HTH domain binded to DNA in protein core structure.The phylogenetic tree showed that CarL-HY and Glycine LHY are closest in molecular evolution distance,followed by Populus LHY and Arabii-dopsis thalana LHY gene.

  11. 中华鲟铁蛋白基因cDNA全长克隆与组织表达分析%Full-length cDNA Cloning and Tissues Expression Analysis of Ferritin Gene from Acipenser sinensis

    Institute of Scientific and Technical Information of China (English)

    陈晓武; 施志仪; 程千千

    2009-01-01

    根据铁蛋白基因的保守序列,搜索GenBank数据库中华鲟的EST数据库得到一条同源序列.通过RT-PCR的方法对该序列进行扩增,修改其测序错误,获得中华鲟铁蛋白亚基cDNA全长,经过注释提交GenBank数据库,获取序列登录号EU348782.该cDNA长度为896bp,包含531bp的完整编码区,推测编码的蛋白质为176aa,分子量为20339.9Mr,理论等电点为5.66.它和大两洋鲑鱼铁蛋白序列同源性最高,达到82.9%.该基闪在中华鲟肝脏、胰脏、肌肉、脑、心脏、鳃和胃粘膜等多种组织表达,在胰脏和心脏中表达量较高,在肌肉组织中表达较低.根据同源模建的方法得到该蛋白质三维结构,其包括5个α螺旋和10个转角结构,和人、蛙和细菌的铁蛋白均能很好的叠合,表现了很高的相似性,表明该蛋白结构和功能在基因进化中的高度保守性.%According to the conserved sequence of the ferritin gene, a homologous sequence was obtained from the EST database through a BLAST search against the GenBank database. This sequence was amplified with the method of RT-PCR, false sequencing was corrected, and full length cDNA of the ferritin subunit from the Chinese sturgeon was obtained. After being submitted to the GenBank database, the sequence accession number EU348782 was assigned. With the length of 896 bp, this cDNA includes entire coding regions of 531 bp, which encodes 176 amino acids (aa). The molecular weight was predicted to be 20339.9Mr and the theoretical isoelectrie point 5.66. It shares 82.9% protein sequence homology with the ferritin of the Atlantic salmon. This gene is expressed in many organs of the Chinese sturgeon, for example, the liver, pancreas, muscle, brain, heart and gastric mucosa. The highest expression level was found in the pancreas and the heart, while the muscular tissue showed the lowest. Homology modeling was used to predict the 3-D structure of the protein, which included 5 alpha helices and 10 turns

  12. Inhibitors of the AAA+ Chaperone p97

    Directory of Open Access Journals (Sweden)

    Eli Chapman

    2015-02-01

    Full Text Available It is remarkable that a pathway as ubiquitous as protein quality control can be targeted to treat cancer. Bortezomib, an inhibitor of the proteasome, was first approved by the US Food and Drug Administration (FDA more than 10 years ago to treat refractory myeloma and later extended to lymphoma. Its use has increased the survival rate of myeloma patients by as much as three years. This success was followed with the recent accelerated approval of the natural product derived proteasome inhibitor carfilzomib (Kyprolis®, which is used to treat patients with bortezomib-resistant multiple myeloma. The success of these two drugs has validated protein quality control as a viable target to fight select cancers, but begs the question why are proteasome inhibitors limited to lymphoma and myeloma? More recently, these limitations have encouraged the search for additional targets within the protein quality control system that might offer heightened cancer cell specificity, enhanced clinical utility, a lower rate of resistance, reduced toxicity, and mitigated side effects. One promising target is p97, an ATPase associated with various cellular activities (AAA+ chaperone. p97 figures prominently in protein quality control as well as serving a variety of other cellular functions associated with cancer. More than a decade ago, it was determined that up-regulation of p97 in many forms of cancer correlates with a poor clinical outcome. Since these initial discoveries, a mechanistic explanation for this observation has been partially illuminated, but details are lacking. Understandably, given this clinical correlation, myriad roles within the cell, and its importance in protein quality control, p97 has emerged as a potential therapeutic target. This review provides an overview of efforts towards the discovery of small molecule inhibitors of p97, offering a synopsis of efforts that parallel the excellent reviews that currently exist on p97 structure, function, and

  13. Single residue modification of only one dimer within the hemoglobin tetramer reveals autonomous dimer function

    Science.gov (United States)

    Ackers, Gary K.; Dalessio, Paula M.; Lew, George H.; Daugherty, Margaret A.; Holt, Jo M.

    2002-07-01

    The mechanism of cooperativity in the human hemoglobin tetramer (a dimer of dimers) has historically been modeled as a simple two-state system in which a low-affinity structural form (T) switches, on ligation, to a high-affinity form (R), yielding a net loss of hydrogen bonds and salt bridges in the dimer-dimer interface. Modifications that weaken these cross-dimer contacts destabilize the quaternary T tetramer, leading to decreased cooperativity and enhanced ligand affinity, as demonstrated in many studies on symmetric double modifications, i.e., a residue site modified in both - or both -subunits. In this work, hybrid tetramers have been prepared with only one modified residue, yielding molecules composed of a wild-type dimer and a modified dimer. It is observed that the cooperative free energy of ligation to the modified dimer is perturbed to the same extent whether in the hybrid tetramer or in the doubly modified tetramer. The cooperative free energy of ligation to the wild-type dimer is unperturbed, even in the hybrid tetramer, and despite the overall destabilization of the T tetramer by the modification. This asymmetric response by the two dimers within the same tetramer shows that loss of dimer-dimer contacts is not communicated across the dimer-dimer interface, but is transmitted through the dimer that bears the modified residue. These observations are interpreted in terms of a previously proposed dimer-based model of cooperativity with an additional quaternary (T/R) component.

  14. Full-length cDNA Cloning and Expression Analysis of Profilin Gene from Sea Cucumber (Apostichopus japonicus)%仿刺参profilin基因全长cDNA的克隆及表达分析

    Institute of Scientific and Technical Information of China (English)

    董颖; 高杉; 陈仲; 杨爱馥; 姜北; 关晓燕; 王摆; 周遵春∗

    2013-01-01

      仿刺参(Apostichopus japonicus)是我国北方沿海重要养殖品种之一.克隆了仿刺参profilin基因全长cDNA序列,并分析了基因的表达规律.该基因cDNA序列全长787 bp,5′-非翻译区(5′-untranslated region, UTR)长205 bp,3′-UTR长204 bp,开放阅读框378 bp,编码125个氨基酸,预测蛋白分子量13.4 kDa.仿刺参Profilin蛋白的PROF保守结构域中含有肌动蛋白互作位点、多聚脯氨酸结合位点和PIP2互作位点.氨基酸序列比对结果显示,仿刺参Profilin与丝盘虫(Trichoplax adhaerens)和囊舌虫(Saccoglossus kowalevskii)相似度最高,为46%. Quantitative real-time PCR结果显示,profilin mRNA在仿刺参未受精卵、受精卵、多细胞期、囊胚期、原肠期、小耳状幼体、中耳状幼体、大耳状幼体、樽型幼体、五触手幼体和稚参11个发育阶段及幼参的体壁、体腔细胞、肠道和呼吸树中均有表达;在仿刺参的不同发育阶段中,未受精卵至原肠期profilin mRNA表达量低,从小耳状幼体至稚参表达量增高;在仿刺参的不同组织中,profilin mRNA在体腔细胞中的表达量最高.经过棘皮动物免疫系统有效激活物脂多糖LPS的刺激,体腔细胞中profilin mRNA表达量显著升高,为仿刺参养殖病害的防控提供科学依据.%Sea cucumber (Apostichopus japonicus) was one of the important aquaculture species in the north coast of China. The studies on function and mechanism of immune-related genes can provide a reference to the prevention of diseases in the cultured A. japonicus. In this study,a full-length cDNA sequence of profilin gene from A. japonicus was obtained by constructing cDNA libraries and sequencing analysis, which was 787 bp and includes a 205 bp 5′-untranslated region (UTR), 378 bp ORF encoding 125 amino acids with a conserved PROF domain, and a 204 bp 3′-UTR;and the predicted molecular weight was 13. 4 kDa. The conserved PROF domain included acting interaction sites, poly

  15. FULL-LENGTH CDNA CLONING AND TISSUE EXPRESSION ANALYSIS OF ARGINASE GENE FROM HYRIOPSIS CUMINGII%三角帆蚌精氨酸酶基因的cDNA克隆与组织表达分析

    Institute of Scientific and Technical Information of China (English)

    刘巧林; 许宝红; 肖调义; 刘敏; 钟蕾; 苏建明

    2011-01-01

    Arginase (Arg) is a sign enzyme among organisms of urea cycle. It is not only related to many diseases in organisms, but also used to treat tumors and cancer as an important tool enzyme. According to the Hyriopsis cumingii (H. Cumingii) expressed sequence tags (EST) obtained by constructing subtractive hybridization cDNA library of H. Cumingii liver, the gene full-length cDNA sequence of arginase from H. Cumingii was cloned by RACE-PCR technique based on the designed gene-special primers. After analyzed by the software DNA Star and the bioinformatics technology, the results showed that the length of arginase gene cDNA sequence was 1720 bp, containing a complete open reading frame (65-1072) which was 1008 bp, encoding a peptide of 335 amino acid residues (aa), flanked by a 64 bp of 5' untranslated region (UTR) and a 648 bp of 3'-UTR. The deduced molecular weight of arginase was about 36.81 kD. At the transcriptional level, quantitative real-time PCR (qPCR) was used to detect the expression of the arginase gene in different tissues. The result revealed that H. Cumingii arginase gene could be expressed in seven kinds of tissue containing liver, stomach, intestine, gill, heart, mantle, axe foot collected from H. Cumingii, especially strongly expressed in digestive organs, such as liver, stomach and intestine, but weakly in heart and mantle. So it concluded that the arginase from the H. Cumingii which belongs to lower invertebrate could possess the same characteristics and functions of arginase type I and II from the higher animals. That means the arginase from the H. Cumingii may not onlyparticipates in urea cycle, but also plays an important role in the processes of physiology and pathology. And the deduction will be verified in the next experiment.%精氨酸酶(Arginase,Arg)是生物体尿素循环当中一种标志性的酶类,它不但与生物体许多疾病相关,而且是目前用于治疗肿瘤和癌症的一种重要的工具酶.根据三角帆蚌消

  16. Review: The HSP90 molecular chaperone-an enigmatic ATPase.

    Science.gov (United States)

    Pearl, Laurence H

    2016-08-01

    The HSP90 molecular chaperone is involved in the activation and cellular stabilization of a range of 'client' proteins, of which oncogenic protein kinases and nuclear steroid hormone receptors are of particular biomedical significance. Work over the last two decades has revealed a conformational cycle critical to the biological function of HSP90, coupled to an inherent ATPase activity that is regulated and manipulated by many of the co-chaperones proteins with which it collaborates. Pharmacological inhibition of HSP90 ATPase activity results in degradation of client proteins in vivo, and is a promising target for development of new cancer therapeutics. Despite this, the actual function that HSP90s conformationally-coupled ATPase activity provides in its biological role as a molecular chaperone remains obscure. © 2016 Wiley Periodicals, Inc. Biopolymers 105: 594-607, 2016. PMID:26991466

  17. Chaperone-assisted translocation of flexible polymers in three dimensions

    CERN Document Server

    Suhonen, P M

    2016-01-01

    Polymer translocation through a nanometer-scale pore assisted by chaperones binding to the polymer is a process encountered in vivo for proteins. Studying the relevant models by computer simulations is computationally demanding. Accordingly, previous studies are either for stiff polymers in three dimensions or flexible polymers in two dimensions. Here, we study chaperone-assisted translocation of flexible polymers in three dimensions using Langevin dynamics. We show that differences in binding mechanisms, more specifically, whether a chaperone can bind to a single or multiple sites on the polymer, lead to substantial differences in translocation dynamics in three dimensions. We show that the single-binding mode leads to dynamics that is very much like that in the constant-force driven translocation and accordingly mainly determined by tension propagation on the cis side. We obtain $\\beta \\approx 1.26$ for the exponent for the scaling of the translocation time with polymer length. This fairly low value can be ...

  18. Pathways of allosteric regulation in Hsp70 chaperones.

    Science.gov (United States)

    Kityk, Roman; Vogel, Markus; Schlecht, Rainer; Bukau, Bernd; Mayer, Matthias P

    2015-01-01

    Central to the protein folding activity of Hsp70 chaperones is their ability to interact with protein substrates in an ATP-controlled manner, which relies on allosteric regulation between their nucleotide-binding (NBD) and substrate-binding domains (SBD). Here we dissect this mechanism by analysing mutant variants of the Escherichia coli Hsp70 DnaK blocked at distinct steps of allosteric communication. We show that the SBD inhibits ATPase activity by interacting with the NBD through a highly conserved hydrogen bond network, and define the signal transduction pathway that allows bound substrates to trigger ATP hydrolysis. We identify variants deficient in only one direction of allosteric control and demonstrate that ATP-induced substrate release is more important for chaperone activity than substrate-stimulated ATP hydrolysis. These findings provide evidence of an unexpected dichotomic allostery mechanism in Hsp70 chaperones and provide the basis for a comprehensive mechanical model of allostery in Hsp70s. PMID:26383706

  19. Universal Stress Protein exhibits a redox-dependent chaperone function in Arabidopsis and enhances plant tolerance to heat shock and oxidative stress

    Directory of Open Access Journals (Sweden)

    Jung eYoung Jun

    2015-12-01

    Full Text Available Although a wide range of physiological information on Universal Stress Proteins (USPs is available from many organisms, their biochemical and molecular functions remain unidentified. The biochemical function of AtUSP (At3g53990 from Arabidopsis thaliana was therefore investigated. Plants over-expressing AtUSP showed a strong resistance to heat shock and oxidative stress, compared with wild-type and Atusp knock-out plants, confirming the crucial role of AtUSP in stress tolerance. AtUSP was present in a variety of structures including monomers, dimers, trimers, and oligomeric complexes, and switched in response to external stresses from low molecular weight (LMW species to high molecular weight (HMW complexes. AtUSP exhibited a strong chaperone function under stress conditions in particular, and this activity was significantly increased by heat treatment. Chaperone activity of AtUSP was critically regulated by the redox status of cells and accompanied by structural changes to the protein. Over-expression of AtUSP conferred a strong tolerance to heat shock and oxidative stress upon Arabidopsis, primarily via its chaperone function.

  20. Mechanism of FGF receptor dimerization and activation

    Science.gov (United States)

    Sarabipour, Sarvenaz; Hristova, Kalina

    2016-01-01

    Fibroblast growth factors (fgfs) are widely believed to activate their receptors by mediating receptor dimerization. Here we show, however, that the FGF receptors form dimers in the absence of ligand, and that these unliganded dimers are phosphorylated. We further show that ligand binding triggers structural changes in the FGFR dimers, which increase FGFR phosphorylation. The observed effects due to the ligands fgf1 and fgf2 are very different. The fgf2-bound dimer structure ensures the smallest separation between the transmembrane (TM) domains and the highest possible phosphorylation, a conclusion that is supported by a strong correlation between TM helix separation in the dimer and kinase phosphorylation. The pathogenic A391E mutation in FGFR3 TM domain emulates the action of fgf2, trapping the FGFR3 dimer in its most active state. This study establishes the existence of multiple active ligand-bound states, and uncovers a novel molecular mechanism through which FGFR-linked pathologies can arise.

  1. Fiber optic D dimer biosensor

    Science.gov (United States)

    Glass, Robert S.; Grant, Sheila A.

    1999-01-01

    A fiber optic sensor for D dimer (a fibrinolytic product) can be used in vivo (e.g., in catheter-based procedures) for the diagnosis and treatment of stroke-related conditions in humans. Stroke is the third leading cause of death in the United States. It has been estimated that strokes and stroke-related disorders cost Americans between $15-30 billion annually. Relatively recently, new medical procedures have been developed for the treatment of stroke. These endovascular procedures rely upon the use of microcatheters. These procedures could be facilitated with this sensor for D dimer integrated with a microcatheter for the diagnosis of clot type, and as an indicator of the effectiveness, or end-point of thrombolytic therapy.

  2. Dimer models and crepant resolutions

    OpenAIRE

    Ishii, Akira; Ueda, Kazushi

    2013-01-01

    We study variations of tautological bundles on moduli spaces of representations of quivers with relations associated with dimer models under a change of stability parameters. We prove that if the tautological bundle induces a derived equivalence for some stability parameter, then the same holds for any generic stability parameter, and any projective crepant resolution can be obtained as the moduli space for some stability parameter. This result is used in 0905.0059 to prove the abelian McKay ...

  3. Stability of the Human Hsp90-p50Cdc37 Chaperone Complex against Nucleotides and Hsp90 Inhibitors, and the Influence of Phosphorylation by Casein Kinase 2

    Directory of Open Access Journals (Sweden)

    Sanne H. Olesen

    2015-01-01

    Full Text Available The molecular chaperone Hsp90 is regulated by co-chaperones such as p50Cdc37, which recruits a wide selection of client protein kinases. Targeted disruption of the Hsp90-p50Cdc37 complex by protein–protein interaction (PPI inhibitors has emerged as an alternative strategy to treat diseases characterized by aberrant Hsp90 activity. Using isothermal microcalorimetry, ELISA and GST-pull down assays we evaluated reported Hsp90 inhibitors and nucleotides for their ability to inhibit formation of the human Hsp90β-p50Cdc37 complex, reconstituted in vitro from full-length proteins. Hsp90 inhibitors, including the proposed PPI inhibitors gedunin and H2-gamendazole, did not affect the interaction of Hsp90 with p50Cdc37 in vitro. Phosphorylation of Hsp90 and p50Cdc37 by casein kinase 2 (CK2 did not alter the thermodynamic signature of complex formation. However, the phosphorylated complex was vulnerable to disruption by ADP (IC50 = 32 µM, while ATP, AMPPNP and Hsp90 inhibitors remained largely ineffective. The differential inhibitory activity of ADP suggests that phosphorylation by CK2 primes the complex for dissociation in response to a drop in ATP/ADP levels. The approach applied herein provides robust assays for a comprehensive biochemical evaluation of potential effectors of the Hsp90-p50Cdc37 complex, such as phosphorylation by a kinase or the interaction with small molecule ligands.

  4. Chaperone-assisted translocation of flexible polymers in three dimensions

    Science.gov (United States)

    Suhonen, P. M.; Linna, R. P.

    2016-01-01

    Polymer translocation through a nanometer-scale pore assisted by chaperones binding to the polymer is a process encountered in vivo for proteins. Studying the relevant models by computer simulations is computationally demanding. Accordingly, previous studies are either for stiff polymers in three dimensions or flexible polymers in two dimensions. Here, we study chaperone-assisted translocation of flexible polymers in three dimensions using Langevin dynamics. We show that differences in binding mechanisms, more specifically, whether a chaperone can bind to a single site or multiple sites on the polymer, lead to substantial differences in translocation dynamics in three dimensions. We show that the single-binding mode leads to dynamics that is very much like that in the constant-force driven translocation and accordingly mainly determined by tension propagation on the cis side. We obtain β ≈1.26 for the exponent for the scaling of the translocation time with polymer length. This fairly low value can be explained by the additional friction due to binding particles. The multiple-site binding leads to translocation the dynamics of which is mainly determined by the trans side. For this process we obtain β ≈1.36 . This value can be explained by our derivation of β =4 /3 for constant-bias translocation, where translocated polymer segments form a globule on the trans side. Our results pave the way for understanding and utilizing chaperone-assisted translocation where variations in microscopic details lead to rich variations in the emerging dynamics.

  5. Hsp100/ClpB Chaperone Function and Mechanism

    Energy Technology Data Exchange (ETDEWEB)

    Vierling, Elizabeth [University of Massachusetts

    2015-01-27

    The supported research investigated the mechanism of action of a unique class of molecular chaperones in higher plants, the Hsp100/ClpB proteins, with the ultimate goal of defining how these chaperones influence plant growth, development, stress tolerance and productivity. Molecular chaperones are essential effectors of cellular “protein quality control”, which comprises processes that ensure the proper folding, localization, activation and turnover of proteins. Hsp100/ClpB proteins are required for temperature acclimation in plants, optimal seed yield, and proper chloroplast development. The model plant Arabidopsis thaliana and genetic and molecular approaches were used to investigate two of the three members of the Hsp100/ClpB proteins in plants, cytosolic AtHsp101 and chloroplast-localized AtClpB-p. Investigating the chaperone activity of the Hsp100/ClpB proteins addresses DOE goals in that this activity impacts how “plants generate and assemble components” as well as “allowing for their self repair”. Additionally, Hsp100/ClpB protein function in plants is directly required for optimal “utilization of biological energy” and is involved in “mechanisms that control the architecture of energy transduction systems”.

  6. Novel covalently linked insulin dimer engineered to investigate the function of insulin dimerization

    DEFF Research Database (Denmark)

    Vinther, Tine N.; Norrman, Mathias; Strauss, Holger M.;

    2012-01-01

    An ingenious system evolved to facilitate insulin binding to the insulin receptor as a monomer and at the same time ensure sufficient stability of insulin during storage. Insulin dimer is the cornerstone of this system. Insulin dimer is relatively weak, which ensures dissociation into monomers in...... oligomerization and insulin stability and function, we engineered a covalently linked insulin dimer in which two monomers were linked by a disulfide bond. The structure of this covalent dimer was identical to the self-association dimer of human insulin. Importantly, this covalent dimer was capable of further...

  7. Synthesis of steroidal dimers: Selective amine catalysed steroidal dimerization

    Indian Academy of Sciences (India)

    Shamsuzzaman; Mohd Gulfam Aalam; Tabassum Siddiqui

    2011-07-01

    Some new dimeric steroids namely cholest-5-en-3-spiro-[6',5'-oxa]-5'-cholest-3'-one (2), cholest-5-en-7-spiro-[4',5'-oxa]-5'-cholest-7'-one (4a) and 3-substitutedcholest-5-en-7-spiro-[4',5'-oxa]-3'-substituted-5'-cholestan-7'-ones (4b, c) are synthesized starting from cholest-5-en-3-one (1), cholest-5-en-7-one (3a) and 3-substituted-cholest-5-en-7-ones (3b, c) respectively by using DMAP and xylene. All the synthesized compounds were characterized by using IR, MS and 1H, 13C NMR spectral and elemental analysis.

