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Sample records for full-length recombinant human

  1. Crystallization and X-ray structure of full-length recombinant human butyrylcholinesterase

    SciTech Connect

    Ngamelue, Michelle N.; Homma, Kohei; Lockridge, Oksana; Asojo, Oluwatoyin A.

    2007-09-01

    The first crystals and the 2.8 Å X-ray structure of full-length recombinant human butyrylcholinesterase are reported. Human butyrylcholinesterase (BChE) has been shown to function as an endogenous scavenger of diverse poisons. BChE is a 340 kDa tetrameric glycoprotein that is present in human serum at a concentration of 5 mg l{sup −1}. The well documented therapeutic effects of BChE on cocaine toxicity and organophosphorus agent poisoning has increased the need for effective methods of producing recombinant therapeutic BChE. In order to be therapeutically useful, BChE must have a long circulatory residence time or associate as tetramers. Full-length recombinant BChE produced in Chinese hamster ovary (CHO) cells or human embryonic kidney cells has been shown to associate as monomers, with a shorter circulatory residence time than the naturally occurring tetrameric serum protein. Based on the preceding observation as well as the need to develop novel methodologies to facilitate the mass production of therapeutic recombinant BChE, studies have been initiated to determine the structural basis of tetramer formation. Towards these ends, full-length monomeric recombinant BChE has been crystallized for the first time. A 2.8 Å X-ray structure was solved in space group P42{sub 1}2, with unit-cell parameters a = b = 156, c = 146 Å.

  2. Characterization of full-length recombinant human Proteoglycan 4 as an ocular surface boundary lubricant.

    PubMed

    Samsom, Michael L; Morrison, Sheila; Masala, Nemanja; Sullivan, Benjamin D; Sullivan, David A; Sheardown, Heather; Schmidt, Tannin A

    2014-10-01

    Proteoglycan 4 (PRG4, or lubricin) is a lubricating mucin-like glycoprotein recently discovered at the ocular surface, where it functions as a boundary lubricant and appears to play a protective role. Recent technological advances have enabled abundant expression of full-length recombinant human PRG4 (rhPRG4). The objectives of this study were to 1) biochemically characterize the gross structure and glycosylations of full-length rhPRG4, and 2) assess the ocular surface boundary lubricating ability of rhPRG4 at both human cornea-eyelid and human cornea-polydimethylsiloxane (PDMS) biointerfaces. rhPRG4 expressed by a Chinese hamster ovary cell line was characterized and compared to native bovine PRG4 by SDS-PAGE western blotting, and protein identity was assessed by tandem mass spectrometry (MS/MS). Human corneas were articulated against PDMS or human eyelids, at effective sliding velocities of 0.3-30 mm/s under physiological loads of ∼15 kPa, to assess and compare the ocular lubricating ability of rhPRG4 to PRG4. Samples were tested serially in PRG4, rhPRG4 (both 300 μg/ml), then saline. Western blotting indicated that rhPRG4 had immunoreactivity at the appropriate apparent molecular weight, and possessed O-linked glycosylation consistent with that of PRG4. rhPRG4 protein identity was confirmed by MS/MS. Both PRG4 and rhPRG4 significantly, and similarly, reduced friction compared to saline at both human cornea - PDMS and human cornea-eyelid biointerfaces. In conclusion, the rhPRG4 studied here demonstrated appropriate higher order structure, O-linked glycosylations, and ocular surface boundary lubricating. Purified rhPRG4 may have clinical utility as a topical treatment of dry eye disease or contact lens biomaterial coating to promote more comfortable wear. PMID:24997456

  3. Production, purification and characterization of recombinant, full-length human claudin-1.

    PubMed

    Bonander, Nicklas; Jamshad, Mohammed; Oberthür, Dominik; Clare, Michelle; Barwell, James; Hu, Ke; Farquhar, Michelle J; Stamataki, Zania; Harris, Helen J; Dierks, Karsten; Dafforn, Timothy R; Betzel, Christian; McKeating, Jane A; Bill, Roslyn M

    2013-01-01

    The transmembrane domain proteins of the claudin superfamily are the major structural components of cellular tight junctions. One family member, claudin-1, also associates with tetraspanin CD81 as part of a receptor complex that is essential for hepatitis C virus (HCV) infection of the liver. To understand the molecular basis of claudin-1/CD81 association we previously produced and purified milligram quantities of functional, full-length CD81, which binds a soluble form of HCV E2 glycoprotein (sE2). Here we report the production, purification and characterization of claudin-1. Both yeast membrane-bound and detergent-extracted, purified claudin-1 were antigenic and recognized by specific antibodies. Analytical ultracentrifugation demonstrated that extraction with n-octyl-β-d-glucopyranoside yielded monodispersed, dimeric pools of claudin-1 while extraction with profoldin-8 or n-decylphosphocholine yielded a dynamic mixture of claudin-1 oligomers. Neither form bound sE2 in line with literature expectations, while further functional analysis was hampered by the finding that incorporation of claudin-1 into proteoliposomes rendered them intractable to study. Dynamic light scattering demonstrated that claudin-1 oligomers associate with CD81 in vitro in a defined molar ratio of 1∶2 and that complex formation was enhanced by the presence of cholesteryl hemisuccinate. Attempts to assay the complex biologically were limited by our finding that claudin-1 affects the properties of proteoliposomes. We conclude that recombinant, correctly-folded, full-length claudin-1 can be produced in yeast membranes, that it can be extracted in different oligomeric forms that do not bind sE2 and that a dynamic preparation can form a specific complex with CD81 in vitro in the absence of any other cellular components. These findings pave the way for the structural characterization of claudin-1 alone and in complex with CD81. PMID:23704991

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

    PubMed Central

    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 transmembrane protein CD20. Methods: In this study, we expressed the extra membrane loop of hCD20 (exCD20) consisting of a non-glycosylated 47-amino acids region. The exCD20 coding sequence was amplified by PCR and cloned in pET32a(+) expression vector. The desired protein was expressed in fusion with thioredoxin and 6× His tag in E. coli Origami strain. ELISA and Western-blotting data were performed to indicate the functionality of this protein. Results: We have obtained the exCD20 recombinant protein which can be detected in ELISA and Western-blot experiments. This recombinant fusion protein was soluble and stable without aggregation and misfolding problems. Conclusion: The recombinant extra membrane loop of human CD20 protein in fusion with thioredoxin (exCD20) can be used in function assays and some applications such as ELISA, immuneblotting, affinity purification, immunization, screening, and development of anti-CD20 antibodies. PMID:23023212

  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

    PubMed Central

    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

  6. Expression, purification, refolding and in vitro recovery of active full length recombinant human gelatinase MMP-9 in Escherichia coli.

    PubMed

    Mohseni, Sara; Moghadam, Tahereh Tohidi; Dabirmanesh, Bahareh; Khajeh, Khosro

    2016-10-01

    Human gelatinase (MMP-9) is a member of matrix metalloproteinases family (MMPs), which has been associated with malignant tumor progression and metastasis by matrix degradation. Herein, active full length recombinant human MMP-9 (amino acid residues 107-707) has been expressed in the form of inclusion bodies in Escherichia coli BL21, using pET21a vector. Solubilization of inclusion bodies was carried out in Tris-HCl buffer with 6 M urea, and refolding was performed using dilution and urea gradient methods. Tris-HCl buffer with 5 mM CaCl2 and 1 μM ZnCl2 at pH 7.8 was used as a refolding buffer. Analysis of the structure by fluorescence and far-UV circular dichroism showed a well-formed structure by urea gradient method. Kinetic parameters in refolding conditions of rhMMP-9 were also analyzed, depicting increase in the enzyme's activity without any aggregation. PMID:27164034

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

    PubMed

    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

  8. Therapeutic effects of recombinant forms of full-length and truncated human surfactant protein D in a murine model of invasive pulmonary aspergillosis.

    PubMed

    Singh, Mamta; Madan, Taruna; Waters, Patrick; Sonar, Sanchaita; Singh, Shiv K; Kamran, Mohammad F; Bernal, Andrés López; Sarma, P Usha; Singh, Vijay K; Crouch, Erika C; Kishore, Uday

    2009-07-01

    Aspergillus fumigatus (Afu) is an opportunistic fungal pathogen that can cause fatal invasive pulmonary aspergillosis (IPA) in immunocompromised individuals. Previously, surfactant protein D (SP-D), a surfactant-associated innate immune molecule, has been shown to enhance phagocytosis and killing of Afu conidia by phagocytic cells in vitro. An intranasal treatment of SP-D significantly increased survival in a murine model of IPA. Here we have examined mechanisms via which recombinant forms of full-length (hSP-D) or truncated human SP-D (rhSP-D) offer protection in a murine model of IPA that were immunosuppressed with hydrocortisone and challenged intranasally with Afu conidia prior to the treatment. SP-D or rhSP-D treatment increased the survival rate to 70% and 80%, respectively (100% mortality on day 7 in IPA mice), with concomitant reduction in the growth of fungal hyphae in the lungs, and increased levels of TNF-alpha and IFN-gamma in the lung suspension supernatants, as compared to untreated IPA mice. The level of macrophage inflammatory protein-1 alpha (MIP-1 alpha) in the lung cell suspension was also raised considerably following treatment with SP-D or rhSP-D. Our results appear to reaffirm the notion that under immunocompromised conditions, human SP-D or its truncated form can offer therapeutic protection against fatal challenge with Afu conidia challenge. Taken together, the SP-D-mediated protective mechanisms include enhanced phagocytosis by recruited macrophages and neutrophils and fungistatic properties, suppression of the levels of pathogenic Th2 cytokines (IL-4 and IL-5), enhanced local production of protective Th1 cytokines, TNF-alpha and IFN-gamma, and that of protective C-C chemokine, MIP-1 alpha. PMID:19403176

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

    NASA Astrophysics Data System (ADS)

    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.

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

    PubMed

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

    2016-03-21

    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. PMID:27004887

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

    PubMed Central

    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 coli and two commercial cell-free (CF) systems, wheat germ extract (WGE) and HeLa cell extract (HCE). Expression was assessed by labeling the fusion proteins specifically and covalently with a fluorescent HaloTag ligand and detecting its fluorescence on a LabChip® GX microfluidic capillary gel electrophoresis instrument. This automated, HT assay provided both qualitative and quantitative assessment of recombinant protein. E. coli was only capable of expressing 20% of the test collection in the supernatant fraction with ≥20 μg yields, whereas CF systems had ≥83% success rates. We purified expressed proteins using an automated HaloTag purification method. We purified 20, 33, and 42% of the test collection from E. coli, WGE, and HCE, respectively, with yields ≥1 μg and ≥90% purity. Based on these observations, we have developed a triage strategy for producing full-length human proteins in these three expression systems. PMID:24806540

  12. Recombinant production of functional full-length and truncated human TRAM/TICAM-2 adaptor protein involved in Toll-like receptor and interferon signaling.

    PubMed

    Ullah, M Obayed; Valkov, Eugene; Ve, Thomas; Williams, Simon; Mas, Caroline; Mansell, Ashley; Kobe, Bostjan

    2015-02-01

    TRAM/TICAM-2 is used by Toll-like receptor 4 (TLR4) as a bridging adaptor during the mammalian innate immune response. It recruits TRIF, another TIR domain-containing adaptor protein, to TLR4 via TIR domain interactions, which leads to the activation of transcription factors responsible for the production of type-1 interferon and cytokines. The molecular mechanisms of these dual interactions mediated by the TRAM TIR domain are not clear. To understand the molecular basis of TIR:TIR domain interactions, structural and biochemical studies of TRAM TIR domain are necessary, and require a functional soluble protein. In this paper, we report a successful purification and characterization of full-length TRAM. Because full-length TRAM likely contains unstructured regions that may be disadvantageous for structural studies, we also carried out a systematic construct design to determine the boundaries of the TRAM TIR domain. The truncated TRAM constructs were designed based on secondary structure predictions and screened by small-scale expression. Selected constructs were subjected to biophysical analyses. We show that the expressed TRAM TIR domain is functional using in vitro GST pull-down assays that demonstrate a physical interaction with the TLR4 TIR domain. We further show, by site-directed mutagenesis, that the "BB loop" regions of both the TRAM TIR domain and the TLR4 TIR domain are crucial for this physical interaction. PMID:25306876

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

    SciTech Connect

    Asojo, Oluwatoyin Ajibola; Asojo, Oluyomi Adebola; Ngamelue, Michelle N.; Homma, Kohei; Lockridge, Oksana

    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. Recombinant production and film properties of full-length hornet silk proteins.

    PubMed

    Kambe, Yusuke; Sutherland, Tara D; Kameda, Tsunenori

    2014-08-01

    Full-length versions of the four main components of silk cocoons of Vespa simillima hornets, Vssilk1-4, were produced as recombinant proteins in Escherichia coli. In shake flasks, the recombinant Vssilk proteins yielded 160-330mg recombinant proteinl(-1). Films generated from solutions of single Vssilk proteins had a secondary structure similar to that of films generated from native hornet silk. The films made from individual recombinant hornet silk proteins had similar or enhanced mechanical performance compared with films generated from native hornet silk, possibly reflecting the homogeneity of the recombinant proteins. The pH-dependent changes in zeta (ζ) potential of each Vssilk film were measured, and isoelectric points (pI) of Vssilk1-4 were determined as 8.9, 9.1, 5.0 and 4.2, respectively. The pI of native hornet silk, a combination of the four Vssilk proteins, was 4.7, a value similar to that of Bombyx mori silkworm silk. Films generated from Vssilk1 and 2 had net positive charge under physiological conditions and showed significantly higher cell adhesion activity. It is proposed that recombinant hornet silk is a valuable new material with potential for cell culture applications. PMID:24862540

  15. The recombinant C-terminus of the human MUC2 mucin forms dimers in Chinese-hamster ovary cells and heterodimers with full-length MUC2 in LS 174T cells.

    PubMed Central

    Lidell, Martin E; Johansson, Malin E V; Mörgelin, Matthias; Asker, Noomi; Gum, James R; Kim, Young S; Hansson, Gunnar C

    2003-01-01

    The entire cDNA corresponding to the C-terminal cysteine-rich domain of the human MUC2 apomucin, after the serine- and threonine-rich tandem repeat, was expressed in Chinese-hamster ovary-K1 cells and in the human colon carcinoma cell line, LS 174T. The C-terminus was expressed as a fusion protein with the green fluorescent protein and mycTag sequences and the murine immunoglobulin kappa-chain signal sequence to direct the protein to the secretory pathway. Pulse-chase studies showed a rapid conversion of the C-terminal monomer into a dimer in both Chinese-hamster ovary-K1 and LS 174T cells. Disulphide-bond-stabilized dimers secreted into the media of both cell lines had a higher apparent molecular mass compared with the intracellular forms. The MUC2 C-terminus was purified from the spent culture medium and visualized by molecular electron microscopy. The dimer nature of the molecule was visible clearly and revealed that each monomer was attached to the other by a large globular domain. Gold-labelled antibodies against the mycTag or green fluorescent protein revealed that these were localized to the ends opposite to the parts responsible for the dimerization. The C-terminus expressed in LS 174T cells formed heterodimers with the full-length wild-type MUC2, but not with the MUC5AC mucin, normally expressed in LS 174T cells. The homodimers of the MUC2 C-termini were secreted continuously from the LS 174T cells, but no wild-type MUC2 secretion has been observed from these cells. This suggests that the information for sorting the MUC2 mucin into the regulated secretory pathway in cells having this ability is present in parts other than the C-terminus of MUC2. PMID:12597771

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

    PubMed Central

    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.

    2016-01-01

    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

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

    PubMed

    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

  18. Milligram Quantities of Homogeneous Recombinant Full-Length Mouse Munc18c from Escherichia coli Cultures

    PubMed Central

    Rehman, Asma; Jarrott, Russell J.; Whitten, Andrew E.; King, Gordon J.; Hu, Shu-Hong; Christie, Michelle P.; Collins, Brett M.; Martin, Jennifer L.

    2013-01-01

    Vesicle fusion is an indispensable cellular process required for eukaryotic cargo delivery. The Sec/Munc18 protein Munc18c is essential for insulin-regulated trafficking of glucose transporter4 (GLUT4) vesicles to the cell surface in muscle and adipose tissue. Previously, our biophysical and structural studies have used Munc18c expressed in SF9 insect cells. However to maximize efficiency, minimize cost and negate any possible effects of post-translational modifications of Munc18c, we investigated the use of Escherichia coli as an expression host for Munc18c. We were encouraged by previous reports describing Munc18c production in E. coli cultures for use in in vitro fusion assay, pulldown assays and immunoprecipitations. Our approach differs from the previously reported method in that it uses a codon-optimized gene, lower temperature expression and autoinduction media. Three N-terminal His-tagged constructs were engineered, two with a tobacco etch virus (TEV) or thrombin protease cleavage site to enable removal of the fusion tag. The optimized protocol generated 1–2 mg of purified Munc18c per L of culture at much reduced cost compared to Munc18c generated using insect cell culture. The purified recombinant Munc18c protein expressed in bacteria was monodisperse, monomeric, and functional. In summary, we developed methods that decrease the cost and time required to generate functional Munc18c compared with previous insect cell protocols, and which generates sufficient purified protein for structural and biophysical studies. PMID:24391775

  19. Near full-length genomic characterization of a HIV type 1 BC recombinant strain from Manipur, India.

    PubMed

    Sarkar, Roni; Sarkar, Kamalesh; Singh, N Brajachand; Singh, Y Manihar; Chakrabarti, Sekhar

    2012-10-01

    Genetic complexity of HIV-1 is brought about by recombination between HIV-1 subtypes which leads to the development of epidemiologically significant founder strains. In the present study, the near full-length genome sequence of an HIV-1 isolate from an injecting drug user of Manipur (India) was determined, which evidenced the presence of a novel HIV-1 BC recombinant strain. Near full-length genome was amplified by polymerase chain reaction using primer walking approach. The recombination break points were detected using bootscan and simplot analyses. This isolate exhibited a mosaic structure consisting of subtype C backbone with subtype B insertions at the upstream of pol gene (3026-3259) and the downstream of env gene which spanned till the nef gene (8183-8961). Phylogenetic relationships determined with neighbor-joining trees, revealed that the subtype C sequences clustered with sequences from Indian subtype C HIV-1 strains, and the subtype B sequences clustered with HIV-1 subtype B strains from Thailand. This finding may create a complex scenario of HIV-1 epidemic among the injecting drug users of Manipur in near future. PMID:22710995

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

    PubMed Central

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

    2007-01-01

    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 protein expression rate and solubility, we chose a subset of 87 plasmids yielding no protein product in E. coli in vivo. These targets were subjected to a more detailed analysis comparing a prokaryotic cell-free E. coli system with an eukaryotic wheat germ system. In addition, we determined the expression rate, yield and solubility of those proteins. After sequence optimisation for the E. coli in vitro system and generating linear templates for wheat germ expression, the success rate of cell-free protein expression reached 93%. Conclusion We have demonstrated that protein expression in cell-free systems is an appropriate technology for the successful expression of soluble full-length proteins. In our study, wheat germ expression using a two compartment system is the method of choice as it shows high solubility and high protein yield. PMID:17915018

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

    PubMed

    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

  2. Complete sequencing and characterization of 21,243 full-length human cDNAs.

    PubMed

    Ota, Toshio; Suzuki, Yutaka; Nishikawa, Tetsuo; Otsuki, Tetsuji; Sugiyama, Tomoyasu; Irie, Ryotaro; Wakamatsu, Ai; Hayashi, Koji; Sato, Hiroyuki; Nagai, Keiichi; Kimura, Kouichi; Makita, Hiroshi; Sekine, Mitsuo; Obayashi, Masaya; Nishi, Tatsunari; Shibahara, Toshikazu; Tanaka, Toshihiro; Ishii, Shizuko; Yamamoto, Jun-ichi; Saito, Kaoru; Kawai, Yuri; Isono, Yuko; Nakamura, Yoshitaka; Nagahari, Kenji; Murakami, Katsuhiko; Yasuda, Tomohiro; Iwayanagi, Takao; Wagatsuma, Masako; Shiratori, Akiko; Sudo, Hiroaki; Hosoiri, Takehiko; Kaku, Yoshiko; Kodaira, Hiroyo; Kondo, Hiroshi; Sugawara, Masanori; Takahashi, Makiko; Kanda, Katsuhiro; Yokoi, Takahide; Furuya, Takako; Kikkawa, Emiko; Omura, Yuhi; Abe, Kumi; Kamihara, Kumiko; Katsuta, Naoko; Sato, Kazuomi; Tanikawa, Machiko; Yamazaki, Makoto; Ninomiya, Ken; Ishibashi, Tadashi; Yamashita, Hiromichi; Murakawa, Katsuji; Fujimori, Kiyoshi; Tanai, Hiroyuki; Kimata, Manabu; Watanabe, Motoji; Hiraoka, Susumu; Chiba, Yoshiyuki; Ishida, Shinichi; Ono, Yukio; Takiguchi, Sumiyo; Watanabe, Susumu; Yosida, Makoto; Hotuta, Tomoko; Kusano, Junko; Kanehori, Keiichi; Takahashi-Fujii, Asako; Hara, Hiroto; Tanase, Tomo-o; Nomura, Yoshiko; Togiya, Sakae; Komai, Fukuyo; Hara, Reiko; Takeuchi, Kazuha; Arita, Miho; Imose, Nobuyuki; Musashino, Kaoru; Yuuki, Hisatsugu; Oshima, Atsushi; Sasaki, Naokazu; Aotsuka, Satoshi; Yoshikawa, Yoko; Matsunawa, Hiroshi; Ichihara, Tatsuo; Shiohata, Namiko; Sano, Sanae; Moriya, Shogo; Momiyama, Hiroko; Satoh, Noriko; Takami, Sachiko; Terashima, Yuko; Suzuki, Osamu; Nakagawa, Satoshi; Senoh, Akihiro; Mizoguchi, Hiroshi; Goto, Yoshihiro; Shimizu, Fumio; Wakebe, Hirokazu; Hishigaki, Haretsugu; Watanabe, Takeshi; Sugiyama, Akio; Takemoto, Makoto; Kawakami, Bunsei; Yamazaki, Masaaki; Watanabe, Koji; Kumagai, Ayako; Itakura, Shoko; Fukuzumi, Yasuhito; Fujimori, Yoshifumi; Komiyama, Megumi; Tashiro, Hiroyuki; Tanigami, Akira; Fujiwara, Tsutomu; Ono, Toshihide; Yamada, Katsue; Fujii, Yuka; Ozaki, Kouichi; Hirao, Maasa; Ohmori, Yoshihiro; Kawabata, Ayako; Hikiji, Takeshi; Kobatake, Naoko; Inagaki, Hiromi; Ikema, Yasuko; Okamoto, Sachiko; Okitani, Rie; Kawakami, Takuma; Noguchi, Saori; Itoh, Tomoko; Shigeta, Keiko; Senba, Tadashi; Matsumura, Kyoka; Nakajima, Yoshie; Mizuno, Takae; Morinaga, Misato; Sasaki, Masahide; Togashi, Takushi; Oyama, Masaaki; Hata, Hiroko; Watanabe, Manabu; Komatsu, Takami; Mizushima-Sugano, Junko; Satoh, Tadashi; Shirai, Yuko; Takahashi, Yukiko; Nakagawa, Kiyomi; Okumura, Koji; Nagase, Takahiro; Nomura, Nobuo; Kikuchi, Hisashi; Masuho, Yasuhiko; Yamashita, Riu; Nakai, Kenta; Yada, Tetsushi; Nakamura, Yusuke; Ohara, Osamu; Isogai, Takao; Sugano, Sumio