  8. The Structure of the MAP2K MEK6 Reveals an Autoinhibitory Dimer

    Energy Technology Data Exchange (ETDEWEB)

    Min, Xiaoshan; Akella, Radha; He, Haixia; Humphreys, John M.; Tsutakawa, Susan E.; Lee, Seung-Jae; Tainer, John A.; Cobb, Melanie H.; Goldsmith, Elizabeth J.

    2009-07-13

    MAP2Ks are dual-specificity protein kinases functioning at the center of three-tiered MAP kinase modules. The structure of the kinase domain of the MAP2K MEK6 with phosphorylation site mimetic aspartic acid mutations (MEK6/{Delta}N/DD) has been solved at 2.3 {angstrom} resolution. The structure reveals an autoinhibited elongated ellipsoidal dimer. The enzyme adopts an inactive conformation, based upon structural queues, despite the phosphomimetic mutations. Gel filtration and small-angle X-ray scattering analysis confirm that the crystallographically observed ellipsoidal dimer is a feature of MEK6/{Delta}N/DD and full-length unphosphorylated wild-type MEK6 in solution. The interface includes the phosphate binding ribbon of each subunit, part of the activation loop, and a rare 'arginine stack' between symmetry-related arginine residues in the N-terminal lobe. The autoinhibited structure likely confers specificity on active MAP2Ks. The dimer may also serve the function in unphosphorylated MEK6 of preventing activation loop phosphorylation by inappropriate kinases.

  9. Cdc13 N-Terminal Dimerization DNA Binding and Telomere Length Regulation

    Energy Technology Data Exchange (ETDEWEB)

    M Mitchell; J Smith; M Mason; S Harper; D Speicher; F Johnson; E Skordalakes

    2011-12-31

    The essential yeast protein Cdc13 facilitates chromosome end replication by recruiting telomerase to telomeres, and together with its interacting partners Stn1 and Ten1, it protects chromosome ends from nucleolytic attack, thus contributing to genome integrity. Although Cdc13 has been studied extensively, the precise role of its N-terminal domain (Cdc13N) in telomere length regulation remains unclear. Here we present a structural, biochemical, and functional characterization of Cdc13N. The structure reveals that this domain comprises an oligonucleotide/oligosaccharide binding (OB) fold and is involved in Cdc13 dimerization. Biochemical data show that Cdc13N weakly binds long, single-stranded, telomeric DNA in a fashion that is directly dependent on domain oligomerization. When introduced into full-length Cdc13 in vivo, point mutations that prevented Cdc13N dimerization or DNA binding caused telomere shortening or lengthening, respectively. The multiple DNA binding domains and dimeric nature of Cdc13 offer unique insights into how it coordinates the recruitment and regulation of telomerase access to the telomeres.

  10. Dimerization of Human Growth Hormone by Zinc

    Science.gov (United States)

    Cunningham, Brian C.; Mulkerrin, Michael G.; Wells, James A.

    1991-08-01

    Size-exclusion chromatography and sedimentation equilibrium studies demonstrated that zinc ion (Zn2+) induced the dimerization of human growth hormone (hGH). Scatchard analysis of 65Zn2+ binding to hGH showed that two Zn2+ ions associate per dimer of hGH in a cooperative fashion. Cobalt (II) can substitute for Zn2+ in the hormone dimer and gives a visible spectrum characteristic of cobalt coordinated in a tetrahedral fashion by oxygen- and nitrogen-containing ligands. Replacement of potential Zn2+ ligands (His18, His21, and Glu174) in hGH with alanine weakened both Zn2+ binding and hGH dimer formation. The Zn2+-hGH dimer was more stable than monomeric hGH to denaturation in guanidine-HCl. Formation of a Zn2+-hGH dimeric complex may be important for storage of hGH in secretory granules.

  11. Functional Significance of Serotonin Receptor Dimerization

    Science.gov (United States)

    Herrick-Davis, Katharine

    2013-01-01

    The original model of G protein activation by a single G-protein-coupled receptor (GPCR) is giving way to a new model wherein two protomers of a GPCR dimer interact with a single G protein. This article will review the evidence suggesting that 5-HT receptors form dimers/oligomers and will compare the findings with results obtained from studies with other biogenic amine receptors. Topics to be covered include the origin or biogenesis of dimer formation, potential dimer interface(s), and oligomer size (dimer versus tetramer or higher order). The functional significance will be discussed in terms of G-protein activation following ligand binding to one or two protomers in a dimeric structure, the formation of heterodimers and the development of bivalent ligands. PMID:23811735

  12. Does Dimeric Melittin Occur in Aqueous Solutions?

    OpenAIRE

    Schubert, D; Pappert, G.; Boss, K.

    1985-01-01

    Melittin, a peptide from bee venom, is known to undergo a monomer / tetramer conversion in aqueous solutions. We have studied the possible participation of dimers in the association equilibrium of melittin by sedimentation equilibrium experiments in the analytical ultracentrifuge and subsequent mathematical analysis of the concentration distributions obtained. It was found that the dimeric state is not significantly populated, the contribution of dimer to the total peptide weight probably bei...

  13. Electronic spectroscopy of transition metal dimer

    OpenAIRE

    Qian, Yue; 钱玥

    2013-01-01

    This thesis reports laser spectroscopic studies of gas-phase transition metal dimers using laser ablation/reaction with free jet expansion and laser-induced fluorescence (LIF) spectroscopy technique. Themolecules studied in this work are palladium dimer (Pd2) and vanadium dimer (V2). Many compounds formed from these transition metals are important and functional catalysts in chemical reactions. Therefore, it is of great significance to start from the fundamental level to understand the prope...

  14. Dimerization of norbornene on zeolite catalysts

    Institute of Scientific and Technical Information of China (English)

    N. G. Grigor’eva; S. V. Bubennov; L. M. Khalilov; B. I. Kutepov

    2015-01-01

    The high activity and selectivity of H‐Beta and H‐ZSM‐12 zeolites in the dimerization of norbornene was established. The norbornene conversion reached 100%in chlorinated paraffin and argon gas medium, with a selectivity of dimer formation of 88%–98%. Four stereo‐isomers of the bis‐2,2’‐norbornylidene structure were identified in the dimer fraction, with the (Z)‐anti‐bis‐2,2’‐norbornylidene prevailing over the others.

  15. Monomer-dimer problem on some networks

    Science.gov (United States)

    Wu, Ruijuan; Yan, Weigen

    2016-09-01

    Zhang et al. (2012) obtained the exact formula for the number of all possible monomer-dimer arrangements and the asymptotic growth constant on a scale-free small-world network. In this note, we generalize this result and obtain the exact solution on the monomer-dimer model on many networks. Particularly, we prove that these networks have the same asymptotic growth constant of the number of monomer-dimer arrangements.

  16. C-terminal truncation of a bovine B12 trafficking chaperone enhances the sensitivity of the glutathione-regulated thermostability

    Directory of Open Access Journals (Sweden)

    Jinju Jeong

    2013-03-01

    Full Text Available The human B12 trafficking chaperone hCblC is well conserved inmammals and non-mammalian eukaryotes. However, the C-terminal∼40 amino acids of hCblC vary significantly and arepredicted to be deleted by alternative splicing of the encodinggene. In this study, we examined the thermostability of the bovineCblC truncated at the C-terminal variable region (t-bCblC and itsregulation by glutathione. t-bCblC is highly thermolabile (Tm =∼42oC similar to the full-length protein (f-bCblC. However,t-bCblC is stabilized to a greater extent than f-bCblC by binding ofreduced glutathione (GSH with increased sensitivity to GSH. Inaddition, binding of oxidized glutathione (GSSG destabilizest-bCblC to a greater extent and with increased sensitivity ascompared to f-bCblC. These results indicate that t-bCblC is a moresensitive form to be regulated by glutathione than the full-lengthform of the protein. [BMB Reports 2013; 46(3: 169-174

  17. Enhanced Chiral Recognition by Cyclodextrin Dimers

    Directory of Open Access Journals (Sweden)

    Bart Jan Ravoo

    2011-07-01

    Full Text Available In this article we investigate the effect of multivalency in chiral recognition. To this end, we measured the host-guest interaction of a β-cyclodextrin dimer with divalent chiral guests. We report the synthesis of carbohydrate-based water soluble chiral guests functionalized with two borneol, menthol, or isopinocampheol units in either (+ or (– configuration. We determined the interaction of these divalent guests with a β-cyclodextrin dimer using isothermal titration calorimetry. It was found that—in spite of a highly unfavorable conformation—the cyclodextrin dimer binds to guest dimers with an increased enantioselectivity, which clearly reflects the effect of multivalency.

  18. Sputtering of dimers off a silicon surface

    Science.gov (United States)

    Nietiadi, Maureen L.; Rosandi, Yudi; Kopnarski, Michael; Urbassek, Herbert M.

    2012-10-01

    We present experimental and molecular-dynamics simulation results of the sputtering of a Si surface by 2 keV Ar ions. Results on both the monomer and dimer distributions are presented. In simulation, these distributions follow a generalized Thompson law with power exponent n=2 and n=3, respectively. The experimental data, obtained via plasma post-ionization in an SNMS (secondary neutral mass spectrometry) apparatus, show good agreement with respect to the dimer fraction, and the relative energy distributions of dimers and monomers. The consequences for the dimer sputtering mechanism are discussed.

  19. Sputtering of dimers off a silicon surface

    Energy Technology Data Exchange (ETDEWEB)

    Nietiadi, Maureen L. [Physics Department, University Kaiserslautern, Erwin-Schroedinger-Strasse, D-67663 Kaiserslautern (Germany); Research Center OPTIMAS, University Kaiserslautern, Erwin-Schroedinger-Strasse, D-67663 Kaiserslautern (Germany); Rosandi, Yudi [Physics Department, University Kaiserslautern, Erwin-Schroedinger-Strasse, D-67663 Kaiserslautern (Germany); Research Center OPTIMAS, University Kaiserslautern, Erwin-Schroedinger-Strasse, D-67663 Kaiserslautern (Germany); Department of Physics, Universitas Padjadjaran, Jatinangor, Sumedang 45363 (Indonesia); Kopnarski, Michael [Physics Department, University Kaiserslautern, Erwin-Schroedinger-Strasse, D-67663 Kaiserslautern (Germany); Research Center OPTIMAS, University Kaiserslautern, Erwin-Schroedinger-Strasse, D-67663 Kaiserslautern (Germany); Institut fuer Oberflaechen- und Schichtanalytik IFOS GmbH, Trippstadter Strasse 120, D-67663 Kaiserslautern (Germany); Urbassek, Herbert M., E-mail: urbassek@rhrk.uni-kl.de [Physics Department, University Kaiserslautern, Erwin-Schroedinger-Strasse, D-67663 Kaiserslautern (Germany); Research Center OPTIMAS, University Kaiserslautern, Erwin-Schroedinger-Strasse, D-67663 Kaiserslautern (Germany)

    2012-10-15

    We present experimental and molecular-dynamics simulation results of the sputtering of a Si surface by 2 keV Ar ions. Results on both the monomer and dimer distributions are presented. In simulation, these distributions follow a generalized Thompson law with power exponent n=2 and n=3, respectively. The experimental data, obtained via plasma post-ionization in an SNMS (secondary neutral mass spectrometry) apparatus, show good agreement with respect to the dimer fraction, and the relative energy distributions of dimers and monomers. The consequences for the dimer sputtering mechanism are discussed.

  20. The chaperone like function of the nonhistone protein HMGB1

    Energy Technology Data Exchange (ETDEWEB)

    Osmanov, Taner; Ugrinova, Iva [Institute of Molecular Biology, Bulgarian Academy of Sciences (Bulgaria); Pasheva, Evdokia, E-mail: eva@bio21.bas.bg [Institute of Molecular Biology, Bulgarian Academy of Sciences (Bulgaria)

    2013-03-08

    Highlights: ► The HMGB1 protein strongly enhanced the formation of nucleosome particles. ► The target of HMGB1 action as a chaperone is the DNA not the histone octamer. ► The acetylation of HMGB1 decreases the stimulating effect of the protein. -- Abstract: Almost all essential nuclear processes as replication, repair, transcription and recombination require the chromatin template to be correctly unwound and than repackaged. The major strategy that the cell uses to overcome the nucleosome barrier is the proper removal of the histone octamer and subsequent deposition onto DNA. Important factors in this multi step phenomenon are the histone chaperones that can assemble nucleosome arrays in vitro in the absence of ATP. The nonhistone protein HMGB1 is a good candidate for a chaperone as its molecule consists of two DNA binding motives, Box’s A and B, and a long nonstructured C tail highly negatively charged. HMGB1 protein is known as a nuclear “architectural” factor for its property to bind preferentially to distorted DNA structures and was reported to kink the double helix. Our experiments show that in the classical stepwise dialysis method for nucleosome assembly the addition of HMGB1 protein stimulates more than two times the formation of middle-positioned nucleosomes. The stimulation effect persists in dialysis free experiment when the reconstitution is possible only in the presence of a chaperone. The addition of HMGB1 protein strongly enhanced the formation of a nucleosome in a dose dependant manner. Our results show that the target of HMGB1 action as a chaperone is the DNA fragment not the histone octamer. One possible explanation for the stimulating effect of HMGB1 is the “architectural” property of the protein to associate with the middle of the DNA fragment and to kink it. The acquired V shaped DNA structure is probably conformationals more favorable to wrap around the prefolded histone octamer. We tested also the role of the post

  1. Chaperone use during intimate examinations in primary care: postal survey of family physicians

    Directory of Open Access Journals (Sweden)

    Upshur Ross EG

    2005-12-01

    Full Text Available Abstract Background Physicians have long been advised to have a third party present during certain parts of a physical examination; however, little is known about the frequency of chaperone use for those specific intimate examinations regularly performed in primary care. We aimed to determine the frequency of chaperone use among family physicians across a variety of intimate physical examinations for both male and female patients, and also to identify the factors associated with chaperone use. Methods Questionnaires were mailed to a randomly selected sample of 500 Ontario members of the College of Family Physicians of Canada. Participants were asked about their use of chaperones when performing a variety of intimate examinations, namely female pelvic, breast, and rectal exams and male genital and rectal exams. Results 276 of 500 were returned (56%, of which 257 were useable. Chaperones were more commonly used with female patients than with males (t = 9.09 [df = 249], p Conclusion Clinical practice concerning the use of chaperones during intimate exams continues to be discordant with the recommendations of medical associations and medico-legal societies. Chaperones are used by only a minority of Ontario family physicians. Chaperone use is higher for examinations of female patients than of male patients and is highest for female pelvic exams. The availability of a nurse in the clinic to act as a chaperone is associated with more frequent use of chaperones.

  2. Metal chaperones: a holistic approach to the treatment of AD

    Directory of Open Access Journals (Sweden)

    PaulAnthonyAdlard

    2012-03-01

    Full Text Available As the burden of proof for the role of metal ion dysregulation in the pathogenesis of multiple CNS disorders grows, it has become important to more precisely identify and differentiate the biological effects of various pharmacological modulators of metal ion homeostasis. This is particularly evident in disorders such as Alzheimer’s disease, where the use of metal chaperones (that transport metals, as opposed to chelators (which exclude metals from biological interactions, may prove to be the first truly disease modifying approach for this condition. The purpose of this mini-review is to highlight the emerging notion that metal chaperones, such as PBT2 (Prana Biotechnology, modulate a variety of critical pathways affecting key aspects of the AD cascade to provide a more “holistic” approach to the treatment of this disease.

  3. Dimerized Mott insulators in hexagonal optical lattices

    International Nuclear Information System (INIS)

    We study bosonic atoms in optical honeycomb lattices with anisotropic tunneling and find dimerized Mott insulator (MI) phases with fractional filling. These incompressible insulating phases are characterized by an interaction-driven localization of particles in respect to the individual dimers and large local particle-number fluctuations within the dimers. We calculate the ground-state phase diagrams and the excitation spectra using an accurate cluster mean-field method. The cluster treatment enables us to probe the fundamental excitations of the dimerized MI where the excitation gap is dominated by the intra-dimer tunneling amplitude. This allows the distinction from normal Mott insulating phases gapped by the on-site interaction. In addition, we present analytical results for the phase diagram derived by a higher-order strong-coupling perturbative expansion approach. By computing finite lattices with large diameters the influence of a harmonic confinement is discussed in detail. It is shown that a large fraction of atoms forms the dimerized MI under experimental conditions. The necessary anisotropic tunneling can be realized either by periodic driving of the optical lattice or by engineering directly a dimerized lattice potential. The dimers can be mapped to their antisymmetric states creating a lattice with coupled p-orbitals. (paper)

  4. Mahler Measure, Eisenstein Series and Dimers

    NARCIS (Netherlands)

    Stienstra, J.

    2007-01-01

    This note reveals a mysterious link between the partition function of certain dimer models on 2-dimensional tori and the L-function of their spectral curves. It also relates the partition function in certain families of dimer models to Eisenstein series. http://www.arxiv.org/abs/math.NT/0502197

  5. Enzymatic oxidative dimerization of silymarin flavonolignans

    Czech Academy of Sciences Publication Activity Database

    Gavezzotti, P.; Vavříková, Eva; Valentová, Kateřina; Fronza, G.; Kudanga, T.; Kuzma, Marek; Riva, S.; Biedermann, David; Křen, Vladimír

    2014-01-01

    Roč. 109, NOV 2014 (2014), s. 24-30. ISSN 1381-1177 R&D Projects: GA MŠk(CZ) LD13041; GA ČR(CZ) GP14-14373P Institutional support: RVO:61388971 Keywords : Silybin dimers * Silydianin dimer * Silymarin Subject RIV: CE - Biochemistry Impact factor: 2.128, year: 2014

  6. Chaperone-assisted translocation of flexible polymers in three dimensions.

    Science.gov (United States)

    Suhonen, P M; Linna, R P

    2016-01-01

    Polymer translocation through a nanometer-scale pore assisted by chaperones binding to the polymer is a process encountered in vivo for proteins. Studying the relevant models by computer simulations is computationally demanding. Accordingly, previous studies are either for stiff polymers in three dimensions or flexible polymers in two dimensions. Here, we study chaperone-assisted translocation of flexible polymers in three dimensions using Langevin dynamics. We show that differences in binding mechanisms, more specifically, whether a chaperone can bind to a single site or multiple sites on the polymer, lead to substantial differences in translocation dynamics in three dimensions. We show that the single-binding mode leads to dynamics that is very much like that in the constant-force driven translocation and accordingly mainly determined by tension propagation on the cis side. We obtain β≈1.26 for the exponent for the scaling of the translocation time with polymer length. This fairly low value can be explained by the additional friction due to binding particles. The multiple-site binding leads to translocation the dynamics of which is mainly determined by the trans side. For this process we obtain β≈1.36. This value can be explained by our derivation of β=4/3 for constant-bias translocation, where translocated polymer segments form a globule on the trans side. Our results pave the way for understanding and utilizing chaperone-assisted translocation where variations in microscopic details lead to rich variations in the emerging dynamics. PMID:26871100

  7. On the facultative requirement of the bacterial RNA chaperone, Hfq.

    OpenAIRE

    Jousselin, Ambre; Metzinger, Laurent; Felden, Brice

    2009-01-01

    The pleiotropic post-transcriptional regulator Hfq is an RNA chaperone that facilitates pairing interactions between small regulatory RNAs (sRNAs) and their mRNA targets in several bacteria. However, this classical pattern, derived from the Escherichia coli model, is not applicable to the whole bacterial kingdom. In this article we discuss the facultative requirement for Hfq for sRNA-mRNA duplex formation among bacteria and the specific features of the Hfq protein and RNA duplexes that might ...

  8. Chaperone-mediated assembly of centromeric chromatin in vitro

    OpenAIRE

    Furuyama, Takehito; Dalal, Yamini; Henikoff, Steven

    2006-01-01

    Every eukaryotic chromosome requires a centromere for attachment to spindle microtubules for chromosome segregation. Although centromeric DNA sequences vary greatly among species, centromeres are universally marked by the presence of a centromeric histone variant, centromeric histone 3 (CenH3), which replaces canonical histone H3 in centromeric nucleosomes. Conventional chromatin is maintained in part by histone chaperone complexes, which deposit the S phase-limited (H3) and constitutive (H3....

  9. Pathways of allosteric regulation in Hsp70 chaperones

    OpenAIRE

    Kityk, Roman; Vogel, Markus; Schlecht, Rainer; Bukau, Bernd; Mayer, Matthias P

    2015-01-01

    Central to the protein folding activity of Hsp70 chaperones is their ability to interact with protein substrates in an ATP-controlled manner, which relies on allosteric regulation between their nucleotide-binding (NBD) and substrate-binding domains (SBD). Here we dissect this mechanism by analysing mutant variants of the Escherichia coli Hsp70 DnaK blocked at distinct steps of allosteric communication. We show that the SBD inhibits ATPase activity by interacting with the NBD through a highly ...

  10. Crystal Structures of Cisplatin Bound to a Human Copper Chaperone

    Energy Technology Data Exchange (ETDEWEB)

    Boal, Amie K.; Rosenzweig, Amy C.; (NWU)

    2010-08-16

    Copper trafficking proteins, including the chaperone Atox1 and the P{sub 1B}-type ATPase ATP7B, have been implicated in cellular resistance to the anticancer drug cisplatin. We have determined two crystal structures of cisplatin-Atox1 adducts that reveal platinum coordination by the conserved CXXC copper-binding motif. Direct interaction of cisplatin with this functionally relevant site has significant implications for understanding the molecular basis for resistance mediated by copper transport pathways.

  11. Oncogenic TPM3-ALK activation requires dimerization through the coiled-coil structure of TPM3

    Energy Technology Data Exchange (ETDEWEB)

    Amano, Yosuke; Ishikawa, Rie; Sakatani, Toshio [Department of Respiratory Medicine, The University of Tokyo Hospital, 7-3-1 Hongo, Bunkyo-ku, Tokyo 113-8655 (Japan); Ichinose, Junji [Department of Cardiothoracic Surgery, The University of Tokyo Hospital, 7-3-1 Hongo, Bunkyo-ku, Tokyo 113-8655 (Japan); Sunohara, Mitsuhiro; Watanabe, Kousuke; Kage, Hidenori [Department of Respiratory Medicine, The University of Tokyo Hospital, 7-3-1 Hongo, Bunkyo-ku, Tokyo 113-8655 (Japan); Nakajima, Jun [Department of Cardiothoracic Surgery, The University of Tokyo Hospital, 7-3-1 Hongo, Bunkyo-ku, Tokyo 113-8655 (Japan); Nagase, Takahide; Ohishi, Nobuya [Department of Respiratory Medicine, The University of Tokyo Hospital, 7-3-1 Hongo, Bunkyo-ku, Tokyo 113-8655 (Japan); Takai, Daiya, E-mail: dtakai-ind@umin.ac.jp [Department of Respiratory Medicine, The University of Tokyo Hospital, 7-3-1 Hongo, Bunkyo-ku, Tokyo 113-8655 (Japan); Department of Clinical Laboratory, The University of Tokyo Hospital, 7-3-1 Hongo, Bunkyo-ku, Tokyo 113-8655 (Japan)

    2015-02-13

    Inflammatory myofibroblastic tumor (IMT) is a mesenchymal tumor that can arise from anywhere in the body. Anaplastic lymphoma kinase (ALK) gene rearrangements, most often resulting in the tropomyosin 3 (TPM3)-ALK fusion gene, are the main causes of IMT. However, the mechanism of malignant transformation in IMT has yet to be elucidated. The purpose of this study was to clarify the role of the TPM3 region in the transformation of IMT via TPM3-ALK. Lentivirus vectors containing a TPM3-ALK fusion gene lacking various lengths of TPM3 were constructed and expressed in HEK293T and NIH3T3 cell lines. Focus formation assay revealed loss of contact inhibition in NIH3T3 cells transfected with full-length TPM3-ALK, but not with ALK alone. Blue-native polyacrylamide gel electrophoresis (BN-PAGE) revealed that TPM3-ALK dimerization increased in proportion to the length of TPM3. Western blot showed phosphorylation of ALK, ERK1/2, and STAT3 in HEK293T cells transfected with TPM3-ALK. Thus, the coiled-coil structure of TPM3 contributes to the transforming ability of the TPM3-ALK fusion protein, and longer TPM3 region leads to higher dimer formation. - Highlights: • TPM3-ALK fusion protein dimerizes through the coiled-coil structure of TPM3. • Longer coiled-coil structure of TPM3 leads to higher TPM3-ALK dimer formation. • Presence of TPM3-ALK dimer leads to ALK, STAT3, and ERK1/2 phosphorylation. • Presence of TPM3-ALK leads to loss of contact inhibition. • BN-PAGE is a simple technique for visualizing oncogenic dimerization.

  12. Oncogenic TPM3-ALK activation requires dimerization through the coiled-coil structure of TPM3

    International Nuclear Information System (INIS)

    Inflammatory myofibroblastic tumor (IMT) is a mesenchymal tumor that can arise from anywhere in the body. Anaplastic lymphoma kinase (ALK) gene rearrangements, most often resulting in the tropomyosin 3 (TPM3)-ALK fusion gene, are the main causes of IMT. However, the mechanism of malignant transformation in IMT has yet to be elucidated. The purpose of this study was to clarify the role of the TPM3 region in the transformation of IMT via TPM3-ALK. Lentivirus vectors containing a TPM3-ALK fusion gene lacking various lengths of TPM3 were constructed and expressed in HEK293T and NIH3T3 cell lines. Focus formation assay revealed loss of contact inhibition in NIH3T3 cells transfected with full-length TPM3-ALK, but not with ALK alone. Blue-native polyacrylamide gel electrophoresis (BN-PAGE) revealed that TPM3-ALK dimerization increased in proportion to the length of TPM3. Western blot showed phosphorylation of ALK, ERK1/2, and STAT3 in HEK293T cells transfected with TPM3-ALK. Thus, the coiled-coil structure of TPM3 contributes to the transforming ability of the TPM3-ALK fusion protein, and longer TPM3 region leads to higher dimer formation. - Highlights: • TPM3-ALK fusion protein dimerizes through the coiled-coil structure of TPM3. • Longer coiled-coil structure of TPM3 leads to higher TPM3-ALK dimer formation. • Presence of TPM3-ALK dimer leads to ALK, STAT3, and ERK1/2 phosphorylation. • Presence of TPM3-ALK leads to loss of contact inhibition. • BN-PAGE is a simple technique for visualizing oncogenic dimerization

  13. Stochastic Flips on Dimer Tilings

    CERN Document Server

    Fernique, Thomas

    2011-01-01

    This paper introduces a Markov process inspired by the problem of quasicrystal growth. It acts over dimer tilings of the triangular grid by randomly performing local transformations, called {\\em flips}, which do not increase the number of identical adjacent tiles (this number can be thought as the tiling energy). Fixed-points of such a process play the role of quasicrystals. We are here interested in the worst-case expected number of flips to converge towards a fixed-point. Numerical experiments suggest a bound quadratic in the number n of tiles of the tiling. We prove a O(n^2.5) upper bound and discuss the gap between this bound and the previous one. We also briefly discuss the average-case.

  14. The New Scene on Studying the Literary Form of Contemporary Novel:Comment on Studying the Literary Form of Full-length Novel in New Century%当代小说研究的新视境--评《新世纪长篇小说文体研究》

    Institute of Scientific and Technical Information of China (English)

    李仲凡; 李晓珺

    2014-01-01

    《新世纪长篇小说文体研究》将宏观理论与微观小说文本有机结合,考证梳理了文体概念的渊源、演变过程与整体特征,并在此基础上对叙述、结构和话语这三大文体的基本范畴进行论述。这是对新世纪长篇小说文体首度进行整体性、系统性研究,可被当作这一时期中国长篇小说的“百科全书”来读。%Studying the Literary Form of Full-length Novel in New Century has done an overall and system-atic research on the literary form of full-length novel in new century. Combining macro theory with microcosmic text of fiction, this book reflects a profound and peculiar analysis about theory as well as text. The writer amply combs the origin of literary form’s concept, evolution process and general characteristic, and at the same time systematically discusses basic category which includes narration, structure and utterance. Furthermore, as an in-fluential work of researching on the literary form of full-length novel in new century, it can be largely regarded as an encyclopedia of Chinese full-length novel in the same period.

  15. Dimerization of VirD2 binding protein is essential for Agrobacterium induced tumor formation in plants.

    Directory of Open Access Journals (Sweden)

    Abhilash Padavannil

    2014-03-01

    Full Text Available The Type IV Secretion System (T4SS is the only bacterial secretion system known to translocate both DNA and protein substrates. The VirB/D4 system from Agrobacterium tumefaciens is a typical T4SS. It facilitates the bacteria to translocate the VirD2-T-DNA complex to the host cell cytoplasm. In addition to protein-DNA complexes, the VirB/D4 system is also involved in the translocation of several effector proteins, including VirE2, VirE3 and VirF into the host cell cytoplasm. These effector proteins aid in the proper integration of the translocated DNA into the host genome. The VirD2-binding protein (VBP is a key cytoplasmic protein that recruits the VirD2-T-DNA complex to the VirD4-coupling protein (VirD4 CP of the VirB/D4 T4SS apparatus. Here, we report the crystal structure and associated functional studies of the C-terminal domain of VBP. This domain mainly consists of α-helices, and the two monomers of the asymmetric unit form a tight dimer. The structural analysis of this domain confirms the presence of a HEPN (higher eukaryotes and prokaryotes nucleotide-binding fold. Biophysical studies show that VBP is a dimer in solution and that the HEPN domain is the dimerization domain. Based on structural and mutagenesis analyses, we show that substitution of key residues at the interface disrupts the dimerization of both the HEPN domain and full-length VBP. In addition, pull-down analyses show that only dimeric VBP can interact with VirD2 and VirD4 CP. Finally, we show that only Agrobacterium harboring dimeric full-length VBP can induce tumors in plants. This study sheds light on the structural basis of the substrate recruiting function of VBP in the T4SS pathway of A. tumefaciens and in other pathogenic bacteria employing similar systems.

  16. Zinc-L-carnosine binds to molecular chaperone HSP70 and inhibits the chaperone activity of the protein.

    Science.gov (United States)

    Haga, Asami; Okamoto, Tomoya; Yamada, Shintaroh; Kubota, Toshihiko; Sanpei, Ann; Takahashi, Shota; Nakayama, Masahiro; Nagai, Miki; Otaka, Michiro; Miyazaki, Toshio; Nunomura, Wataru; Grave, Ewa; Itoh, Hideaki

    2013-09-01

    In this study, we have investigated the specific binding proteins of Zinc-L-carnosine (Polaprezinc) using Polaprezinc-affinity column chromatography in vitro. A protein having a 70-kDa molecular mass was eluted by the linear gradient of 0-1.0 mM Polaprezinc from the affinity column and the protein was identified as the molecular chaperone HSP70 by immunoblotting. The chaperone activity of HSP70 was completely suppressed by Polaprezinc. The ATPase activity of HSP70 was affected to some extent by the reagent. In the circular dichroism (CD) spectrum, the secondary structure of HSP70 was changed in the presence of Polaprezinc, i.e. it decreased in the α-helix. We have determined the Polaprezinc-binding domain of HSP70 by using recombinant HSP70N- and C-domains. Although Polaprezinc could bind to both the N-terminal and the C-terminal of HSP70, the HSP70N-domain has a high affinity to the drug. Regarding the peptide cleavage of the HSP70N- and C-domains with proteinase K, the intact HSP70N still remained in the presence of Polaprezinc. On the other hand, the quantity of the intact C-domain slightly decreased under the same conditions along with the newly digested small peptides appeared. It has been suggested that Polaprezinc binds to HSP70 especially in the N-domains, suppresses the chaperone activity and delays an ATPase activities of HSP70. PMID:23687308

  17. Conformational Selection Underlies Recognition of a Molybdoenzyme by Its Dedicated Chaperone

    OpenAIRE

    Lorenzi, Magali; Sylvi, Léa; Gerbaud, Guillaume; Mileo, Elisabetta; Halgand, Frédéric; Walburger, Anne; Vezin, Hervé; Belle, Valérie; Guigliarelli, Bruno; Magalon, Axel

    2012-01-01

    Molecular recognition is central to all biological processes. Understanding the key role played by dedicated chaperones in metalloprotein folding and assembly requires the knowledge of their conformational ensembles. In this study, the NarJ chaperone dedicated to the assembly of the membrane-bound respiratory nitrate reductase complex NarGHI, a molybdenum-iron containing metalloprotein, was taken as a model of dedicated chaperone. The combination of two techniques ie site-directed spin labeli...

  18. Cooperation of multiple chaperones required for the assembly of mammalian 20S proteasomes

    DEFF Research Database (Denmark)

    Hirano, Y.; Hayashi, H.; Iemura, S.; Hendil, K. B.; Niwa, S.; Kishimoto, T.; Natsume, T.; Tanaka, K.; Murata, S.

    2006-01-01

    proteasomes is assisted by proteasome-specific chaperones, named PAC1, PAC2, and hUmp1, but the details are still unknown. Here, we report the identification of a chaperone, designated PAC3, as a component of a rings. Although it can intrinsically bind directly to both a and ß subunits, PAC3 dissociates...... describe a cooperative system of multiple chaperones involved in the correct assembly of mammalian 20S proteasomes....

  19. Full-length genomes of 16 hepatitis C virus genotype 1 isolates representing subtypes 1c, 1d, 1e, 1g, 1h, 1i, 1j and 1k, and two new subtypes 1m and 1n, and four unclassified variants reveal ancestral relationships among subtypes.

    Science.gov (United States)

    Lu, Ling; Li, Chunhua; Xu, Yan; Murphy, Donald G

    2014-07-01

    We characterized the full-length genomes of 16 distinct hepatitis C virus genotype 1 (HCV-1) isolates. Among them, four represented the first full-length genomes for subtypes 1d (QC103), 1i (QC181), 1j (QC329) and 1k (QC82), and another four corresponded to subtypes 1c (QC165), 1g (QC78), 1h (QC156) and 1e (QC172). Both QC196 and QC87 were assigned into a new subtype 1m, and QC113 and QC74 into another new subtype 1n. The remaining four (QC60, QC316, QC152 and QC180) did not classify among the established subtypes and corresponded to four new lineages. Subtypes 1j, 1k, 1m, 1n and the unclassified isolate QC60 were identified in Haitian immigrants. In the updated HCV nomenclature of 2005, a total of 12 subtypes of HCV-1 were designated. Including the data from the present study, all but subtype 1f now have their full-length genomes defined. Further analysis of partial NS5B sequences available in GenBank denoted a total of 21 unclassified lineages, indicating the taxonomic complexity of HCV-1. Among them, six have had their full-length genomes characterized. Based on the available full-length genome sequences, a timescale phylogenetic tree was reconstructed which estimated important time points in the evolution of HCV-1. It revealed that subtype 1a diverged from its nearest relatives 135 years ago and subtype 1b diverged from its nearest relatives 112 years ago. When subtypes 1a, 1j, 1k, 1m, 1n and six close relatives (all but one from Haitian immigrants) were considered as a whole, the divergence time was 176 years ago. This diversification was concurrent with the time period when the transatlantic slave trade was active. When taking all the HCV-1 isolates as a single lineage, the divergence time was 326 years ago. This analysis suggested the existence of a recent common ancestor for subtype 1a and the Haitian variants; a co-origin for subtypes 1b, 1i and 1d was also implied. PMID:24718832

  20. Improving recombinant Rubisco biogenesis, plant photosynthesis and growth by coexpressing its ancillary RAF1 chaperone.

    Science.gov (United States)

    Whitney, Spencer M; Birch, Rosemary; Kelso, Celine; Beck, Jennifer L; Kapralov, Maxim V

    2015-03-17

    Enabling improvements to crop yield and resource use by enhancing the catalysis of the photosynthetic CO2-fixing enzyme Rubisco has been a longstanding challenge. Efforts toward realization of this goal have been greatly assisted by advances in understanding the complexities of Rubisco's biogenesis in plastids and the development of tailored chloroplast transformation tools. Here we generate transplastomic tobacco genotypes expressing Arabidopsis Rubisco large subunits (AtL), both on their own (producing tob(AtL) plants) and with a cognate Rubisco accumulation factor 1 (AtRAF1) chaperone (producing tob(AtL-R1) plants) that has undergone parallel functional coevolution with AtL. We show AtRAF1 assembles as a dimer and is produced in tob(AtL-R1) and Arabidopsis leaves at 10-15 nmol AtRAF1 monomers per square meter. Consistent with a postchaperonin large (L)-subunit assembly role, the AtRAF1 facilitated two to threefold improvements in the amount and biogenesis rate of hybrid L8(A)S8(t) Rubisco [comprising AtL and tobacco small (S) subunits] in tob(AtL-R1) leaves compared with tob(AtL), despite >threefold lower steady-state Rubisco mRNA levels in tob(AtL-R1). Accompanying twofold increases in photosynthetic CO2-assimilation rate and plant growth were measured for tob(AtL-R1) lines. These findings highlight the importance of ancillary protein complementarity during Rubisco biogenesis in plastids, the possible constraints this has imposed on Rubisco adaptive evolution, and the likely need for such interaction specificity to be considered when optimizing recombinant Rubisco bioengineering in plants. PMID:25733857

  1. A genomewide analysis of genes for the heat shock protein 70 chaperone system in the ascidian Ciona intestinalis

    OpenAIRE

    Wada, Shuichi; Hamada, Mayuko; Satoh, Nori

    2006-01-01

    Molecular chaperones play crucial roles in various aspects of the biogenesis and maintenance of proteins in the cell. The heat shock protein 70 (HSP70) chaperone system, in which HSP70 proteins act as chaperones, is one of the major molecular chaperone systems conserved among a variety of organisms. To shed light on the evolutionary history of the constituents of the chordate HSP70 chaperone system and to identify all of the components of the HSP70 chaperone system in ascidians, we carried ou...

  2. Structural Bioinformatics and Protein Docking Analysis of the Molecular Chaperone-Kinase Interactions: Towards Allosteric Inhibition of Protein Kinases by Targeting the Hsp90-Cdc37 Chaperone Machinery

    Directory of Open Access Journals (Sweden)

    Gennady Verkhivker

    2013-11-01

    Full Text Available A fundamental role of the Hsp90-Cdc37 chaperone system in mediating maturation of protein kinase clients and supporting kinase functional activity is essential for the integrity and viability of signaling pathways involved in cell cycle control and organism development. Despite significant advances in understanding structure and function of molecular chaperones, the molecular mechanisms and guiding principles of kinase recruitment to the chaperone system are lacking quantitative characterization. Structural and thermodynamic characterization of Hsp90-Cdc37 binding with protein kinase clients by modern experimental techniques is highly challenging, owing to a transient nature of chaperone-mediated interactions. In this work, we used experimentally-guided protein docking to probe the allosteric nature of the Hsp90-Cdc37 binding with the cyclin-dependent kinase 4 (Cdk4 kinase clients. The results of docking simulations suggest that the kinase recognition and recruitment to the chaperone system may be primarily determined by Cdc37 targeting of the N-terminal kinase lobe. The interactions of Hsp90 with the C-terminal kinase lobe may provide additional “molecular brakes” that can lock (or unlock kinase from the system during client loading (release stages. The results of this study support a central role of the Cdc37 chaperone in recognition and recruitment of the kinase clients. Structural analysis may have useful implications in developing strategies for allosteric inhibition of protein kinases by targeting the Hsp90-Cdc37 chaperone machinery.

  3. Stochastic Optimization Based Study of Dimerization Kinetics

    CERN Document Server

    Talukder, Srijeeta; Metzler, Ralf; Banik, Suman K; Chaudhury, Pinaki

    2013-01-01

    We investigate the potential of numerical algorithms to decipher the kinetic parameters involved in multi-step chemical reactions. To this end we study a dimerization kinetics of protein as a model system. We follow the dimerization kinetics using a stochastic simulation algorithm and combine it with three different optimization techniques (Genetic Algorithm, Simulated Annealing and Parallel Tempering) to obtain the rate constants involved in each reaction step. We find good convergence of the numerical scheme to the rate constants of the process. We also perform a sensitivity test on the reaction kinetic parameters to see the relative effects of the parameters for the associated profile of the monomer/dimer distribution.

  4. T-shaped dimer of coronene

    OpenAIRE

    Obolensky, O. I.; Semenikhina, V. V.; Solov'yov, A. V.; Greiner, W.

    2005-01-01

    An evidence of importance of the T-shaped configuration of coronene dimer is presented. That is, the dimer's lowest energy configuration is not necessarily a stack, as it might had been expected a priori. This is a surprising result for dimer of such a large polycyclic aromatic hydrocarbon (PAH) as coronene. The energy of the T-shaped configuration at all considered levels of density functional theory (B3LYP,PBE/6-31+G(d),D95,cc-pVDZ,cc-pVTZ) was systematically lower than the energies of thre...

  5. Rearrangements of α-helical structures of FlgN chaperone control the binding affinity for its cognate substrates during flagellar type III export.

    Science.gov (United States)

    Kinoshita, Miki; Nakanishi, Yuki; Furukawa, Yukio; Namba, Keiichi; Imada, Katsumi; Minamino, Tohru

    2016-08-01

    The bacterial flagellar type III export chaperones not only act as bodyguards to protect their cognate substrates from aggregation and proteolysis in the cytoplasm but also ensure the order of export through their interactions with an export gate protein FlhA. FlgN chaperone binds to FlgK and FlgL with nanomolar affinity and transfers them to FlhA for their efficient and rapid transport for the formation of the hook-filament junction zone. However, it remains unknown how FlgN releases FlgK and FlgL at the FlhA export gate platform in a timely manner. Here, we have solved the crystal structure of Salmonella FlgN at 2.3 Å resolution and carried out structure-based functional analyses. FlgN consists of three α helices, α1, α2 and α3. Helix α1 adopts two distinct, extended and bent conformations through the conformational change of N-loop between α1 and α2. The N-loop deletion not only increases the probability of FlgN dimer formation but also abolish the interaction between FlgN and FlgK. Highly conserved Asn-92, Asn-95 and Ile-103 residues in helix α3 are involved in the strong interaction with FlgK. We propose that the N-loop coordinates helical rearrangements of FlgN with the association and dissociation of its cognate substrates during their export. PMID:27178222

  6. Environment assisted energy transfer in dimer system

    Energy Technology Data Exchange (ETDEWEB)

    Khan, Salman, E-mail: sksafi@comsats.edu.pk [Department of Physics, COMSATS Institute of Information Technology, Chak Shahzad, Islamabad (Pakistan); Ibrahim, M.; Khan, M.K. [Department of Physics, Quaid-i-Azam University, Islamabad (Pakistan)

    2014-02-15

    The influence of collective and multilocal environments on the energy transfer between the levels of a dimer is studied. The dynamics of energy transfer are investigated by considering coupling of collective environment with the levels of the dimer in the presence of both two individuals and mutually correlated multilocal environments. It is shown that every way of coupling we consider assists, though differently, the probability of transition between the levels of dimer. The probability of transition is strongly enhanced when the two local environments are mutually correlated. -- Highlights: • The dynamics of energy transfer between the levels of a dimer are studied. • Coupling of collective as well as individual environments are considered. • The environments are in spin star configurations. • The environment assists the energy transfer between the levels. • For correlated multilocal environments, the transition probability is almost 100%.

  7. Synthesis of new dimeric carvacrol compounds

    OpenAIRE

    Uttam B. More; Hemant P. Narkhede; Mahulikar, Pramod P.