    2004-01-01

    As a base for human transcriptome and functional genomics, we created the "full-length long Japan" (FLJ) collection of sequenced human cDNAs. We determined the entire sequence of 21,243 selected clones and found that 14,490 cDNAs (10,897 clusters) were unique to the FLJ collection. About half of them (5,416) seemed to be protein-coding. Of those, 1,999 clusters had not been predicted by computational methods. The distribution of GC content of nonpredicted cDNAs had a peak at approximately 58% compared with a peak at approximately 42%for predicted cDNAs. Thus, there seems to be a slight bias against GC-rich transcripts in current gene prediction procedures. The rest of the cDNAs unique to the FLJ collection (5,481) contained no obvious open reading frames (ORFs) and thus are candidate noncoding RNAs. About one-fourth of them (1,378) showed a clear pattern of splicing. The distribution of GC content of noncoding cDNAs was narrow and had a peak at approximately 42%, relatively low compared with that of protein-coding cDNAs. PMID:14702039

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

    PubMed

    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

  4. Sequence Variability, Gene Structure, and Expression of Full-Length Human Endogenous Retrovirus H

    PubMed Central

    Jern, Patric; Sperber, Göran O.; Ahlsén, Göran; Blomberg, Jonas

    2005-01-01

    Recently, we identified and classified 926 human endogenous retrovirus H (HERV-H)-like proviruses in the human genome. In this paper, we used the information to, in silico, reconstruct a putative ancestral HERV-H. A calculated consensus sequence was nearly open in all genes. A few manual adjustments resulted in a putative 9-kb HERV-H provirus with open reading frames (ORFs) in gag, pro, pol, and env. Long terminal repeats (LTRs) differed by 1.1%, indicating proximity to an integration event. The gag ORF was extended upstream of the normal myristylation start site. There was a long leader (including a “pre-gag” ORF) region positioned like the N terminus of murine leukemia virus (MLV) “glyco-Gag,” potentially encoding a proline- and serine-rich domain remotely similar to MLV pp12. Another ORF, starting inside the 5′ LTR, had no obvious similarity to known protein domains. Unlike other hitherto described gammaretroviruses, the reconstructed Gag had two zinc finger motifs. Alternative splicing of sequences related to the HERV-H consensus was confirmed using dbEST data. env transcripts were most prevalent in colon tumors, but also in normal testis. We found no evidence for full length env transcripts in the dbEST. HERV-H had a markedly skewed nucleotide composition, disfavoring guanine and favoring cytidine. We conclude that the HERV-H consensus shared a gene arrangement common to gammaretroviruses with gag separated by stop codon from pro-pol in the same reading frame, while env resides in another reading frame. There was also alternative splicing. HERV-H consensus yielded new insights in gammaretroviral evolution and will be useful as a model in studies on expression and function. PMID:15858016

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

    PubMed Central

    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

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

    PubMed

    Noe, Amy R; Espinosa, Diego; Li, Xiangming; Coelho-Dos-Reis, Jordana G A; Funakoshi, Ryota; Giardina, Steve; Jin, Hongfan; Retallack, Diane M; Haverstock, Ryan; Allen, Jeffrey R; Vedvick, Thomas S; Fox, Christopher B; Reed, Steven G; Ayala, Ramses; Roberts, Brian; Winram, Scott B; Sacci, John; Tsuji, Moriya; Zavala, Fidel; Gutierrez, Gabriel M

    2014-01-01

    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/boost strategy

  7. Full-length dysferlin expression driven by engineered human dystrophic blood derived CD133+ stem cells.

    PubMed

    Meregalli, Mirella; Navarro, Claire; Sitzia, Clementina; Farini, Andrea; Montani, Erica; Wein, Nicolas; Razini, Paola; Beley, Cyriaque; Cassinelli, Letizia; Parolini, Daniele; Belicchi, Marzia; Parazzoli, Dario; Garcia, Luis; Torrente, Yvan

    2013-12-01

    The protein dysferlin is abundantly expressed in skeletal and cardiac muscles, where its main function is membrane repair. Mutations in the dysferlin gene are involved in two autosomal recessive muscular dystrophies: Miyoshi myopathy and limb-girdle muscular dystrophy type 2B. Development of effective therapies remains a great challenge. Strategies to repair the dysferlin gene by skipping mutated exons, using antisense oligonucleotides (AONs), may be suitable only for a subset of mutations, while cell and gene therapy can be extended to all mutations. AON-treated blood-derived CD133+ stem cells isolated from patients with Miyoshi myopathy led to partial dysferlin reconstitution in vitro but failed to express dysferlin after intramuscular transplantation into scid/blAJ dysferlin null mice. We thus extended these experiments producing the full-length dysferlin mediated by a lentiviral vector in blood-derived CD133+ stem cells isolated from the same patients. Transplantation of engineered blood-derived CD133+ stem cells into scid/blAJ mice resulted in sufficient dysferlin expression to correct functional deficits in skeletal muscle membrane repair. Our data suggest for the first time that lentivirus-mediated delivery of full-length dysferlin in stem cells isolated from Miyoshi myopathy patients could represent an alternative therapeutic approach for treatment of dysferlinopathies. PMID:24028392

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

    SciTech Connect

    Rangarajan, Erumbi S.; Izard, Tina

    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}.

  9. Full-Length Recombinant Plasmodium falciparum VAR2CSA Binds Specifically to CSPG and Induces Potent Parasite Adhesion-Blocking Antibodies

    PubMed Central

    Khunrae, Pongsak; Dahlbäck, Madeleine; Nielsen, Morten A.; Andersen, Gorm; Ditlev, Sisse B.; Resende, Mafalda; Pinto, Vera V.; Theander, Thor G.; Higgins, Matthew K.; Salanti, Ali

    2010-01-01

    Plasmodium falciparum malaria remains one of the world's leading causes of human suffering and poverty. Each year, the disease takes 1–3 million lives, mainly in sub-Saharan Africa. The adhesion of infected erythrocytes (IEs) to vascular endothelium or placenta is the key event in the pathogenesis of severe P. falciparum infection. In pregnant women, the parasites express a single and unique member of the P. falciparum erythrocyte membrane protein 1 (PfEMP1) family named VAR2CSA, which is associated with the ability of the IEs to adhere specifically to chondroitin sulphate A (CSA) in the placenta. Several Duffy-binding-like domains from VAR2CSA molecules have been shown in vitro to bind to CSA, but it has also been demonstrated that Duffy-binding-like domains from PfEMP1 proteins other than VAR2CSA can bind CSA. In addition, the specificity of the binding of VAR2CSA domains to glycosaminoglycans does not match that of VAR2CSA-expressing IEs. This has led to speculation that the domains of native VAR2CSA need to come together to form a specific binding site or that VAR2CSA might bind to CSA through a bridging molecule. Here, we describe the expression and purification of the complete extracellular region of VAR2CSA secreted at high yields from insect cells. Using surface plasmon resonance, we demonstrate that VAR2CSA alone binds with nanomolar affinity to human chondroitin sulphate proteoglycan and with significantly weaker affinity to other glycosaminoglycans, showing a specificity similar to that observed for IEs. Antibodies raised against full-length VAR2CSA completely inhibit recombinant VAR2CSA binding, as well as parasite binding to chondroitin sulphate proteoglycan. This is the first study to describe the successful production and functionality of a full-length PfEMP1. The specificity of the binding and anti-adhesion potency of induced IgG, together with high-yield production, encourages the use of full-length PfEMP1 in vaccine development strategies. PMID

  10. Near Full-Length Genomic Characterization of a Novel CRF 01_AE/C Recombinant from Western India.

    PubMed

    Karade, Santosh; Pandey, Sudhanshu; Gianchandani, Sheetal; Kurle, Swarali N; Ghate, Manisha; Gaikwad, Nitin S; Rewari, Bharat B; Gangakhedkar, Raman R

    2015-12-01

    HIV is known for its genetic variability across the globe. The HIV epidemic in India is primarily driven by subtype C, although sporadic circulating and unique recombinant forms are also reported from a few metropolitan cities in which genotyping facilities are available. Here we report a novel CRF01_AE/C recombinant from a multicenter study on the effectiveness of antiretroviral therapy (ART), 12 months after its initiation. Our subject is a 32-year-old heterosexual female, a native of Pune city in western India. Identification and analyses of recombination breakpoints using jpHMM@Gobics and SimPlot bootscanning revealed six recombination breakpoints, indicating insertion of the CRF01_AE genome at three points in the backbone of subtype C. Both subtype C and CRF01_AE are commonly seen in the population at risk of heterosexual HIV transmission, thereby providing an opportunity for cocirculation and recombination. The emergence of a novel recombinant of CRF01_AE/C is indicative of the increasing genetic diversity of the HIV epidemic in India. PMID:26323027

  11. Identification and Functional Analyses of 11 769 Full-length Human cDNAs Focused on Alternative Splicing

    PubMed Central

    Wakamatsu, Ai; Kimura, Kouichi; Yamamoto, Jun-ichi; Nishikawa, Tetsuo; Nomura, Nobuo; Sugano, Sumio; Isogai, Takao

    2009-01-01

    We analyzed diversity of mRNA produced as a result of alternative splicing in order to evaluate gene function. First, we predicted the number of human genes transcribed into protein-coding mRNAs by using the sequence information of full-length cDNAs and 5′-ESTs and obtained 23 241 of such human genes. Next, using these genes, we analyzed the mRNA diversity and consequently sequenced and identified 11 769 human full-length cDNAs whose predicted open reading frames were different from other known full-length cDNAs. Especially, 30% of the cDNAs we identified contained variation in the transcription start site (TSS). Our analysis, which particularly focused on multiple variable first exons (FEVs) formed due to the alternative utilization of TSSs, led to the identification of 261 FEVs expressed in the tissue-specific manner. Quantification of the expression profiles of 13 genes by real-time PCR analysis further confirmed the tissue-specific expression of FEVs, e.g. OXR1 had specific TSS in brain and tumor tissues, and so on. Finally, based on the results of our mRNA diversity analysis, we have created the FLJ Human cDNA Database. From our result, it has been understood mechanisms that one gene produces suitable protein-coding transcripts responding to the situation and the environment. PMID:19880432

  12. A combined computational and structural model of the full-length human prolactin receptor

    NASA Astrophysics Data System (ADS)

    Bugge, Katrine; Papaleo, Elena; Haxholm, Gitte W.; Hopper, Jonathan T. S.; Robinson, Carol V.; Olsen, Johan G.; Lindorff-Larsen, Kresten; Kragelund, Birthe B.

    2016-05-01

    The prolactin receptor is an archetype member of the class I cytokine receptor family, comprising receptors with fundamental functions in biology as well as key drug targets. Structurally, each of these receptors represent an intriguing diversity, providing an exceptionally challenging target for structural biology. Here, we access the molecular architecture of the monomeric human prolactin receptor by combining experimental and computational efforts. We solve the NMR structure of its transmembrane domain in micelles and collect structural data on overlapping fragments of the receptor with small-angle X-ray scattering, native mass spectrometry and NMR spectroscopy. Along with previously published data, these are integrated by molecular modelling to generate a full receptor structure. The result provides the first full view of a class I cytokine receptor, exemplifying the architecture of more than 40 different receptor chains, and reveals that the extracellular domain is merely the tip of a molecular iceberg.

  13. A combined computational and structural model of the full-length human prolactin receptor

    PubMed Central

    Bugge, Katrine; Papaleo, Elena; Haxholm, Gitte W.; Hopper, Jonathan T. S.; Robinson, Carol V.; Olsen, Johan G.; Lindorff-Larsen, Kresten; Kragelund, Birthe B.

    2016-01-01

    The prolactin receptor is an archetype member of the class I cytokine receptor family, comprising receptors with fundamental functions in biology as well as key drug targets. Structurally, each of these receptors represent an intriguing diversity, providing an exceptionally challenging target for structural biology. Here, we access the molecular architecture of the monomeric human prolactin receptor by combining experimental and computational efforts. We solve the NMR structure of its transmembrane domain in micelles and collect structural data on overlapping fragments of the receptor with small-angle X-ray scattering, native mass spectrometry and NMR spectroscopy. Along with previously published data, these are integrated by molecular modelling to generate a full receptor structure. The result provides the first full view of a class I cytokine receptor, exemplifying the architecture of more than 40 different receptor chains, and reveals that the extracellular domain is merely the tip of a molecular iceberg. PMID:27174498

  14. Efficient assembly of full-length infectious clone of Brazilian IBDV isolate by homologous recombination in yeast.

    PubMed

    Silva, J V J; Arenhart, S; Santos, H F; Almeida-Queiroz, S R; Silva, A N M R; Trevisol, I M; Bertani, G R; Gil, L H V G

    2014-01-01

    The Infectious Bursal Disease Virus (IBDV) causes immunosuppression in young chickens. Advances in molecular virology and vaccines for IBDV have been achieved by viral reverse genetics (VRG). VRG for IBDV has undergone changes over time, however all strategies used to generate particles of IBDV involves multiple rounds of amplification and need of in vitro ligation and restriction sites. The aim of this research was to build the world's first VRG for IBDV by yeast-based homologous recombination; a more efficient, robust and simple process than cloning by in vitro ligation. The wild type IBDV (Wt-IBDV-Br) was isolated in Brazil and had its genome cloned in pJG-CMV-HDR vector by yeast-based homologous recombination. The clones were transfected into chicken embryo fibroblasts and the recovered virus (IC-IBDV-Br) showed genetic stability and similar phenotype to Wt-IBDV-Br, which were observed by nucleotide sequence, focus size/morphology and replication kinetics, respectively. Thus, IBDV reverse genetics by yeast-based homologous recombination provides tools to IBDV understanding and vaccines/viral vectors development. PMID:25763067

  15. Efficient assembly of full-length infectious clone of Brazilian IBDV isolate by homologous recombination in yeast

    PubMed Central

    Silva, J.V.J.; Arenhart, S.; Santos, H.F.; Almeida-Queiroz, S.R.; Silva, A.N.M.R.; Trevisol, I.M.; Bertani, G.R.; Gil, L.H.V.G.

    2014-01-01

    The Infectious Bursal Disease Virus (IBDV) causes immunosuppression in young chickens. Advances in molecular virology and vaccines for IBDV have been achieved by viral reverse genetics (VRG). VRG for IBDV has undergone changes over time, however all strategies used to generate particles of IBDV involves multiple rounds of amplification and need of in vitro ligation and restriction sites. The aim of this research was to build the world’s first VRG for IBDV by yeast-based homologous recombination; a more efficient, robust and simple process than cloning by in vitro ligation. The wild type IBDV (Wt-IBDV-Br) was isolated in Brazil and had its genome cloned in pJG-CMV-HDR vector by yeast-based homologous recombination. The clones were transfected into chicken embryo fibroblasts and the recovered virus (IC-IBDV-Br) showed genetic stability and similar phenotype to Wt-IBDV-Br, which were observed by nucleotide sequence, focus size/morphology and replication kinetics, respectively. Thus, IBDV reverse genetics by yeast-based homologous recombination provides tools to IBDV understanding and vaccines/viral vectors development. PMID:25763067

  16. Identification of HBsAg-specific antibodies from a mammalian cell displayed full-length human antibody library of healthy immunized donor.

    PubMed

    Li, Chang-Zheng; Liang, Zhong-Kun; Chen, Zhen-Rui; Lou, Hai-Bo; Zhou, Ye; Zhang, Zhe-Huan; Yu, Fei; Liu, Shuwen; Zhou, Yuanping; Wu, Shuguang; Zheng, Wenling; Tan, Wanlong; Jiang, Shibo; Zhou, Chen

    2012-03-01

    Hepatitis B immunoglobulin (HBIG) is important in the management of hepatitis B virus (HBV) infection. Aiming to develop recombinant monoclonal antibodies as an alternative to HBIG, we report the successful identification of HBV surface antigen (HBsAg)-specific antibodies from a full-length human antibody library displayed on mammalian cell surface. Using total RNA of peripheral blood mononuclear cells of a natively immunized donor as template, the antibody repertoire was amplified. Combining four-way ligation and the Flp recombinase-mediated integration (Flp-In) system, we constructed a mammalian cell-based, fully human, full-length antibody display library in which each cell displayed only one kind of antibody molecule. By screening the cell library using fluorescence-activated cell sorting (FACS), eight cell clones that displayed HBsAg-specific antibodies on cell surfaces were identified. DNA sequence analysis of the antibody genes revealed three unique antibodies. FACS data indicated that fluorescent strength of expression (FSE), fluorescent strength of binding (FSB) and relative binding ability (RBA) were all different among them. These results demonstrated that by using our antibody mammalian display and screening platform, we can successfully identify antigen-specific antibodies from an immunized full-length antibody library. Therefore, this platform is very useful for the development of therapeutic antibodies. PMID:22179672

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

    SciTech Connect

    Imanishi, Tadashi; Itoh, Takeshi; Suzuki, Yutaka; O'Donovan, Claire; Fukuchi, Satoshi; Koyanagi, Kanako O.; Barrero, Roberto A.; Tamura, Takuro; Yamaguchi-Kabata, Yumi; Tanino, Motohiko; Yura, Kei; Miyazaki, Satoru; Ikeo, Kazuho; Homma, Keiichi; Kasprzyk, Arek; Nishikawa, Tetsuo; Hirakawa, Mika; Thierry-Mieg, Jean; Thierry-Mieg, Danielle; Ashurst, Jennifer; Jia, Libin; Nakao, Mitsuteru; Thomas, Michael A.; Mulder, Nicola; Karavidopoulou, Youla; Jin, Lihua; Kim, Sangsoo; Yasuda, Tomohiro; Lenhard, Boris; Eveno, Eric; Suzuki, Yoshiyuki; Yamasaki, Chisato; Takeda, Jun-ichi; Gough, Craig; Hilton, Phillip; Fujii, Yasuyuki; Sakai, Hiroaki; Tanaka, Susumu; Amid, Clara; Bellgard, Matthew; de Fatima Bonaldo, Maria; Bono Hidemasa; Bromberg, Susan K.; Brookes, Anthony J.; Bruford, Elspeth; Carninci Piero; Chelala, Claude; Couillault, Christine; de Souza, Sandro J.; Debily, Marie-Anne; Devignes, Marie-Dominique; Dubchak, Inna; Endo, Toshinori; Estreicher, Anne; Eyras, Eduardo; Fukami-Kobayashi, Kaoru; Gopinath, Gopal R.; Graudens, Esther; Hahn, Yoonsoo; Han, Michael; Han, Ze-Guang; Hanada, Kousuke; Hanaoka, Hideki; Harada, Erimi; Hashimoto, Katsuyuki; Hinz, Ursula; Hirai, Momoki; Hishiki, Teruyoshi; Hopkinson, Ian; Imbeaud, Sandrine; Inoko, Hidetoshi; Kanapin, Alexander; Kaneko, Yayoi; Kasukawa, Takeya; Kelso, Janet; Kersey, Paul; Kikuno Reiko; Kimura, Kouichi; Korn, Bernhard; Kuryshev, Vladimir; Makalowska, Izabela; Makino Takashi; Mano, Shuhei; Mariage-Samson, Regine; Mashima, Jun; Matsuda, Hideo; Mewes, Hans-Werner; Minoshima, Shinsei; Nagai, Keiichi; Nagasaki, Hideki; Nagata, Naoki; Nigam, Rajni; Ogasawara, Osamu; Ohara, Osamu; Ohtsubo, Masafumi; Okada, Norihiro; Okido, Toshihisa; Oota, Satoshi; Ota, Motonori; Ota, Toshio; Otsuki, Tetsuji; Piatier-Tonneau, Dominique; Poustka, Annemarie; Ren, Shuang-Xi; Saitou, Naruya; Sakai, Katsunaga; Sakamoto, Shigetaka; Sakate, Ryuichi; Schupp, Ingo; Servant, Florence; Sherry, Stephen; Shiba Rie; et al.

    2004-01-15

    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 percent of the human genome sequence (National Center for Biotechnology Information build 34 assembly) may contain misassembled or missing regions. We found that 6.5 percent 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 nonprotein-coding RNA

  18. Near Full-Length Genome Identification of a Novel HIV-1 Recombinant Form (CRF01_AE/CRF07_BC) in Zhejiang, China.

    PubMed

    Peng, Xiaorong; Li-Jun, Xu; Xie, Tiansheng; Liu, Fumin; Wu, Nanping

    2016-09-01

    CRF01_AE and CRF07_BC are the two major circulating recombinant forms (CRFs) in China. Furthermore, many kinds of unique recombinant forms (URFs) between CRF01_AE and CRF07_BC were recently identified in China. Here we detected a novel recombinant of CRF07_BC/CRF01_AE, whose genome structure is distinctly different from other URFs reported before. The phylogenetic analysis of the near full-length sequence of 15zj032 reveals that three regions of CRF01_AE insert into the CRF07_BC backbone. Recently, the continued emergence of novel URFs implies that super infections of different subtypes of HIV-1 are common in China and should be given enough importance. PMID:27353182

  19. A Novel Drug-Resistant HIV-1 Circulating Recombinant Form CRF76_01B Identified by Near Full-Length Genome Analysis.

    PubMed

    Ogawa, Satoko; Hachiya, Atsuko; Hosaka, Masumi; Matsuda, Masakazu; Ode, Hirotaka; Shigemi, Urara; Okazaki, Reiko; Sadamasu, Kenji; Nagashima, Mami; Toyokawa, Takao; Tateyama, Masao; Tanaka, Yasuhito; Sugiura, Wataru; Yokomaku, Yoshiyuki; Iwatani, Yasumasa

    2016-03-01

    HIV-1 CRF01_AE and subtype B (B) have dominated and their different circulating recombinant forms (CRFs) have emerged in East and Southeast Asian countries. Here, we report a novel drug-resistant HIV-1 CRF. Five independent recombinant specimens exhibiting discordant subtype results for the gag, pol, and env sequences were isolated. These recombinants had the CRF01_AE (gag p17)/B (pol PR-RT and IN)/CRF01_AE (env C2-V3) pattern similar to CRF69_01B. Sequence analysis of four near full-length HIV-1 genomes revealed a unique phylogenetic cluster distinct from previously reported CRFs. Of the four recombinants, three shared an identical mosaic structure including seven breakpoints in the gag, pol, vif, and env regions, designated CRF76_01B. The one remaining recombinant had additional recombination breakpoints in the vpu region and exhibited another unique recombinant form composed of CRF76_01B and B. These findings provide important insight into the transmission dynamics of HIV-1 in Asia that may be important for its effective prevention. PMID:26528581

  20. First full-length genomic sequence of a hepatitis A virus isolated in Argentina shows recombination between subgenotypes IA and IB.