    2008-01-01

    The polymer supported carvacrol anion was reacted with 1,2-dibromoethane, 1,4-dibromoethane, oxalyl dichloride, malonyl dichloride, succinyl dichloride, glutaroyl dichloride, and adipoyl dichloride to afford the corresponding dimeric carvacryl ethers or esters

  8. Synthesis of new dimeric carvacrol compounds

    Directory of Open Access Journals (Sweden)

    Uttam B. More

    2008-12-01

    Full Text Available The polymer supported carvacrol anion was reacted with 1,2-dibromoethane, 1,4-dibromoethane, oxalyl dichloride, malonyl dichloride, succinyl dichloride, glutaroyl dichloride, and adipoyl dichloride to afford the corresponding dimeric carvacryl ethers or esters

  9. Modeling dimer structure for efficient singlet fission

    Czech Academy of Sciences Publication Activity Database

    Havlas, Zdeněk; Jovanovic, M.; Michl, Josef

    Kyoto: -, 2013. Oa1. [JCS International Symposium on Theoretical Chemistry /5./. 02.12.2013-06.12.2013, Nara] Institutional support: RVO:61388963 Keywords : singlet fission * dimer structure Subject RIV: CF - Physical ; Theoretical Chemistry

  10. Formation of cystine slipknots in dimeric proteins.

    Science.gov (United States)

    Sikora, Mateusz; Cieplak, Marek

    2013-01-01

    We consider mechanical stability of dimeric and monomeric proteins with the cystine knot motif. A structure based dynamical model is used to demonstrate that all dimeric and some monomeric proteins of this kind should have considerable resistance to stretching that is significantly larger than that of titin. The mechanisms of the large mechanostability are elucidated. In most cases, it originates from the induced formation of one or two cystine slipknots. Since there are four termini in a dimer, there are several ways of selecting two of them to pull by. We show that in the cystine knot systems, there is strong anisotropy in mechanostability and force patterns related to the selection. We show that the thermodynamic stability of the dimers is enhanced compared to the constituting monomers whereas machanostability is either lower or higher. PMID:23520470

  11. Water dimer and the atmospheric continuum

    International Nuclear Information System (INIS)

    The physical origin of humidity-related atmospheric continuum absorption is examined. The existence of double water molecules (dimers) in equilibrium water vapor at room temperature is proved by direct spectroscopic experiments supported by ab initio calculations. It is demonstrated that diluting water vapor with air does not significantly reduce the abundance of dimers. Numerous previous studies have predicted the presence of water dimers in the atmosphere and their influence on chemical reactions, homogeneous condensation, and Earth's radiation balance. Our results provide experimental proof of the presence of dimers in the atmosphere, thus enabling a detailed study of their role in natural processes. Prospects for future research are discussed. (reviews of topical problems)

  12. Reductive dimerization mechanisms of some streptocyanine dyes

    International Nuclear Information System (INIS)

    Cyclic voltammetric studies of streptocyanine dyes were carried out on a glassy carbon electrode. For dye electroreduction, logarithmic analysis of the convoluted current indicates an EC2 mechanism with dimerization following electron transfer. Relevant kinetic and thermodynamic values are reported

  13. Genomic organization of ATOX1, a human copper chaperone

    Directory of Open Access Journals (Sweden)

    Kaler Stephen G

    2003-02-01

    Full Text Available Abstract Background Copper is an essential trace element that plays a critical role in the survival of all living organisms. Menkes disease and occipital horn syndrome (OHS are allelic disorders of copper transport caused by defects in a X-linked gene (ATP7A that encodes a P-type ATPase that transports copper across cellular membranes, including the trans-Golgi network. Genetic studies in yeast recently revealed a new family of cytoplasmic proteins called copper chaperones which bind copper ions and deliver them to specific cellular pathways. Biochemical studies of the human homolog of one copper chaperone, ATOX1, indicate direct interaction with the Menkes/OHS protein. Although no disease-associated mutations have been reported in ATOX1, mice with disruption of the ATOX1 locus demonstrate perinatal mortality similar to that observed in the brindled mice (Mobr, a mouse model of Menkes disease. The cDNA sequence for ATOX1 is known, and the genomic organization has not been reported. Results We determined the genomic structure of ATOX1. The gene contains 4 exons spanning a genomic distance of approximately 16 kb. The translation start codon is located in the 3' end of exon 1 and the termination codon in exon 3. We developed a PCR-based assay to amplify the coding regions and splice junctions from genomic DNA. We screened for ATOX1 mutations in two patients with classical Menkes disease phenotypes and one individual with occipital horn syndrome who had no alterations detected in ATP7A, as well as an adult female with chronic anemia, low serum copper and evidence of mild dopamine-beta-hydroxylase deficiency and no alterations in the ATOX1 coding or splice junction sequences were found. Conclusions In this study, we characterized the genomic structure of the human copper chaperone ATOX1 to facilitate screening of this gene from genomic DNA in patients whose clinical or biochemical phenotypes suggest impaired copper transport.

  14. Designing Stable Antiparallel Coiled Coil Dimers

    Institute of Scientific and Technical Information of China (English)

    曾宪纲; 周海梦

    2001-01-01

    The history of antiparallel coiled coil dimer design is briefly reviewed and the main principles governing the successful designs are explained. They include analysis of the inter-subunit electrostatic repulsion for determining partners for dimerization and of the buried polar interaction for determining the relative orientation of the partners. A theory is proposed to explain the lack of antiparallel coiled coil homodimers in nature.

  15. Formation of Cystine Slipknots in Dimeric Proteins

    OpenAIRE

    Sikora, Mateusz; Cieplak, Marek

    2013-01-01

    We consider mechanical stability of dimeric and monomeric proteins with the cystine knot motif. A structure based dynamical model is used to demonstrate that all dimeric and some monomeric proteins of this kind should have considerable resistance to stretching that is significantly larger than that of titin. The mechanisms of the large mechanostability are elucidated. In most cases, it originates from the induced formation of one or two cystine slipknots. Since there are four termini in a dim...

  16. Residue conservation and dimer-interface analysis of olfactory receptor molecular models

    Directory of Open Access Journals (Sweden)

    Ramanathan Sowdhamini

    2012-10-01

    Full Text Available Olfactory Receptors (ORs are members of the Class A rhodopsin like G-protein coupled receptors (GPCRs which are the initial players in the signal transduction cascade, leading to the generation of nerve impulses transmitted to the brain and resulting in the detection of odorant molecules. Despite the accumulation of thousands of olfactory receptor sequences, no crystal structures of ORs are known tο date. However, the recent availability of crystallographic models of a few GPCRs allows us to generate homology models of ORs and analyze their amino acid patterns, as there is a huge diversity in OR sequences. In this study, we have generated three-dimensional models of 100 representative ORs from Homo sapiens, Mus musculus, Drosophila melanogaster, Caenorhabditis elegans and Sacharomyces cerevisiae which were selected on the basis of a composite classification scheme and phylogenetic analysis. The crystal structure of bovine rhodopsin was used as a template and it was found that the full-length models have more than 90% of their residues in allowed regions of the Ramachandran plot. The structures were further used for analysis of conserved residues in the transmembrane and extracellular loop regions in order to identify functionally important residues. Several ORs are known to be functional as dimers and hence dimer interfaces were predicted for OR models to analyse their oligomeric functional state.

  17. Single residue modification of only one dimer within the hemoglobin tetramer reveals autonomous dimer function

    OpenAIRE

    Ackers, Gary K.; Dalessio, Paula M.; Lew, George H.; Daugherty, Margaret A.; Holt, Jo M.

    2002-01-01

    The mechanism of cooperativity in the human hemoglobin tetramer (a dimer of αβ dimers) has historically been modeled as a simple two-state system in which a low-affinity structural form (T) switches, on ligation, to a high-affinity form (R), yielding a net loss of hydrogen bonds and salt bridges in the dimer–dimer interface. Modifications that weaken these cross-dimer contacts destabilize the quaternary T tetramer, leading to decreased cooperativity and enhanced ligand affinity, as demonstrat...

  18. Model systems of protein-misfolding diseases reveal chaperone modifiers of proteotoxicity

    Science.gov (United States)

    2016-01-01

    ABSTRACT Chaperones and co-chaperones enable protein folding and degradation, safeguarding the proteome against proteotoxic stress. Chaperones display dynamic responses to exogenous and endogenous stressors and thus constitute a key component of the proteostasis network (PN), an intricately regulated network of quality control and repair pathways that cooperate to maintain cellular proteostasis. It has been hypothesized that aging leads to chronic stress on the proteome and that this could underlie many age-associated diseases such as neurodegeneration. Understanding the dynamics of chaperone function during aging and disease-related proteotoxic stress could reveal specific chaperone systems that fail to respond to protein misfolding. Through the use of suppressor and enhancer screens, key chaperones crucial for proteostasis maintenance have been identified in model organisms that express misfolded disease-related proteins. This review provides a literature-based analysis of these genetic studies and highlights prominent chaperone modifiers of proteotoxicity, which include the HSP70-HSP40 machine and small HSPs. Taken together, these studies in model systems can inform strategies for therapeutic regulation of chaperone functionality, to manage aging-related proteotoxic stress and to delay the onset of neurodegenerative diseases. PMID:27491084

  19. Natural products triptolide, celastrol, and withaferin A inhibit the chaperone activity of peroxiredoxin i

    NARCIS (Netherlands)

    Zhao, Qian; Ding, Yu; Deng, Zhangshuang; Lee, On Yi; Gao, Peng; Chen, Pin; Rose, Rebecca J.; Zhao, Hong; Zhang, Zhehao; Tao, Xin Pei; Heck, Albert J R; Kao, Richard; Yang, Dan

    2015-01-01

    Peroxiredoxin I (Prx I) plays an important role in cancer development and inflammation. It is a dual-functional protein which acts as both an antioxidant enzyme and a molecular chaperone. While there have been intensive studies on its peroxidase activity, Prx I's chaperone activity remains elusive,

  20. Targeting Molecular Chaperones for the Treatment of Cystic Fibrosis: Is It a Viable Approach?

    Science.gov (United States)

    Heard, Ashley; Thompson, Jake; Carver, Jessica; Bakey, Michelle; Wang, X Robert

    2015-01-01

    Cystic Fibrosis (CF) is largely caused by protein misfolding and the loss of function of a plasma membrane anion channel known as the cystic fibrosis transmembrane conductance regulator (CFTR). The most common CF-causing mutation, F508del, leads to severe conformational defect in CFTR. The cellular chaperone machinery plays an important role in CFTR biogenesis and quality control. Multiple attempts have been made to improve the cell surface functional expression of the mutant CFTR by modulating the expression of components of the cellular chaperone machinery. The efficacy of such an approach has been low largely due to the severe intrinsic folding defects of the F508del CFTR. Moreover, the impact of chaperone perturbation on the chaperone machinery itself and on other physiologically important proteins might lead to potentially severe side effects. Approaches aimed at disrupting chaperone-CFTR interactions show greater efficacy, and are compatible with small-molecule drug discovery and gene therapy. Combination between chaperone modulators and F508del correctors might further enhance potency and specificity. As molecular chaperones play important roles in regulating inflammation and immunity, they can be potential targets for controlling airway infection and inflammation in patients. If such effects can be synergized with chaperone-mediated regulation of CFTR biogenesis and quality control, more efficacious therapeutics will be developed to combat CF lung disease. PMID:25981601

  1. The aggregation and neurotoxicity of TDP-43 and its ALS-associated 25 kDa fragment are differentially affected by molecular chaperones in Drosophila.

    Directory of Open Access Journals (Sweden)

    Jenna M Gregory

    Full Text Available Almost all cases of sporadic amyotrophic lateral sclerosis (ALS, and some cases of the familial form, are characterised by the deposition of TDP-43, a member of a family of heteronuclear ribonucleoproteins (hnRNP. Although protein misfolding and deposition is thought to be a causative feature of many of the most prevalent neurodegenerative diseases, a link between TDP-43 aggregation and the dysfunction of motor neurons has yet to be established, despite many correlative neuropathological studies. We have investigated this relationship in the present study by probing the effect of altering TDP-43 aggregation behaviour in vivo by modulating the levels of molecular chaperones in a Drosophila model. More specifically, we quantify the effect of either pharmacological upregulation of the heat shock response or specific genetic upregulation of a small heat shock protein, CG14207, on the neurotoxicity of both TDP-43 and of its disease associated 25 kDa fragment (TDP-25 in a Drosophila model. Inhibition of the aggregation of TDP-43 by either method results in a partial reduction of its neurotoxic effects on both photoreceptor and motor neurons, whereas inhibition of the aggregation of TDP-25 results not only in a complete suppression of its toxicity but also its clearance from the brain in both neuronal subtypes studied. The results demonstrate, therefore, that aggregation plays a crucial role in mediating the neurotoxic effects of both full length and truncated TDP-43, and furthermore reveal that the in vivo propensity of these two proteins to aggregate and their susceptibility to molecular chaperone mediated clearance are quite distinct.

  2. Copper transporters and chaperones: Their function on angiogenesis and cellular signalling

    Indian Academy of Sciences (India)

    SR BHARATHI DEVI; DHIVYA M ALOYSIUS; KN SULOCHANA

    2016-09-01

    Copper, although known as a micronutrient, has a pivotal role in modulating the cellular metabolism. Many studieshave reported the role of copper in angiogenesis. Copper chaperones are intracellular proteins that mediate coppertrafficking to various cell organelles. However, the role and function of copper chaperones in relation to angiogenesishas to be further explored. The intracellular copper levels when in excess are deleterious and certain mutations ofcopper chaperones have been shown to induce cell death and influence various cellular metabolisms. The study ofthese chaperones will be helpful in understanding the players in the cascade of events in angiogenesis and their role incellular metabolic pathways. In this review we have briefly listed the copper chaperones associated with angiogenicand metabolic signalling and their function.

  3. Kinetic analysis reveals the diversity of microscopic mechanisms through which molecular chaperones suppress amyloid formation

    Science.gov (United States)

    Arosio, Paolo; Michaels, Thomas C. T.; Linse, Sara; Månsson, Cecilia; Emanuelsson, Cecilia; Presto, Jenny; Johansson, Jan; Vendruscolo, Michele; Dobson, Christopher M.; Knowles, Tuomas P. J.

    2016-03-01

    It is increasingly recognized that molecular chaperones play a key role in modulating the formation of amyloid fibrils, a process associated with a wide range of human disorders. Understanding the detailed mechanisms by which they perform this function, however, has been challenging because of the great complexity of the protein aggregation process itself. In this work, we build on a previous kinetic approach and develop a model that considers pairwise interactions between molecular chaperones and different protein species to identify the protein components targeted by the chaperones and the corresponding microscopic reaction steps that are inhibited. We show that these interactions conserve the topology of the unperturbed reaction network but modify the connectivity weights between the different microscopic steps. Moreover, by analysing several protein-molecular chaperone systems, we reveal the striking diversity in the microscopic mechanisms by which molecular chaperones act to suppress amyloid formation.

  4. Small intestinal mucosa expression of putative chaperone fls485

    Directory of Open Access Journals (Sweden)

    Raupach Kerstin

    2010-03-01

    Full Text Available Abstract Background Maturation of enterocytes along the small intestinal crypt-villus axis is associated with significant changes in gene expression profiles. fls485 coding a putative chaperone protein has been recently suggested as a gene involved in this process. The aim of the present study was to analyze fls485 expression in human small intestinal mucosa. Methods fls485 expression in purified normal or intestinal mucosa affected with celiac disease was investigated with a molecular approach including qRT-PCR, Western blotting, and expression strategies. Molecular data were corroborated with several in situ techniques and usage of newly synthesized mouse monoclonal antibodies. Results fls485 mRNA expression was preferentially found in enterocytes and chromaffine cells of human intestinal mucosa as well as in several cell lines including Rko, Lovo, and CaCo2 cells. Western blot analysis with our new anti-fls485 antibodies revealed at least two fls485 proteins. In a functional CaCo2 model, an increase in fls485 expression was paralleled by cellular maturation stage. Immunohistochemistry demonstrated fls485 as a cytosolic protein with a slightly increasing expression gradient along the crypt-villus axis which was impaired in celiac disease Marsh IIIa-c. Conclusions Expression and synthesis of fls485 are found in surface lining epithelia of normal human intestinal mucosa and deriving epithelial cell lines. An interdependence of enterocyte differentiation along the crypt-villus axis and fls485 chaperone activity might be possible.

  5. Degradation of AF1Q by chaperone-mediated autophagy

    Energy Technology Data Exchange (ETDEWEB)

    Li, Peng; Ji, Min; Lu, Fei; Zhang, Jingru [Department of Hematology, Key Laboratory of Cardiovascular Remodeling and Function Research, Qilu Hospital, Shandong University, Jinan 250012 (China); Li, Huanjie; Cui, Taixing; Li Wang, Xing [Research Center for Cell Therapy, Key Laboratory of Cardiovascular Remodeling and Function Research, Qilu Hospital, Shandong University, Jinan 250012 (China); Tang, Dongqi, E-mail: tangdq@sdu.edu.cn [Research Center for Cell Therapy, Key Laboratory of Cardiovascular Remodeling and Function Research, Qilu Hospital, Shandong University, Jinan 250012 (China); Center for Stem Cell and Regenerative Medicine, The Second Hospital of Shandong University, Jinan 250033 (China); Ji, Chunyan, E-mail: jichunyan@sdu.edu.cn [Department of Hematology, Key Laboratory of Cardiovascular Remodeling and Function Research, Qilu Hospital, Shandong University, Jinan 250012 (China)

    2014-09-10

    AF1Q, a mixed lineage leukemia gene fusion partner, is identified as a poor prognostic biomarker for pediatric acute myeloid leukemia (AML), adult AML with normal cytogenetic and adult myelodysplastic syndrome. AF1Q is highly regulated during hematopoietic progenitor differentiation and development but its regulatory mechanism has not been defined clearly. In the present study, we used pharmacological and genetic approaches to influence chaperone-mediated autophagy (CMA) and explored the degradation mechanism of AF1Q. Pharmacological inhibitors of lysosomal degradation, such as chloroquine, increased AF1Q levels, whereas activators of CMA, including 6-aminonicotinamide and nutrient starvation, decreased AF1Q levels. AF1Q interacts with HSPA8 and LAMP-2A, which are core components of the CMA machinery. Knockdown of HSPA8 or LAMP-2A increased AF1Q protein levels, whereas overexpression showed the opposite effect. Using an amino acid deletion AF1Q mutation plasmid, we identified that AF1Q had a KFERQ-like motif which was recognized by HSPA8 for CMA-dependent proteolysis. In conclusion, we demonstrate for the first time that AF1Q can be degraded in lysosomes by CMA. - Highlights: • Chaperone-mediated autophagy (CMA) is involved in the degradation of AF1Q. • Macroautophagy does not contribute to the AF1Q degradation. • AF1Q has a KFERQ-like motif that is recognized by CMA core components.

  6. Transduction of the Hedgehog signal through the dimerization of Fused and the nuclear translocation of Cubitus interruptus

    Institute of Scientific and Technical Information of China (English)

    Yanyan Zhang; Feifei Mao; Yi Lu; Wenqing Wu; Lei Zhang; Yun Zhao

    2011-01-01

    The Hedgehog (Hh) family of secreted proteins is essential for development in both vertebrates and invertebrates.As one of main morphogens during metazoan development,the graded Hh signal is transduced across the plasma membrane by Smoothened (Smo) through the differential phosphorylation of its cytoplasmic tail,leading to pathway activation and the differential expression of target genes.However,how Smo transduces the graded Hh signal via the Costal2 (Cos2)/Fused (Fu) complex remains poorly understood.Here we present a model of the cell response to a Hh gradient by translating Smo phosphorylation information to Fu dimerization and Cubitus interruptus (Ci)nuclear localization information.Our findings suggest that the phosphorylated C-terminus of Smo recruits the Cos2/Fu complex to the membrane through the interaction between Smo and Cos2,which further induces Fu dimerization.Dimerized Fu is phosphorylated and transduces the Hh signal by phosphorylating Cos2 and Suppressor of Fu (Su(fu)).We further show that this process promotes the dissociation of the full-length Ci (Ci155) and Cos2 or Su(fu),and results in the translocation of Ci155 into the nucleus,activating the expression of target genes.

  7. Two-way molecular ligation for efficient conversion of monomeric hepatitis B virus DNA constructs into tandem dimers.

    Science.gov (United States)

    Zong, Li; Qin, Yanli; Jia, Haodi; Zhou, Huailiang; Chen, Chaoyang; Qiao, Ke; Zhang, Jiming; Wang, Yongxiang; Li, Jisu; Tong, Shuping

    2016-07-01

    Replication of the 3.2-kb hepatitis B virus (HBV) genome is driven by the covalently closed circular (ccc) DNA in the nucleus, from which four classes of co-terminal RNAs are transcribed. Genome replication requires just the 3.5-kb pregenomic RNA, which is terminally redundant. Cloning the full-length HBV genome into a vector disrupts its continuity, thus preventing genome replication at the step of pregenomic RNA transcription. This can be overcome by converting the monomeric construct into a tandem dimer, yet the need to ligate two molecules of the HBV genome with vector DNA makes it inefficient and even unsuccessful. To overcome this problem we partially digested the monomeric construct with the unique restriction enzyme used for cloning, and dephosphorylated the linearized monomer before its ligation with another copy of the HBV genome. Alternatively, the monomer was linearized at another unique restriction site inside the HBV genome, followed by its dephosphorylation and ligation with another copy of the HBV genome linearized at the same site. These approaches of two-way molecular ligation greatly improved the efficiency of dimer formation with about 50% of the bacterial colonies screened harboring tandem dimers. PMID:27025357

  8. Camelliin B and nobotanin I, macrocyclic ellagitannin dimers and related dimers, and their antitumor activity.

    Science.gov (United States)

    Yoshida, T; Chou, T; Haba, K; Okano, Y; Shingu, T; Miyamoto, K; Koshiura, R; Okuda, T

    1989-11-01

    Camelliin B and nobotanin I, dimeric hydrolyzable tannins of a new class having macrocyclic structures, were isolated from Camellia japonica and Heterocentron roseum, respectively. Nobotanin G and H of the structures related to nobotanin I, were also obtained from H. roseum. Camelliin B and also woodfordin C, a macrocyclic dimer from Woodfordia fruticosa, exhibited marked host-mediated antitumor activities. PMID:2632067

  9. Dimerization of Protegrin-1 in Different Environments

    Directory of Open Access Journals (Sweden)

    Yiannis N. Kaznessis

    2010-09-01

    Full Text Available The dimerization of the cationic β-hairpin antimicrobial peptide protegrin-1 (PG1 is investigated in three different environments: water, the surface of a lipid bilayer membrane, and the core of the membrane. PG1 is known to kill bacteria by forming oligomeric membrane pores, which permeabilize the cells. PG1 dimers are found in two distinct, parallel and antiparallel, conformations, known as important intermediate structural units of the active pore oligomers. What is not clear is the sequence of events from PG1 monomers in solution to pores inside membranes. The step we focus on in this work is the dimerization of PG1. In particular, we are interested in determining where PG1 dimerization is most favorable. We use extensive molecular dynamics simulations to determine the potential of mean force as a function of distance between two PG1 monomers in the aqueous subphase, the surface of model lipid bilayers and the interior of these bilayers. We investigate the two known distinct modes of dimerization that result in either a parallel or an antiparallel β-sheet orientation. The model bilayer membranes are composed of anionic palmitoyl-oleoyl-phosphatidylglycerol (POPG and palmitoyl-oleoyl-phosphatidylethanolamine (POPE in a 1:3 ratio (POPG:POPE. We find the parallel PG1 dimer association to be more favorable than the antiparallel one in water and inside the membrane. However, we observe that the antiparallel PG1 β-sheet dimer conformation is somewhat more stable than the parallel dimer association at the surface of the membrane. We explore the role of hydrogen bonds and ionic bridges in peptide dimerization in the three environments. Detailed knowledge of how networks of ionic bridges and hydrogen bonds contribute to peptide stability is essential for the purpose of understanding the mechanism of action for membrane-active peptides as well as for designing peptides which can modulate membrane properties. The findings are suggestive of the

  10. Radiation chemistry of aromatic dimer radical cations

    International Nuclear Information System (INIS)

    π-π Interactions of aromatic molecules are paid attention much in many fields, especially biology, chemistry, and applied physics, represented as protein, DNA, electron donor-accepter complexes, charge transfers, and self assembly molecules. Aromatic molecules including benzene rings are the simplest case to study the π-π interactions. To interpret the charge resonance (CR) structure in the dimer radical cations, spectroscopic and ESR methods have been carried out. The spectroscopic study on the dimer radical ion of molecules with two chromophores would be profitable to identify the electronic and configurational properties. In this article, dynamics of the dimer radical cation of benzenes, polystyrenes, and resist polymers is described on the basis of direct observation of CR band by the nanosecond pulse radiolysis and low temperature γ-radiolysis methods. (author)

  11. Photoionization of helium dimers; Photoionisation von Heliumdimeren

    Energy Technology Data Exchange (ETDEWEB)

    Havermeier, Tilo

    2010-06-09

    The helium dimer is one of the most weakly bound systems in the universe. This makes it an interesting quantum mechanical object for investigation. These Van der Waals Clusters can be produced in an expansion of a cryogenic gas jet through a small nozzle into vacuum. In the present experiment we examine the interaction of He dimers with synchrotron radiation at an energy range from 64 to 78 eV. We observed different pathways leading to single ionization of both He atoms of the dimer compound. This two close standing ions begin now to dissociate in cause of their coulomb potential. All charged fragments were detected in coincidence with a COLTRIMS system. Especially Interatomic Coulombic Decay (ICD) and the two step process (TS1) were clearly identified. Furthermore a distribution of the internuclear distance was obtained from the measured Kinetic Energy Release (KER). (orig.)

  12. The nucleotide exchange factors of Hsp70 molecular chaperone

    Directory of Open Access Journals (Sweden)

    Andreas eBracher

    2015-04-01

    Full Text Available Molecular chaperones of the Hsp70 family form an important hub in the cellular protein folding networks in bacteria and eukaryotes, connecting translation with the downstream machineries of protein folding and degradation. The Hsp70 folding cycle is driven by two types of cochaperones: J-domain proteins stimulate ATP hydrolysis by Hsp70, while nucleotide exchange factors (NEFs promote replacement of Hsp70-bound ADP with ATP. Bacteria and organelles of bacterial origin have only one known NEF type for Hsp70, GrpE. In contrast, a large diversity of Hsp70 NEFs has been discovered in the eukaryotic cell. These NEFs belong to the Hsp110/Grp170, HspBP1/Sil1 and BAG domain protein families. In this short review we compare the structures and molecular mechanisms of nucleotide exchange factors for Hsp70 and discuss how these cochaperones contribute to protein folding and quality control in the cell.

  13. Modulation of deregulated chaperone-mediated autophagy by a phosphopeptide.

    Science.gov (United States)

    Macri, Christophe; Wang, Fengjuan; Tasset, Inmaculada; Schall, Nicolas; Page, Nicolas; Briand, Jean-Paul; Cuervo, Ana Maria; Muller, Sylviane

    2015-01-01

    The P140 peptide, a 21-mer linear peptide (sequence 131-151) generated from the spliceosomal SNRNP70/U1-70K protein, contains a phosphoserine residue at position 140. It significantly ameliorates clinical manifestations in autoimmune patients with systemic lupus erythematosus and enhances survival in MRL/lpr lupus-prone mice. Previous studies showed that after P140 treatment, there is an accumulation of autophagy markers sequestosome 1/p62 and MAP1LC3-II in MRL/lpr B cells, consistent with a downregulation of autophagic flux. We now identify chaperone-mediated autophagy (CMA) as a target of P140 and demonstrate that its inhibitory effect on CMA is likely tied to its ability to alter the composition of HSPA8/HSC70 heterocomplexes. As in the case of HSPA8, expression of the limiting CMA component LAMP2A, which is increased in MRL/lpr B cells, is downregulated after P140 treatment. We also show that P140, but not the unphosphorylated peptide, uses the clathrin-dependent endo-lysosomal pathway to enter into MRL/lpr B lymphocytes and accumulates in the lysosomal lumen where it may directly hamper lysosomal HSPA8 chaperoning functions, and also destabilize LAMP2A in lysosomes as a result of its effect on HSP90AA1. This dual effect may interfere with the endogenous autoantigen processing and loading to major histocompatibility complex class II molecules and as a consequence, lead to lower activation of autoreactive T cells. These results shed light on mechanisms by which P140 can modulate lupus disease and exert its tolerogenic activity in patients. The unique selective inhibitory effect of the P140 peptide on CMA may be harnessed in other pathological conditions in which reduction of CMA activity would be desired. PMID:25719862

  14. Cardiomyocyte ryanodine receptor degradation by chaperone-mediated autophagy

    Science.gov (United States)

    Pedrozo, Zully; Torrealba, Natalia; Fernández, Carolina; Gatica, Damian; Toro, Barbra; Quiroga, Clara; Rodriguez, Andrea E.; Sanchez, Gina; Gillette, Thomas G.; Hill, Joseph A.; Donoso, Paulina; Lavandero, Sergio

    2013-01-01

    Time for primary review: 15 days Aims Chaperone-mediated autophagy (CMA) is a selective mechanism for the degradation of soluble cytosolic proteins bearing the sequence KFERQ. These proteins are targeted by chaperones and delivered to lysosomes where they are translocated into the lysosomal lumen and degraded via the lysosome-associated membrane protein type 2A (LAMP-2A). Mutations in LAMP2 that inhibit autophagy result in Danon disease characterized by hypertrophic cardiomyopathy. The ryanodine receptor type 2 (RyR2) plays a key role in cardiomyocyte excitation–contraction and its dysfunction can lead to cardiac failure. Whether RyR2 is degraded by CMA is unknown. Methods and results To induce CMA, cultured neonatal rat cardiomyocytes were treated with geldanamycin (GA) to promote protein degradation through this pathway. GA increased LAMP-2A levels together with its redistribution and colocalization with Hsc70 in the perinuclear region, changes indicative of CMA activation. The inhibition of lysosomes but not proteasomes prevented the loss of RyR2. The recovery of RyR2 content after incubation with GA by siRNA targeting LAMP-2A suggests that RyR2 is degraded via CMA. In silico analysis also revealed that the RyR2 sequence harbours six KFERQ motifs which are required for the recognition Hsc70 and its degradation via CMA. Our data suggest that presenilins are involved in RyR2 degradation by CMA. Conclusion These findings are consistent with a model in which oxidative damage of the RyR2 targets it for turnover by presenilins and CMA, which could lead to removal of damaged or leaky RyR2 channels. PMID:23404999

  15. Phosphorylation Dependence of Hsp27 Multimeric Size and Molecular Chaperone Function*

    OpenAIRE

    Hayes, David; Napoli, Vanessa; Mazurkie, Andrew; Stafford, Walter F.; Graceffa, Philip

    2009-01-01

    The molecular chaperone Hsp27 exists as a distribution of large oligomers that are disassembled by phosphorylation at Ser-15, -78, and -82. It is controversial whether the unphosphorylated Hsp27 or the widely used triple Ser-to-Asp phospho-mimic mutant is the more active molecular chaperone in vitro. This question was investigated here by correlating chaperone activity, as measured by the aggregation of reduced insulin or α-lactalbumin, with Hsp27 self-association as monitored by analytical u...

  16. HEP-Flury株病毒辅助质粒包装CTN株病毒基因组拯救狂犬病病毒%The function of helper plasmids from HEP-Flury strain rabies virus on encapsidating the full-length genome of CTN strain

    Institute of Scientific and Technical Information of China (English)

    黄莹; 唐青; 扈荣良

    2009-01-01

    Objective To identify the helper plasmids from HEP-Flury strain rabies virus that could encapsidate the full-length genome of CTN strain. Methods Four overlapped fragments covering the full-length genome of rabies virus CTN strain were cloned into expression vector. A recombinant full-length genome plasmid (pCTN-GFP) contained the full-length genome of the CTN strain expect for ψ gene which was replaced by GFP gene was then constructed using restriction enzyme cleavage and ligation in vitro. In order to obtain the recombinant rabies virus CTN-GFP, the pCTN-GFP was transfected with helper plasmids carrying N, P, L gene of HEP-Flury strain. Results The four gene fragments of the genome were amplified and cloned into the expression vector. The recombinant genome cDNA plasmid pCTN-GFP was constructed and subjected to restriction endonuclease digestions. After sequenced to assure no absence and mutations compared with their parental viruses, it was ready for virus rescue. After the transfection of both pCTN-GFP and the helper plasmids from HEP-Flury strain into BHK-21 cells, the recombinant rabies virus CTN-GFP was rescued and confirmed by fluorescence analysis and RT-PCR, which demonstrated that the CTN-GFP was recovered from cloned cDNA. Conclusion The proteins of HEP-Flury strain rabies virus could encapsidate and transcribe the CTN strain rabies virus RNA genome.%目的 以CTN株狂犬病病毒全长基因组cDNA重组质粒(pCTN-GFP)和HEP-Flury株病毒N、P、L辅助质粒共转染拯救具有活性的CTN株重组狂犬病病毒(CTN-GFP).方法 将CTN株狂犬病病毒全长基因组分4段进行扩增,体外连接扩增片段并克隆入表达载体中,构建CTN株狂犬病病毒全长基因组cDNA重组质粒,基因组ψ基因被标识基因GFP取代,通过与HEP-Flury株病毒N、P、L3个辅助质粒共转染BHK-21细胞,拯救能够稳定表达GFP的重组病毒CTN-GFP.结果 成功扩增全长基因组的4个基因片段,并将其克隆入表达载体,构

  17. Isolation and Primary Expression Analysis of a Full-Length cDNA Clone Encoding Small GTP-Binding Protein Gene CaRab8 in Pepper%辣椒小G蛋白CaRab8基因全长cDNA的分离及表达特征的初步分析

    Institute of Scientific and Technical Information of China (English)

    赖燕; 肖翔; 林菁; 虞露; 陈桂信; 官德义; 何水林

    2011-01-01

    通过对辣椒均一化cDNA文库的筛选,分离获得了一个与烟草RAB8-2基因编码的蛋白有着高度同源性的cDNA阳性克隆,命名为CaR ab8.测序结果表明,该cDNA长度为961 bp,包含有651 bp的完整的开放阅读框,推测编码一个217个氨基酸的蛋白,该蛋白具有GTP/GDP结合活性必需的保守域和包括YYRGA在内的Rab家族成员特有的5个结构域.实时定量RT-PCR结果表明,CaRab8基因的表达水平在茉莉酸甲酯(MeJA)的诱导下在短时间内呈上调表达,在相应时间点受水杨酸(SA)诱导下调表达.%One 961bp full -length cDNA clone was isolated from pepper normalized cDNA library, which containing a complete 651 bp open reading frame and encoding a putative protein composed of 217 amino acids. Amino acid sequence deduced by this cDNA clone showed high similarity to NtRAB8-2 protein from tobacco. The full-length cDNA was named CaRab8. The protein encoded by this cDNA clone included conserved domains for guanine nucleotide binding and GTPase activities and five domains specific to Rab family including YYRGA domain. Real time quantitative PCR analysis showed the expression level of CaRab8 was induced by MeJA while was repressed by SA.

  18. A dimeric peptide with erythropoiesis-stimulating activity uniquely affects erythropoietin receptor ligation and cell surface expression.

    Science.gov (United States)

    Verma, Rakesh; Green, Jennifer M; Schatz, Peter J; Wojchowski, Don M

    2016-08-01

    Erythropoiesis-stimulating agents (ESAs) that exert long-acting antianemia effects have been developed recently, but their mechanisms are poorly understood. Analyses reveal unique erythropoietin receptor (EPOR)-binding properties for one such ESA, the synthetic EPOR agonist peginesatide. Compared with recombinant human EPO and darbepoietin, peginesatide exhibited a slow on rate, but sustained EPOR residency and resistant displacement. In EPO-dependent human erythroid progenitor UT7epo cells, culture in peginesatide unexpectedly upmodulated endogenous cell surface EPOR levels with parallel increases in full-length EPOR-68K levels. These unique properties are suggested to contribute to the durable activity of this (and perhaps additional) dimeric peptide hematopoietic growth factor receptor agonist. PMID:27174804

  19. Construction and preliminary analysis for a full length cDNA library of the dominant strain of Penicillium marneffei isolated from AIDS patient in yeast phase%艾滋病患者马尔尼菲青霉菌优势株酵母相全长cDNA文库的构建和初步分析

    Institute of Scientific and Technical Information of China (English)

    李凌华; 胡凤玉; 陈万山; 宋伟南; 邝燕玲; 蔡卫平; 唐小平

    2010-01-01

    Objective To construct a full length cDNA library of the dominant strain of Penicillium marneffei (PM) in yeast phase isolated from AIDS patients in Guangdong province and screen UniGenes as well as full-length genes, so as to establish the foundation for the study of PM's functional genes and pathogenic mechanisms. Methods CloneMiner cDNA construction kit was utilized to extract mRNA of the dominant PM strain isolated from AIDS patients in Guangdong province. The mRNA was reversed into cDNA, then cloned into a pDONR222 vector by BP recombination to obtain an Uncut cDNA library, which was homogenized later to construct a normalized cDNA library with the principal of saturation hybridization for DNA genome. 2000 clones were chosen randomly to make a bi-directional sequencing and analyzed with bioinformatics for screening UniGenes and full-length genes. Results The total clone number of the Uncut cDNA library was 1.16 × 107 cfu/mL, with a recombination rate of 95% and an average insertion element being over 1 kb. The total clone number of the normalized cDNA library was 1.18 × 106 cfu/mL, with a recombination rate of 95% and an average insertion element being over 1 kb as well. 1945 genes which DNA length were longer than 1 kb were obtained by sequencing and merged into 1360 UniG enes, of which 632 genes were full-length ones. Conclusions The full-length cDNA library of the dominant strain of PM from AIDS patients in Guangdong province possesses good quality.Meanwhile, the technical routine presents high efficiency in obtaining full-length genes and establishing a gene expression spectrum, which can contentedly meet the needs of future experiments.%目的 构建广东地区艾滋病患者马尔尼菲青霉菌(PM)优势株酵母相全长cDNA文库,筛选UniGene和全长基因,为PM的功能基因组学研究和致病机制的探讨奠定基础.方法 应用CloneMiner cDNA construction kit提取广东地区艾滋病患者PM优势株酵母相mRNA,反转录成cDNA后

  20. A Solvent-Exposed Patch in Chaperone-Bound YopE Is Required for Translocation by the Type III Secretion System▿

    OpenAIRE

    Rodgers, Loren; Mukerjea, Romila; Birtalan, Sara; Friedberg, Devorah; Ghosh, Partho

    2010-01-01

    Most effector proteins of bacterial type III secretion (T3S) systems require chaperone proteins for translocation into host cells. Such effectors are bound by chaperones in a conserved and characteristic manner, with the chaperone-binding (Cb) region of the effector wound around the chaperone in a highly extended conformation. This conformation has been suggested to serve as a translocation signal in promoting the association between the chaperone-effector complex and a bacterial component re...

  1. A New Diterpenoid Dimer from Annona glabra

    Institute of Scientific and Technical Information of China (English)

    2003-01-01

    Anew diterpenoid dimer annonebinide A has been isolated from the stems ofAnnona glabra. Its structure was determined to be ent-16α-hydroxykauran-17-yl ent- 16β-kauran- 17-oate on the basis ofspectroscopic and chemical evidence.

  2. The hyperfine spectrum of hydrogen dimers

    International Nuclear Information System (INIS)

    The authors' aim was to obtain the level scheme for the hydrogen dimers and to investigate the angle dependent interactions by analyzing the zero magnetic field hyperfine spectrum of the ortho-ortho and ortho-para species. The results were tested by several recent semi-empirical and ab initio potentials. (Auth.)

  3. Molecular mechanisms of asymmetric RAF dimer activation.

    Science.gov (United States)

    Jambrina, Pablo G; Bohuszewicz, Olga; Buchete, Nicolae-Viorel; Kolch, Walter; Rosta, Edina

    2014-08-01

    Protein phosphorylation is one of the most common post-translational modifications in cell regulatory mechanisms. Dimerization plays also a crucial role in the kinase activity of many kinases, including RAF, CDK2 (cyclin-dependent kinase 2) and EGFR (epidermal growth factor receptor), with heterodimers often being the most active forms. However, the structural and mechanistic details of how phosphorylation affects the activity of homo- and hetero-dimers are largely unknown. Experimentally, synthesizing protein samples with fully specified and homogeneous phosphorylation states remains a challenge for structural biology and biochemical studies. Typically, multiple changes in phosphorylation lead to activation of the same protein, which makes structural determination methods particularly difficult. It is also not well understood how the occurrence of phosphorylation and dimerization processes synergize to affect kinase activities. In the present article, we review available structural data and discuss how MD simulations can be used to model conformational transitions of RAF kinase dimers, in both their phosphorylated and unphosphorylated forms. PMID:25109958

  4. Participation of the extracellular domain in (pro)renin receptor dimerization

    International Nuclear Information System (INIS)

    Highlights: • The (pro)renin receptor [(P)RR] is a regulator of the renin–angiotensin system. • The region responsible for (P)RR dimerization was investigated. • (P)RR extracellular domain constructs were retained intracellularly. • The extracellular domain of (P)RR is responsible for its dimerization. • Novel insight into the regulatory mechanism of soluble (P)RR secretion is provided. - Abstract: The (pro)renin receptor [(P)RR] induces the catalytic activation of prorenin, as well as the activation of the mitogen-activated protein kinase (MAPK) signaling pathway; as such, it plays an important regulatory role in the renin–angiotensin system. (P)RR is known to form a homodimer, but the region participating in its dimerization is unknown. Using glutathione S-transferase (GST) as a carrier protein and a GST pull-down assay, we investigated the interaction of several (P)RR constructs with full-length (FL) (P)RR in mammalian cells. GST fusion proteins with FL (P)RR (GST-FL), the C-terminal M8-9 fragment (GST-M8-9), the extracellular domain (ECD) of (P)RR (GST-ECD), and the (P)RR ECD with a deletion of 32 amino acids encoded by exon 4 (GST-ECDd4) were retained intracellularly, whereas GST alone was efficiently secreted into the culture medium when transiently expressed in COS-7 cells. Immunofluorescence microscopy showed prominent localization of GST-ECD to the endoplasmic reticulum. The GST pull-down analysis revealed that GST-FL, GST-ECD, and GST-ECDd4 bound FLAG-tagged FL (P)RR, whereas GST-M8-9 showed little or no binding when transiently co-expressed in HEK293T cells. Furthermore, pull-down analysis using His-tag affinity resin showed co-precipitation of soluble (P)RR with FL (P)RR from a stable CHO cell line expressing FL h(P)RR with a C-terminal decahistidine tag. These results indicate that the (P)RR ECD participates in dimerization

  5. Participation of the extracellular domain in (pro)renin receptor dimerization

    Energy Technology Data Exchange (ETDEWEB)

    Suzuki-Nakagawa, Chiharu [The United Graduate School of Agricultural Sciences, Gifu University, Gifu (Japan); Nishimura, Misa; Tsukamoto, Tomoko; Aoyama, Sho [Department of Applied Life Science, Faculty of Applied Biological Sciences, Gifu University, Gifu (Japan); Ebihara, Akio; Suzuki, Fumiaki [The United Graduate School of Agricultural Sciences, Gifu University, Gifu (Japan); Department of Applied Life Science, Faculty of Applied Biological Sciences, Gifu University, Gifu (Japan); Nakagawa, Tsutomu, E-mail: nakagawa@gifu-u.ac.jp [The United Graduate School of Agricultural Sciences, Gifu University, Gifu (Japan); Department of Applied Life Science, Faculty of Applied Biological Sciences, Gifu University, Gifu (Japan)

    2014-02-21

    Highlights: • The (pro)renin receptor [(P)RR] is a regulator of the renin–angiotensin system. • The region responsible for (P)RR dimerization was investigated. • (P)RR extracellular domain constructs were retained intracellularly. • The extracellular domain of (P)RR is responsible for its dimerization. • Novel insight into the regulatory mechanism of soluble (P)RR secretion is provided. - Abstract: The (pro)renin receptor [(P)RR] induces the catalytic activation of prorenin, as well as the activation of the mitogen-activated protein kinase (MAPK) signaling pathway; as such, it plays an important regulatory role in the renin–angiotensin system. (P)RR is known to form a homodimer, but the region participating in its dimerization is unknown. Using glutathione S-transferase (GST) as a carrier protein and a GST pull-down assay, we investigated the interaction of several (P)RR constructs with full-length (FL) (P)RR in mammalian cells. GST fusion proteins with FL (P)RR (GST-FL), the C-terminal M8-9 fragment (GST-M8-9), the extracellular domain (ECD) of (P)RR (GST-ECD), and the (P)RR ECD with a deletion of 32 amino acids encoded by exon 4 (GST-ECDd4) were retained intracellularly, whereas GST alone was efficiently secreted into the culture medium when transiently expressed in COS-7 cells. Immunofluorescence microscopy showed prominent localization of GST-ECD to the endoplasmic reticulum. The GST pull-down analysis revealed that GST-FL, GST-ECD, and GST-ECDd4 bound FLAG-tagged FL (P)RR, whereas GST-M8-9 showed little or no binding when transiently co-expressed in HEK293T cells. Furthermore, pull-down analysis using His-tag affinity resin showed co-precipitation of soluble (P)RR with FL (P)RR from a stable CHO cell line expressing FL h(P)RR with a C-terminal decahistidine tag. These results indicate that the (P)RR ECD participates in dimerization.