    PubMed

    Aguirre, Sebastian; Malirat, Viviana; Scodeller, Eduardo; Mattion, Nora

    2011-01-01

    A hepatitis A virus (HAV) recovered in Argentina from a stool sample of a sick child in the year 2006 (HAV-Arg/06) was entirely sequenced. Phylogenetic analysis included the HAV-Arg/06 sequence in subgenotype IA, either considering the usual VP1-2A variable junction fragment or the full length nucleotide sequence. Interestingly, a recombination event with subgenotype IB, involving a portion of the 2C-3A nonstructural proteins coding region (nucleotides 4961-5140) was detected using specific software. Only subgenotype IA strains have been detected in Argentina or Uruguay, whereas subgenotype IA and IB strains have been reported to circulate in Brazil. Although recombination has been given an important role in the evolution of picornaviruses, there have been only a few reports of its involvement in the evolution of HAV, probably due to the limited number of complete HAV sequences available. This study constitutes the first report of a full-length HAV sequence in Argentina and the third in South America, after the sequence of the IA isolate HAV5 from Uruguay and the IB isolate HAF-203 from Brazil. The availability of new sequence data covering the complete HAV genome will help establish a more consistent genetic relatedness among HAV isolates and the role of recombination in its evolution. PMID:21056065

  1. Responses of Nontransformed Human Hepatocytes to Conditional Expression of Full-Length Hepatitis C Virus Open Reading Frame

    PubMed Central

    Tang, Weiliang; Lázaro, Catherine A.; Campbell, Jean S.; Parks, W. Tony; Katze, Michael G.; Fausto, Nelson

    2007-01-01

    Hepatitis C virus (HCV) is a major cause of chronic hepatitis that can lead to cirrhosis and hepatocellular carcinoma. To study the effects of HCV protein expression on host cells, we established conditional expression of the full-length open reading frame (ORF) of an infectious cDNA clone of HCV (genotype 1a, H77 strain) in the nontransformed human hepatocyte line cell HH4 using the ecdysone receptor regulatory system. Treatment with the ecdysone analog ponasterone-A induced tightly regulated and dose-dependent full-length HCV ORF expression and properly processed HCV proteins. HCV Core, NS3, and NS5A colocalized in perinuclear regions and associated with the early endosomal protein EEA1. HCV ORF expression caused marked growth inhibition, increased intracellular reactive oxygen species, up-regulation of glutamate-l-cysteine ligase activity, increased glutathione level, and activation of nuclear factor κB. Although it was not directly cytotoxic, HCV ORF expression sensitized HH4 cells to Fas at certain concentrations but not to tumor necrosis factor-related apoptosis-inducing ligand. HCV ORF expression in HH4 cells up-regulated genes involved in innate immune response/inflammation and oxidative stress responses and down-regulated cell growth-related genes. Expression of HCV ORF in host cells may contribute to HCV pathogenesis by producing oxidative stress and increasing the expression of genes related to the innate immune response and inflammation. PMID:17991716

  2. Structure of full-length human anti-PD1 therapeutic IgG4 antibody pembrolizumab.

    PubMed

    Scapin, Giovanna; Yang, Xiaoyu; Prosise, Winifred W; McCoy, Mark; Reichert, Paul; Johnston, Jennifer M; Kashi, Ramesh S; Strickland, Corey

    2015-12-01

    Immunoglobulin G4 antibodies exhibit unusual properties with important biological consequences. We report the structure of the human full-length IgG4 S228P anti-PD1 antibody pembrolizumab, solved to 2.3-Å resolution. Pembrolizumab is a compact molecule, consistent with the presence of a short hinge region. The Fc domain is glycosylated at the CH2 domain on both chains, but one CH2 domain is rotated 120° with respect to the conformation observed in all reported structures to date, and its glycan chain faces the solvent. We speculate that this new conformation is driven by the shorter hinge. The structure suggests a role for the S228P mutation in preventing the IgG4 arm exchange. In addition, this unusual Fc conformation suggests possible structural diversity between IgG subclasses and shows that use of isolated antibody fragments could mask potentially important interactions, owing to molecular flexibility. PMID:26595420

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

    SciTech Connect

    Pierce, Anson; Wei, Rochelle; Halade, Dipti; Yoo, Si-Eun; Ran, Qitao; Richardson, Arlan

    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.

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

    PubMed Central

    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

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

    PubMed

    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

  6. Near-Full-Length Genome Sequences of a Novel HIV-1 Circulating Recombinant Form, CRF01_AE/B'/C (CRF78_cpx), in Yunnan, China.

    PubMed

    Song, Yindi; Feng, Yue; Miao, Zhijiang; Wang, Binghui; Yang, Ming; Zhang, A-Mei; Liu, Li; Xia, Xueshan

    2016-06-01

    We report a novel HIV circulating recombinant form (CRF78_cpx) composed mainly of CRF01_AE with inserts from subtypes B and C identified from three epidemiologically unlinked individuals in Yunnan province, China. Two of the subjects are heterosexual men and one is a male intravenous drug user. Sequencing and analyzing the near-full-length genome of these three isolates (YNTC88, YNTC19, and YNTC35) revealed identical recombination breakpoints in all three viruses, but considerable genetic diversity between them, across the genomes, indicating that this is not a newly created CRF, only newly detected. CRF78_cpx differs from previously documented CRF01-AE/B'/C forms in its distinct backbone, inserted fragment size, and breakpoints, and is not related to other described recombinants in the region such as CRF07_BC or CRF65_cpx (also composed of CRF01_AE, B', and C). Our present findings further enrich the diversity of the prevalent HIV-1 CRFs in Yunnan, which is considered as an epicenter of HIV-1 infections in China. PMID:26885715

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

    PubMed

    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

  8. Computational insights into the inhibition and destabilization of morin on the oligomer of full-length human islet amyloid polypeptide.

    PubMed

    Wang, Qianqian; Zhou, Shuangyan; Wei, Wei; Yao, Xiaojun; Liu, Huanxiang; Hu, Zhide

    2015-11-21

    The aggregation of human islet amyloid polypeptide (hIAPP) is closely related with the occurrence of type 2 diabetes (T2D). Natural flavonoid morin was confirmed to not only inhibit the amyloid formation of hIAPP, but disaggregate its preformed amyloid fibrils. In this study, with the goal of elucidating the molecular mechanism of inhibition and destabilization of morin on the full-length hIAPP(1-37) oligomer, molecular dynamics simulations were performed for hIAPP(1-37) pentamer in the presence and absence of morin. The obtained results show that during the protein-inhibitor interaction, morin can notably alter the structural properties of hIAPP(1-37) pentamer, such as morphology, solvent accessible surface area and secondary structure. Moreover, we identified three possible binding sites of morin on hIAPP, all of which located near the amyloidogenic region of this protein. From the binding free energy calculations, we found that Site II was the most possible one. Further conformational analysis together with energy decomposition showed that the residues His18, Phe23 and Ile26 play a key role in the binding with morin by hydrogen bond, π-π and hydrophobic interactions. The proposal of the theoretical mechanism of morin against hIAPP aggregation will provide valuable information for the development of new drugs to inhibit hIAPP aggregation. PMID:26460729

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

    PubMed Central

    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

  10. Human wild-type full-length tau accumulation disrupts mitochondrial dynamics and the functions via increasing mitofusins

    PubMed Central

    Li, Xia-Chun; Hu, Yu; Wang, Zhi-hao; Luo, Yu; Zhang, Yao; Liu, Xiu-Ping; Feng, Qiong; Wang, Qun; Ye, Keqiang; Liu, Gong-Ping; Wang, Jian-Zhi

    2016-01-01

    Intracellular accumulation of tau protein is hallmark of sporadic Alzheimer’s disease (AD), however, the cellular mechanism whereby tau accumulation causes neurodegeneration is poorly understood. Here we report that overexpression of human wild-type full-length tau (termed htau) disrupted mitochondrial dynamics by enhancing fusion and induced their perinuclear accumulation in HEK293 cells and rat primary hippocampal neurons. The htau accumulation at later stage inhibited mitochondrial functions shown by the decreased ATP level, the ratio of ATP/ADP and complex I activity. Simultaneously, the cell viability was decreased with retraction of the cellular/neuronal processes. Further studies demonstrated that htau accumulation increased fusion proteins, including OPA1 and mitofusins (Mfn1, Mfn2) and reduced the ubiquitination of Mfn2. Downregulation of the mitofusins by shRNA to ~45% or ~52% of the control levels attenuated the htau-enhanced mitochondrial fusion and restored the functions, while downregulation of OPA1 to ~50% of the control level did not show rescue effects. Finally, abnormal mitochondrial accumulation and dysfunction were also observed in the brains of htau transgenic mice. Taken together, our data demonstrate that htau accumulation decreases cell viability and causes degeneration via enhancing mitofusin-associated mitochondrial fusion, which provides new insights into the molecular mechanisms underlying tauopathies. PMID:27099072

  11. Sequencing and Phylogenetic Analysis of Near Full-Length HIV-1 Subtypes A, B, G and Unique Recombinant AC and AD Viral Strains Identified in South Africa

    PubMed Central

    Wilkinson, Eduan; Holzmayer, Vera; Jacobs, Graeme B.; de Oliveira, Tulio; Brennan, Catherine A.; Hackett, John; van Rensburg, Estrelita Janse

    2015-01-01

    Abstract By the end of 2012, more than 6.1 million people were infected with HIV-1 in South Africa. Subtype C was responsible for the majority of these infections and more than 300 near full-length genomes (NFLGs) have been published. Currently very few non-subtype C isolates have been identified and characterized within the country, particularly full genome non-C isolates. Seven patients from the Tygerberg Virology (TV) cohort were previously identified as possible non-C subtypes and were selected for further analyses. RNA was isolated from five individuals (TV047, TV096, TV101, TV218, and TV546) and DNA from TV016 and TV1057. The NFLGs of these samples were amplified in overlapping fragments and sequenced. Online subtyping tools REGA version 3 and jpHMM were used to screen for subtypes and recombinants. Maximum likelihood (ML) phylogenetic analysis (phyML) was used to infer subtypes and SimPlot was used to confirm possible intersubtype recombinants. We identified three subtype B (TV016, TV047, and TV1057) isolates, one subtype A1 (TV096), one subtype G (TV546), one unique AD (TV101), and one unique AC (TV218) recombinant form. This is the first NFLG of subtype G that has been described in South Africa. The subtype B sequences described also increased the NFLG subtype B sequences in Africa from three to six. There is a need for more NFLG sequences, as partial HIV-1 sequences may underrepresent viral recombinant forms. It is also necessary to continue monitoring the evolution and spread of HIV-1 in South Africa, because understanding viral diversity may play an important role in HIV-1 prevention strategies. PMID:25492033

  12. Full-length transcriptome analysis of human retina-derived cell lines ARPE-19 and Y79 using the vector-capping method.

    PubMed

    Oshikawa, Mio; Tsutsui, Chihiro; Ikegami, Tomoko; Fuchida, Yuki; Matsubara, Maki; Toyama, Shigeru; Usami, Ron; Ohtoko, Kuniyo; Kato, Seishi

    2011-08-01

    PURPOSE. To collect an entire set of full-length cDNA clones derived from human retina-derived cell lines and to identify full-length transcripts for retinal preferentially expressed genes. METHODS. The full-length cDNA libraries were constructed from a retinoblastoma cell line, Y79, and a retinal pigment epithelium cell line, ARPE-19, using the vector-capping method, which generates a genuine full-length cDNA. By single-pass sequencing of the 5'-end of cDNA clones and subsequent mapping to the human genome, the authors determined their transcriptional start sites and annotated the cDNA clones. RESULTS. Of the 23,616 clones isolated from Y79-derived cDNA libraries, 19,229 full-length cDNA clones were identified and classified into 4808 genes, including genes of >10 kbp. Of the 7067 genes obtained from the Y79 and ARPE-19 libraries, the authors selected 72 genes that were preferentially expressed in the eye, of which 131 clones corresponding to 57 genes were fully sequenced. As a result, we discovered many variants that were produced by different transcriptional start sites, alternative splicing, and alternative polyadenylation. CONCLUSIONS. The bias-free, full-length cDNA libraries constructed using the vector-capping method were shown to be useful for collecting an entire set of full-length cDNA clones for these retinal cell lines. Full-length transcriptome analysis of these cDNA libraries revealed that there were, unexpectedly, many transcript variants for each gene, indicating that obtaining the full-length cDNA for each variant is indispensable for analyzing its function. The full-length cDNA clones (approximately 80,000 clones each for ARPE-19 and Y79) will be useful as a resource for investigating the human retina. PMID:21697133

  13. Full-length spleen tyrosine kinase inhibits the invasion and metastasis of human laryngeal squamous cell carcinoma

    PubMed Central

    Li, Zhihai; Cai, Zhiyi; Tao, Baohong; Jin, Qiaozhi

    2015-01-01

    Objective: This study aimed to investigate correlation between full-length spleen tyrosine kinase [SYK (L)] expression and clinical characteristics of laryngeal squamous cell carcinoma (LSCC), and explore effects of SYK (L) on invasion and metastasis of LSCC. Methods: The human laryngeal cancer Hep-2 cells with low SYK (L) expression were transfected with pIRES2-EGFP-SYK (L) vector and empty vector pIRES2-EGFP to generate Hep-2-SYK (L) cells and Hep-2-neo cells. The cell invasion and migration abilities were determined. Results: The SYK (L) positive expression rate in LSCC tissues was significantly lower than in vocal cord dysplasia tissues and normal laryngeal tissues (P < 0.05). There was a significant correlation between SYK (L) expression and LSCC T stage, histopathological grade and lymph node metastasis (P < 0.05). mRNA expression of SYK (L) in Hep-2-SYK (L) cells was significantly higher than in Hep-2-neo cells and Hep-2 cells (P < 0.01). The protein expression of SYK (L) in Hep-2-SYK (L) cells was markedly higher than in Hep-2-neo cells and Hep-2 cells (P < 0.01). The number of invasive cells was significantly lower in Hep-2-SYK (L) group than in Hep-2-neo group and Hep-2 group (P < 0.01). The average number of migrating cells in Hep-2-SYK (L) group also markedly reduced as compared to Hep-2-neo group and Hep-2 group (P < 0.01). Conclusion: The SYK (L) expression was down-regulated in LSCC, which was closely correlated with cancer growth and lymph node metastasis. SYK (L) up-regulation was able to inhibit the invasion and metastasis of LSCC, therefore suppressing tumor development. Thus, SYK (L) may be a potential target for the LSCC treatment. PMID:26884848

  14. Three-Dimensional Structures of Full-Length, Membrane-Embedded Human α(IIb)β(3) Integrin Complexes.

    PubMed

    Xu, Xiao-Ping; Kim, Eldar; Swift, Mark; Smith, Jeffrey W; Volkmann, Niels; Hanein, Dorit

    2016-02-23

    Integrins are bidirectional, allosteric transmembrane receptors that play a central role in hemostasis and arterial thrombosis. Using cryo-electron microscopy, multireference single-particle reconstruction methods, and statistics-based computational fitting approaches, we determined three-dimensional structures of human integrin αIIbβ3 embedded in a lipid bilayer (nanodiscs) while bound to domains of the cytosolic regulator talin and to extracellular ligands. We also determined the conformations of integrin in solution by itself to localize the membrane and the talin-binding site. To our knowledge, our data provide unprecedented three-dimensional information about the conformational states of intact, full-length integrin within membrane bilayers under near-physiological conditions and in the presence of cytosolic activators and extracellular ligands. We show that αIIbβ3 integrins exist in a conformational equilibrium clustered around four main states. These conformations range from a compact bent nodule to two partially extended intermediate conformers and finally to a fully upright state. In the presence of nanodiscs and the two ligands, the equilibrium is significantly shifted toward the upright conformation. In this conformation, the receptor extends ∼20 nm upward from the membrane. There are no observable contacts between the two subunits other than those in the headpiece near the ligand-binding pocket, and the α- and β-subunits are well separated with their cytoplasmic tails ∼8 nm apart. Our results indicate that extension of the ectodomain is possible without separating the legs or extending the hybrid domain, and that the ligand-binding pocket is not occluded by the membrane in any conformations of the equilibrium. Further, they suggest that integrin activation may be influenced by equilibrium shifts. PMID:26910421

  15. Computational Study on Full-length Human Ku70 with Double Stranded DNA: Dynamics, Interactions and Functional Implications

    NASA Technical Reports Server (NTRS)

    Hu, Shaowen; Cucinotta, Francis A.

    2009-01-01

    The Ku70/80 heterodimer is the first repair protein in the initial binding of double-strand break (DSB) ends following DNA damage, and is a component of nonhomologous end joining repair, the primary pathway for DSB repair in mammalian cells. In this study we constructed a full-length human Ku70 structure based on its crystal structure, and performed 20 ns conventional molecular dynamic (CMD) simulations on this protein and several other complexes with short DNA duplexes of different sequences. The trajectories of these simulations indicated that, without the topological support of Ku80, the residues in the bridge and C-terminal arm of Ku70 are more flexible than other experimentally identified domains. We studied the two missing loops in the crystal structure and predicted that they are also very flexible. Simulations revealed that they make an important contribution to the Ku70 interaction with DNA. Dislocation of the previously studied SAP domain was observed in several systems, implying its role in DNA binding. Targeted molecular dynamic (TMD) simulation was also performed for one system with a far-away 14bp DNA duplex. The TMD trajectory and energetic analysis disclosed detailed interactions of the DNA-binding residues during the DNA dislocation, and revealed a possible conformational transition for a DSB end when encountering Ku70 in solution. Compared to experimentally based analysis, this study identified more detailed interactions between DNA and Ku70. Free energy analysis indicated Ku70 alone is able to bind DNA with relatively high affinity, with consistent contributions from various domains of Ku70 in different systems. The functional implications of these domains in the processes of Ku heterodimerization and DNA damage recognition and repair can be characterized in detail based upon this analysis.

  16. Amyloid Core Formed of Full-Length Recombinant Mouse Prion Protein Involves Sequence 127–143 but Not Sequence 107–126

    PubMed Central

    Chatterjee, Biswanath; Lee, Chung-Yu; Lin, Chen; Chen, Eric H.-L.; Huang, Chao-Li; Yang, Chien-Chih; Chen, Rita P.-Y.

    2013-01-01

    The principal event underlying the development of prion disease is the conversion of soluble cellular prion protein (PrPC) into its disease-causing isoform, PrPSc. This conversion is associated with a marked change in secondary structure from predominantly α-helical to a high β-sheet content, ultimately leading to the formation of aggregates consisting of ordered fibrillar assemblies referred to as amyloid. In vitro, recombinant prion proteins and short prion peptides from various species have been shown to form amyloid under various conditions and it has been proposed that, theoretically, any protein and peptide could form amyloid under appropriate conditions. To identify the peptide segment involved in the amyloid core formed from recombinant full-length mouse prion protein mPrP(23–230), we carried out seed-induced amyloid formation from recombinant prion protein in the presence of seeds generated from the short prion peptides mPrP(107–143), mPrP(107–126), and mPrP(127–143). Our results showed that the amyloid fibrils formed from mPrP(107–143) and mPrP(127–143), but not those formed from mPrP(107–126), were able to seed the amyloidogenesis of mPrP(23–230), showing that the segment residing in sequence 127–143 was used to form the amyloid core in the fibrillization of mPrP(23–230). PMID:23844138

  17. Full-length CD4 electroinserted in the erythrocyte membrane as a long-lived inhibitor of infection by human immunodeficiency virus

    SciTech Connect

    Zeira, M.; Volsky, D.J. ); Tosi, P.F.; Mouneimne, Y.; Lazarte, J.; Sneed, L.; Nicolau, C. )

    1991-05-15

    Recombinant full-length CD4 expressed in Spodoptera frugiperda 9 cells with the baculovirus system was electroinserted in erythrocyte (RBC) membranes. Of the inserted CD4, 70% was correctly oriented as shown by fluorescence quenching experiments with fluorescein-labeled CD4. The inserted CD4 displayed the same epitopes as the naturally occurring CD4 in human T4 cells. Double-labeling experiments ({sup 125}I-CD4 and {sup 51}Cr-RBC) showed that the half-life of CD4 electroinserted in RBC membrane in rabbits was approximately 7 days. Using the fluorescence dequenching technique with octadecylrhodamine B-labeled human immunodeficiency virus (HIV)-1, the authors showed fusion of the HIV envelope with the plasma membrane of RBC-CD4, whereas no such fusion could be detected with RBC. The dequenching efficiency of RBC-CD4 is the same as that of CEM cells. Exposure to anti-CD4 monoclonal antibody OKT4A, which binds to the CD4 region that attaches to envelope glycoprotein gp120, caused a significant decrease in the dequenching of fluorescence. In vitro infectivity studies showed that preincubation of HIV-1 with RBC-CD4 reduced by 80-90% the appearance of HIV antigens in target cells, the amount of viral reverse transcriptase, and the amount of p24 core antigen produced by the target cells. RBC-CD4, but not RBCs, aggregated with chronically HIV-1-infected T cells and caused formation of giant cells. These data show that the RBC-CD4 reagent is relatively long lived in circulation and efficient in attaching to HIV-1 and HIV-infected cells, and thus it may have value as a therapeutic agent against AIDS.

  18. The full-length E1-circumflexE4 protein of human papillomavirus type 18 modulates differentiation-dependent viral DNA amplification and late gene expression

    SciTech Connect

    Wilson, Regina; Ryan, Gordon B.; Knight, Gillian L.; Laimins, Laimonis A.; Roberts, Sally . E-mail: s.roberts@bham.ac.uk

    2007-06-05

    Activation of the productive phase of the human papillomavirus (HPV) life cycle in differentiated keratinocytes is coincident with high-level expression of E1-circumflexE4 protein. To determine the role of E1-circumflexE4 in the HPV replication cycle, we constructed HPV18 mutant genomes in which expression of the full-length E1-circumflexE4 protein was abrogated. Undifferentiated keratinocytes containing mutant genomes showed enhanced proliferation when compared to cells containing wildtype genomes, but there were no differences in maintenance of viral episomes. Following differentiation, cells with mutant genomes exhibited reduced levels of viral DNA amplification and late gene expression, compared to wildtype genome-containing cells. This indicates that HPV18 E1-circumflexE4 plays an important role in regulating HPV late functions, and it may also function in the early phase of the replication cycle. Our finding that full-length HPV18 E1-circumflexE4 protein plays a significant role in promoting viral genome amplification concurs with a similar report with HPV31, but is in contrast to an HPV11 study where viral DNA amplification was not dependent on full-length E1-circumflexE4 expression, and to HPV16 where only C-terminal truncations in E1-circumflexE4 abrogated vegetative genome replication. This suggests that type-specific differences exist between various E1-circumflexE4 proteins.

  19. An ancestral host defence peptide within human β-defensin 3 recapitulates the antibacterial and antiviral activity of the full-length molecule

    PubMed Central

    Nigro, Ersilia; Colavita, Irene; Sarnataro, Daniela; Scudiero, Olga; Zambrano, Gerardo; Granata, Vincenzo; Daniele, Aurora; Carotenuto, Alfonso; Galdiero, Stefania; Folliero, Veronica; Galdiero, Massimiliano; Urbanowicz, Richard A.; Ball, Jonathan K.; Salvatore, Francesco; Pessi, Antonello

    2015-01-01

    Host defence peptides (HDPs) are critical components of innate immunity. Despite their diversity, they share common features including a structural signature, designated “γ-core motif”. We reasoned that for each HDPs evolved from an ancestral γ-core, the latter should be the evolutionary starting point of the molecule, i.e. it should represent a structural scaffold for the modular construction of the full-length molecule, and possess biological properties. We explored the γ-core of human β-defensin 3 (HBD3) and found that it: (a) is the folding nucleus of HBD3; (b) folds rapidly and is stable in human serum; (c) displays antibacterial activity; (d) binds to CD98, which mediates HBD3 internalization in eukaryotic cells; (e) exerts antiviral activity against human immunodeficiency virus and herpes simplex virus; and (f) is not toxic to human cells. These results demonstrate that the γ-core within HBD3 is the ancestral core of the full-length molecule and is a viable HDP per se, since it is endowed with the most important biological features of HBD3. Notably, the small, stable scaffold of the HBD3 γ-core can be exploited to design disease-specific antimicrobial agents. PMID:26688341

  20. Recombinant production and characterization of full-length and truncated β-1,3-glucanase PglA from Paenibacillus sp. S09

    PubMed Central

    2013-01-01

    of non-catalytic modules on enzymatic properties of β-1,3-glucanase. Activity comparison of full-length PglA and truncated forms revealed the negative effect of C-terminal region on thermal stability of the enzyme. Both the N-and C-terminal domains exerted strong binding activity toward insoluble β-1,3-glucan, and could be classified into CBM families. PMID:24283345

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

    PubMed Central

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

    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 the amino acid sequence of the protein and then by codon optimization, in order to ensure the maximum expression in Escherichia coli. In the next step, the synthetic DNA encoding IGFBP3 was inserted into the pUC57 vector, at the appropriate restriction sites and then subcloned in the pET-11a expression vector in the same restriction sites. The constructed vector was transformed to E. coli BL21 as an expression host and induced in the presence of IPTG for expression of the IGFBP3 protein. Protein expression was evaluated by sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE). Results: Double digestion of the new plasmid (pET-11a -IGBP3) with NdeI and BamHI showed two bands in 873 bp and 5700 bp. To study the accurate cloning procedure, the plasmid was sequenced and its authenticity was confirmed. Also the expected protein band (31.6 kDa) was observed in SDS-PAGE analysis. 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. PMID:25878991

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

    SciTech Connect

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

    1987-05-01

    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. 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.