  6. Peptides interfering 3A protein dimerization decrease FMDV multiplication

    OpenAIRE

    Mónica González-Magaldi; Ángela Vázquez-Calvo; de la Torre, Beatriz G; Javier Valle; David Andreu; Francisco Sobrino

    2015-01-01

    Nonstructural protein 3A is involved in relevant functions in foot-and-mouth disease virus (FMDV) replication. FMDV 3A can form homodimers and preservation of the two hydrophobic ??-helices (??1 and ??2) that stabilize the dimer interface is essential for virus replication. In this work, small peptides mimicking residues involved in the dimer interface were used to interfere with dimerization and thus gain insight on its biological function. The dimer interface peptides ??1, ??2 and that span...

  7. Double-dimer pairings and skew Young diagrams

    OpenAIRE

    Kenyon, Richard W.; Wilson, David B.

    2010-01-01

    We study the number of tilings of skew Young diagrams by ribbon tiles shaped like Dyck paths, in which the tiles are "vertically decreasing". We use these quantities to compute pairing probabilities in the double-dimer model: Given a planar bipartite graph $G$ with special vertices, called nodes, on the outer face, the double-dimer model is formed by the superposition of a uniformly random dimer configuration (perfect matching) of $G$ together with a random dimer configuration of the graph fo...

  8. Universal bosonic tetramers of dimer-atom-atom structure

    OpenAIRE

    Deltuva, A.

    2012-01-01

    Unstable four-boson states having an approximate dimer-atom-atom structure are studied using momentum-space integral equations for the four-particle transition operators. For a given Efimov trimer the universal properties of the lowest associated tetramer are determined. The impact of this tetramer on the atom-trimer and dimer-dimer collisions is analyzed. The reliability of the three-body dimer-atom-atom model is studied.

  9. The histone chaperones Vps75 and Nap1 form ring-like, tetrameric structures in solution

    Science.gov (United States)

    Bowman, Andrew; Hammond, Colin M.; Stirling, Andrew; Ward, Richard; Shang, Weifeng; El-Mkami, Hassane; Robinson, David A.; Svergun, Dmitri I.; Norman, David G.; Owen-Hughes, Tom

    2014-01-01

    NAP-1 fold histone chaperones play an important role in escorting histones to and from sites of nucleosome assembly and disassembly. The two NAP-1 fold histone chaperones in budding yeast, Vps75 and Nap1, have previously been crystalized in a characteristic homodimeric conformation. In this study, a combination of small angle X-ray scattering, multi angle light scattering and pulsed electron–electron double resonance approaches were used to show that both Vps75 and Nap1 adopt ring-shaped tetrameric conformations in solution. This suggests that the formation of homotetramers is a common feature of NAP-1 fold histone chaperones. The tetramerisation of NAP-1 fold histone chaperones may act to shield acidic surfaces in the absence of histone cargo thus providing a ‘self-chaperoning’ type mechanism. PMID:24688059

  10. A Common Structural Motif in the Binding of Virulence Factors to Bacterial Secretion Chaperones

    International Nuclear Information System (INIS)

    Salmonella invasion protein A (SipA) is translocated into host cells by a type III secretion system (T3SS) and comprises two regions: one domain binds its cognate type III secretion chaperone, InvB, in the bacterium to facilitate translocation, while a second domain functions in the host cell, contributing to bacterial uptake by polymerizing actin. We present here the crystal structures of the SipA chaperone binding domain (CBD) alone and in complex with InvB. The SipA CBD is found to consist of a nonglobular polypeptide as well as a large globular domain, both of which are necessary for binding to InvB. We also identify a structural motif that may direct virulence factors to their cognate chaperones in a diverse range of pathogenic bacteria. Disruption of this structural motif leads to a destabilization of several chaperone-substrate complexes from different species, as well as an impairment of secretion in Salmonella

  11. Specificity of Lipoprotein Chaperones for the Characteristic Lipidated Structural Motifs of their Cognate Lipoproteins.

    Science.gov (United States)

    Mejuch, Tom; van Hattum, Hilde; Triola, Gemma; Jaiswal, Mamta; Waldmann, Herbert

    2015-11-01

    Lipoprotein-binding chaperones mediate intracellular transport of lipidated proteins and determine their proper localisation and functioning. Understanding of the exact structural parameters that determine recognition and transport by different chaperones is of major interest. We have synthesised several lipid-modified peptides, representative of different lipoprotein classes, and have investigated their binding to the relevant chaperones PDEδ, UNC119a, UNC119b, and galectins-1 and -3. Our results demonstrate that PDEδ recognises S-isoprenylated C-terminal peptidic structures but not N-myristoylated peptides. In contrast, UNC119 proteins bind only mono-N-myristoylated, but do not recognise doubly lipidated and S-isoprenylated peptides at the C terminus. For galectins-1 and -3, neither binding to N-acylated, nor to C-terminally prenylated peptides could be determined. These results shed light on the specificity of the chaperone-mediated cellular lipoprotein transport systems. PMID:26503308

  12. Histone chaperones FACT and Spt6 prevent histone variants from turning into histone deviants.

    Science.gov (United States)

    Jeronimo, Célia; Robert, François

    2016-05-01

    Histone variants are specialized histones which replace their canonical counterparts in specific nucleosomes. Together with histone post-translational modifications and DNA methylation, they contribute to the epigenome. Histone variants are incorporated at specific locations by the concerted action of histone chaperones and ATP-dependent chromatin remodelers. Recent studies have shown that the histone chaperone FACT plays key roles in preventing pervasive incorporation of two histone variants: H2A.Z and CenH3/CENP-A. In addition, Spt6, another histone chaperone, was also shown to be important for appropriate H2A.Z localization. FACT and Spt6 are both associated with elongating RNA polymerase II. Based on these two examples, we propose that the establishment and maintenance of histone variant genomic distributions depend on a transcription-coupled epigenome editing (or surveillance) function of histone chaperones. PMID:26990181

  13. Dimerization of visual pigments in vivo.

    Science.gov (United States)

    Zhang, Tao; Cao, Li-Hui; Kumar, Sandeep; Enemchukwu, Nduka O; Zhang, Ning; Lambert, Alyssia; Zhao, Xuchen; Jones, Alex; Wang, Shixian; Dennis, Emily M; Fnu, Amrita; Ham, Sam; Rainier, Jon; Yau, King-Wai; Fu, Yingbin

    2016-08-01

    It is a deeply engrained notion that the visual pigment rhodopsin signals light as a monomer, even though many G protein-coupled receptors are now known to exist and function as dimers. Nonetheless, recent studies (albeit all in vitro) have suggested that rhodopsin and its chromophore-free apoprotein, R-opsin, may indeed exist as a homodimer in rod disk membranes. Given the overwhelmingly strong historical context, the crucial remaining question, therefore, is whether pigment dimerization truly exists naturally and what function this dimerization may serve. We addressed this question in vivo with a unique mouse line (S-opsin(+)Lrat(-/-)) expressing, transgenically, short-wavelength-sensitive cone opsin (S-opsin) in rods and also lacking chromophore to exploit the fact that cone opsins, but not R-opsin, require chromophore for proper folding and trafficking to the photoreceptor's outer segment. In R-opsin's absence, S-opsin in these transgenic rods without chromophore was mislocalized; in R-opsin's presence, however, S-opsin trafficked normally to the rod outer segment and produced functional S-pigment upon subsequent chromophore restoration. Introducing a competing R-opsin transmembrane helix H1 or helix H8 peptide, but not helix H4 or helix H5 peptide, into these transgenic rods caused mislocalization of R-opsin and S-opsin to the perinuclear endoplasmic reticulum. Importantly, a similar peptide-competition effect was observed even in WT rods. Our work provides convincing evidence for visual pigment dimerization in vivo under physiological conditions and for its role in pigment maturation and targeting. Our work raises new questions regarding a potential mechanistic role of dimerization in rhodopsin signaling. PMID:27462111

  14. A Nove Asymmetric ent—Kauranoid Dimer from Isodon enanderianus

    Institute of Scientific and Technical Information of China (English)

    纳智; 黎胜红; 等

    2002-01-01

    Further investigation on the aerial parts of Isodon enanderianus afforded a novel asymmetric ent-kauranoid dimer,enanderi-nanin J(1).The structure of the dimer was elucidated by means of spectroscopic methods (including 2D NMR tecniques ),Enanderinanin J was a dimer of xerophilusin A and probably formed by [4+2] cycloaddition.

  15. A Novel Asymmetric ent-Kauranoid Dimer from Isodon enanderianus

    Institute of Scientific and Technical Information of China (English)

    NA,Zhi(纳智); LI,Sheng-Hong(黎胜红); XIANG,Wei(项伟); ZHAO,Ai-Hua(赵爱华); LI,Chao-Ming(李朝明); SUN,Han-Dong(孙汉董)

    2002-01-01

    Further investigation on the aerial parts of Isodon enanderianus afforded a novel asymmetric ent-kauranoid dimer, enanuderinaninJ (1). The structure of the dimer was elucidated by means of spectroscopic methods (including 2D NMR techniques ). Enanderinanin J was a dimer of xerophilusin A and probably formed by [ 4 + 2] cycloaddition.

  16. Capturing a Dynamic Chaperone-Substrate Interaction Using NMR-Informed Molecular Modeling.

    Science.gov (United States)

    Salmon, Loïc; Ahlstrom, Logan S; Horowitz, Scott; Dickson, Alex; Brooks, Charles L; Bardwell, James C A

    2016-08-10

    Chaperones maintain a healthy proteome by preventing aggregation and by aiding in protein folding. Precisely how chaperones influence the conformational properties of their substrates, however, remains unclear. To achieve a detailed description of dynamic chaperone-substrate interactions, we fused site-specific NMR information with coarse-grained simulations. Our model system is the binding and folding of a chaperone substrate, immunity protein 7 (Im7), with the chaperone Spy. We first used an automated procedure in which NMR chemical shifts inform the construction of system-specific force fields that describe each partner individually. The models of the two binding partners are then combined to perform simulations on the chaperone-substrate complex. The binding simulations show excellent agreement with experimental data from multiple biophysical measurements. Upon binding, Im7 interacts with a mixture of hydrophobic and hydrophilic residues on Spy's surface, causing conformational exchange within Im7 to slow down as Im7 folds. Meanwhile, the motion of Spy's flexible loop region increases, allowing for better interaction with different substrate conformations, and helping offset losses in Im7 conformational dynamics that occur upon binding and folding. Spy then preferentially releases Im7 into a well-folded state. Our strategy has enabled a residue-level description of a dynamic chaperone-substrate interaction, improving our understanding of how chaperones facilitate substrate folding. More broadly, we validate our approach using two other binding partners, showing that this approach provides a general platform from which to investigate other flexible biomolecular complexes through the integration of NMR data with efficient computational models. PMID:27415450

  17. Probing molecular mechanisms of the Hsp90 chaperone: biophysical modeling identifies key regulators of functional dynamics.

    Directory of Open Access Journals (Sweden)

    Anshuman Dixit

    Full Text Available Deciphering functional mechanisms of the Hsp90 chaperone machinery is an important objective in cancer biology aiming to facilitate discovery of targeted anti-cancer therapies. Despite significant advances in understanding structure and function of molecular chaperones, organizing molecular principles that control the relationship between conformational diversity and functional mechanisms of the Hsp90 activity lack a sufficient quantitative characterization. We combined molecular dynamics simulations, principal component analysis, the energy landscape model and structure-functional analysis of Hsp90 regulatory interactions to systematically investigate functional dynamics of the molecular chaperone. This approach has identified a network of conserved regions common to the Hsp90 chaperones that could play a universal role in coordinating functional dynamics, principal collective motions and allosteric signaling of Hsp90. We have found that these functional motifs may be utilized by the molecular chaperone machinery to act collectively as central regulators of Hsp90 dynamics and activity, including the inter-domain communications, control of ATP hydrolysis, and protein client binding. These findings have provided support to a long-standing assertion that allosteric regulation and catalysis may have emerged via common evolutionary routes. The interaction networks regulating functional motions of Hsp90 may be determined by the inherent structural architecture of the molecular chaperone. At the same time, the thermodynamics-based "conformational selection" of functional states is likely to be activated based on the nature of the binding partner. This mechanistic model of Hsp90 dynamics and function is consistent with the notion that allosteric networks orchestrating cooperative protein motions can be formed by evolutionary conserved and sparsely connected residue clusters. Hence, allosteric signaling through a small network of distantly connected

  18. The histone chaperones Vps75 and Nap1 form ring-like, tetrameric structures in solution

    OpenAIRE

    Bowman, A.; Hammond, C. M.; Stirling, A.; Ward, R.; Shang, W.; El-Mkami, H.; Robinson, D A; Svergun, Dmitri; Norman, D. G.; Owen-Hughes, T.

    2014-01-01

    MRC [G1100021]; Wellcome Senior Fellowship [095062]. Source of open access funding: The Wellcome Trust [094090]. NAP-1 fold histone chaperones play an important role in escorting histones to and from sites of nucleosome assembly and disassembly. The two NAP-1 fold histone chaperones in budding yeast, Vps75 and Nap1, have previously been crystalized in a characteristic homodimeric conformation. In this study, a combination of small angle X-ray scattering, multi angle light scattering and pu...

  19. Quantifying the role of chaperones in protein translocation by computational modeling

    OpenAIRE

    Assenza, Salvatore; De Los Rios, Paolo; Barducci, Alessandro

    2015-01-01

    The molecular chaperone Hsp70 plays a central role in the import of cytoplasmic proteins into organelles, driving their translocation by binding them from the organellar interior. Starting from the experimentally-determined structure of the E. coli Hsp70, we computed, by means of molecular simulations, the effective free-energy profile for substrate translocation upon chaperone binding. We then used the resulting free energy to quantitatively characterize the kinetics of the import process, w...

  20. Quantifying the role of chaperones in protein translocation by computational modelling

    OpenAIRE

    Assenza, Salvatore; Rios, Paolo De Los; Barducci, Alessandro

    2014-01-01

    The molecular chaperone Hsp70 plays a central role in the import of cytoplasmic proteins into organelles, driving their translocation by binding them from the organellar interior. Starting from the experimentally-determined structure of the \\textit{E. coli} Hsp70, we computed, by means of molecular simulations, the effective free-energy profile for substrate translocation upon chaperone binding. We then used the resulting free energy to quantitatively characterize the kinetics of the import p...

  1. CrAgDb--a database of annotated chaperone repertoire in archaeal genomes.

    Science.gov (United States)

    Rani, Shikha; Srivastava, Abhishikha; Kumar, Manish; Goel, Manisha

    2016-03-01

    Chaperones are a diverse class of ubiquitous proteins that assist other cellular proteins in folding correctly and maintaining their native structure. Many different chaperones cooperate to constitute the 'proteostasis' machinery in the cells. It has been proposed earlier that archaeal organisms could be ideal model systems for deciphering the basic functioning of the 'protein folding machinery' in higher eukaryotes. Several chaperone families have been characterized in archaea over the years but mostly one protein at a time, making it difficult to decipher the composition and mechanistics of the protein folding system as a whole. In order to deal with these lacunae, we have developed a database of all archaeal chaperone proteins, CrAgDb (Chaperone repertoire in Archaeal genomes). The data have been presented in a systematic way with intuitive browse and search facilities for easy retrieval of information. Access to these curated datasets should expedite large-scale analysis of archaeal chaperone networks and significantly advance our understanding of operation and regulation of the protein folding machinery in archaea. Researchers could then translate this knowledge to comprehend the more complex protein folding pathways in eukaryotic systems. The database is freely available at http://14.139.227.92/mkumar/cragdb/. PMID:26862144

  2. Enhancement of Chaperone Activity of Plant-Specific Thioredoxin through γ-Ray Mediated Conformational Change

    Directory of Open Access Journals (Sweden)

    Seung Sik Lee

    2015-11-01

    Full Text Available AtTDX, a thioredoxin-like plant-specific protein present in Arabidospis is a thermo-stable and multi-functional enzyme. This enzyme is known to act as a thioredoxin and as a molecular chaperone depending upon its oligomeric status. The present study examines the effects of γ-irradiation on the structural and functional changes of AtTDX. Holdase chaperone activity of AtTDX was increased and reached a maximum at 10 kGy of γ-irradiation and declined subsequently in a dose-dependent manner, together with no effect on foldase chaperone activity. However, thioredoxin activity decreased gradually with increasing irradiation. Electrophoresis and size exclusion chromatography analysis showed that AtTDX had a tendency to form high molecular weight (HMW complexes after γ-irradiation and γ-ray-induced HMW complexes were tightly associated with a holdase chaperone activity. The hydrophobicity of AtTDX increased with an increase in irradiation dose till 20 kGy and thereafter decreased further. Analysis of the secondary structures of AtTDX using far UV-circular dichroism spectra revealed that the irradiation remarkably increased the exposure of β-sheets and random coils with a dramatic decrease in α-helices and turn elements in a dose-dependent manner. The data of the present study suggest that γ-irradiation may be a useful tool for increasing holdase chaperone activity without adversely affecting foldase chaperone activity of thioredoxin-like proteins.

  3. Nucleosome adaptability conferred by sequence and structural variations in histone H2A-H2B dimers.

    Science.gov (United States)

    Shaytan, Alexey K; Landsman, David; Panchenko, Anna R

    2015-06-01

    Nucleosome variability is essential for their functions in compacting the chromatin structure and regulation of transcription, replication and cell reprogramming. The DNA molecule in nucleosomes is wrapped around an octamer composed of four types of core histones (H3, H4, H2A, H2B). Nucleosomes represent dynamic entities and may change their conformation, stability and binding properties by employing different sets of histone variants or by becoming post-translationally modified. There are many variants of histones H2A and H2B. Specific H2A and H2B variants may preferentially associate with each other resulting in different combinations of variants and leading to the increased combinatorial complexity of nucleosomes. In addition, the H2A-H2B dimer can be recognized and substituted by chaperones/remodelers as a distinct unit, can assemble independently and is stable during nucleosome unwinding. In this review we discuss how sequence and structural variations in H2A-H2B dimers may provide necessary complexity and confer the nucleosome functional variability. PMID:25731851

  4. Crystal structure of the 500-kDa yeast acetyl-CoA carboxylase holoenzyme dimer.

    Science.gov (United States)

    Wei, Jia; Tong, Liang

    2015-10-29

    Acetyl-CoA carboxylase (ACC) has crucial roles in fatty acid metabolism and is an attractive target for drug discovery against diabetes, cancer and other diseases. Saccharomyces cerevisiae ACC (ScACC) is crucial for the production of very-long-chain fatty acids and the maintenance of the nuclear envelope. ACC contains biotin carboxylase (BC) and carboxyltransferase (CT) activities, and its biotin is linked covalently to the biotin carboxyl carrier protein (BCCP). Most eukaryotic ACCs are 250-kilodalton (kDa), multi-domain enzymes and function as homodimers and higher oligomers. They contain a unique, 80-kDa central region that shares no homology with other proteins. Although the structures of the BC, CT and BCCP domains and other biotin-dependent carboxylase holoenzymes are known, there is currently no structural information on the ACC holoenzyme. Here we report the crystal structure of the full-length, 500-kDa holoenzyme dimer of ScACC. The structure is remarkably different from that of the other biotin-dependent carboxylases. The central region contains five domains and is important for positioning the BC and CT domains for catalysis. The structure unexpectedly reveals a dimer of the BC domain and extensive conformational differences compared to the structure of the BC domain alone, which is a monomer. These structural changes reveal why the BC domain alone is catalytically inactive and define the molecular mechanism for the inhibition of eukaryotic ACC by the natural product soraphen A and by phosphorylation of a Ser residue just before the BC domain core in mammalian ACC. The BC and CT active sites are separated by 80 Å, and the entire BCCP domain must translocate during catalysis. PMID:26458104

  5. Histone Chaperone HIRA in Regulation of Transcription Factor RUNX1.

    Science.gov (United States)

    Majumder, Aditi; Syed, Khaja Mohieddin; Joseph, Sunu; Scambler, Peter J; Dutta, Debasree

    2015-05-22

    RUNX1 (Runt-related transcription factor 1) is indispensable for the generation of hemogenic endothelium. However, the regulation of RUNX1 during this developmental process is poorly understood. We investigated the role of the histone chaperone HIRA (histone cell cycle regulation-defective homolog A) from this perspective and report that HIRA significantly contributes toward the regulation of RUNX1 in the transition of differentiating mouse embryonic stem cells from hemogenic to hematopoietic stage. Direct interaction of HIRA and RUNX1 activates the downstream targets of RUNX1 implicated in generation of hematopoietic stem cells. At the molecular level, HIRA-mediated incorporation of histone H3.3 variant within the Runx1 +24 mouse conserved noncoding element is essential for the expression of Runx1 during endothelial to hematopoietic transition. An inactive chromatin at the intronic enhancer of Runx1 in absence of HIRA significantly repressed the transition of cells from hemogenic to hematopoietic fate. We expect that the HIRA-RUNX1 axis might open up a novel approach in understanding leukemogenesis in future. PMID:25847244

  6. FKBP immunophilins and Alzheimer's disease: A chaperoned affair

    Indian Academy of Sciences (India)

    Weihuan Cao; Mary Konsolaki

    2011-08-01

    The FK506-binding protein (FKBP) family of immunophilins consists of proteins with a variety of protein–protein interaction domains and versatile cellular functions. Analysis of the functions of immunophilins has been the focus of studies in recent years and has led to the identification of various molecular pathways in which FKBPs play an active role. All FKBPs contain a domain with prolyl cis/trans isomerase (PPIase) activity. Binding of the immunosuppressant molecule FK506 to this domain inhibits their PPIase activity while mediating immune suppression through inhibition of calcineurin. The larger members, FKBP51 and FKBP52, interact with Hsp90 and exhibit chaperone activity that is shown to regulate steroid hormone signalling. From these studies it is clear that FKBP proteins are expressed ubiquitously but show relatively high levels of expression in the nervous system. Consistent with this expression, FKBPs have been implicated with both neuroprotection and neurodegeneration. This review will focus on recent studies involving FKBP immunophilins in Alzheimer’s-disease-related pathways.