  3. Quantitative measurement of full-length and C-terminal proteolyzed RBP4 in serum of normal and insulin-resistant humans using a novel mass spectrometry immunoassay.

    PubMed

    Yang, Qin; Eskurza, Iratxe; Kiernan, Urban A; Phillips, David A; Blüher, Matthias; Graham, Timothy E; Kahn, Barbara B

    2012-03-01

    Serum retinol-binding protein 4 (RBP4) levels are increased in insulin-resistant humans and correlate with severity of insulin resistance in metabolic syndrome. Quantitative Western blotting (qWestern) has been the most accurate method for serum RBP4 measurements, but qWestern is technically complex and labor intensive. The lack of a reliable, high-throughput method for RBP4 measurements has resulted in variability in findings in insulin-resistant humans. Many commonly used ELISAs have limited dynamic range. Neither the current ELISAs nor qWestern distinguish among full-length and carboxyl terminus proteolyzed forms of circulating RBP4 that are altered in different medical conditions. Here, we report the development of a novel quantitative mass spectrometry immunoaffinity assay (qMSIA) to measure full-length and proteolyzed forms of RBP4. qMSIA and qWestern of RBP4 were performed in identical serum aliquots from insulin-sensitive/normoglycemic or insulin-resistant humans with impaired glucose tolerance or type 2 diabetes. Total RBP4 qMSIA measurements were highly similar to qWestern and correlated equally well with clinical severity of insulin resistance (assessed by clamp glucose disposal rate, r = -0.74), hemoglobin A1c (r = 0.63), triglyceride/high-density lipoprotein (r = 0.55), waist/hip (r = 0.61), and systolic blood pressure (r = 0.53, all P < 0.001). Proteolyzed forms of RBP4 accounted for up to 50% of total RBP4 in insulin-resistant subjects, and des(Leu)-RBP4 (cleavage of last leucine) correlated highly with insulin resistance (assessed by glucose disposal rate, r = -0.69). In multiple regression analysis, insulin resistance but not glomerular filtration rate was the strongest, independent predictor of serum RBP4 levels. Thus, qMSIA provides a novel tool for accurately measuring serum RBP4 levels as a biomarker for severity of insulin resistance and risk for type 2 diabetes and metabolic syndrome. PMID:22253430

  4. Structure of the Full-Length Human RPA14/32 Complex Gives Insights Into the Mechanism of DNA Binding And Complex Formation

    SciTech Connect

    Deng, X.; Habel, J.E.; Kabaleeswaran, V.; Snell, E.H.; Wold, M.S.; Borgstahl, G.E.O.

    2009-06-03

    Replication protein A (RPA) is the ubiquitous, eukaryotic single-stranded DNA (ssDNA) binding protein and is essential for DNA replication, recombination, and repair. Here, crystal structures of the soluble RPA heterodimer, composed of the RPA14 and RPA32 subunits, have been determined for the full-length protein in multiple crystal forms. In all crystals, the electron density for the N-terminal (residues 1--42) and C-terminal (residues 175--270) regions of RPA32 is weak and of poor quality indicating that these regions are disordered and/or assume multiple positions in the crystals. Hence, the RPA32 N terminus, that is hyperphosphorylated in a cell-cycle-dependent manner and in response to DNA damaging agents, appears to be inherently disordered in the unphosphorylated state. The C-terminal, winged helix-loop-helix, protein-protein interaction domain adopts several conformations perhaps to facilitate its interaction with various proteins. Although the ordered regions of RPA14/32 resemble the previously solved protease-resistant core crystal structure, the quaternary structures between the heterodimers are quite different. Thus, the four-helix bundle quaternary assembly noted in the original core structure is unlikely to be related to the quaternary structure of the intact heterotrimer. An organic ligand binding site between subunits RPA14 and RPA32 was identified to bind dioxane. Comparison of the ssDNA binding surfaces of RPA70 with RPA14/32 showed that the lower affinity of RPA14/32 can be attributed to a shallower binding crevice with reduced positive electrostatic charge.

  5. 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

    PubMed Central

    Martín-Valls, G. E.; Kvisgaard, L. K.; Tello, M.; Darwich, L.; Cortey, M.; Burgara-Estrella, A. J.; Hernández, J.; Larsen, L. E.

    2014-01-01

    ABSTRACT 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 potential recombination breakpoints along the viral genome. For ORF5, 11 sets of genotype 1 sequences from different geographical areas, including 2 Asian, 1 American, and 7 European regions, and three sets of genotype 2, including sets from China, Mexico, and the United States, were analyzed separately. Potential recombination breakpoints were detected in 10/11 genotype 1 sets, including 9 cases in which the clustering of at least one isolate was different before and after the breakpoints. In genotype 2, potential breakpoints and different tree clustering of at least one strain before and after the breakpoint were observed in 2 out of 3 sets. The results indicated that most of the ORF5 data sets contained at least one recombinant sequence. When the full-length genome sequences were examined, both genotype 1 and 2 sets presented breakpoints (10 and 9, respectively), resulting in significantly different topologies before and after the breakpoints. Mosaic genomes were detected in genotype 1 sequences. These results may have significant implications for the understanding of the molecular epidemiology of PRRSV. IMPORTANCE PRRSV is one of the most important viruses affecting swine production worldwide, causing big economic losses and sanitary problems. One of the key questions on PRRSV arises from its genetic diversity, which is thought to have a direct impact on immunobiology, epidemiology, diagnosis, and vaccine efficacy. One of the causes of this genetic diversity is

  6. Near Full-Length Genomic Characterization of a Novel HIV Type 1 CRF01_AE/CRF07_BC Recombinant Form Transmitted Between a Heterosexual Couple in Guangxi, China.

    PubMed

    Dong, Zhe; Shen, Zhiyong; Xiong, Runsong; Liang, Fuxiong; Liang, Shujia; Li, Jianjun; Tan, Guangjie; Jiang, He; Yang, Xiaoyi; Li, Fan; Liao, Lingjie; Shao, Yiming; Feng, Yi; Zhu, Qiuying

    2016-07-01

    In this research, we reported a new second generation recombinant form (GXDY460B) between circulating recombinant form (CRF)01_AE and CRF07_BC in a seroconversion couple who obtained the virus from her husband by heterosexual behavior. The analysis result of the near full-length genomic characterization showed that the genome comprises at least 12 interlaced segments, including six CRF07_BC and six CRF01_AE segments, with CRF07_BC as the main framework. Cocirculation of multiple virus subtypes and multiple infection routes have existed for a long time in Guangxi, but the recombinant strain was rarely reported among heterosexual transmission population because of its lower crowd confounding degree than men who have sex with men and injecting drug user population. It is the first time that the unique recombinant form (URF) between CRF01_AE and CRF07_BC was identified among heterosexual transmission in Guangxi. The emergence of the novel recombinant helps to understand the pattern of the URF virus. PMID:26892263

  7. 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

    SciTech Connect

    Toral-Barza, Lourdes; Zhang Weiguo; Lamison, Craig; LaRocque, James; Gibbons, James; Yu, Ker . E-mail: yuk@wyeth.com

    2005-06-24

    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 {sub m}) values of TOR for ATP and the His6-S6K substrate were shown to be 50 and 0.8 {mu}M, 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.

  8. Identification of genes expressed in human CD34+ hematopoietic stem/progenitor cells by expressed sequence tags and efficient full-length cDNA cloning

    PubMed Central

    Mao, Mao; Fu, Gang; Wu, Ji-Sheng; Zhang, Qing-Hua; Zhou, Jun; Kan, Li-Xin; Huang, Qiu-Hua; He, Kai-Li; Gu, Bai-Wei; Han, Ze-Guang; Shen, Yu; Gu, Jian; Yu, Ya-Ping; Xu, Shu-Hua; Wang, Ya-Xin; Chen, Sai-Juan; Chen, Zhu

    1998-01-01

    Hematopoietic stem/progenitor cells (HSPCs) possess the potentials of self-renewal, proliferation, and differentiation toward different lineages of blood cells. These cells not only play a primordial role in hematopoietic development but also have important clinical application. Characterization of the gene expression profile in CD34+ HSPCs may lead to a better understanding of the regulation of normal and pathological hematopoiesis. In the present work, genes expressed in human umbilical cord blood CD34+ cells were catalogued by partially sequencing a large amount of cDNA clones [or expressed sequence tags (ESTs)] and analyzing these sequences with the tools of bioinformatics. Among 9,866 ESTs thus obtained, 4,697 (47.6%) showed identity to known genes in the GenBank database, 2,603 (26.4%) matched to the ESTs previously deposited in a public domain database, 1,415 (14.3%) were previously undescribed ESTs, and the remaining 1,151 (11.7%) were mitochondrial DNA, ribosomal RNA, or repetitive (Alu or L1) sequences. Integration of ESTs of known genes generated a profile including 855 genes that could be divided into different categories according to their functions. Some (8.2%) of the genes in this profile were considered related to early hematopoiesis. The possible function of ESTs corresponding to so far unknown genes were approached by means of homology and functional motif searches. Moreover, attempts were made to generate libraries enriched for full-length cDNAs, to better explore the genes in HSPCs. Nearly 60% of the cDNA clones of mRNA under 2 kb in our libraries had 5′ ends upstream of the first ATG codon of the ORF. With this satisfactory result, we have developed an efficient working system that allowed fast sequencing of 32 full-length cDNAs, 16 of them being mapped to the chromosomes with radiation hybrid panels. This work may lay a basis for the further research on the molecular network of hematopoietic regulation. PMID:9653160

  9. Near full-length genome sequence of a novel HIV-1 recombinant form (CRF01_AE/B) detected among men who have sex with men in Jilin Province, China.

    PubMed

    Li, Xingguang; Feng, Yi; Yang, Yao; Chen, Yanli; Guo, Qi; Sun, Liuyan; Zang, Xihui; Xing, Hui; Shao, Yiming

    2014-07-01

    We report here a novel HIV-1 recombinant form (CRF01_AE/B) detected from a comprehensive HIV-1 molecular epidemiologic study among men who have sex with men (MSM) in Jilin province of northeastern China. The near full-length genome (NFLG) analyses showed that the novel HIV-1 recombinant isolate (JL.RF07) was composed of CRF01_AE cluster 5 (northeastern China origin) and subtype B (U.S. and European origin), with six recombinant breakpoints observed in the pol, vif, tat, rev, and env gene regions. To the best of our knowledge, this is the first detection of a novel HIV-1 recombinant form (CRF01_AE/B) in Jilin, which may indicate an active transmission network of HIV-1 infection among MSM in the region. Further studies of the molecular epidemiology of the HIV-1 epidemic among MSM in northeastern China are necessary to gain a fuller understanding of the transmission network and potential public health impact of HIV-1 among MSM in this region. PMID:24521207

  10. TALENs-directed knockout of the full-length transcription factor Nrf1α that represses malignant behaviour of human hepatocellular carcinoma (HepG2) cells.

    PubMed

    Ren, Yonggang; Qiu, Lu; Lü, Fenglin; Ru, Xufang; Li, Shaojun; Xiang, Yuancai; Yu, Siwang; Zhang, Yiguo

    2016-01-01

    The full-length Nrf1α is processed into distinct isoforms, which together regulate genes essential for maintaining cellular homeostasis and organ integrity, and liver-specific loss of Nrf1 in mice results in spontaneous hepatoma. Herein, we report that the human constitutive Nrf1α, rather than smaller Nrf1β/γ, expression is attenuated or abolished in the case of low-differentiated high-metastatic hepatocellular carcinomas. Therefore, Nrf1α is of importance in the physio-pathological origin and development, but its specific pathobiological function(s) remains elusive. To address this, TALENs-directed knockout of Nrf1α, but not Nrf1β/γ, is created in the human hepatocellular carcinoma (HepG2) cells. The resulting Nrf1α(-/-) cells are elongated, with slender spindle-shapes and enlarged gaps between cells observed under scanning electron microscope. When compared with wild-type controls, the invasive and migratory abilities of Nrf1α(-/-) cells are increased significantly, along with the cell-cycle G2-M arrest and S-phase reduction, as accompanied by suppressed apoptosis. Despite a modest increase in the soft-agar colony formation of Nrf1α(-/-) cells, its loss-of-function markedly promotes malgrowth of the subcutaneous carcinoma xenograft in nude mice with hepatic metastasis. Together with molecular expression results, we thus suppose requirement of Nrf1α (and major derivates) for gene regulatory mechanisms repressing cancer cell process (e.g. EMT) and malignant behaviour (e.g. migration). PMID:27065079

  11. TALENs-directed knockout of the full-length transcription factor Nrf1α that represses malignant behaviour of human hepatocellular carcinoma (HepG2) cells

    PubMed Central

    Ren, Yonggang; Qiu, Lu; Lü, Fenglin; Ru, Xufang; Li, Shaojun; Xiang, Yuancai; Yu, Siwang; Zhang, Yiguo

    2016-01-01

    The full-length Nrf1α is processed into distinct isoforms, which together regulate genes essential for maintaining cellular homeostasis and organ integrity, and liver-specific loss of Nrf1 in mice results in spontaneous hepatoma. Herein, we report that the human constitutive Nrf1α, rather than smaller Nrf1β/γ, expression is attenuated or abolished in the case of low-differentiated high-metastatic hepatocellular carcinomas. Therefore, Nrf1α is of importance in the physio-pathological origin and development, but its specific pathobiological function(s) remains elusive. To address this, TALENs-directed knockout of Nrf1α, but not Nrf1β/γ, is created in the human hepatocellular carcinoma (HepG2) cells. The resulting Nrf1α−/− cells are elongated, with slender spindle-shapes and enlarged gaps between cells observed under scanning electron microscope. When compared with wild-type controls, the invasive and migratory abilities of Nrf1α−/− cells are increased significantly, along with the cell-cycle G2-M arrest and S-phase reduction, as accompanied by suppressed apoptosis. Despite a modest increase in the soft-agar colony formation of Nrf1α−/− cells, its loss-of-function markedly promotes malgrowth of the subcutaneous carcinoma xenograft in nude mice with hepatic metastasis. Together with molecular expression results, we thus suppose requirement of Nrf1α (and major derivates) for gene regulatory mechanisms repressing cancer cell process (e.g. EMT) and malignant behaviour (e.g. migration). PMID:27065079

  12. Strategies for the production of difficult-to-express full-length eukaryotic proteins using microbial cell factories: production of human alpha-galactosidase A.

    PubMed

    Unzueta, Ugutz; Vázquez, Felicitas; Accardi, Giulia; Mendoza, Rosa; Toledo-Rubio, Verónica; Giuliani, Maria; Sannino, Filomena; Parrilli, Ermenegilda; Abasolo, Ibane; Schwartz, Simo; Tutino, Maria L; Villaverde, Antonio; Corchero, José L; Ferrer-Miralles, Neus

    2015-07-01

    Obtaining high levels of pure proteins remains the main bottleneck of many scientific and biotechnological studies. Among all the available recombinant expression systems, Escherichia coli facilitates gene expression by its relative simplicity, inexpensive and fast cultivation, well-known genetics and the large number of tools available for its biotechnological application. However, recombinant expression in E. coli is not always a straightforward procedure and major obstacles are encountered when producing many eukaryotic proteins and especially membrane proteins, linked to missing posttranslational modifications, proteolysis and aggregation. In this context, many conventional and unconventional eukaryotic hosts are under exploration and development, but in some cases linked to complex culture media or processes. In this context, alternative bacterial systems able to overcome some of the limitations posed by E. coli keeping the simplicity of prokaryotic manipulation are currently emerging as convenient hosts for protein production. We have comparatively produced a "difficult-to-express" human protein, the lysosomal enzyme alpha-galactosidase A (hGLA) in E. coli and in the psychrophilic bacterium Pseudoalteromonas haloplanktis TAC125 cells (P. haloplanktis TAC125). While in E. coli the production of active hGLA was unreachable due to proteolytic instability and/or protein misfolding, the expression of hGLA gene in P. haloplanktis TAC125 allows obtaining active enzyme. These results are discussed in the context of emerging bacterial systems for protein production that represent appealing alternatives to the regular use of E. coli and also of more complex eukaryotic systems. PMID:25616525

  13. A Method to Produce and Purify Full-Length Recombinant Alpha Dystroglycan: Analysis of N- and O-Linked Monosaccharide Composition in CHO Cells with or without LARGE Overexpression

    PubMed Central

    Yoon, Jung Hae; Xu, Rui; Martin, Paul

    2013-01-01

    α dystroglycan (αDG) is part of the dystrophin-associated glycoprotein (DAG) complex, a series of cytoskeletal, transmembrane, and membrane-associated proteins that serve to link the extracellular matrix (ECM) surrounding individual skeletal myofibers to the intracellular F-actin cytoskeleton. Glycosylation and ECM protein binding to αDG are regulated by a number of genes that, when defective, give rise to congenital or limb-girdle forms of muscular dystrophy termed dystroglycanopathies. One such dystroglycanopathy gene is LARGE. Here, we describe a method to produce and purify full-length, furin-resistant, recombinant αDG from CHO cells and CHO cells overexpressing LARGE (CHO-LARGE). In addition, we analyze the O- and N-linked monosaccharide composition of such proteins. αDG purified from CHO-LARGE cells had increased molar content of xylose and fucose relative to CHO, while no significant changes were found in N-linked monosaccharides. Glucuronic acid could not be quantified by the methods used. These studies describe a method to produce and purify the milligram amounts of αDG needed for certain biochemical methods, including monosaccharide analysis. Key words: Dystroglycan, muscular dystrophy, xylose, fucose, laminin, LARGE Correspondence: Paul.Martin@nationwidechildrens.org PMID:23390591

  14. Evidence supporting a critical contribution of intrinsically disordered regions to the biochemical behavior of full-length human HP1γ.

    PubMed

    Velez, Gabriel; Lin, Marisa; Christensen, Trace; Faubion, William A; Lomberk, Gwen; Urrutia, Raul

    2016-01-01

    HP1γ, a non-histone chromatin protein, has elicited significant attention because of its role in gene silencing, elongation, splicing, DNA repair, cell growth, differentiation, and many other cancer-associated processes, including therapy resistance. These characteristics make it an ideal target for developing small drugs for both mechanistic experimentation and potential therapies. While high-resolution structures of the two globular regions of HP1γ, the chromo- and chromoshadow domains, have been solved, little is currently known about the conformational behavior of the full-length protein. Consequently, in the current study, we use threading, homology-based molecular modeling, molecular mechanics calculations, and molecular dynamics simulations to develop models that allow us to infer properties of full-length HP1γ at an atomic resolution level. HP1γ appears as an elongated molecule in which three Intrinsically Disordered Regions (IDRs, 1, 2, and 3) endow this protein with dynamic flexibility, intermolecular recognition properties, and the ability to integrate signals from various intracellular pathways. Our modeling also suggests that the dynamic flexibility imparted to HP1γ by the three IDRs is important for linking nucleosomes with PXVXL motif-containing proteins, in a chromatin environment. The importance of the IDRs in intermolecular recognition is illustrated by the building and study of both IDR2 HP1γ-importin-α and IDR1 and IDR2 HP1γ-DNA complexes. The ability of the three IDRs for integrating cell signals is demonstrated by combined linear motif analyses and molecular dynamics simulations showing that posttranslational modifications can generate a histone mimetic sequence within the IDR2 of HP1γ, which when bound by the chromodomain can lead to an autoinhibited state. Combined, these data underscore the importance of IDRs 1, 2, and 3 in defining the structural and dynamic properties of HP1γ, discoveries that have both mechanistic and

  15. Nanoscale insights into full-length prion protein aggregation on model lipid membranes.

    PubMed

    Pan, Yangang; Wang, Bin; Zhang, Tong; Zhang, Yanan; Wang, Hongda; Xu, Bingqian

    2016-06-30

    The aggregates of the full-length human recombinant prion protein (PrP) (23-231) on model membranes were investigated by combining the atomic force microscopy (AFM) measurements and theoretical calculations at pH 5.0, showing the great effect of PrP concentration on its supramolecular assemblies on the lipid bilayer. PMID:27284592

  16. Analysis of a cDNA clone expressing a human autoimmune antigen: full-length sequence of the U2 small nuclear RNA-associated B antigen

    SciTech Connect

    Habets, W.J.; Sillekens, P.T.G.; Hoet, M.H.; Schalken, J.A.; Roebroek, A.J.M.; Leunissen, J.A.M.; Van de Ven, W.J.M.; Van Venrooij, W.J.

    1987-04-01

    A U2 small nuclear RNA-associated protein, designated B'', was recently identified as the target antigen for autoimmune sera from certain patients with systemic lupus erythematosus and other rheumatic diseases. Such antibodies enabled them to isolate cDNA clone lambdaHB''-1 from a phage lambdagt11 expression library. This clone appeared to code for the B'' protein as established by in vitro translation of hybrid-selected mRNA. The identity of clone lambdaHB''-1 was further confirmed by partial peptide mapping and analysis of the reactivity of the recombinant antigen with monospecific and monoclonal antibodies. Analysis of the nucleotide sequence of the 1015-base-pair cDNA insert of clone lambdaHB''-1 revealed a large open reading frame of 800 nucleotides containing the coding sequence for a polypeptide of 25,457 daltons. In vitro transcription of the lambdaHB''-1 cDNA insert and subsequent translation resulted in a protein product with the molecular size of the B'' protein. These data demonstrate that clone lambdaHB''-1 contains the complete coding sequence of this antigen. The deduced polypeptide sequence contains three very hydrophilic regions that might constitute RNA binding sites and/or antigenic determinants. These findings might have implications both for the understanding of the pathogenesis of rheumatic diseases as well as for the elucidation of the biological function of autoimmune antigens.

  17. Expression of active iron regulatory factor from a full-length human cDNA by in vitro transcription/translation.

    PubMed Central

    Hirling, H; Emery-Goodman, A; Thompson, N; Neupert, B; Seiser, C; Kühn, L C

    1992-01-01

    Iron regulatory factor (IRF), also called iron responsive element-binding protein (IRE-BP), is a cytoplasmic RNA-binding protein which regulates post-transcriptionally transferrin receptor mRNA stability and ferritin mRNA translation. By using the polymerase chain reaction (PCR) and the sequence published by Rouault et al. (1990) a probe was derived which permitted the isolation of three human IRF cDNA clones. Hybridization to genomic DNA and mRNA, as well as sequencing data indicated a single copy gene of about 40 kb specifying a 4.0 kb mRNA that translates into a protein of 98,400 dalton. By in vitro transcription of a assembled IRF cDNA coupled to in vitro translation in a wheat germ extract, we obtained full sized IRF that bound specifically to a human ferritin IRE. In vitro translated IRF retained sensitivity to sulfhydryl oxidation by diamide and could be reactivated by beta-mercaptoethanol in the same way as native placental IRF. An IRF deletion mutant shortened by 132 amino acids at the COOH-terminus was no longer able to bind to an IRE, indicating that this region of the protein plays a role in RNA recognition. Placental IRF has previously been shown to migrate as a doublet on SDS-polyacrylamide gels. After V8 protease digestion the heterogeneity was located in a 65/70 kDa NH2-terminal doublet. The liberated 31 kDa COOH-terminal polypeptide was found to be homogeneous by amino acid sequencing supporting the conclusion of a single IRF gene. Images PMID:1738601

  18. Structural transitions in full-length human prion protein detected by xenon as probe and spin labeling of the N-terminal domain.