  7. The histone chaperone CAF-1 safeguards somatic cell identity.

    Science.gov (United States)

    Cheloufi, Sihem; Elling, Ulrich; Hopfgartner, Barbara; Jung, Youngsook L; Murn, Jernej; Ninova, Maria; Hubmann, Maria; Badeaux, Aimee I; Euong Ang, Cheen; Tenen, Danielle; Wesche, Daniel J; Abazova, Nadezhda; Hogue, Max; Tasdemir, Nilgun; Brumbaugh, Justin; Rathert, Philipp; Jude, Julian; Ferrari, Francesco; Blanco, Andres; Fellner, Michaela; Wenzel, Daniel; Zinner, Marietta; Vidal, Simon E; Bell, Oliver; Stadtfeld, Matthias; Chang, Howard Y; Almouzni, Genevieve; Lowe, Scott W; Rinn, John; Wernig, Marius; Aravin, Alexei; Shi, Yang; Park, Peter J; Penninger, Josef M; Zuber, Johannes; Hochedlinger, Konrad

    2015-12-10

    Cellular differentiation involves profound remodelling of chromatic landscapes, yet the mechanisms by which somatic cell identity is subsequently maintained remain incompletely understood. To further elucidate regulatory pathways that safeguard the somatic state, we performed two comprehensive RNA interference (RNAi) screens targeting chromatin factors during transcription-factor-mediated reprogramming of mouse fibroblasts to induced pluripotent stem cells (iPS cells). Subunits of the chromatin assembly factor-1 (CAF-1) complex, including Chaf1a and Chaf1b, emerged as the most prominent hits from both screens, followed by modulators of lysine sumoylation and heterochromatin maintenance. Optimal modulation of both CAF-1 and transcription factor levels increased reprogramming efficiency by several orders of magnitude and facilitated iPS cell formation in as little as 4 days. Mechanistically, CAF-1 suppression led to a more accessible chromatin structure at enhancer elements early during reprogramming. These changes were accompanied by a decrease in somatic heterochromatin domains, increased binding of Sox2 to pluripotency-specific targets and activation of associated genes. Notably, suppression of CAF-1 also enhanced the direct conversion of B cells into macrophages and fibroblasts into neurons. Together, our findings reveal the histone chaperone CAF-1 to be a novel regulator of somatic cell identity during transcription-factor-induced cell-fate transitions and provide a potential strategy to modulate cellular plasticity in a regenerative setting. PMID:26659182

  8. Mechanism of Amyloidogenesis of a Bacterial AAA+ Chaperone.

    Science.gov (United States)

    Chan, Sze Wah Samuel; Yau, Jason; Ing, Christopher; Liu, Kaiyin; Farber, Patrick; Won, Amy; Bhandari, Vaibhav; Kara-Yacoubian, Nareg; Seraphim, Thiago V; Chakrabarti, Nilmadhab; Kay, Lewis E; Yip, Christopher M; Pomès, Régis; Sharpe, Simon; Houry, Walid A

    2016-07-01

    Amyloids are fibrillar protein superstructures that are commonly associated with diseases in humans and with physiological functions in various organisms. The precise mechanisms of amyloid formation remain to be elucidated. Surprisingly, we discovered that a bacterial Escherichia coli chaperone-like ATPase, regulatory ATPase variant A (RavA), and specifically the LARA domain in RavA, forms amyloids under acidic conditions at elevated temperatures. RavA is involved in modulating the proper assembly of membrane respiratory complexes. LARA contains an N-terminal loop region followed by a β-sandwich-like folded core. Several approaches, including nuclear magnetic resonance spectroscopy and molecular dynamics simulations, were used to determine the mechanism by which LARA switches to an amyloid state. These studies revealed that the folded core of LARA is amyloidogenic and is protected by its N-terminal loop. At low pH and high temperatures, the interaction of the N-terminal loop with the folded core is disrupted, leading to amyloid formation. PMID:27265850

  9. Fibrillar dimer formation of islet amyloid polypeptides

    Energy Technology Data Exchange (ETDEWEB)

    Chiu, Chi-cheng [Univ. of Chicago, IL (United States); Argonne National Lab. (ANL), Argonne, IL (United States); de Pablo, Juan J. [Univ. of Chicago, IL (United States); Argonne National Lab. (ANL), Argonne, IL (United States)

    2015-05-08

    Amyloid deposits of human islet amyloid polypeptide (hIAPP), a 37-residue hormone co-produced with insulin, have been implicated in the development of type 2 diabetes. Residues 20 – 29 of hIAPP have been proposed to constitute the amyloidogenic core for the aggregation process, yet the segment is mostly unstructured in the mature fibril, according to solid-state NMR data. Here we use molecular simulations combined with bias-exchange metadynamics to characterize the conformational free energies of hIAPP fibrillar dimer and its derivative, pramlintide. We show that residues 20 – 29 are involved in an intermediate that exhibits transient β-sheets, consistent with recent experimental and simulation results. By comparing the aggregation of hIAPP and pramlintide, we illustrate the effects of proline residues on inhibition of the dimerization of IAPP. The mechanistic insights presented here could be useful for development of therapeutic inhibitors of hIAPP amyloid formation.

  10. Fibrillar dimer formation of islet amyloid polypeptides

    Science.gov (United States)

    Chiu, Chi-cheng; de Pablo, Juan J.

    2015-09-01

    Amyloid deposits of human islet amyloid polypeptide (hIAPP), a 37-residue hormone co-produced with insulin, have been implicated in the development of type 2 diabetes. Residues 20 - 29 of hIAPP have been proposed to constitute the amyloidogenic core for the aggregation process, yet the segment is mostly unstructured in the mature fibril, according to solid-state NMR data. Here we use molecular simulations combined with bias-exchange metadynamics to characterize the conformational free energies of hIAPP fibrillar dimer and its derivative, pramlintide. We show that residues 20 - 29 are involved in an intermediate that exhibits transient β-sheets, consistent with recent experimental and simulation results. By comparing the aggregation of hIAPP and pramlintide, we illustrate the effects of proline residues on inhibition of the dimerization of IAPP. The mechanistic insights presented here could be useful for development of therapeutic inhibitors of hIAPP amyloid formation.

  11. An introduction to the dimer model

    International Nuclear Information System (INIS)

    A perfect matching of a graph is a subset of edges which covers every vertex exactly once, that is, for every vertex there is exactly one edge in the set with that vertex as endpoint. The dimer model is the study of the set of perfect matchings of a (possibly infinite) graph. The most well-known example is when the graph is Z2, for which perfect matchings are equivalent (via a simple duality) to domino tilings, that is, tilings of the plane with 2 x 1 and 1 x 2 rectangles. In the first three sections we study domino tilings of the plane and of finite polygonal regions, or equivalently, perfect matchings on Z2 and subgraphs of Z2. In the last two sections we study the FK-percolation model and the dimer model on a more general family of planar graphs

  12. Revisiting the optical $PT$-symmetric dimer

    CERN Document Server

    Morales, J D Huerta; López-Aguayo, S; Rodríguez-Lara, B M

    2016-01-01

    Optics has proved a fertile ground for the experimental simulation of quantum mechanics. Most recently, optical realizations of $\\mathcal{PT}$-symmetric quantum mechanics have been shown, both theoretically and experimentally, opening the door to international efforts aiming at the design of practical optical devices exploiting this symmetry. Here, we focus on the optical $\\mathcal{PT}$-symmetric dimer, a two-waveguide coupler were the materials show symmetric effective gain and loss, and provide a review of the linear and nonlinear optical realizations from a symmetry based point of view. We go beyond a simple review of the literature and show that the dimer is just the smallest of a class of planar $N$-waveguide couplers that are the optical realization of Lorentz group in 2+1 dimensions. Furthermore, we provide a formulation to describe light propagation through waveguide couplers described by non-Hermitian mode coupling matrices based on a non-Hermitian generalization of Ehrenfest theorem.

  13. Experimentally guided structural modeling and dynamics analysis of Hsp90-p53 interactions: allosteric regulation of the Hsp90 chaperone by a client protein.

    Science.gov (United States)

    Blacklock, Kristin; Verkhivker, Gennady M

    2013-11-25

    A fundamental role of the Hsp90 chaperone system in mediating maturation of protein clients is essential for the integrity of signaling pathways involved in cell cycle control and organism development. Molecular characterization of Hsp90 interactions with client proteins is fundamental to understanding the activity of many tumor-inducing signaling proteins and presents an active area of structural and biochemical studies. In this work, we have probed mechanistic aspects of allosteric regulation of Hsp90 by client proteins via a detailed computational study of Hsp90 interactions with the tumor suppressor protein p53. Experimentally guided protein docking and molecular dynamics structural refinement have reconstructed the recognition-competent states of the Hsp90-p53 complexes that are consistent with the NMR studies. Protein structure network analysis has identified critical interacting networks and specific residues responsible for structural integrity and stability of the Hsp90-p53 complexes. Coarse-grained modeling was used to characterize the global dynamics of the regulatory complexes and map p53-induced changes in the conformational equilibrium of Hsp90. The variations in the functional dynamics profiles of the Hsp90-p53 complexes are consistent with the NMR studies and could explain differences in the functional role of the alternative binding sites. Despite the overall similarity of the collective movements and the same global interaction footprint, p53 binding at the C-terminal interaction site of Hsp90 may have a more significant impact on the chaperone dynamics, which is consistent with the stronger allosteric effect of these interactions revealed by the experimental studies. The results suggest that p53-induced modulation of the global dynamics and structurally stable interaction networks can target the regulatory hinge regions and facilitate stabilization of the closed Hsp90 dimer that underlies the fundamental stimulatory effect of the p53 client. PMID

  14. Alcohol dimers - how much diagonal OH anharmonicity?

    OpenAIRE

    Kollipost, Franz; Papendorf, Kim; Lee, Yu-Fang; Lee, Yuan-Pern; Suhm, Martin A

    2014-01-01

    The OH bond of methanol, ethanol and t-butyl alcohol becomes more anharmonic upon hydrogen bonding and the infrared intensity ratio between the overtone and the fundamental transition of the bridging OH stretching mode decreases drastically. FTIR spectroscopy of supersonic slit jet expansions allows to quantify these effects for isolated alcohol dimers, enabling a direct comparison to anharmonic vibrational predictions. The diagonal anharmonicity increase amounts to 15-18%, growing with incre...

  15. Dimer models and quiver gauge theories

    Science.gov (United States)

    Pichai, Ramadevi

    2013-12-01

    = 1 quiver gauge theories on coincident D3 branes placed at a tip of a Calabi-Yau singularity C are dual to string theories on AdS5×X5 where X5 are Sasaki-Einstein spaces. We present a neat combinatorial approach called dimer model to understand interrelations between toric quiver gauge theories and toric data representing the Calabi-Yau singularities.

  16. Entanglement and decoherence in a quantum dimer

    Institute of Scientific and Technical Information of China (English)

    Hou Xi-Wen; Hui Zi; Ding Rui-Min; Chen Xiao-Yang; Gao Yu

    2006-01-01

    The dynamical properties of quantum entanglement in an integrable quantum dimer are studied in terms of the reduced-density linear entropy with various coupling parameters and total boson numbers. The characteristic time of decoherence process in the early-time evolution of the linear entropy is obtained, indicating that the characteristic time and the corresponding entropy exhibit a maximum near the position of the corresponding classical separatrix energy.

  17. Temperature-sensitive protein–DNA dimerizers

    OpenAIRE

    Hauschild, Karl E.; Metzler, Renee E.; Arndt, Hans-Dieter; Moretti, Rocco; Raffaelle, Marni; Dervan, Peter B.; Ansari, Aseem Z.

    2005-01-01

    Programmable DNA-binding polyamides coupled to short peptides have led to the creation of synthetic artificial transcription factors. A hairpin polyamide–YPWM tetrapeptide conjugate facilitates the binding of a natural transcription factor Exd to an adjacent DNA site. Such small molecules function as protein–DNA dimerizers that stabilize complexes at composite DNA binding sites. Here we investigate the role of the linker that connects the polyamide to the peptide. We find that a substantial d...

  18. Repair of DNA-containing pyrimidine dimers

    International Nuclear Information System (INIS)

    Ultraviolet light-induced pyrimidine dimers in DNA are recognized and repaired by a number of unique cellular surveillance systems. The most direct biochemical mechanism responding to this kind of genotoxicity involves direct photoreversal by flavin enzymes that specifically monomerize pyrimidine:pyrimidine dimers monophotonically in the presence of visible light. Incision reactions are catalyzed by a combined pyrimidine dimer DNA-glycosylase:apyrimidinic endonuclease found in some highly UV-resistant organisms. At a higher level of complexity, Escherichia coli has a uvr DNA repair system comprising the UvrA, UvrB, and UvrC proteins responsible for incision. There are several preincision steps governed by this pathway, which includes an ATP-dependent UvrA dimerization reaction required for UvrAB nucleoprotein formation. This complex formation driven by ATP binding is associated with localized topological unwinding of DNA. This same protein complex can catalyze an ATPase-dependent 5'----3'-directed strand displacement of D-loop DNA or short single strands annealed to a single-stranded circular or linear DNA. This putative translocational process is arrested when damaged sites are encountered. The complex is now primed for dual incision catalyzed by UvrC. The remainder of the repair process involves UvrD (helicase II) and DNA polymerase I for a coordinately controlled excision-resynthesis step accompanied by UvrABC turnover. Furthermore, it is proposed that levels of repair proteins can be regulated by proteolysis. UvrB is converted to truncated UvrB* by a stress-induced protease that also acts at similar sites on the E. coli Ada protein. Although UvrB* can bind with UvrA to DNA, it cannot participate in helicase or incision reactions. It is also a DNA-dependent ATPase.21 references

  19. Repair of DNA-containing pyrimidine dimers

    Energy Technology Data Exchange (ETDEWEB)

    Grossman, L.; Caron, P.R.; Mazur, S.J.; Oh, E.Y.

    1988-08-01

    Ultraviolet light-induced pyrimidine dimers in DNA are recognized and repaired by a number of unique cellular surveillance systems. The most direct biochemical mechanism responding to this kind of genotoxicity involves direct photoreversal by flavin enzymes that specifically monomerize pyrimidine:pyrimidine dimers monophotonically in the presence of visible light. Incision reactions are catalyzed by a combined pyrimidine dimer DNA-glycosylase:apyrimidinic endonuclease found in some highly UV-resistant organisms. At a higher level of complexity, Escherichia coli has a uvr DNA repair system comprising the UvrA, UvrB, and UvrC proteins responsible for incision. There are several preincision steps governed by this pathway, which includes an ATP-dependent UvrA dimerization reaction required for UvrAB nucleoprotein formation. This complex formation driven by ATP binding is associated with localized topological unwinding of DNA. This same protein complex can catalyze an ATPase-dependent 5'----3'-directed strand displacement of D-loop DNA or short single strands annealed to a single-stranded circular or linear DNA. This putative translocational process is arrested when damaged sites are encountered. The complex is now primed for dual incision catalyzed by UvrC. The remainder of the repair process involves UvrD (helicase II) and DNA polymerase I for a coordinately controlled excision-resynthesis step accompanied by UvrABC turnover. Furthermore, it is proposed that levels of repair proteins can be regulated by proteolysis. UvrB is converted to truncated UvrB* by a stress-induced protease that also acts at similar sites on the E. coli Ada protein. Although UvrB* can bind with UvrA to DNA, it cannot participate in helicase or incision reactions. It is also a DNA-dependent ATPase.21 references.

  20. Nanoradar based on nonlinear dimer nanoantenna.

    Science.gov (United States)

    Lapshina, Nadezhda; Noskov, Roman; Kivshar, Yuri

    2012-09-15

    We introduce the concept of a nanoradar based on the operation of a nonlinear plasmonic nanoantenna. The nanoradar action originates from modulational instability occurring in a dimer nanoantenna consisting of two subwavelength nonlinear nanoparticles. Modulation instability causes a dynamical energy exchange between the nanoantenna eigenmodes resulting in periodic scanning of the nanoantenna scattering pattern. Such nanoradar demonstrates a wide scanning sector, low operation threshold, and ultrafast time response being potentially useful for many applications in nanophotonics circuitry. PMID:23041904

  1. Fractionalization in dimerized graphene and graphene bilayer

    OpenAIRE

    Milovanović, M. V.

    2008-01-01

    We show that the fractional statistics of quasiparticles in dimerized graphene, in recent proposals for charge and statistics fractionalization, can have two realizations depending whether elementary objects can be considered as point-like or extended objects. Therefore, there are two phases of proposed excitations and we give their topological descriptions with their respective statistics. We propose that a natural setting for fractionalization are certain systems with excitonic instabilitie...

  2. On the dimerization of linear polymers

    International Nuclear Information System (INIS)

    We use the continuum limit of the Su-Schrieffer-Heeger model for linear polymers to construct its effective potential (Gibbs free energy) both at zero and finite temperature. We study both trans and cis-polymers. Our results show that, depending on a renormalization condition to be extracted from experiment, there are several possibilities for the minima of the dimerized ground state of cis-polymers. All calculations are done in the one-loop approximation. (author). 16 refs, 3 figs

  3. THE FULL-LENGTH cDNA LIBRARY OF HEMOCYTE INDUCED BY AEROMONAS HYDROPHILA AND MOLECULAR CHARACTERISTICS OF CYCLOPHILINA FROM HYRIOPSIS SCHLEGELH%嗜水气单胞菌诱导的池蝶蚌血细胞cDNA文库的构建和亲环蛋白基因序列的初步分析

    Institute of Scientific and Technical Information of China (English)

    谢凯; 徐灵; 盛军庆; 曾柳根; 王军花; 洪一江

    2011-01-01

    实验利用灭活的嗜水气单胞菌(Aeromonas hydrophila)诱导处于四龄池蝶蚌(Hyriopsis schlegelii)14h,将诱导后的池蝶蚌血细胞的总RNA进行逆转录,用LD-PCR法合成双链cDNA,从而首次成功构建池蝶蚌血细胞的全长cDNA文库.原始文库的滴度为4× 106cfu/cm3,重组率为90%,扩增后文库的滴度为3.55× 109pfu/mL.目前文库已随机测序672个样品,将所得双向序列进行拼接,去除载体,并多序列比对去除重复序列后,发现436条为已知功能序列,其余为未知功能序列.序列中最小长度270 bp,最大长度为2153 bp,平均大小608.6 bp,表明插入片段大小理想.从文库中筛选获得免疫相关基因池蝶蚌亲环蛋白A(HsCypA)全长基因并进行序列分析.结果显示,HsCypA全长1229 bp,序列包括52 bp的5’非编码区、495 bp的开放阅读框、682 bp的3’非编码区和29 bp的poly(A)尾,没有明显的加尾信号.对Cyp A氨基酸序列二级结构进行了较详细的分析并进行了三维建模,同时构建了其系统进化树,分析表明亲环蛋白家族是一个在进化上非常保守的蛋白家族.综合分析,Cyp A在水生动物中不仅仅只是一种组成型蛋白,而是可能在病原感染防御中发挥重要作用.%Hyriopsis schlegelii, originated from the Lake BIWA of Japan, was introduced into China in 1997. In order to seek for genes related to the freshwater mussel on immune system, a full-length haemocyts cDNA library was constructed by using SMART (switching mechanism at 5' end of the RNA transcript) technique. The total RNA was isolated from the four years old mussel blood hemocyte induced by Aeromonas hydrophila for 14 hours. The "anchor first-strand cDNA" containing a sites (A & B) of symmetrical sfi I restriction enzyme was synthesized by reverse transcription, and the double-strand cDNA was synthesized and amplified by LD-PCR (long-distance PCR). After digested by the proteinase K and sfi I restriction enzyme, size

  4. High affinity binding of hydrophobic and autoantigenic regions of proinsulin to the 70 kDa chaperone DnaK

    OpenAIRE

    Schloot Nanette C; Fingberg Waltraud; Alloza Iraide; Vandenbroeck Koen; Blasius Elias; Siegenthaler Rahel K; Burkart Volker; Christen Philipp; Kolb Hubert

    2010-01-01

    Abstract Background Chaperones facilitate proper folding of peptides and bind to misfolded proteins as occurring during periods of cell stress. Complexes of peptides with chaperones induce peptide-directed immunity. Here we analyzed the interaction of (pre)proinsulin with the best characterized chaperone of the hsp70 family, bacterial DnaK. Results Of a set of overlapping 13-mer peptides of human preproinsulin high affinity binding to DnaK was found for the signal peptide and one further regi...

  5. T-shaped dimer of coronene

    CERN Document Server

    Obolensky, O I; Semenikhina, V V; Solovyov, A V

    2005-01-01

    An evidence of importance of the T-shaped configuration of coronene dimer is presented. Thus, the dimer's lowest energy configuration is not necessarily a stack, as it might had been expected a priori. This is a surprising result for the dimer of such a large polycyclic aromatic hydrocarbon (PAH) as coronene for which a graphite-like stack.The energy of the T-shaped configuration at all considered levels of density functional theory (B3LYP,PBE/6-31+G(d),D95,cc-pVDZ,cc-pVTZ) was systematically lower than the energy of the three plausible stack configurations. In order to get a better description of the van der Waals interaction,the density functional theory (DFT) results were adjusted by adding a phenomenological Lennard-Jones-type term into the total energy of the system. However, the van der Waals correction is somewhat arbitrary and its magnitude can not be rigorously justified. Depending on the choice of the parameters in the phenomenological term both the T-shaped and the parallel-displaced (PD) stack con...

  6. Probing the HIV-1 genomic RNA trafficking pathway and dimerization by genetic recombination and single virion analyses.

    Directory of Open Access Journals (Sweden)

    Michael D Moore

    2009-10-01

    Full Text Available Once transcribed, the nascent full-length RNA of HIV-1 must travel to the appropriate host cell sites to be translated or to find a partner RNA for copackaging to form newly generated viruses. In this report, we sought to delineate the location where HIV-1 RNA initiates dimerization and the influence of the RNA transport pathway used by the virus on downstream events essential to viral replication. Using a cell-fusion-dependent recombination assay, we demonstrate that the two RNAs destined for copackaging into the same virion select each other mostly within the cytoplasm. Moreover, by manipulating the RNA export element in the viral genome, we show that the export pathway taken is important for the ability of RNA molecules derived from two viruses to interact and be copackaged. These results further illustrate that at the point of dimerization the two main cellular export pathways are partially distinct. Lastly, by providing Gag in trans, we have demonstrated that Gag is able to package RNA from either export pathway, irrespective of the transport pathway used by the gag mRNA. These findings provide unique insights into the process of RNA export in general, and more specifically, of HIV-1 genomic RNA trafficking.

  7. Dimer monomer transition and dimer re-formation play important role for ATM cellular function during DNA repair

    Energy Technology Data Exchange (ETDEWEB)

    Du, Fengxia [Laboratory of Genome Variations and Precision Bio-Medicine, Beijing Institute of Genomics, Chinese Academy of Sciences, Beijing 100101 (China); Zhang, Minjie [Laboratory of Genome Variations and Precision Bio-Medicine, Beijing Institute of Genomics, Chinese Academy of Sciences, Beijing 100101 (China); University of Chinese Academy of Sciences, Beijing 100049 (China); Li, Xiaohua; Yang, Caiyun [Laboratory of Genome Variations and Precision Bio-Medicine, Beijing Institute of Genomics, Chinese Academy of Sciences, Beijing 100101 (China); Meng, Hao; Wang, Dong; Chang, Shuang [Laboratory of Genome Variations and Precision Bio-Medicine, Beijing Institute of Genomics, Chinese Academy of Sciences, Beijing 100101 (China); University of Chinese Academy of Sciences, Beijing 100049 (China); Xu, Ye [Department of Radiation Oncology, Division of Genomic Stability, Dana Farber Cancer Institute, Harvard Medical School, MA 02134 (United States); Price, Brendan, E-mail: Brendan_Price@dfci.harvard.edu [Department of Radiation Oncology, Division of Genomic Stability, Dana Farber Cancer Institute, Harvard Medical School, MA 02134 (United States); Sun, Yingli, E-mail: sunyl@big.ac.cn [Laboratory of Genome Variations and Precision Bio-Medicine, Beijing Institute of Genomics, Chinese Academy of Sciences, Beijing 100101 (China)

    2014-10-03

    Highlights: • ATM phosphorylates the opposite strand of the dimer in response to DNA damage. • The PETPVFRLT box of ATM plays a key role in its dimer dissociation in DNA repair. • The dephosphorylation of ATM is critical for dimer re-formation after DNA repair. - Abstract: The ATM protein kinase, is a serine/threonine protein kinase that is recruited and activated by DNA double-strand breaks, mediates responses to ionizing radiation in mammalian cells. Here we show that ATM is held inactive in unirradiated cells as a dimer and phosphorylates the opposite strand of the dimer in response to DNA damage. Cellular irradiation induces rapid intermolecular autophosphorylation of serine 1981 that causes dimer dissociation and initiates cellular ATM kinase activity. ATM cannot phosphorylate the substrates when it could not undergo dimer monomer transition. After DNA repair, the active monomer will undergo dephosphorylation to form dimer again and dephosphorylation is critical for dimer re-formation. Our work reveals novel function of ATM dimer monomer transition and explains why ATM dimer monomer transition plays such important role for ATM cellular activity during DNA repair.

  8. Dimer monomer transition and dimer re-formation play important role for ATM cellular function during DNA repair

    International Nuclear Information System (INIS)

    Highlights: • ATM phosphorylates the opposite strand of the dimer in response to DNA damage. • The PETPVFRLT box of ATM plays a key role in its dimer dissociation in DNA repair. • The dephosphorylation of ATM is critical for dimer re-formation after DNA repair. - Abstract: The ATM protein kinase, is a serine/threonine protein kinase that is recruited and activated by DNA double-strand breaks, mediates responses to ionizing radiation in mammalian cells. Here we show that ATM is held inactive in unirradiated cells as a dimer and phosphorylates the opposite strand of the dimer in response to DNA damage. Cellular irradiation induces rapid intermolecular autophosphorylation of serine 1981 that causes dimer dissociation and initiates cellular ATM kinase activity. ATM cannot phosphorylate the substrates when it could not undergo dimer monomer transition. After DNA repair, the active monomer will undergo dephosphorylation to form dimer again and dephosphorylation is critical for dimer re-formation. Our work reveals novel function of ATM dimer monomer transition and explains why ATM dimer monomer transition plays such important role for ATM cellular activity during DNA repair

  9. Molecular and biochemical characterization of a unique mutation in CCS, the human copper chaperone to superoxide dismutase

    DEFF Research Database (Denmark)

    Huppke, Peter; Brendel, Cornelia; Korenke, Georg Christoph; Marquardt, Iris; Donsante, Anthony; Yi, Ling; Hicks, Julia D; Steinbach, Peter J; Wilson, Callum; Elpeleg, Orly; Møller, Lisbeth Birk; Christodoulou, John; Kaler, Stephen G; Gärtner, Jutta

    2012-01-01

    Copper (Cu) is a trace metal that readily gains and donates electrons, a property that renders it desirable as an enzyme cofactor but dangerous as a source of free radicals. To regulate cellular Cu metabolism, an elaborate system of chaperones and transporters has evolved, although no human Cu...... chaperone mutations have been described to date. We describe a child from a consanguineous family who inherited homozygous mutations in the SLC33A1, encoding an acetyl CoA transporter, and in CCS, encoding the Cu chaperone for superoxide dismutase. The CCS mutation, p.Arg163Trp, predicts substitution of a...... gene coding for a Cu chaperone....

  10. AR-12 Inhibits Multiple Chaperones Concomitant With Stimulating Autophagosome Formation Collectively Preventing Virus Replication.

    Science.gov (United States)

    Booth, Laurence; Roberts, Jane L; Ecroyd, Heath; Tritsch, Sarah R; Bavari, Sina; Reid, St Patrick; Proniuk, Stefan; Zukiwski, Alexander; Jacob, Abraham; Sepúlveda, Claudia S; Giovannoni, Federico; García, Cybele C; Damonte, Elsa; González-Gallego, Javier; Tuñón, María J; Dent, Paul

    2016-10-01

    We have recently demonstrated that AR-12 (OSU-03012) reduces the function and ATPase activities of multiple HSP90 and HSP70 family chaperones. Combined knock down of chaperones or AR-12 treatment acted to reduce the expression of virus receptors and essential glucosidase proteins. Combined knock down of chaperones or AR-12 treatment inactivated mTOR and elevated ATG13 S318 phosphorylation concomitant with inducing an endoplasmic reticulum stress response that in an eIF2α-dependent fashion increased Beclin1 and LC3 expression and autophagosome formation. Over-expression of chaperones prevented the reduction in receptor/glucosidase expression, mTOR inactivation, the ER stress response, and autophagosome formation. AR-12 reduced the reproduction of viruses including Mumps, Influenza, Measles, Junín, Rubella, HIV (wild type and protease resistant), and Ebola, an effect replicated by knock down of multiple chaperone proteins. AR-12-stimulated the co-localization of Influenza, EBV and HIV virus proteins with LC3 in autophagosomes and reduced viral protein association with the chaperones HSP90, HSP70, and GRP78. Knock down of Beclin1 suppressed drug-induced autophagosome formation and reduced the anti-viral protection afforded by AR-12. In an animal model of hemorrhagic fever virus, a transient exposure of animals to low doses of AR-12 doubled animal survival from ∼30% to ∼60% and suppressed liver damage as measured by ATL, GGT and LDH release. Thus through inhibition of chaperone protein functions; reducing the production, stability and processing of viral proteins; and stimulating autophagosome formation/viral protein degradation, AR-12 acts as a broad-specificity anti-viral drug in vitro and in vivo. We argue future patient studies with AR-12 are warranted. J. Cell. Physiol. 231: 2286-2302, 2016. © 2016 Wiley Periodicals, Inc. PMID:27187154

  11. Structural basis for recognition of H3K56-acetylated histone H3-H4 by the chaperone Rtt106

    Energy Technology Data Exchange (ETDEWEB)

    Su, Dan; Hu, Qi; Li, Qing; Thompson, James R; Cui, Gaofeng; Fazly, Ahmed; Davies, Brian A; Botuyan, Maria Victoria; Zhang, Zhiguo; Mer, Georges [Mayo

    2013-04-08

    Dynamic variations in the structure of chromatin influence virtually all DNA-related processes in eukaryotes and are controlled in part by post-translational modifications of histones. One such modification, the acetylation of lysine 56 (H3K56ac) in the amino-terminal α-helix (αN) of histone H3, has been implicated in the regulation of nucleosome assembly during DNA replication and repair, and nucleosome disassembly during gene transcription. In Saccharomyces cerevisiae, the histone chaperone Rtt106 contributes to the deposition of newly synthesized H3K56ac-carrying H3-H4 complex on replicating DNA, but it is unclear how Rtt106 binds H3-H4 and specifically recognizes H3K56ac as there is no apparent acetylated lysine reader domain in Rtt106. Here, we show that two domains of Rtt106 are involved in a combinatorial recognition of H3-H4. An N-terminal domain homodimerizes and interacts with H3-H4 independently of acetylation while a double pleckstrin-homology (PH) domain binds the K56-containing region of H3. Affinity is markedly enhanced upon acetylation of K56, an effect that is probably due to increased conformational entropy of the αN helix of H3. Our data support a mode of interaction where the N-terminal homodimeric domain of Rtt106 intercalates between the two H3-H4 components of the (H3-H4)2 tetramer while two double PH domains in the Rtt106 dimer interact with each of the two H3K56ac sites in (H3-H4)2. We show that the Rtt106-(H3-H4)2 interaction is important for gene silencing and the DNA damage response.

  12. MspA nanopores from subunit dimers.

    Directory of Open Access Journals (Sweden)

    Mikhail Pavlenok

    Full Text Available Mycobacterium smegmatis porin A (MspA forms an octameric channel and represents the founding member of a new family of pore proteins. Control of subunit stoichiometry is important to tailor MspA for nanotechnological applications. In this study, two MspA monomers were connected by linkers ranging from 17 to 62 amino acids in length. The oligomeric pore proteins were purified from M. smegmatis and were shown to form functional channels in lipid bilayer experiments. These results indicated that the peptide linkers did not prohibit correct folding and localization of MspA. However, expression levels were reduced by 10-fold compared to wild-type MspA. MspA is ideal for nanopore sequencing due to its unique pore geometry and its robustness. To assess the usefulness of MspA made from dimeric subunits for DNA sequencing, we linked two M1-MspA monomers, whose constriction zones were modified to enable DNA translocation. Lipid bilayer experiments demonstrated that this construct also formed functional channels. Voltage gating of MspA pores made from M1 monomers and M1-M1 dimers was identical indicating similar structural and dynamic channel properties. Glucose uptake in M. smegmatis cells lacking porins was restored by expressing the dimeric mspA M1 gene indicating correct folding and localization of M1-M1 pores in their native membrane. Single-stranded DNA hairpins produced identical ionic current blockades in pores made from monomers and subunit dimers demonstrating that M1-M1 pores are suitable for DNA sequencing. This study provides the proof of principle that production of single-chain MspA pores in M. smegmatis is feasible and paves the way for generating MspA pores with altered stoichiometries. Subunit dimers enable better control of the chemical and physical properties of the constriction zone of MspA. This approach will be valuable both in understanding transport across the outer membrane in mycobacteria and in tailoring MspA for nanopore

  13. MspA nanopores from subunit dimers.

    Science.gov (United States)

    Pavlenok, Mikhail; Derrington, Ian M; Gundlach, Jens H; Niederweis, Michael

    2012-01-01

    Mycobacterium smegmatis porin A (MspA) forms an octameric channel and represents the founding member of a new family of pore proteins. Control of subunit stoichiometry is important to tailor MspA for nanotechnological applications. In this study, two MspA monomers were connected by linkers ranging from 17 to 62 amino acids in length. The oligomeric pore proteins were purified from M. smegmatis and were shown to form functional channels in lipid bilayer experiments. These results indicated that the peptide linkers did not prohibit correct folding and localization of MspA. However, expression levels were reduced by 10-fold compared to wild-type MspA. MspA is ideal for nanopore sequencing due to its unique pore geometry and its robustness. To assess the usefulness of MspA made from dimeric subunits for DNA sequencing, we linked two M1-MspA monomers, whose constriction zones were modified to enable DNA translocation. Lipid bilayer experiments demonstrated that this construct also formed functional channels. Voltage gating of MspA pores made from M1 monomers and M1-M1 dimers was identical indicating similar structural and dynamic channel properties. Glucose uptake in M. smegmatis cells lacking porins was restored by expressing the dimeric mspA M1 gene indicating correct folding and localization of M1-M1 pores in their native membrane. Single-stranded DNA hairpins produced identical ionic current blockades in pores made from monomers and subunit dimers demonstrating that M1-M1 pores are suitable for DNA sequencing. This study provides the proof of principle that production of single-chain MspA pores in M. smegmatis is feasible and paves the way for generating MspA pores with altered stoichiometries. Subunit dimers enable better control of the chemical and physical properties of the constriction zone of MspA. This approach will be valuable both in understanding transport across the outer membrane in mycobacteria and in tailoring MspA for nanopore sequencing of DNA. PMID

  14. Peptides Interfering 3A Protein Dimerization Decrease FMDV Multiplication.

    Directory of Open Access Journals (Sweden)

    Mónica González-Magaldi

    Full Text Available Nonstructural protein 3A is involved in relevant functions in foot-and-mouth disease virus (FMDV replication. FMDV 3A can form homodimers and preservation of the two hydrophobic α-helices (α1 and α2 that stabilize the dimer interface is essential for virus replication. In this work, small peptides mimicking residues involved in the dimer interface were used to interfere with dimerization and thus gain insight on its biological function. The dimer interface peptides α1, α2 and that spanning the two hydrophobic α-helices, α12, impaired in vitro dimer formation of a peptide containing the two α-helices, this effect being higher with peptide α12. To assess the effect of dimer inhibition in cultured cells, the interfering peptides were N-terminally fused to a heptaarginine (R7 sequence to favor their intracellular translocation. Thus, when fused to R7, interference peptides (100 μM were able to inhibit dimerization of transiently expressed 3A, the higher inhibitions being found with peptides α1 and α12. The 3A dimerization impairment exerted by the peptides correlated with significant, specific reductions in the viral yield recovered from peptide-treated FMDV infected cells. In this case, α2 was the only peptide producing significant reductions at concentrations lower than 100 μM. Thus, dimer interface peptides constitute a tool to understand the structure-function relationship of this viral protein and point to 3A dimerization as a potential antiviral target.

  15. Molecular Chaperones of Leishmania: Central Players in Many Stress-Related and -Unrelated Physiological Processes

    Directory of Open Access Journals (Sweden)

    Jose M. Requena

    2015-01-01

    Full Text Available Molecular chaperones are key components in the maintenance of cellular homeostasis and survival, not only during stress but also under optimal growth conditions. Folding of nascent polypeptides is supported by molecular chaperones, which avoid the formation of aggregates by preventing nonspecific interactions and aid, when necessary, the translocation of proteins to their correct intracellular localization. Furthermore, when proteins are damaged, molecular chaperones may also facilitate their refolding or, in the case of irreparable proteins, their removal by the protein degradation machinery of the cell. During their digenetic lifestyle, Leishmania parasites encounter and adapt to harsh environmental conditions, such as nutrient deficiency, hypoxia, oxidative stress, changing pH, and shifts in temperature; all these factors are potential triggers of cellular stress. We summarize here our current knowledge on the main types of molecular chaperones in Leishmania and their functions. Among them, heat shock proteins play important roles in adaptation and survival of this parasite against temperature changes associated with its passage from the poikilothermic insect vector to the warm-blooded vertebrate host. The study of structural features and the function of chaperones in Leishmania biology is providing opportunities (and challenges for drug discovery and improving of current treatments against leishmaniasis.

  16. ATP-dependent molecular chaperones in plastids--More complex than expected.

    Science.gov (United States)

    Trösch, Raphael; Mühlhaus, Timo; Schroda, Michael; Willmund, Felix

    2015-09-01

    Plastids are a class of essential plant cell organelles comprising photosynthetic chloroplasts of green tissues, starch-storing amyloplasts of roots and tubers or the colorful pigment-storing chromoplasts of petals and fruits. They express a few genes encoded on their organellar genome, called plastome, but import most of their proteins from the cytosol. The import into plastids, the folding of freshly-translated or imported proteins, the degradation or renaturation of denatured and entangled proteins, and the quality-control of newly folded proteins all require the action of molecular chaperones. Members of all four major families of ATP-dependent molecular chaperones (chaperonin/Cpn60, Hsp70, Hsp90 and Hsp100 families) have been identified in plastids from unicellular algae to higher plants. This review aims not only at giving an overview of the most current insights into the general and conserved functions of these plastid chaperones, but also into their specific plastid functions. Given that chloroplasts harbor an extreme environment that cycles between reduced and oxidized states, that has to deal with reactive oxygen species and is highly reactive to environmental and developmental signals, it can be presumed that plastid chaperones have evolved a plethora of specific functions some of which are just about to be discovered. Here, the most urgent questions that remain unsolved are discussed, and guidance for future research on plastid chaperones is given. This article is part of a Special Issue entitled: Chloroplast Biogenesis. PMID:25596449

  17. Engineering and Evolution of Molecular Chaperones and Protein Disaggregases with Enhanced Activity

    Science.gov (United States)

    Mack, Korrie L.; Shorter, James

    2016-01-01

    Cells have evolved a sophisticated proteostasis network to ensure that proteins acquire and retain their native structure and function. Critical components of this network include molecular chaperones and protein disaggregases, which function to prevent and reverse deleterious protein misfolding. Nevertheless, proteostasis networks have limits, which when exceeded can have fatal consequences as in various neurodegenerative disorders, including Parkinson's disease and amyotrophic lateral sclerosis. A promising strategy is to engineer proteostasis networks to counter challenges presented by specific diseases or specific proteins. Here, we review efforts to enhance the activity of individual molecular chaperones or protein disaggregases via engineering and directed evolution. Remarkably, enhanced global activity or altered substrate specificity of various molecular chaperones, including GroEL, Hsp70, ClpX, and Spy, can be achieved by minor changes in primary sequence and often a single missense mutation. Likewise, small changes in the primary sequence of Hsp104 yield potentiated protein disaggregases that reverse the aggregation and buffer toxicity of various neurodegenerative disease proteins, including α-synuclein, TDP-43, and FUS. Collectively, these advances have revealed key mechanistic and functional insights into chaperone and disaggregase biology. They also suggest that enhanced chaperones and disaggregases could have important applications in treating human disease as well as in the purification of valuable proteins in the pharmaceutical sector. PMID:27014702

  18. Heterogeneity in the properties of NEFL mutants causing Charcot-Marie-Tooth disease results in differential effects on neurofilament assembly and susceptibility to intervention by the chaperone-inducer, celastrol.

    Science.gov (United States)

    Gentil, Benoit J; Mushynski, Walter E; Durham, Heather D

    2013-07-01

    Aberrant aggregation of neurofilament proteins is a common feature of neurodegenerative diseases. For example, neurofilament light protein (NEFL) mutants causing Charcot-Marie-Tooth disease induce misassembly of neurofilaments. This study demonstrated that mutations in different functional domains of NEFL have different effects on filament assembly and susceptibility to interventions to restore function. The mouse NEFL mutants, NEFL(Q333P) and NEFL(P8R), exhibited different assembly properties in SW13-cells, cells lacking endogenous intermediate filaments, indicating different consequences of these mutations on the biochemical properties of NEFL. The p.Q333P mutation caused reversible misfolding of the protein. NEFL(Q333P) could be refolded and form coil-coiled dimers, in vitro using chaotropic agent, and in cultured cells by induction of HSPA1 and HSPB1. Celastrol, an inducer of chaperone proteins, induced HSPA1 expression in motor neurons and prevented the formation of neurofilament inclusions and mitochondrial shortening induced by expression of NEFL(Q333P), but not in sensory neurons. Conversely, celastrol had a protective effect against the toxicity of NEFL(P8R), a mutant which is sensitive to HSBP1 but not HSPA1 chaperoning, only in large-sized sensory neurons, not in motor neurons. Importantly, sensory and motor neurons do not respond identically to celastrol and different chaperones are upregulated by the same treatment. Thus, effective therapy of CMT not only depends on the identity of the mutated gene, but the consequences of the specific mutation on the properties of the protein and the neuronal population targeted. PMID:23618875

  19. SMARTer技术构建辣椒黄绿苗突变体叶片全长cDNA文库%Construction of full-length cDNA library of yellow bud mutant leaves in Capsicum annuum L.using SMARTer technique

    Institute of Scientific and Technical Information of China (English)

    马志虎; 孙国胜; 张昌伟; 杨玉霞; 潘跃平

    2013-01-01

    本研究以辣椒黄绿苗嫩叶为材料,提取总RNA,采用LD-PCR技术合成First-strand cDNA和ds cDNA.将分级纯化后的ds cDNA连接到载体pSMART2IFD上,用电穿孔法将重组子转化到大肠杆菌感受态细胞DH5α中,构建辣椒全长cDNA文库.文库质量检测结果显示:原始文库滴度为1.76×106 PFU/ml,重组率为94%,插入片段长度为500~2 000 bp,平均长度为1 170 bp,表明构建的辣椒叶片cDNA文库较为理想,可用于目的基因筛选.%Total RNA was extracted from yellow bud mutant leaves of Capsicum annuum L. , and first-strand cDNA and ds cDNA were synthesized by LD-PCR technology. The purified ds cDNA was connected to vector pSMART2IFD, and the recombinant vectors were transformed into competent Escherichia coli cells DH5a by electroporation to construct full-length cDNA library of Capsicum annuum L_ The library quality test results showed the titer of original library was 1.76× 106PFU/ml, the recombination rate was 94% , and the inserted fragment length was 500-2 000 bp, indicating that the library was ideal for target genes selection.

  20. E2 polypeptides encoded by bovine papillomavirus type 1 form dimers through the common carboxyl-terminal domain: transactivation is mediated by the conserved amino-terminal domain.

    Science.gov (United States)

    McBride, A A; Byrne, J C; Howley, P M

    1989-01-01

    The E2 open reading frame (ORF) of bovine papillomavirus type 1 (BPV-1) encodes positive- and negative-acting factors that regulate viral gene expression. The full-length ORF encodes a transactivator, and two transcriptional repressors are expressed from the 3' half of the ORF. Previous analysis has shown that a conserved C-terminal region of 101 amino acids, which is shared by E2 transactivator and repressor proteins, contains the specific DNA binding activity. Further analysis of the E2 transactivator shows that a conserved N-terminal domain of approximately 220 amino acids is crucial for the transcriptional activation function, whereas the variable internal region is not required. The E2 proteins bind to a sequence, ACCGN4CGGT, several copies of which are sufficient to constitute an E2-dependent enhancer. By using a gel retardation assay and proteins derived by in vitro transcription and translation, we were able to show that the E2 polypeptides bind as dimers to a single DNA binding site. The dimeric E2 proteins are stable in the absence of DNA and dimerization is mediated through the DNA binding domain. This may reveal an additional mechanism of repression that could potentially result from the formation of inactive heterodimers between transactivator and repressor species. PMID:2536165