    PubMed

    Narayanan, Sunilkumar Puthenpurackal; Nair, Divya Gopalakrishnan; Schaal, Daniel; Barbosa de Aguiar, Marisa; Wenzel, Sabine; Kremer, Werner; Schwarzinger, Stephan; Kalbitzer, Hans Robert

    2016-01-01

    Fatal neurodegenerative disorders termed transmissible spongiform encephalopathies (TSEs) are associated with the accumulation of fibrils of misfolded prion protein PrP. The noble gas xenon accommodates into four transiently enlarged hydrophobic cavities located in the well-folded core of human PrP(23-230) as detected by [(1)H, (15)N]-HSQC spectroscopy. In thermal equilibrium a fifth xenon binding site is formed transiently by amino acids A120 to L125 of the presumably disordered N-terminal domain and by amino acids K185 to T193 of the well-folded domain. Xenon bound PrP was modelled by restraint molecular dynamics. The individual microscopic and macroscopic dissociation constants could be derived by fitting the data to a model including a dynamic opening and closing of the cavities. As observed earlier by high pressure NMR spectroscopy xenon binding influences also other amino acids all over the N-terminal domain including residues of the AGAAAAGA motif indicating a structural coupling between the N-terminal domain and the core domain. This is in agreement with spin labelling experiments at positions 93 or 107 that show a transient interaction between the N-terminus and the start of helix 2 and the end of helix 3 of the core domain similar to that observed earlier by Zn(2+)-binding to the octarepeat motif. PMID:27341298

  19. Structural transitions in full-length human prion protein detected by xenon as probe and spin labeling of the N-terminal domain

    PubMed Central

    Narayanan, Sunilkumar Puthenpurackal; Nair, Divya Gopalakrishnan; Schaal, Daniel; Barbosa de Aguiar, Marisa; Wenzel, Sabine; Kremer, Werner; Schwarzinger, Stephan; Kalbitzer, Hans Robert

    2016-01-01

    Fatal neurodegenerative disorders termed transmissible spongiform encephalopathies (TSEs) are associated with the accumulation of fibrils of misfolded prion protein PrP. The noble gas xenon accommodates into four transiently enlarged hydrophobic cavities located in the well-folded core of human PrP(23–230) as detected by [1H, 15N]-HSQC spectroscopy. In thermal equilibrium a fifth xenon binding site is formed transiently by amino acids A120 to L125 of the presumably disordered N-terminal domain and by amino acids K185 to T193 of the well-folded domain. Xenon bound PrP was modelled by restraint molecular dynamics. The individual microscopic and macroscopic dissociation constants could be derived by fitting the data to a model including a dynamic opening and closing of the cavities. As observed earlier by high pressure NMR spectroscopy xenon binding influences also other amino acids all over the N-terminal domain including residues of the AGAAAAGA motif indicating a structural coupling between the N-terminal domain and the core domain. This is in agreement with spin labelling experiments at positions 93 or 107 that show a transient interaction between the N-terminus and the start of helix 2 and the end of helix 3 of the core domain similar to that observed earlier by Zn2+-binding to the octarepeat motif. PMID:27341298

  20. Full-Length Human Placental sFlt-1-e15a Isoform Induces Distinct Maternal Phenotypes of Preeclampsia in Mice

    PubMed Central

    Szalai, Gabor; Romero, Roberto; Chaiworapongsa, Tinnakorn; Xu, Yi; Wang, Bing; Ahn, Hyunyoung; Xu, Zhonghui; Chiang, Po Jen; Sundell, Birgitta; Wang, Rona; Jiang, Yang; Plazyo, Olesya; Olive, Mary; Tarca, Adi L.; Dong, Zhong; Qureshi, Faisal; Papp, Zoltan; Hassan, Sonia S.; Hernandez-Andrade, Edgar; Than, Nandor Gabor

    2015-01-01

    Objective Most anti-angiogenic preeclampsia models in rodents utilized the overexpression of a truncated soluble fms-like tyrosine kinase-1 (sFlt-1) not expressed in any species. Other limitations of mouse preeclampsia models included stressful blood pressure measurements and the lack of postpartum monitoring. We aimed to 1) develop a mouse model of preeclampsia by administering the most abundant human placental sFlt-1 isoform (hsFlt-1-e15a) in preeclampsia; 2) determine blood pressures in non-stressed conditions; and 3) develop a survival surgery that enables the collection of fetuses and placentas and postpartum (PP) monitoring. Methods Pregnancy status of CD-1 mice was evaluated with high-frequency ultrasound on gestational days (GD) 6 and 7. Telemetry catheters were implanted in the carotid artery on GD7, and their positions were verified by ultrasound on GD13. Mice were injected through tail-vein with adenoviruses expressing hsFlt-1-e15a (n = 11) or green fluorescent protein (GFP; n = 9) on GD8/GD11. Placentas and pups were delivered by cesarean section on GD18 allowing PP monitoring. Urine samples were collected with cystocentesis on GD6/GD7, GD13, GD18, and PPD8, and albumin/creatinine ratios were determined. GFP and hsFlt-1-e15a expression profiles were determined by qRT-PCR. Aortic ring assays were performed to assess the effect of hsFlt-1-e15a on endothelia. Results Ultrasound predicted pregnancy on GD7 in 97% of cases. Cesarean section survival rate was 100%. Mean arterial blood pressure was higher in hsFlt-1-e15a-treated than in GFP-treated mice (∆MAP = 13.2 mmHg, p = 0.00107; GD18). Focal glomerular changes were found in hsFlt-1-e15a -treated mice, which had higher urine albumin/creatinine ratios than controls (109.3±51.7μg/mg vs. 19.3±5.6μg/mg, p = 4.4x10-2; GD18). Aortic ring assays showed a 46% lesser microvessel outgrowth in hsFlt-1-e15a-treated than in GFP-treated mice (p = 1.2x10-2). Placental and fetal weights did not differ between the

  1. Expression and characterization of full-length human heme oxygenase-1: the presence of intact membrane-binding region leads to increased binding affinity for NADPH cytochrome P450 reductase.

    PubMed

    Huber, Warren J; Backes, Wayne L

    2007-10-30

    Heme oxygenase-1 (HO-1) is the chief regulatory enzyme in the oxidative degradation of heme to biliverdin. In the process of heme degradation, HO-1 receives the electrons necessary for catalysis from the flavoprotein NADPH cytochrome P450 reductase (CPR), releasing free iron and carbon monoxide. Much of the recent research involving heme oxygenase has been done using a 30 kDa soluble form of the enzyme, which lacks the membrane binding region (C-terminal 23 amino acids). The goal of this study was to express and purify a full-length human HO-1 (hHO-1) protein; however, due to the lability of the full-length form, a rapid purification procedure was required. This was accomplished by use of a glutathione-s-transferase (GST)-tagged hHO-1 construct. Although the procedure permitted the generation of a full-length HO-1, this form was contaminated with a 30 kDa degradation product that could not be eliminated. Therefore, attempts were made to remove a putative secondary thrombin cleavage site by a conservative mutation of amino acid 254, which replaces arginine with lysine. This mutation allowed the expression and purification of a full-length hHO-1 protein. Unlike wild type (WT) HO-1, the R254K mutant could be purified to a single 32 kDa protein capable of degrading heme at the same rate as the WT enzyme. The R254K full-length form had a specific activity of approximately 200-225 nmol of bilirubin h-1 nmol-1 HO-1 as compared to approximately 140-150 nmol of bilirubin h-1 nmol-1 for the WT form, which contains the 30 kDa contaminant. This is a 2-3-fold increase from the previously reported soluble 30 kDa HO-1, suggesting that the C-terminal 23 amino acids are essential for maximal catalytic activity. Because the membrane-spanning domain is present, the full-length hHO-1 has the potential to incorporate into phospholipid membranes, which can be reconstituted at known concentrations, in combination with other endoplasmic reticulum resident enzymes. PMID:17915953

  2. Near full-length genome sequence of a novel HIV type 1 second-generation recombinant form (CRF01_AE/CRF07_BC) identified among men who have sex with men in Jilin, China.

    PubMed

    Li, Xingguang; Ning, Chuanyi; He, Xiang; Yang, Yao; Xing, Hui; Hong, Kunxue; Shao, Yiming; Yang, Rongge

    2013-12-01

    We report here a novel HIV-1 second-generation recombinant form (CRF01_AE/CRF07_BC) composed of CRF01_AE and CRF07_BC, identified among men who have sex with men (MSM) in Jilin, with four breakpoints observed in the pol, vif, and vpr genes. The CRF01_AE regions of the recombinant were clustered with the CRF01_AE lineage, which is mainly circulating among MSM in northern China, with the support of 100% bootstrap value, indicating that the parental origin of the CRF01_AE regions was from MSM, in which recombination events may be more likely to occur. To the best of our knowledge, this is the first detection of a novel HIV-1 second-generation recombinant form (CRF01AE/CRF07_BC) in Jilin, which indicates active transmission networks of HIV-1 infection among MSM in the region. Therefore, it is necessary to continue monitoring the molecular epidemiology of HIV-1 among MSM in Jilin to obtain a better understanding of the transmission and potential public health impact of HIV-1 among MSM in the region. PMID:23809010

  3. Recombinant Human Erythropoietin

    PubMed Central

    Bartels, Claudia; Späte, Kira; Krampe, Henning

    2008-01-01

    Treatment of multiple sclerosis (MS) is still unsatisfactory and essentially non-existing for the progressive course of the disease. Recombinant human erythropoietin (EPO) may be a promising neuroprotective/neuroregenerative treatment of MS. In the nervous system, EPO acts anti-apoptotic, antioxidative, anti-inflammatory, neurotrophic and plasticity-modulating. Beneficial effects have been shown in animal models of various neurological and psychiatric diseases, including different models of experimental autoimmune encephalomyelitis. EPO is also effective in human brain disease, as shown in double-blind placebo-controlled clinical studies on ischemic stroke and chronic schizophrenia. An exploratory study on chronic progressive MS yielded lasting improvement in motor and cognitive performance upon high-dose long-term EPO treatment. PMID:21180577

  4. Apical expression of human full-length hCEACAM1-4L protein renders the Madin Darby Canine Kidney cells responsive to lipopolysaccharide leading to TLR4-dependent Erk1/2 and p38 MAPK signalling.

    PubMed

    Liévin-Le Moal, Vanessa; Beau, Isabelle; Rougeaux, Clémence; Kansau, Imad; Fabrega, Sylvie; Brice, Cédric; Korotkova, Natalia; Moseley, Steve L; Servin, Alain L

    2011-05-01

    CEACAM1 expressed by granulocytes and epithelial cells is recognized as a membrane-associated receptor by some Gram-negative pathogens. Here we report a previously unsuspected role of human CEACAM1-4L (hCEACAM1-4L) in polarized epithelial cells. We find that in contrast with non-transfected cells, Madin Darby Canine Kidney strain II (MDCK) engineered for the apical expression of the long cytoplasmic chain protein hCEACAM1-4L showed a serum-independent increase in the phosphorylation of the extracellular signal-regulated kinase 1/2 (Erk1/2) and p38 mitogen-activated protein kinases (MAPKs) after treatment with lipopolysaccharide (LPS) of wild-type, diffusely adhering Afa/Dr Escherichia coli (Afa/Dr DAEC) strain IH11128. Aggregates of FITC-LPS bind the apical domain of MDCK-hCEACAM1-4L cells colocalizing with the apically expressed hCEACAM1-4L protein and do not bind MDCK-pCEP cells, and surface plasmon resonance analysis shows that LPS binds to the extracellular domain of the CEACAM1-4L protein. We showed that cell polarization and lipid rafts positively control the LPS-IH11128-induced phosphorylation of Erk1/2 in MDCK-hCEACAM1-4L cells. Structure-function analysis using mutated hCEACAM1-4L protein shows that the cytoplasmic domain of the protein is needed for LPS-induced MAPK signalling, and that phosphorylation of Tyr-residues is not increased in association with MAPK signalling. The hCEACAM1-4L-dependent Erk1/2 phosphorylation develops in the presence of lipid A and does not develop in the presence of penta-acylated LPS. Finally, small interfering RNA (siRNA) silencing of canine TLR4 abolishes the hCEACAM1-4L-dependent, LPS-induced phosphorylation of Erk1/2. Collectively, our results support the notion that the apically expressed, full-length hCEACAM1-4L protein functions as a novel LPS-conveying molecule at the mucosal surface of polarized epithelial cells for subsequent MD-2/TLR4 receptor-dependent MAPK Erk1/2 and p38 signalling. PMID:21352462

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

    PubMed

    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

  6. International Validation of Two Human Recombinant Estrogen Receptor (ERa) Binding Assays

    EPA Science Inventory

    An international validation study has been successfully completed for 2 competitive binding assays using human recombinant ERa. Assays evaluated included the Freyberger-Wilson (FW) assay using a full length human ER, and the Chemical Evaluation and Research Institute (CERI) assay...

  7. Selection of Recombinant Human Antibodies.

    PubMed

    Tomszak, Florian; Weber, Susanne; Zantow, Jonas; Schirrmann, Thomas; Hust, Michael; Frenzel, André

    2016-01-01

    Since the development of therapeutic antibodies the demand of recombinant human antibodies is steadily increasing. Traditionally, therapeutic antibodies were generated by immunization of rat or mice, the generation of hybridoma clones, cloning of the antibody genes and subsequent humanization and engineering of the lead candidates. In the last few years, techniques were developed that use transgenic animals with a human antibody gene repertoire. Here, modern recombinant DNA technologies can be combined with well established immunization and hybridoma technologies to generate already affinity maturated human antibodies. An alternative are in vitro technologies which enabled the generation of fully human antibodies from antibody gene libraries that even exceed the human antibody repertoire. Specific antibodies can be isolated from these libraries in a very short time and therefore reduce the development time of an antibody drug at a very early stage.In this review, we describe different technologies that are currently used for the in vitro and in vivo generation of human antibodies. PMID:27236551

  8. Crystal Structure of a Full-Length [beta]-Catenin

    SciTech Connect

    Xing, Yi; Takemaru, Ken-Ichi; Liu, Jing; Berndt, Jason D.; Zheng, Jie J.; Moon, Randall T.; Xu, Wenqing

    2008-08-19

    {beta}-catenin plays essential roles in cell adhesion and Wnt signaling, while deregulation of {beta}-catenin is associated with multiple diseases including cancers. Here, we report the crystal structures of full-length zebrafish {beta}-catenin and a human {beta}-catenin fragment that contains both the armadillo repeat and the C-terminal domains. Our structures reveal that the N-terminal region of the C-terminal domain, a key component of the C-terminal transactivation domain, forms a long {alpha} helix that packs on the C-terminal end of the armadillo repeat domain, and thus forms part of the {beta}-catenin superhelical core. The existence of this helix redefines our view of interactions of {beta}-catenin with some of its critical partners, including ICAT and Chibby, which may form extensive interactions with this C-terminal domain {alpha} helix. Our crystallographic and NMR studies also suggest that the unstructured N-terminal and C-terminal tails interact with the ordered armadillo repeat domain in a dynamic and variable manner.

  9. Full-length infectious clone of a low passage dengue virus serotype 2 from Brazil

    PubMed Central

    Santos, Jefferson José da Silva; Magalhães, Tereza; Silva, José Valter Joaquim; da Silva, Andréa Nazaré Monteiro Rangel; Cordeiro, Marli Tenório; Gil, Laura Helena Vega Gonzales

    2015-01-01

    Full-length dengue virus (DENV) cDNA clones are an invaluable tool for many studies, including those on the development of attenuated or chimeric vaccines and on host-virus interactions. Furthermore, the importance of low passage DENV infectious clones should be highlighted, as these may harbour critical and unique strain-specific viral components from field-circulating isolates. The successful construction of a functional Brazilian low passage DENV serotype 2 full-length clone through homologous recombination reported here supports the use of a strategy that has been shown to be highly useful by our group for the development of flavivirus infectious clones and replicons. PMID:26200712

  10. Management of full-length complete ureteral avulsion

    PubMed Central

    Tang, Kaifa; Sun, Fa; Tian, Yuan; Zhao, Yili

    2016-01-01

    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. PMID:27136483

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

    SciTech Connect

    Lombardo, N J; Lanning, D D; Panisko, F E

    1992-12-01

    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.

  12. Isolation of full-length RNA from a thermophilic cyanobacterium.

    PubMed

    Luo, X Z; Stevens, S E

    1997-11-01

    Isolation of full-length mRNA without degradation is critical in the study of in vivo gene regulation and transcription, cDNA synthesis and reverse transcription (RT)-PCR. It is particularly difficult to isolate full-length mRNA from thermophiles, which have higher turnover rates of mRNA degradation. Mastigocladus laminosus is a thermophilic heterocystous cyanobacterium. The assay of M. laminosus cell lysates showed that RNase activity was high and was resistant to the conventional guanidine thiocyanate and 2-mercaptoethanol denaturation methods. The mRNA isolated by several conventional methods was completely degraded. A method was developed to purify full-length mRNA by a combination of fast cooling, vanadyl-ribonucleoside-complex inhibition, phenol-chloroform-isoamyl alcohol extraction, lithium chloride precipitation and the lysing of cells with the French Press. This method produced high-quality, full-length mRNA in high yield. Purified mRNA was suitable for Northern blotting, cDNA synthesis and RT-PCR. This method could be applicable to other thermophiles in which the RNase activity is high and/or is resistant to guanidine thiocyanate. PMID:9383558

  13. Recovering full-length viral genomes from metagenomes

    PubMed Central

    Smits, Saskia L.; Bodewes, Rogier; Ruiz-González, Aritz; Baumgärtner, Wolfgang; Koopmans, Marion P.; Osterhaus, Albert D. M. E.; Schürch, Anita C.

    2015-01-01

    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. PMID:26483782

  14. Renal Agenesis with Full Length Ipsilateral Refluxing Ureter.

    PubMed

    Pal, Dilip Kumar; 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. PMID:27170916

  15. Renal Agenesis with Full Length Ipsilateral Refluxing Ureter

    PubMed Central

    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. PMID:27170916

  16. Stable preparations of tyrosine hydroxylase provide the solution structure of the full-length enzyme

    PubMed Central

    Bezem, Maria T.; Baumann, Anne; Skjærven, Lars; Meyer, Romain; Kursula, Petri; Martinez, Aurora; Flydal, Marte I.

    2016-01-01

    Tyrosine hydroxylase (TH) catalyzes the rate-limiting step in the biosynthesis of catecholamine neurotransmitters. TH is a highly complex enzyme at mechanistic, structural, and regulatory levels, and the preparation of kinetically and conformationally stable enzyme for structural characterization has been challenging. Here, we report on improved protocols for purification of recombinant human TH isoform 1 (TH1), which provide large amounts of pure, stable, active TH1 with an intact N-terminus. TH1 purified through fusion with a His-tagged maltose-binding protein on amylose resin was representative of the iron-bound functional enzyme, showing high activity and stabilization by the natural feedback inhibitor dopamine. TH1 purified through fusion with a His-tagged ZZ domain on TALON is remarkably stable, as it was partially inhibited by resin-derived cobalt. This more stable enzyme preparation provided high-quality small-angle X-ray scattering (SAXS) data and reliable structural models of full-length tetrameric TH1. The SAXS-derived model reveals an elongated conformation (Dmax = 20 nm) for TH1, different arrangement of the catalytic domains compared with the crystal structure of truncated forms, and an N-terminal region with an unstructured tail that hosts the phosphorylation sites and a separated Ala-rich helical motif that may have a role in regulation of TH by interacting with binding partners. PMID:27462005

  17. Stable preparations of tyrosine hydroxylase provide the solution structure of the full-length enzyme.

    PubMed

    Bezem, Maria T; Baumann, Anne; Skjærven, Lars; Meyer, Romain; Kursula, Petri; Martinez, Aurora; Flydal, Marte I

    2016-01-01

    Tyrosine hydroxylase (TH) catalyzes the rate-limiting step in the biosynthesis of catecholamine neurotransmitters. TH is a highly complex enzyme at mechanistic, structural, and regulatory levels, and the preparation of kinetically and conformationally stable enzyme for structural characterization has been challenging. Here, we report on improved protocols for purification of recombinant human TH isoform 1 (TH1), which provide large amounts of pure, stable, active TH1 with an intact N-terminus. TH1 purified through fusion with a His-tagged maltose-binding protein on amylose resin was representative of the iron-bound functional enzyme, showing high activity and stabilization by the natural feedback inhibitor dopamine. TH1 purified through fusion with a His-tagged ZZ domain on TALON is remarkably stable, as it was partially inhibited by resin-derived cobalt. This more stable enzyme preparation provided high-quality small-angle X-ray scattering (SAXS) data and reliable structural models of full-length tetrameric TH1. The SAXS-derived model reveals an elongated conformation (Dmax = 20 nm) for TH1, different arrangement of the catalytic domains compared with the crystal structure of truncated forms, and an N-terminal region with an unstructured tail that hosts the phosphorylation sites and a separated Ala-rich helical motif that may have a role in regulation of TH by interacting with binding partners. PMID:27462005

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

    SciTech Connect

    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.

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

    PubMed

    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

  20. A drosophila full-length cDNA resource

    SciTech Connect

    Stapleton, Mark; Carlson, Joseph; Brokstein, Peter; Yu, Charles; Champe, Mark; George, Reed; Guarin, Hannibal; Kronmiller, Brent; Pacleb, Joanne; Park, Soo; Rubin, Gerald M.; Celniker, Susan E.

    2003-05-09

    Background: A collection of sequenced full-length cDNAs is an important resource both for functional genomics studies and for the determination of the intron-exon structure of genes. Providing this resource to the Drosophila melanogaster research community has been a long-term goal of the Berkeley Drosophila Genome Project. We have previously described the Drosophila Gene Collection (DGC), a set of putative full-length cDNAs that was produced by generating and analyzing over 250,000 expressed sequence tags (ESTs) derived from a variety of tissues and developmental stages. Results: We have generated high-quality full-insert sequence for 8,921 clones in the DGC. We compared the sequence of these clones to the annotated Release 3 genomic sequence, and identified more than 5,300 cDNAs that contain a complete and accurate protein-coding sequence. This corresponds to at least one splice form for 40 percent of the predicted D. melanogaster genes. We also identified potential new cases of RNA editing. Conclusions: We show that comparison of cDNA sequences to a high-quality annotated genomic sequence is an effective approach to identifying and eliminating defective clones from a cDNA collection and ensure its utility for experimentation. Clones were eliminated either because they carry single nucleotide discrepancies, which most probably result from reverse transcriptase errors, or because they are truncated and contain only part of the protein-coding sequence.

  1. Structural photoactivation of a full-length bacterial phytochrome

    PubMed Central

    Björling, Alexander; Berntsson, Oskar; Lehtivuori, Heli; Takala, Heikki; Hughes, Ashley J.; Panman, Matthijs; Hoernke, Maria; Niebling, Stephan; Henry, Léocadie; Henning, Robert; Kosheleva, Irina; Chukharev, Vladimir; Tkachenko, Nikolai V.; Menzel, Andreas; Newby, Gemma; Khakhulin, Dmitry; Wulff, Michael; Ihalainen, Janne A.; Westenhoff, Sebastian

    2016-01-01

    Phytochromes are light sensor proteins found in plants, bacteria, and fungi. They function by converting a photon absorption event into a conformational signal that propagates from the chromophore through the entire protein. However, the structure of the photoactivated state and the conformational changes that lead to it are not known. We report time-resolved x-ray scattering of the full-length phytochrome from Deinococcus radiodurans on micro- and millisecond time scales. We identify a twist of the histidine kinase output domains with respect to the chromophore-binding domains as the dominant change between the photoactivated and resting states. The time-resolved data further show that the structural changes up to the microsecond time scales are small and localized in the chromophore-binding domains. The global structural change occurs within a few milliseconds, coinciding with the formation of the spectroscopic meta-Rc state. Our findings establish key elements of the signaling mechanism of full-length bacterial phytochromes. PMID:27536728

  2. Conformational states of the full-length glucagon receptor

    NASA Astrophysics Data System (ADS)

    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.

  3. Conformational states of the full-length glucagon receptor

    PubMed Central

    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

    2015-01-01

    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. PMID:26227798

  4. Full-length minor ampullate spidroin gene sequence.

    PubMed

    Chen, Gefei; Liu, Xiangqin; Zhang, Yunlong; Lin, Senzhu; Yang, Zijiang; Johansson, Jan; Rising, Anna; Meng, Qing

    2012-01-01

    Spider silk includes seven protein based fibers and glue-like substances produced by glands in the spider's abdomen. Minor ampullate silk is used to make the auxiliary spiral of the orb-web and also for wrapping prey, has a high tensile strength and does not supercontract in water. So far, only partial cDNA sequences have been obtained for minor ampullate spidroins (MiSps). Here we describe the first MiSp full-length gene sequence from the spider species Araneus ventricosus, using a multidimensional PCR approach. Comparative analysis of the sequence reveals regulatory elements, as well as unique spidroin gene and protein architecture including the presence of an unusually large intron. The spliced full-length transcript of MiSp gene is 5440 bp in size and encodes 1766 amino acid residues organized into conserved nonrepetitive N- and C-terminal domains and a central predominantly repetitive region composed of four units that are iterated in a non regular manner. The repeats are more conserved within A. ventricosus MiSp than compared to repeats from homologous proteins, and are interrupted by two nonrepetitive spacer regions, which have 100% identity even at the nucleotide level. PMID:23251707

  5. Full-Length Minor Ampullate Spidroin Gene Sequence

    PubMed Central

    Chen, Gefei; Liu, Xiangqin; Zhang, Yunlong; Lin, Senzhu; Yang, Zijiang; Johansson, Jan; Rising, Anna; Meng, Qing

    2012-01-01

    Spider silk includes seven protein based fibers and glue-like substances produced by glands in the spider's abdomen. Minor ampullate silk is used to make the auxiliary spiral of the orb-web and also for wrapping prey, has a high tensile strength and does not supercontract in water. So far, only partial cDNA sequences have been obtained for minor ampullate spidroins (MiSps). Here we describe the first MiSp full-length gene sequence from the spider species Araneus ventricosus, using a multidimensional PCR approach. Comparative analysis of the sequence reveals regulatory elements, as well as unique spidroin gene and protein architecture including the presence of an unusually large intron. The spliced full-length transcript of MiSp gene is 5440 bp in size and encodes 1766 amino acid residues organized into conserved nonrepetitive N- and C-terminal domains and a central predominantly repetitive region composed of four units that are iterated in a non regular manner. The repeats are more conserved within A. ventricosus MiSp than compared to repeats from homologous proteins, and are interrupted by two nonrepetitive spacer regions, which have 100% identity even at the nucleotide level. PMID:23251707

  6. Chinese hamster ovary cells contain transcriptionally active full-length type C proviruses.

    PubMed Central

    Lie, Y S; Penuel, E M; Low, M A; Nguyen, T P; Mangahas, J O; Anderson, K P; Petropoulos, C J

    1994-01-01

    We have isolated a genomic locus from Chinese hamster ovary (CHO) cells that contains a full-length provirus. Nucleotide sequence analysis indicates that it is a defective member of the rodent type C retrovirus family with an env region that is similar to those of mouse amphotropic retrovirus and subgroup B feline leukemia virus. We were able to demonstrate that this provirus is a member of a closely related family of full-length proviruses in CHO cells and Chinese hamster liver. Hybridization probes generated from this genomic clone were used to characterize type C retrovirus RNA expression in CHO cells. Full-length genomic RNA and subgenomic envelope mRNA were detected in CHO cell lines but not in the human-derived 293 cell line. Interestingly, we discovered that the site of retrovirus integration lies within a G repeat sequence belonging to the short interspersed element family of retroposons. Images PMID:7966574

  7. Chinese hamster ovary cells contain transcriptionally active full-length type C proviruses.

    PubMed

    Lie, Y S; Penuel, E M; Low, M A; Nguyen, T P; Mangahas, J O; Anderson, K P; Petropoulos, C J

    1994-12-01

    We have isolated a genomic locus from Chinese hamster ovary (CHO) cells that contains a full-length provirus. Nucleotide sequence analysis indicates that it is a defective member of the rodent type C retrovirus family with an env region that is similar to those of mouse amphotropic retrovirus and subgroup B feline leukemia virus. We were able to demonstrate that this provirus is a member of a closely related family of full-length proviruses in CHO cells and Chinese hamster liver. Hybridization probes generated from this genomic clone were used to characterize type C retrovirus RNA expression in CHO cells. Full-length genomic RNA and subgenomic envelope mRNA were detected in CHO cell lines but not in the human-derived 293 cell line. Interestingly, we discovered that the site of retrovirus integration lies within a G repeat sequence belonging to the short interspersed element family of retroposons. PMID:7966574

  8. Simulations of The Dalles Dam Proposed Full Length Spillwall

    SciTech Connect

    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

  9. Analysis and Optimization of "Full-Length" Diodes

    SciTech Connect

    Schock, Alfred

    2012-01-19

    A method of analyzing the axial variation of the heat generation rate, temperature, voltage, current density and emitter heat flux in a thermionic converter is described. The method is particularly useful for the case of "long" diodes, each extending over the full length of the reactor core. For a given diode geometry and fuel distribution, the analysis combines a nuclear solution of the axial fission density profile with the iterative solution of four differential equations representing the thermal, electrical, and thermionic interactions within the diode. The digital computer program developed to solve these equations can also perform a design optimization with respect to lead resistance, load voltage, and emitter thickness, for a specified maximum emitter temperature. Typical results are presented, and the use of this analysis for predicting the diode operating characteristics is illustrated.

  10. Full length talin stimulates integrin activation and axon regeneration

    PubMed Central

    Tan, Chin Lik; Kwok, Jessica C.F.; Heller, Janosch P.D.; Zhao, Rongrong; Eva, Richard; Fawcett, James W.

    2015-01-01

    Integrin function is regulated by activation involving conformational changes that modulate ligand-binding affinity and downstream signaling. Activation is regulated through inside-out signaling which is controlled by many signaling pathways via a final common pathway through kindlin and talin, which bind to the intracellular tail of beta integrins. Previous studies have shown that the axon growth inhibitory molecules NogoA and chondroitin sulfate proteoglycans (CSPGs) inactivate integrins. Overexpressing kindlin-1 in dorsal root ganglion (DRG) neurons activates integrins, enabling their axons to overcome inhibitory molecules in the environment, and promoting regeneration in vivo following dorsal root crush. Other studies have indicated that expression of the talin head alone or with kindlin can enhance integrin activation. Here, using adult rat DRG neurons, we investigate the effects of overexpressing various forms of talin on axon growth and integrin signaling. We found that overexpression of the talin head activated axonal integrins but inhibited downstream signaling via FAK, and did not promote axon growth. Similarly, co-expression of the talin head and kindlin-1 prevented the growth-promoting effect of kindlin-1, suggesting that the talin head acts as a form of dominant negative for integrin function. Using full-length talin constructs in PC12 cells we observed that neurite growth was enhanced by the expression of wild-type talin and more so by two ‘activated’ forms of talin produced by point mutation (on laminin and aggrecan–laminin substrates). Nevertheless, co-expression of full-length talin with kindlin did not promote neurite growth more than either molecule alone. In vivo, we find that talin is present in PNS axons (sciatic nerve), and also in CNS axons of the corticospinal tract. PMID:25771432

  11. Full-Length Semaphorin-3C Is an Inhibitor of Tumor Lymphangiogenesis and Metastasis.

    PubMed

    Mumblat, Yelena; Kessler, Ofra; Ilan, Neta; Neufeld, Gera

    2015-06-01

    Semaphorins play important regulatory roles in diverse processes such as axon guidance, angiogenesis, and immune responses. We find that semaphorin-3C (sema3C) induces the collapse of the cytoskeleton of lymphatic endothelial cells (LEC) in a neuropilin-2-, plexin-D1-, and plexin-A1-dependent manner, while most other semaphorins, including antiangiogenic semaphorins such as sema3A do not. Sema3C is cleaved, like other class-3 semaphorins, by furin-like pro-protein convertases (FPPC). Cleaved sema3C (p65-Sema3C) was unable to induce the collapse of the cytoskeleton of LEC. FPPC are strongly upregulated in tumor cells. In order to examine the effects of full-length sema3C on tumor progression, we therefore generated an active point mutated furin cleavage-resistant sema3C (FR-sema3C). FR-sema3C inhibited potently proliferation of LEC and to a lesser extent proliferation of human umbilical vein-derived endothelial cells. FR-sema3C also inhibited VEGF-C-induced phosphorylation of VEGFR-3, ERK1/2, and AKT. Expression of recombinant FR-sema3C in metastatic, triple-negative LM2-4 breast cancer cells did not affect their migration or proliferation in vitro. However, tumors derived from FR-sema3C-expressing LM2-4 cells implanted in mammary fat pads developed at a slower rate, contained a lower concentration of blood vessels and lymph vessels, and metastasized much less effectively to lymph nodes. Interestingly, p65-Sema3C, but not FR-sema3C, rendered A549 lung cancer cells resistant to serum deprivation, suggesting that previously reported protumorigenic activities of sema3C may be due to p65-Sema3C produced by tumor cells. Our observations suggest that FR-sema3C may be further developed into a novel antitumorigenic drug. PMID:25808871

  12. Phylogenetic Analysis of Near Full-Length HIV Type 1 Genomic Sequences from 21 Korean Individuals

    PubMed Central

    Kim, Jung-Eun; Foley, Brian T.

    2013-01-01

    Abstract The Korean subclade of subtype B (KSB) is the most prevalent HIV-1 strain found in Korea. To date, only two near full-length HIV-1 sequences from Korean patients have been reported. Here, we analyzed a total of 24 near full-length genomes of HIV-1 strains that were isolated from 17 antiretroviral therapy (ART)-naive patients and four ART-exposed patients. Proviral DNA from peripheral blood mononuclear cells was PCR amplified and directly sequenced. Phylogenetic analyses were used to classify viruses from 19 patients as KSB, from one patient as subtype B, from one patient as subtype D, and three viruses from one patient as CRF02_AG. All KSB viruses demonstrated TAAAA instead of TATAA at the TATA box in the LTR. Of the 19 KSB patients, their sequence identities at the nucleotide level ranged from 89.8% to 97.1% from the lowest env gene to the highest pol gene. Other than the CRF02_AG viruses, no recombination events were noted in any of the 19 KSB patients, which is consistent with our previous studies on the pol, vif, and nef genes. Except for one strain, all of the strains were classified as non-syncytium-inducing strains. This is the first report to describe near full-length KSB. PMID:23199052

  13. Synaptonemal complex extension from clustered telomeres mediates full-length chromosome pairing in Schmidtea mediterranea

    PubMed Central

    Xiang, Youbin; Miller, Danny E.; Ross, Eric J.; Sánchez Alvarado, Alejandro; Hawley, R. Scott

    2014-01-01

    In the 1920s, József Gelei proposed that chromosome pairing in flatworms resulted from the formation of a telomere bouquet followed by the extension of synapsis from telomeres at the base of the bouquet, thus facilitating homolog pairing in a processive manner. A modern interpretation of Gelei’s model postulates that the synaptonemal complex (SC) is nucleated close to the telomeres and then extends progressively along the full length of chromosome arms. We used the easily visible meiotic chromosomes, a well-characterized genome, and RNAi in the sexual biotype of the planarian Schmidtea mediterranea to test that hypothesis. By identifying and characterizing S. mediterranea homologs of genes encoding synaptonemal complex protein 1 (SYCP1), the topoisomerase-like protein SPO11, and RAD51, a key player in homologous recombination, we confirmed that SC formation begins near the telomeres and progresses along chromosome arms during zygotene. Although distal regions pair at the time of bouquet formation, pairing of a unique interstitial locus is not observed until the formation of full-length SC at pachytene. Moreover, neither full extension of the SC nor homologous pairing is dependent on the formation of double-strand breaks. These findings validate Gelei’s speculation that full-length pairing of homologous chromosomes is mediated by the extension of the SC formed near the telomeres. S. mediterranea thus becomes the first organism described (to our knowledge) that forms a canonical telomere bouquet but does not require double-strand breaks for synapsis between homologous chromosomes. However, the initiation of SC formation at the base of the telomere bouquet, which then is followed by full-length homologous pairing in planarian spermatocytes, is not observed in other species and may not be conserved. PMID:25404302

  14. Crystal Structure of a Full-Length Autotransporter

    SciTech Connect

    van den Berg, B.

    2010-01-01

    The autotransporter (AT) secretion mechanism is the most common mechanism for the secretion of virulence factors across the outer membrane (OM) from pathogenic Gram-negative bacteria. In addition, ATs have attracted biotechnological and biomedical interest for protein display on bacterial cell surfaces. Despite their importance, the mechanism by which passenger domains of ATs pass the OM is still unclear. The classical view is that the {beta}-barrel domain provides the conduit through which the unfolded passenger moves, with the energy provided by vectorial folding of the {beta}-strand-rich passenger on the extracellular side of the OM. We present here the first structure of a full-length AT, the esterase EstA from Pseudomonas aeruginosa, at a resolution of 2.5 {angstrom}. EstA has a relatively narrow, 12-stranded {beta}-barrel that is covalently attached to the passenger domain via a long, curved helix that occupies the lumen of the {beta}-barrel. The passenger has a structure that is dramatically different from that of other known passengers, with a globular fold that is dominated by {alpha}-helices and loops. The arrangement of secondary-structure elements suggests that the passenger can fold sequentially, providing the driving force for passenger translocation. The esterase active-site residues are located at the apical surface of the passenger, at the entrance of a large hydrophobic pocket that contains a bound detergent molecule that likely mimics substrate. The EstA structure provides insight into AT mechanism and will facilitate the design of fusion proteins for cell surface display.

  15. Generation of a Mouse Full-length Balancer with Versatile Cassette-shuttling Selection Strategy

    PubMed Central

    Ye, Zhisheng; Sun, Lei; Li, Rongbo; Han, Min; Zhuang, Yuan; Wu, Xiaohui; Xu, Tian

    2016-01-01

    Balancer chromosomes are important tools for a variety of genetic manipulations in lower model organisms, owing to their ability to suppress recombination. In mouse, however, such effort has not been accomplished, mostly due to the size of the chromosomes and the complexity of multiple step chromosomal engineering. We developed an effective and versatile cassette-shuttling selection (CASS) strategy involving only two selection markers to achieve the sequential production of multiple large inversions along the chromosome. Using this strategy, we successfully generated the first full-length balancer in mice and showed that Balancer 17M-GFP can efficiently suppress recombination. Our study has not only generated a useful genetic resource, but also provided a strategy for constructing mammalian balancer chromosomes. PMID:27489495

  16. Human Insulin from Recombinant DNA Technology

    NASA Astrophysics Data System (ADS)

    Johnson, Irving S.

    1983-02-01

    Human insulin produced by recombinant DNA technology is the first commercial health care product derived from this technology. Work on this product was initiated before there were federal guidelines for large-scale recombinant DNA work or commercial development of recombinant DNA products. The steps taken to facilitate acceptance of large-scale work and proof of the identity and safety of such a product are described. While basic studies in recombinant DNA technology will continue to have a profound impact on research in the life sciences, commercial applications may well be controlled by economic conditions and the availability of investment capital.

  17. Recombinant Human Papillomavirus (HPV) Nonavalent Vaccine

    Cancer.gov

    This page contains brief information about recombinant human papillomavirus (HPV) nonavalent vaccine and a collection of links to more information about the use of this vaccine, research results, and ongoing clinical trials.

  18. Recombinant Human Papillomavirus (HPV) Bivalent Vaccine

    Cancer.gov

    This page contains brief information about recombinant human papillomavirus (HPV) bivalent vaccine and a collection of links to more information about the use of this vaccine, research results, and ongoing clinical trials.

  19. Recombinant Human Papillomavirus (HPV) Quadrivalent Vaccine

    Cancer.gov

    This page contains brief information about recombinant human papillomavirus (HPV) quadrivalent vaccine and a collection of links to more information about the use of this vaccine, research results, and ongoing clinical trials.

  20. Blueprint for a High-Performance Biomaterial: Full-Length Spider Dragline Silk Genes

    PubMed Central

    Ayoub, Nadia A.; Garb, Jessica E.; Tinghitella, Robin M.; Collin, Matthew A.; Hayashi, Cheryl Y.

    2007-01-01

    Spider dragline (major ampullate) silk outperforms virtually all other natural and manmade materials in terms of tensile strength and toughness. For this reason, the mass-production of artificial spider silks through transgenic technologies has been a major goal of biomimetics research. Although all known arthropod silk proteins are extremely large (>200 kiloDaltons), recombinant spider silks have been designed from short and incomplete cDNAs, the only available sequences. Here we describe the first full-length spider silk gene sequences and their flanking regions. These genes encode the MaSp1 and MaSp2 proteins that compose the black widow's high-performance dragline silk. Each gene includes a single enormous exon (>9000 base pairs) that translates into a highly repetitive polypeptide. Patterns of variation among sequence repeats at the amino acid and nucleotide levels indicate that the interaction of selection, intergenic recombination, and intragenic recombination governs the evolution of these highly unusual, modular proteins. Phylogenetic footprinting revealed putative regulatory elements in non-coding flanking sequences. Conservation of both upstream and downstream flanking sequences was especially striking between the two paralogous black widow major ampullate silk genes. Because these genes are co-expressed within the same silk gland, there may have been selection for similarity in regulatory regions. Our new data provide complete templates for synthesis of recombinant silk proteins that significantly improve the degree to which artificial silks mimic natural spider dragline fibers. PMID:17565367

  1. Crystal structures of 7-methylguanosine 5'-triphosphate (m(7)GTP)- and P(1)-7-methylguanosine-P(3)-adenosine-5',5'-triphosphate (m(7)GpppA)-bound human full-length eukaryotic initiation factor 4E: biological importance of the C-terminal flexible region.

    PubMed Central

    Tomoo, Koji; Shen, Xu; Okabe, Koumei; Nozoe, Yoshiaki; Fukuhara, Shoichi; Morino, Shigenobu; Ishida, Toshimasa; Taniguchi, Taizo; Hasegawa, Hiroshi; Terashima, Akira; Sasaki, Masahiro; Katsuya, Yoshio; Kitamura, Kunihiro; Miyoshi, Hiroshi; Ishikawa, Masahide; Miura, Kin-ichiro

    2002-01-01

    The crystal structures of the full-length human eukaryotic initiation factor (eIF) 4E complexed with two mRNA cap analogues [7-methylguanosine 5'-triphosphate (m(7)GTP) and P(1)-7-methylguanosine-P(3)-adenosine-5',5'-triphosphate (m(7)GpppA)] were determined at 2.0 A resolution (where 1 A=0.1 nm). The flexibility of the C-terminal loop region of eIF4E complexed with m(7)GTP was significantly reduced when complexed with m(7)GpppA, suggesting the importance of the second nucleotide in the mRNA cap structure for the biological function of eIF4E, especially the fixation and orientation of the C-terminal loop region, including the eIF4E phosphorylation residue. The present results provide the structural basis for the biological function of both N- and C-terminal mobile regions of eIF4E in translation initiation, especially the regulatory function through the switch-on/off of eIF4E-binding protein-eIF4E phosphorylation. PMID:11879179

  2. Production of functional human insulin-like growth factor binding proteins (IGFBPs) using recombinant expression in HEK293 cells.

    PubMed

    Wanscher, Anne Sofie Molsted; Williamson, Michael; Ebersole, Tasja Wainani; Streicher, Werner; Wikström, Mats; Cazzamali, Giuseppe

    2015-04-01

    Insulin-like growth factor binding proteins (IGFBPs) display many functions in humans including regulation of the insulin-like growth factor (IGF) signaling pathway. The various roles of human IGFBPs make them attractive protein candidates in drug discovery. Structural and functional knowledge on human proteins with therapeutic relevance is needed to design and process the next generation of protein therapeutics. In order to conduct structural and functional investigations large quantities of recombinant proteins are needed. However, finding a suitable recombinant production system for proteins such as full-length human IGFBPs, still remains a challenge. Here we present a mammalian HEK293 expression method suitable for over-expression of secretory full-length human IGFBP-1 to -7. Protein purification of full-length human IGFBP-1, -2, -3 and -5 was conducted using a two-step chromatography procedure and the final protein yields were between 1 and 12mg protein per liter culture media. The recombinant IGFBPs contained PTMs and exhibited high-affinity interactions with their natural ligands IGF-1 and IGF-2. PMID:25448590

  3. Full-length soluble urokinase plasminogen activator receptor down-modulates nephrin expression in podocytes.

    PubMed

    Alfano, Massimo; Cinque, Paola; Giusti, Guido; Proietti, Silvia; Nebuloni, Manuela; Danese, Silvio; D'Alessio, Silvia; Genua, Marco; Portale, Federica; Lo Porto, Manuela; Singhal, Pravin C; Rastaldi, Maria Pia; Saleem, Moin A; Mavilio, Domenico; Mikulak, Joanna

    2015-01-01

    Increased plasma level of soluble urokinase-type plasminogen activator receptor (suPAR) was associated recently with focal segmental glomerulosclerosis (FSGS). In addition, different clinical studies observed increased concentration of suPAR in various glomerular diseases and in other human pathologies with nephrotic syndromes such as HIV and Hantavirus infection, diabetes and cardiovascular disorders. Here, we show that suPAR induces nephrin down-modulation in human podocytes. This phenomenon is mediated only by full-length suPAR, is time-and dose-dependent and is associated with the suppression of Wilms' tumor 1 (WT-1) transcription factor expression. Moreover, an antagonist of αvβ3 integrin RGDfv blocked suPAR-induced suppression of nephrin. These in vitro data were confirmed in an in vivo uPAR knock out Plaur(-/-) mice model by demonstrating that the infusion of suPAR inhibits expression of nephrin and WT-1 in podocytes and induces proteinuria. This study unveiled that interaction of full-length suPAR with αvβ3 integrin expressed on podocytes results in down-modulation of nephrin that may affect kidney functionality in different human pathologies characterized by increased concentration of suPAR. PMID:26380915

  4. Protein Crystal Recombinant Human Insulin

    NASA Technical Reports Server (NTRS)

    1994-01-01

    The comparison of protein crystal, Recombiant Human Insulin; space-grown (left) and earth-grown (right). On STS-60, Spacehab II indicated that space-grown crystals are larger and of greater optical clarity than their earth-grown counterparts. Recombiant Human Insulin facilitates the incorporation of glucose into cells. In diabetics, there is either a decrease in or complete lack of insulin, thereby leading to several harmful complications. Principal Investigator is Larry DeLucas.

  5. Human recombinant lysosomal enzymes produced in microorganisms.

    PubMed

    Espejo-Mojica, Ángela J; Alméciga-Díaz, Carlos J; Rodríguez, Alexander; Mosquera, Ángela; Díaz, Dennis; Beltrán, Laura; Díaz, Sergio; Pimentel, Natalia; Moreno, Jefferson; Sánchez, Jhonnathan; Sánchez, Oscar F; Córdoba, Henry; Poutou-Piñales, Raúl A; Barrera, Luis A

    2015-01-01

    Lysosomal storage diseases (LSDs) are caused by accumulation of partially degraded substrates within the lysosome, as a result of a function loss of a lysosomal protein. Recombinant lysosomal proteins are usually produced in mammalian cells, based on their capacity to carry out post-translational modifications similar to those observed in human native proteins. However, during the last years, a growing number of studies have shown the possibility to produce active forms of lysosomal proteins in other expression systems, such as plants and microorganisms. In this paper, we review the production and characterization of human lysosomal proteins, deficient in several LSDs, which have been produced in microorganisms. For this purpose, Escherichia coli, Saccharomyces cerevisiae, Pichia pastoris, Yarrowia lipolytica, and Ogataea minuta have been used as expression systems. The recombinant lysosomal proteins expressed in these hosts have shown similar substrate specificities, and temperature and pH stability profiles to those produced in mammalian cells. In addition, pre-clinical results have shown that recombinant lysosomal enzymes produced in microorganisms can be taken-up by cells and reduce the substrate accumulated within the lysosome. Recently, metabolic engineering in yeasts has allowed the production of lysosomal enzymes with tailored N-glycosylations, while progresses in E. coli N-glycosylations offer a potential platform to improve the production of these recombinant lysosomal enzymes. In summary, microorganisms represent convenient platform for the production of recombinant lysosomal proteins for biochemical and physicochemical characterization, as well as for the development of ERT for LSD. PMID:26071627

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

    PubMed

    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

  7. Characterization of full-length HIV-1 CRF17_BF genomes and comparison to the prototype CRF12_BF strains.

    PubMed

    Aulicino, Paula C; Gómez-Carrillo, Manuel; Bello, Gonzalo; Rocco, Carlos; Mangano, Andrea; Carr, Jean; Sen, Luisa; Foley, Brian

    2012-03-01

    The aim of this work is to characterize the full-length intersubtype recombinant structure of the HIV-1 Circulating Recombinant Form CRF17_BF. A single genome of CRF17_BF was originally described in 2001 as being largely similar to CRF12_BF. Since then, more genomes of CRF17_BF have been sequenced but not adequately described in publications. Here we describe CRF17_BF as a genuine CRF, and analyze its recombination pattern based on bootscan analyses, subtype signature patterns, and phylogenetic reconstruction of subtype-delimited segments. We show that CRF17_BF can be distinguished from CRF12_BF in several regions of the genome, including vpu, pol, env and nef. A complete and accurate characterization and description of recombination breakpoints in CRFs is required for a proper surveillance of HIV-1 genotypes, and important for epidemiological purposes. PMID:22266022

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

    PubMed

    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

  9. Cutaneous allergy to human (recombinant DNA) insulin.

    PubMed

    Grammer, L C; Metzger, B E; Patterson, R

    1984-03-16

    p6 report two cases of cutaneous allergy to human (recombinant DNA) insulin. Each patient had a history of systemic allergic reactions to porcine insulin and was at least as reactive to human as to porcine insulin by end-point cutaneous titration. Both patients' insulin allergy was managed with animal insulins and both have done well. Our experience with these two patients indicates that human insulin (rDNA) should not be expected to be efficacious in all patients with systemic allergy to insulin. PMID:6366262

  10. Infectious RNA transcripts from full-length dengue virus type 2 cDNA clones made in yeast.

    PubMed Central

    Polo, S; Ketner, G; Levis, R; Falgout, B

    1997-01-01

    The dengue virus type 2 genomic RNA was amplified by reverse transcription-PCR and cloned as four cDNA fragments. We could not assemble these four fragments into full-length cDNA in Escherichia coli. The full-length dengue virus cDNA was constructed by homologous recombination in yeast, either as part of a yeast artificial chromosome or in a yeast-E. coli shuttle vector. Full-length cDNA clones were propagated once in E. coli to prepare useful quantities of DNA. In vitro transcription of these clones produced full-length RNA transcripts. Introduction of these transcripts into LLC-MK2 cells produced typical dengue infection, as judged by cytopathic effects and indirect immunofluorescence. Infectivity was sensitive to RNase digestion and was dependent on the presence of cap analog in the transcription reaction mixture. Virus in the medium was passaged on C6-36 cells to produce stocks, and these stocks had titers and plaque morphologies similar to those of the parental dengue virus type 2. Intracellular dengue virus RNA from cells infected with transcript-derived virus contained an introduced BstEII site, proving that infectivity was derived from RNA transcripts and not from contamination with parental dengue virus. Transcript-derived virus was comparable to dengue virus type 2 for growth and protein expression in tissue culture cells. Sequence analysis of the dengue virus cDNA in one full-length clone revealed only one unexpected silent mutation. By using yeast technology, it will be easy to introduce specific mutations into the dengue virus cDNA, allowing analysis of the virus phenotype in cells transfected with mutant transcripts. PMID:9188607

  11. Human DNA repair and recombination genes

    SciTech Connect

    Thompson, L.H.; Weber, C.A.; Jones, N.J.

    1988-09-01

    Several genes involved in mammalian DNA repair pathways were identified by complementation analysis and chromosomal mapping based on hybrid cells. Eight complementation groups of rodent mutants defective in the repair of uv radiation damage are now identified. At least seven of these genes are probably essential for repair and at least six of them control the incision step. The many genes required for repair of DNA cross-linking damage show overlap with those involved in the repair of uv damage, but some of these genes appear to be unique for cross-link repair. Two genes residing on human chromosome 19 were cloned from genomic transformants using a cosmid vector, and near full-length cDNA clones of each gene were isolated and sequenced. Gene ERCC2 efficiently corrects the defect in CHO UV5, a nucleotide excision repair mutant. Gene XRCC1 normalizes repair of strand breaks and the excessive sister chromatid exchange in CHO mutant EM9. ERCC2 shows a remarkable /approximately/52% overall homology at both the amino acid and nucleotide levels with the yeast RAD3 gene. Evidence based on mutation induction frequencies suggests that ERCC2, like RAD3, might also be an essential gene for viability. 100 refs., 4 tabs.

  12. The C-Terminal Region Mesd Peptide Mimics Full-Length Mesd and Acts as an Inhibitor of Wnt/β-Catenin Signaling in Cancer Cells

    PubMed Central

    Lin, Cuihong; Lu, Wenyan; Zhang, Wei; Londoño-Joshi, Angelina I.; Buchsbaum, Donald J.; Bu, Guojun; Li, Yonghe

    2013-01-01

    While Mesd was discovered as a specialized molecular endoplasmic reticulum chaperone for the Wnt co-receptors LRP5 and LRP6, recombinant Mesd protein is able to bind to mature LRP5 and LRP6 on the cell surface and acts as a universal antagonist of LRP5/6 modulators. In our previous study, we found that the C-terminal region of Mesd, which is absent in sequences from invertebrates, is necessary and sufficient for binding to mature LRP6 on the cell surface. In the present studies, we further characterized the interaction between the C-terminal region Mesd peptide and LRP5/6. We found that Mesd C-terminal region-derived peptides block Mesd binding to LRP5 at the cell surface too. We also showed that there are two LRP5/6 binding sites within Mesd C-terminal region which contain several positively charged residues. Moreover, we demonstrated that the Mesd C-terminal region peptide, like the full-length Mesd protein, blocked Wnt 3A- and Rspodin1-induced Wnt/β-catenin signaling in LRP5- and LRP6- expressing cells, suppressed Wnt/β-catenin signaling in human breast HS578T cells and prostate cancer PC-3 cells, and inhibited cancer cell proliferation, although the full-length Mesd protein is more potent than its peptide. Finally, we found that treatment of the full-length Mesd protein and its C-terminal region peptide significantly increased chemotherapy agent Adriamycin-induced cytotoxicity in HS578T and PC-3 cells. Together, our results suggest that Mesd C-terminal region constitutes the major LRP5/6-binding domain, and that Mesd protein and its C-terminal region peptide have a potential therapeutic value in cancer. PMID:23469146

  13. Directed Evolution of a Secretory Leader for the Improved Expression of Heterologous Proteins and Full-Length Antibodies in S. cerevisiae

    PubMed Central

    Rakestraw, J. Andy; Sazinsky, Stephen L.; Piatesi, Andrea; Antipov, Eugene; Wittrup, K. Dane

    2010-01-01

    Because of its eukaryotic nature, simple fermentation requirements, and pliable genetics, there have been many attempts at improving recombinant protein production in S. cerevisiae. These strategies typically involve altering the expression of a native protein thought to be involved in heterologous protein trafficking. Usually, these approaches yield three to ten-fold improvements over wild-type strains and are almost always specific to one type of protein. In this study, a library of mutant alpha mating factor 1 leader peptides (MFα1pp) is screened for the enhanced secretion of a single-chain antibody. One of the isolated mutants is shown to enhance the secretion of the scFv up to sixteen-fold over wild-type. These leaders also confer a secretory improvement to two other scFvs as well as two additional, structurally unrelated proteins. Moreover, the improved leader sequences, combined with strain engineering, allow for a one-hundred eighty fold improvement over previous reports in the secretion of full length, functional, glycosylated human IgG1. The production of full-length IgG1 at milligram per liter titers in a simple, laboratory-scale system will significantly expedite drug discovery and reagent synthesis while reducing antibody cloning, production, and characterization costs. PMID:19459139

  14. Evaluation of liver fluke recombinant cathepsin B-1 protease as a serodiagnostic antigen for human opisthorchiasis.

    PubMed

    Sripa, Jittiyawadee; Brindley, Paul J; Sripa, Banchob; Loukas, Alex; Kaewkes, Sasithorn; Laha, Thewarach

    2012-03-01

    A cathepsin B-like cysteine protease belonging to family C1 is abundantly expressed in the transcriptome and proteome of the carcinogenic liver fluke of humans, Opisthorchis viverrini. This enzyme is present in excretory/secretory (ES) products released by parasites cultured in vitro. This study evaluated the performance of recombinant O. viverrini cathepsin B1 (rOv-CB-1) as an antigen for immunodiagnosis of opisthorchiasis. The full length Ov-CB-1 cDNA was cloned and recombinant protein was produced in catalytically active form in Pichia pastoris. The recombinant Ov-CB-1 (rOv-CB-1) was affinity purified via nickel-NTA chromatography and tested in enzyme-linked immunosorbent assays (ELISA) with human sera from an opisthorchiasis endemic area. Sera from egg-positive O. viverrini infections produced a strong IgG antibody response to rOv-CB-1 both in ELISA and immunoblot analysis. The sensitivity and specificity of the ELISA test was 67% and 81%, respectively. These findings support the feasibility of using recombinant Ov-CB-1 in ELISA for the serodiagnosis of human opisthorchiasis. PMID:21704728

  15. Quasispecies Analyses of the HIV-1 Near-full-length Genome With Illumina MiSeq

    PubMed Central

    Ode, Hirotaka; Matsuda, Masakazu; Matsuoka, Kazuhiro; Hachiya, Atsuko; Hattori, Junko; Kito, Yumiko; Yokomaku, Yoshiyuki; Iwatani, Yasumasa; Sugiura, Wataru

    2015-01-01

    Human immunodeficiency virus type-1 (HIV-1) exhibits high between-host genetic diversity and within-host heterogeneity, recognized as quasispecies. Because HIV-1 quasispecies fluctuate in terms of multiple factors, such as antiretroviral exposure and host immunity, analyzing the HIV-1 genome is critical for selecting effective antiretroviral therapy and understanding within-host viral coevolution mechanisms. Here, to obtain HIV-1 genome sequence information that includes minority variants, we sought to develop a method for evaluating quasispecies throughout the HIV-1 near-full-length genome using the Illumina MiSeq benchtop deep sequencer. To ensure the reliability of minority mutation detection, we applied an analysis method of sequence read mapping onto a consensus sequence derived from de novo assembly followed by iterative mapping and subsequent unique error correction. Deep sequencing analyses of aHIV-1 clone showed that the analysis method reduced erroneous base prevalence below 1% in each sequence position and discarded only < 1% of all collected nucleotides, maximizing the usage of the collected genome sequences. Further, we designed primer sets to amplify the HIV-1 near-full-length genome from clinical plasma samples. Deep sequencing of 92 samples in combination with the primer sets and our analysis method provided sufficient coverage to identify >1%-frequency sequences throughout the genome. When we evaluated sequences of pol genes from 18 treatment-naïve patients' samples, the deep sequencing results were in agreement with Sanger sequencing and identified numerous additional minority mutations. The results suggest that our deep sequencing method would be suitable for identifying within-host viral population dynamics throughout the genome. PMID:26617593

  16. Quasispecies Analyses of the HIV-1 Near-full-length Genome With Illumina MiSeq.

    PubMed

    Ode, Hirotaka; Matsuda, Masakazu; Matsuoka, Kazuhiro; Hachiya, Atsuko; Hattori, Junko; Kito, Yumiko; Yokomaku, Yoshiyuki; Iwatani, Yasumasa; Sugiura, Wataru

    2015-01-01

    Human immunodeficiency virus type-1 (HIV-1) exhibits high between-host genetic diversity and within-host heterogeneity, recognized as quasispecies. Because HIV-1 quasispecies fluctuate in terms of multiple factors, such as antiretroviral exposure and host immunity, analyzing the HIV-1 genome is critical for selecting effective antiretroviral therapy and understanding within-host viral coevolution mechanisms. Here, to obtain HIV-1 genome sequence information that includes minority variants, we sought to develop a method for evaluating quasispecies throughout the HIV-1 near-full-length genome using the Illumina MiSeq benchtop deep sequencer. To ensure the reliability of minority mutation detection, we applied an analysis method of sequence read mapping onto a consensus sequence derived from de novo assembly followed by iterative mapping and subsequent unique error correction. Deep sequencing analyses of aHIV-1 clone showed that the analysis method reduced erroneous base prevalence below 1% in each sequence position and discarded only < 1% of all collected nucleotides, maximizing the usage of the collected genome sequences. Further, we designed primer sets to amplify the HIV-1 near-full-length genome from clinical plasma samples. Deep sequencing of 92 samples in combination with the primer sets and our analysis method provided sufficient coverage to identify >1%-frequency sequences throughout the genome. When we evaluated sequences of pol genes from 18 treatment-naïve patients' samples, the deep sequencing results were in agreement with Sanger sequencing and identified numerous additional minority mutations. The results suggest that our deep sequencing method would be suitable for identifying within-host viral population dynamics throughout the genome. PMID:26617593

  17. Methionine oxidation induces amyloid fibril formation by full-length apolipoprotein A-I

    PubMed Central

    Wong, Yuan Qi; Binger, Katrina J.; Howlett, Geoffrey J.; Griffin, Michael D. W.

    2010-01-01

    Apolipoprotein A-I (apoA-I) is the major protein component of HDL, where it plays an important role in cholesterol transport. The deposition of apoA-I derived amyloid is associated with various hereditary systemic amyloidoses and atherosclerosis; however, very little is known about the mechanism of apoA-I amyloid formation. Methionine residues in apoA-I are oxidized via several mechanisms in vivo to form methionine sulfoxide (MetO), and significant levels of methionine oxidized apoA-I (MetO-apoA-I) are present in normal human serum. We investigated the effect of methionine oxidation on the structure, stability, and aggregation of full-length, lipid-free apoA-I. Circular dichrosim spectroscopy showed that oxidation of all three methionine residues in apoA-I caused partial unfolding of the protein and decreased its thermal stability, reducing the melting temperature (Tm) from 58.7 °C for native apoA-I to 48.2 °C for MetO-apoA-I. Analytical ultracentrifugation revealed that methionine oxidation inhibited the native self association of apoA-I to form dimers and tetramers. Incubation of MetO-apoA-I for extended periods resulted in aggregation of the protein, and these aggregates bound Thioflavin T and Congo Red. Inspection of the aggregates by electron microscopy revealed fibrillar structures with a ribbon-like morphology, widths of approximately 11 nm, and lengths of up to several microns. X-ray fibre diffraction studies of the fibrils revealed a diffraction pattern with orthogonal peaks at spacings of 4.64 Å and 9.92 Å, indicating a cross-β amyloid structure. This systematic study of fibril formation by full-length apoA-I represents the first demonstration that methionine oxidation can induce amyloid fibril formation. PMID:20133843

  18. Infectious full-length clones of Calibrachoa Mottle Virus (CbMV)

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Full-length cDNA clones derived from genomic RNA (gRNA) and subgenomic RNAs (sgRNAs) of Calibrachoa mottle virus (CbMV) were constructed under the control of the T7 RNA promoter and ligated into plasmid pUC-18. The capped and uncapped in vitro transcripts, synthesized from full length genomic cDNA...

  19. Expression, Purification, and Identification of Associated Proteins of the Full-length hCDK12/CyclinK Complex*

    PubMed Central

    Bartkowiak, Bartlomiej; Greenleaf, Arno L.

    2015-01-01

    The coupling of transcription and associated processes has been shown to be dependent on the RNA polymerase II (RNAPII) C-terminal repeat domain (CTD) and the phosphorylation of the heptad repeats of which it is composed (consensus sequence Y1S2P3T4S5P6S7). Two primary S2 position CTD kinases have been identified in higher eukaryotes: P-TEFb and CDK12/CyclinK. The more recently discovered CDK12 appears to act at the 3′-end of the transcription unit and has been identified as a tumor suppressor for ovarian cancer; however much is still unknown about the in vivo roles of CDK12/CyclinK. In an effort to further characterize these roles we have purified to near homogeneity and characterized, full-length, active, human CDK12/CyclinK, and identified hCDK12-associated proteins via mass spectrometry. We find that employing a “2A” peptide-linked multicistronic construct containing CDK12 and CyclinK results in the efficient production of active, recombinant enzyme in the baculovirus/Sf9 expression system. Using GST-CTD fusion protein substrates we find that CDK12/CyclinK prefers a substrate with unmodified repeats or one that mimics prephosphorylation at the S7 position of the CTD; also the enzyme is sensitive to the inhibitor flavopiridol at higher concentrations. Identification of CDK12-associating proteins reveals a strong enrichment for RNA-processing factors suggesting that CDK12 affects RNA processing events in two distinct ways: Indirectly through generating factor-binding phospho-epitopes on the CTD of elongating RNAPII and directly through binding to specific factors. PMID:25429106

  20. High-yield bacterial expression and structural characterization of recombinant human insulin-like growth factor binding protein-2

    PubMed Central

    Swain, Monalisa; Slomiany, Mark G.; Rosenzweig, Steven A.; Atreya, Hanudatta S.

    2010-01-01

    The diverse biological activities of the insulin-like growth factors (IGF-1 and IGF-2) are mediated by the IGF-1 receptor (IGF-IR). These actions are modulated by a family of six IGF-binding proteins (IGFBP-1–6; 22–31 kDa) that via high affinity binding to the IGFs (KD ~ 300–700 pM) both protect the IGFs in the circulation and attenuate IGF action by blocking their receptor access. In recent years, IGFBPs have been implicated in a variety of cancers. However, the structural basis of their interaction with IGFs and/or other proteins is not completely understood. A critical challenge in the structural characterization of full-length IGFBPs has been the difficulty in expressing these proteins at levels suitable for NMR/X-ray crystallography analysis. Here we describe the high-yield expression of full-length recombinant human IGFBP-2 (rhIGFBP-2) in E. coli. Using a single step purification protocol, rhIGFBP-2 was obtained with >95% purity and structurally characterized using NMR spectroscopy. The protein was found to exist as a monomer at the high concentrations required for structural studies and to exist in a single conformation exhibiting a unique intra-molecular disulfide-bonding pattern. The protein retained full biologic activity. This study represents the first high-yield expression of wild-type recombinant human IGFBP-2 in E. coli and first structural characterization of a full-length IGFBP. PMID:20541521

  1. Isolation, characterization and functional analysis of full length p53 cDNA from Bubalus bubalis.

    PubMed

    Singh, Minu; Aggarwal, Suruchi; Mohanty, Ashok K; Mukhopadhyay, Tapas

    2015-09-01

    p53 plays a pivotal role in maintaining the genomic integrity of the cell and has an important role in cellular transformation. We isolated and cloned a full length p53 cDNA (Bp53) from water buffalo in expression vectors designed to generate tagged proteins with FLAG or GFP. Bp53 was found to be 1161 nucleotide long and codes for 386 amino acid residues with 79% homology with human p53 containing 393 amino acids. Although Bp53 has some inherent differences in amino acid composition in different functional domains as compared to human p53 but the total electrostatic charge of amino acids has been maintained. Bp53 cDNA was transiently transfected in a p53 null human NSCLC cell line and as expected, it was predominantly localized in the nucleus. Besides, Bp53 effectively transactivates a number of target genes similar to human p53 and exerts most of its anti-tumorigenic potential in culture as observed in clonogenic and cell viability assays. Like human p53 mutants, core domain mutant version of Bp53 was found to be mis-localized to cytoplasm with diminished tumor suppressor activity. However, Bp53 appeared to be more sensitive to mdm2 mediated degradation and as a result, this protein was less stable as compared to human p53. For the first time we have characterized a functionally efficient wild-type p53 from buffalo having lower stability than human p53 and thus, buffalo p53 could be used as a model system for further insight to the molecular basis of wild-type p53 instability. PMID:26003295

  2. Retrotransposon mdg3 of Drosophila: General structure and functional domains of the full-length copy

    SciTech Connect

    Avedisov, S.N.; Ilyin, Yu.V.

    1995-09-01

    A full-length copy of the transposable element mdg3 from the plasmid clone Dm38 of Drosophila melanogaster was obtained by screening the DNA library of the cell culture 67J25D. Previous work demonstrated that only full-length copies of mdg3 (5.5 kb) are amplified in this culture, whereas the number of deleted copies probably has not changed since the cell line was established. We sequenced the full-length copy of mdg3 from cDm38 by the method described by Sanger. 10 refs., 2 figs., 2 tabs.

  3. Patterns of recombination on human chromosome 22

    SciTech Connect

    Schlumpf, K.S.; Kim, D.; Haines, J.L.

    1994-09-01

    Virtually all genetic linkage maps generated to date are gross averages across individuals, ages, and (often) sexes. In addition, although some level of positive interference has been assumed, until recently little evidence to support this in humans has been available. The major stumbling block has been the quality of the data available, since even a few genotypic errors can have drastic effects on both the map length and the number of apparent recombinants. In addition, variation in recombination by factors other than sex have pretty much been ignored. To explore recombination in more detail, we have generated a microsatellite marker map of human chromosome 22. This map includes 32 markers genotyped through 46 sibships of the Venezuelan Reference Pedigree (VRP). Extensive error checking and regenotyping was performed to remove as many genotypic errors as possible, but no genotypes were removed simply because they created unlikely events. The following 1000:1 odds map has been obtained: cen--F8VWFP1--11--S264--3-S311--4--S257--2--TOP1P2--3--S156--1--CRYB2--1--S258--2--S310--6--S193--1--S275--3--S268--1--S280--4--S304--3--S283--2--LiR1--3--IL2RB--3--S299--1--S302--1--S537--2--S270--4--PDGF--8--S274--qter. The female map (91 cM) is twice as long as the male map (46 cM) and the log-likelihood difference in the maps (22.3) is highly significant (P=0.001, df=22) and appears constant across the chromosome. Analysis of recombination with age showed no particular trends for either males or females when chromosomes were grouped into three categories (20, 20-30, 30+) by parental age at birth of child. Positive interference was found in maternally derived chromosomes ({chi}{sup 2}=30.5 (4), p<0.005), but not in paternally derived chromosomes ({chi}{sup 2}=6.24 (3), P=0.10). This contrasts to data from chromosomes 9 and 21 where positive interference was found for both sexes. More detailed analyses are in progress.

  4. Species-Specific Expression of Full-Length and Alternatively Spliced Variant Forms of CDK5RAP2.

    PubMed

    Park, John S Y; Lee, Marie-Katrina; Kang, SungMyung; Jin, Yan; Fu, Songbin; Rosales, Jesusa L; Lee, Ki-Young

    2015-01-01

    CDK5RAP2 is one of the primary microcephaly genes that are associated with reduced brain size and mental retardation. We have previously shown that human CDK5RAP2 exists as a full-length form (hCDK5RAP2) or an alternatively spliced variant form (hCDK5RAP2-V1) that is lacking exon 32. The equivalent of hCDK5RAP2-V1 has been reported in rat and mouse but the presence of full-length equivalent hCDK5RAP2 in rat and mouse has not been examined. Here, we demonstrate that rat expresses both a full length and an alternatively spliced variant form of CDK5RAP2 that are equivalent to our previously reported hCDK5RAP2 and hCDK5RAP2-V1, repectively. However, mouse expresses only one form of CDK5RAP2 that is equivalent to the human and rat alternatively spliced variant forms. Knowledge of this expression of different forms of CDK5RAP2 in human, rat and mouse is essential in selecting the appropriate model for studies of CDK5RAP2 and primary microcephaly but our findings further indicate the evolutionary divergence of mouse from the human and rat species. PMID:26550838

  5. Species-Specific Expression of Full-Length and Alternatively Spliced Variant Forms of CDK5RAP2

    PubMed Central

    Park, John S. Y.; Lee, Marie-Katrina; Kang, SungMyung; Jin, Yan; Fu, Songbin; Rosales, Jesusa L.; Lee, Ki-Young

    2015-01-01

    CDK5RAP2 is one of the primary microcephaly genes that are associated with reduced brain size and mental retardation. We have previously shown that human CDK5RAP2 exists as a full-length form (hCDK5RAP2) or an alternatively spliced variant form (hCDK5RAP2-V1) that is lacking exon 32. The equivalent of hCDK5RAP2-V1 has been reported in rat and mouse but the presence of full-length equivalent hCDK5RAP2 in rat and mouse has not been examined. Here, we demonstrate that rat expresses both a full length and an alternatively spliced variant form of CDK5RAP2 that are equivalent to our previously reported hCDK5RAP2 and hCDK5RAP2-V1, repectively. However, mouse expresses only one form of CDK5RAP2 that is equivalent to the human and rat alternatively spliced variant forms. Knowledge of this expression of different forms of CDK5RAP2 in human, rat and mouse is essential in selecting the appropriate model for studies of CDK5RAP2 and primary microcephaly but our findings further indicate the evolutionary divergence of mouse from the human and rat species. PMID:26550838

  6. [Comparison of methods to construct a full-length cDNA library].

    PubMed

    Mao, Xin-Guo; Jing, Rui-Lian; Kong, Xiu-Ying; Zhao, Guang-Yao; Jia, Ji-Zeng

    2006-07-01

    The use of full-length cDNA libraries is an effective tool to obtain complete gene information in a high-efficiency, high-throughput manner, especially in organisms with huge genomes that are not amenable to whole genome sequencing. In this review, we outlined several methods of full-length cDNA library construction and compared their advantages and disadvantages based on their respective principles. Drawing on our own experience, we described the Cap-trapper method in detail, with an emphasis on its application in wheat full-length cDNA library construction as well as the determination of the ratio of full-length cDNA in a library. PMID:16825176

  7. Fabrication and Testing of Full-Length Single-Cell Externally Fueled Converters for Thermionic Reactors

    SciTech Connect

    Schock, Alfred

    1995-08-01

    Paper presented at the 29th IECEC in Monterey, CA in August 1994. The present paper describes the fabrication and testing of full-length prototypcial converters, both unfueled and fueled, and presents parametric results of electrically heated tests.

  8. Monitoring recombinant human erythropoietin abuse among athletes.

    PubMed

    Citartan, Marimuthu; Gopinath, Subash C B; Chen, Yeng; Lakshmipriya, Thangavel; Tang, Thean-Hock

    2015-01-15

    The illegal administration of recombinant human erythropoietin (rHuEPO) among athletes is largely preferred over blood doping to enhance stamina. The advent of recombinant DNA technology allowed the expression of EPO-encoding genes in several eukaryotic hosts to produce rHuEPO, and today these performance-enhancing drugs are readily available. As a mimetic of endogenous EPO (eEPO), rHuEPO augments the oxygen carrying capacity of blood. Thus, monitoring the illicit use of rHuEPO among athletes is crucial in ensuring an even playing field and maintaining the welfare of athletes. A number of rHuEPO detection methods currently exist, including measurement of hematologic parameters, gene-based detection methods, glycomics, use of peptide markers, electrophoresis, isoelectric focusing (IEF)-double immunoblotting, aptamer/antibody-based methods, and lateral flow tests. This review gleans these different strategies and highlights the leading molecular recognition elements that have potential roles in rHuEPO doping detection. PMID:25058943

  9. Single-Molecule Studies of Unlabeled Full-Length p53 Protein Binding to DNA.

    PubMed

    Nuttall, Philippa; Lee, Kidan; Ciccarella, Pietro; Carminati, Marco; Ferrari, Giorgio; Kim, Ki-Bum; Albrecht, Tim

    2016-03-10

    p53 is an antitumor protein that plays an important role in apoptosis, preserving genomic stability and preventing angiogenesis, and it has been implicated in a large number of human cancers. For this reason it is an interesting target for both fundamental studies, such as the mechanism of interaction with DNA, and applications in biosensing. Here, we report a comprehensive study of label-free, full length p53 (flp53) and its interaction with engineered double-stranded DNA in vitro, at the single-molecule level, using atomic force microscopy (AFM) imaging and solid-state nanopore sensing. AFM data show that dimeric and tetrameric p53 bind to the DNA in a sequence-specific manner, confirming previously reported relative binding affinities. The statistical significance is tested using both the Grubbs test and stochastic simulations. For the first time, ultralow noise solid-state nanopore sensors are employed for the successful differentiation between bare DNA and p53/DNA complexes. Furthermore, translocation statistics reflect the binding affinities of different DNA sequences, in accordance with AFM data. Our results thus highlight the potential of solid-state nanopore sensors for single-molecule biosensing, especially when labeling is either not possible or at least not a viable option. PMID:26855037

  10. High-quality full-length immunoglobulin profiling with unique molecular barcoding.

    PubMed

    Turchaninova, M A; Davydov, A; Britanova, O V; Shugay, M; Bikos, V; Egorov, E S; Kirgizova, V I; Merzlyak, E M; Staroverov, D B; Bolotin, D A; Mamedov, I Z; Izraelson, M; Logacheva, M D; Kladova, O; Plevova, K; Pospisilova, S; Chudakov, D M

    2016-09-01

    High-throughput sequencing analysis of hypermutating immunoglobulin (IG) repertoires remains a challenging task. Here we present a robust protocol for the full-length profiling of human and mouse IG repertoires. This protocol uses unique molecular identifiers (UMIs) introduced in the course of cDNA synthesis to control bottlenecks and to eliminate PCR and sequencing errors. Using asymmetric 400+100-nt paired-end Illumina sequencing and UMI-based assembly with the new version of the MIGEC software, the protocol allows up to 750-nt lengths to be sequenced in an almost error-free manner. This sequencing approach should also be applicable to various tasks beyond immune repertoire studies. In IG profiling, the achieved length of high-quality sequence covers the variable region of even the longest chains, along with the fragment of a constant region carrying information on the antibody isotype. The whole protocol, including preparation of cells and libraries, sequencing and data analysis, takes 5 to 6 d. PMID:27490633

  11. Recombinant Human Elastase Treatment of Cephalic Veins

    PubMed Central

    Wong, Marco D; Bingham, Karen; Moss, Emma; Warn, J Donald; Smirnov, Igor; Bland, Kimberly S; Starcher, Barry; Franano, F Nicholas; Burke, Steven K

    2016-01-01

    Background Vessel injury at the time of Arteriovenous Fistula (AVF) creation may lead to neointimal hyperplasia that impairs AVF maturation. Vonapanitase, a recombinant human chymotrypsin-like elastase family member 1, is an investigational drug under development to improve AVF maturation and patency. The current studies were designed to document vonapanitase effects in human cephalic veins that are used in AVF creation. Methods Human cephalic veins were mounted on a perfusion myograph. Vonapanitase 1.2, 4, 13.2, and 40 μg/ml or saline was applied drop wise on the vein followed by saline rinse. Vein segments were cut into rings for elastin content determination by desmosine radioimmunoassay and histology. Fluorescently-labelled vonapanitase was applied to veins and adventitial imaging was performed using laser scanning confocal microscopy. In vivo time course experiments were performed by treating rabbit jugular veins and harvesting 1 h and 4 h after vonapanitase treatment. Results / Conclusion Vonapanitase reduced desmosine content in a dose-related manner. Histology also confirmed a dose-related reduction in elastic fiber staining. Fluorescently-labelled vonapanitase persistently localized to elastic fibers in the vein adventitia. In vivo experiments showed a reduction in desmosine content in jugular veins from 1 h to 4 h following treatment. These data suggest that vonapanitase targets elastin in elastic fibers in a dose related manner and that elastase remains in the vessel wall and has catalytic activity for at least 1 h.

  12. In vitro translation of the full-length RNA transcript of figwort mosaic virus (Caulimovirus).

    PubMed

    Ranu, R S; Gowda, S; Scholthof, H; Wu, F C; Shepherd, R J

    1996-01-01

    The circular DNA genome of FMV consists of seven tandemly arranged genes placed successively on a full-length RNA transcript that spans the entire circular viral genome. This transcript is a tentative mRNA for at least five of the six major conserved genes of this virus (genes I-V) that are positioned on this transcript. The sixth major gene (gene VI) is expressed as a separate monocistronic transcript. A long 5'-nontranslated leader (598 nucleotides), a small nonconserved gene (VII), and a short intergenic region (57 nucleotides) precede the five major conserved genes (I through V) on the full-length transcript. A reporter gene (CAT), as a separate cistron or fused in-frame, to viral cistrons in various downstream positions in cloned versions of the viral genome was used in a transcription vector to generate artificial full-length transcripts of FMV. When these mRNAs were translated in vitro (rabbit reticulocyte lysate system), the reporter gene was translated efficiently in all positions. Translation of internal native viral gene positioned on the full-length transcript of FMV was also determined (the gene VI product). These observations suggest that the full-length FMV transcript functions as a polycistronic mRNA in plants. Results are best explained on the basis of translational coupling/relay race model. PMID:8882638

  13. Reversal of a full-length mutant huntingtin neuronal cell phenotype by chemical inhibitors of polyglutamine-mediated aggregation

    PubMed Central

    Wang, Jin; Gines, Silvia; MacDonald, Marcy E; Gusella, James F

    2005-01-01

    Background Huntington's disease (HD) is an inherited neurodegenerative disorder triggered by an expanded polyglutamine tract in huntingtin that is thought to confer a new conformational property on this large protein. The propensity of small amino-terminal fragments with mutant, but not wild-type, glutamine tracts to self-aggregate is consistent with an altered conformation but such fragments occur relatively late in the disease process in human patients and mouse models expressing full-length mutant protein. This suggests that the altered conformational property may act within the full-length mutant huntingtin to initially trigger pathogenesis. Indeed, genotype-phenotype studies in HD have defined genetic criteria for the disease initiating mechanism, and these are all fulfilled by phenotypes associated with expression of full-length mutant huntingtin, but not amino-terminal fragment, in mouse models. As the in vitro aggregation of amino-terminal mutant huntingtin fragment offers a ready assay to identify small compounds that interfere with the conformation of the polyglutamine tract, we have identified a number of aggregation inhibitors, and tested whether these are also capable of reversing a phenotype caused by endogenous expression of mutant huntingtin in a striatal cell line from the HdhQ111/Q111 knock-in mouse. Results We screened the NINDS Custom Collection of 1,040 FDA approved drugs and bioactive compounds for their ability to prevent in vitro aggregation of Q58-htn 1–171 amino terminal fragment. Ten compounds were identified that inhibited aggregation with IC50 < 15 μM, including gossypol, gambogic acid, juglone, celastrol, sanguinarine and anthralin. Of these, both juglone and celastrol were effective in reversing the abnormal cellular localization of full-length mutant huntingtin observed in mutant HdhQ111/Q111 striatal cells. Conclusions At least some compounds identified as aggregation inhibitors also prevent a neuronal cellular phenotype caused

  14. Modelling the structure of full-length Epstein-Barr virus nuclear antigen 1.

    PubMed

    Hussain, Mushtaq; Gatherer, Derek; Wilson, Joanna B

    2014-12-01

    Epstein-Barr virus is a clinically important human virus associated with several cancers and is the etiologic agent of infectious mononucleosis. The viral nuclear antigen-1 (EBNA1) is central to the replication and propagation of the viral genome and likely contributes to tumourigenesis. We have compared EBNA1 homologues from other primate lymphocryptoviruses and found that the central glycine/alanine repeat (GAr) domain as well as predicted cellular protein (USP7 and CK2) binding sites are present in homologues in the Old World primates, but not the marmoset, suggesting that these motifs may have co-evolved. Using the resolved structure of the C-terminal one-third of EBNA1 (homodimerization and DNA binding domain), we have gone on to develop monomeric and dimeric models in silico of the full-length protein. The C-terminal domain is predicted to be structurally highly similar between homologues, indicating conserved function. Zinc could be stably incorporated into the model, bonding with two N-terminal cysteines predicted to facilitate multimerisation. The GAr contains secondary structural elements in the models, while the protein binding regions are unstructured, irrespective of the prediction approach used and sequence origin. These intrinsically disordered regions may facilitate the diversity observed in partner interactions. We hypothesize that the structured GAr could mask the disordered regions, thereby protecting the protein from default degradation. In the dimer conformation, the C-terminal tails of each monomer wrap around a proline-rich protruding loop of the partner monomer, providing dimer stability, a feature which could be exploited in therapeutic design. PMID:25011696

  15. Full-Length cDNA, Prokaryotic Expression, and Antimicrobial Activity of UuHb-F-I from Urechis unicinctus

    PubMed Central

    Niu, Rongli; Chen, Xiang

    2016-01-01

    Hemoglobin, which widely exists in all vertebrates and in some invertebrates, is possibly a precursor of antimicrobial peptides (AMPs). However, AMPs in the hemoglobin of invertebrates have been rarely investigated. This study is the first to report the full-length cDNA, prokaryotic expression, and antimicrobial activity of UuHb-F-I from Urechis unicinctus. The full-length cDNA sequence of UuHb-F-I was 780 bp with an open-reading frame of 429 bp encoding 142 amino acids. MALDI-TOF-MS suggested that the recombinant protein of UuHb-F-I (rUuHb-F-I) yielded a molecular weight of 15,168.01 Da, and its N-terminal amino acid sequence was MGLTGAQIDAIK. rUuHb-F-I exhibited different antimicrobial activities against microorganisms. The lowest minimum inhibitory concentration against Micrococcus luteus was 2.78–4.63 μM. Our results may help elucidate the immune defense mechanism of U. unicinctus and may provide insights into new AMPs in drug discovery. PMID:27471730

  16. Full-Length cDNA, Prokaryotic Expression, and Antimicrobial Activity of UuHb-F-I from Urechis unicinctus.

    PubMed

    Niu, Rongli; Chen, Xiang

    2016-01-01

    Hemoglobin, which widely exists in all vertebrates and in some invertebrates, is possibly a precursor of antimicrobial peptides (AMPs). However, AMPs in the hemoglobin of invertebrates have been rarely investigated. This study is the first to report the full-length cDNA, prokaryotic expression, and antimicrobial activity of UuHb-F-I from Urechis unicinctus. The full-length cDNA sequence of UuHb-F-I was 780 bp with an open-reading frame of 429 bp encoding 142 amino acids. MALDI-TOF-MS suggested that the recombinant protein of UuHb-F-I (rUuHb-F-I) yielded a molecular weight of 15,168.01 Da, and its N-terminal amino acid sequence was MGLTGAQIDAIK. rUuHb-F-I exhibited different antimicrobial activities against microorganisms. The lowest minimum inhibitory concentration against Micrococcus luteus was 2.78-4.63 μM. Our results may help elucidate the immune defense mechanism of U. unicinctus and may provide insights into new AMPs in drug discovery. PMID:27471730

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

    PubMed

    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

  18. New strategy for expression of recombinant hydroxylated human collagen α1(III) chains in Pichia pastoris GS115.

    PubMed

    He, Jing; Ma, Xiaoxuan; Zhang, Fenglong; Li, Linbo; Deng, Jianjun; Xue, Wenjiao; Zhu, Chenhui; Fan, Daidi

    2015-01-01

    Type III collagen is one of the most abundant proteins in the human body, which forms collagen fibrils and provides the stiff, resilient characteristics of many tissues. In this paper, a new method for secretory expression of recombinant hydroxylated human collagen α1(III) chain in Pichia pastoris GS115 was applied. The gene encoding for full-length human collagen α1(III) chain (COL3A1) without N-terminal propeptide and C-terminal propeptide was cloned in the pPIC9K expression vector. The prolyl 4-hydroxylase (P4H, EC 1.14.11.2) α-subunit (P4Hα) and β-subunit (P4Hβ) genes were cloned in the same expression vector, pPICZB. Fluorogenic quantitative PCR indicates that COL3A1 and P4H genes have been expressed in mRNA level. SDS-PAGE shows that secretory expression of recombinant human collagen α1(III) chain was successfully achieved in P. pastoris GS115. In addition, the result of amino acids composition analysis shows that the recombinant human collagen α1(III) chain contains hydroxyproline by coexpression with the P4H. Furthermore, liquid chromatography coupled with tandem mass spectrometry analysis demonstrates that proline residues of the recombinant human collagen α1(III) chain were hydroxylated in the X or Y positions of Gly-X-Y triplets. PMID:24953863

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

    PubMed

    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

  20. rhEPO (recombinant human eosinophil peroxidase): expression in Pichia pastoris and biochemical characterization

    PubMed Central

    Ciaccio, Chiara; Gambacurta, Alessandra; Sanctis, Giampiero DE; Spagnolo, Domenico; Sakarikou, Christina; Petrella, Giovanni; Coletta, Massimo

    2006-01-01

    A Pichia pastoris expression system has for the first time been successfully developed to produce rhEPO (recombinant human eosinophil peroxidase). The full-length rhEPO coding sequence was cloned into the pPIC9 vector in frame with the yeast α-Factor secretion signal under the transcriptional control of the AOX (acyl-CoA oxidase) promoter, and transformed into P. pastoris strain GS115. Evidence for the production of rhEPO by P. pastoris as a glycosylated dimer precursor of approx. 80 kDa was determined by SDS/PAGE and gel filtration chromatography. Recombinant hEPO undergoes proteolytic processing, similar to that in the native host, to generate two chains of approx. 50 and 20 kDa. A preliminary biochemical characterization of purified rhEPO demonstrated that the spectral and kinetic properties of the recombinant wild-type EPO are comparable with those of the native enzyme and are accompanied by oxidizing activity towards several physiological anionic substrates such as SCN−, Br− and Cl−. On the basis of the estimated Km and kcat values it is evident that the pseudohalide SCN− is the most specific substrate for rhEPO, consistent with the catalytic properties of other mammalian EPOs purified from blood. PMID:16396635

  1. Optimizing Production of Antigens and Fabs in the Context of Generating Recombinant Antibodies to Human Proteins

    PubMed Central

    Zhong, Nan; Loppnau, Peter; Seitova, Alma; Ravichandran, Mani; Fenner, Maria; Jain, Harshika; Bhattacharya, Anandi; Hutchinson, Ashley; Paduch, Marcin; Lu, Vincent; Olszewski, Michal; Kossiakoff, Anthony A.; Dowdell, Evan; Koide, Akiko; Koide, Shohei; Huang, Haiming; Nadeem, Vincent; Sidhu, Sachdev S.; Greenblatt, Jack F.; Marcon, Edyta; Arrowsmith, Cheryl H.; Edwards, Aled M.; Gräslund, Susanne

    2015-01-01

    We developed and optimized a high-throughput project workflow to generate renewable recombinant antibodies to human proteins involved in epigenetic signalling. Three different strategies to produce phage display compatible protein antigens in bacterial systems were compared, and we found that in vivo biotinylation through the use of an Avi tag was the most productive method. Phage display selections were performed on 265 in vivo biotinylated antigen domains. High-affinity Fabs (<20nM) were obtained for 196. We constructed and optimized a new expression vector to produce in vivo biotinylated Fabs in E. coli. This increased average yields up to 10-fold, with an average yield of 4 mg/L. For 118 antigens, we identified Fabs that could immunoprecipitate their full-length endogenous targets from mammalian cell lysates. One Fab for each antigen was converted to a recombinant IgG and produced in mammalian cells, with an average yield of 15 mg/L. In summary, we have optimized each step of the pipeline to produce recombinant antibodies, significantly increasing both efficiency and yield, and also showed that these Fabs and IgGs can be generally useful for chromatin immunoprecipitation (ChIP) protocols. PMID:26437229

  2. Therapeutic use of recombinant methionyl human leptin.

    PubMed

    Vatier, Camille; Gautier, Jean-François; Vigouroux, Corinne

    2012-10-01

    Recombinant methionyl human leptin (r-metHuLeptin) was first used as a replacement therapy in patients bearing inactivating mutations in the leptin gene. In this indication, it was shown since 1999 to be very efficient in inducing a dramatic weight loss in rare children and adults with severe obesity due to the lack of leptin. These first clinical trials clearly showed that r-metHuLeptin acted centrally to reduce food intake, inducing loss of fat mass, and to correct metabolic alterations, immune and neuroendocrine defects. A few years later, r-metHuLeptin was also shown to reverse the metabolic complications associated with lipodystrophic syndromes, due to primary defects in fat storage, which induce leptin deficiency. The beneficial effects, which could be mediated by central and/or peripheral mechanisms, are thought to mainly involve the lowering effects of leptin on ectopic lipid storage, in particular in liver and muscles, reducing insulin resistance. Interestingly, r-metHuLeptin therapy also reversed the hypothalamic-pituitary-gonadal axis dysfunctions associated with hypothalamic amenorrhea. However, if r-metHuLeptin treatment has been shown to be dramatically efficient in leptin-deficient states, its very limited effect in inducing weight loss in common obese patients revealed that, in patients with adequate leptin secretion, mechanisms of leptin resistance and leptin tolerance prevent r-metHuLeptin from inducing any additional effects. This review will present the current data about the effects of r-metHuLeptin therapy in humans, and discuss the recent perspectives of this therapy in new indications. PMID:22464954

  3. Expression of Functional Recombinant Human Tissue Transglutaminase (TG2) Using the Bac-to-Bac Baculovirus Expression System

    PubMed Central

    Yazdani, Yaghoub; Azari, Shahram; Kalhor, Hamid Reza

    2016-01-01

    Purpose: Tissue transglutaminase (TG2) is a unique multifunctional enzyme. The enzyme possesses enzymatic activities such as transamidation/crosslinking and non-enzymatic functions such as cell migration and signal transduction. TG2 has been shown to be involved in molecular mechanisms of cancers and several neurodegenerative diseases such as Alzheimer’s disease. The present study aimed at cloning and expression of full length human TG2 in Bac-to-Bac baculovirus expression system and evaluation of its activity. Methods: pFastBac HTA donor vector containing coding sequence of human TG2 was constructed. The construct was transformed to DH10Bac for generating recombinant bacmid. The verified bacmid was transfected to insect cell line (Sf9). Expression of recombinant TG2 was examined by RT-PCR, SDS-PAGE and western blot analysis. Functional analysis was evaluated by fluorometric assay and gel electrophoresis. Results: Recombinant bacmid was verified by amplification of a band near to 4500 bp. Expression analysis showed that the enzyme was expressed as a protein with a molecular weight near 80 kDa. Western blot confirmed the presence of TG2 and the activity assays including flurometric assay indicated that the recombinant TG2 was functional. The electrophoresis assay conformed that the expressed TG2 was the indeed capable of crosslinking in the presence of physiological concentration calcium ions. Conclusion: Human TG2 was expressed efficiently in the active biological form in the Bac-to-Bac baculovirus expression system. The expressed enzyme could be used for medical diagnostic, or studies which aim at finding novel inhibitors of the enzymes . To best of our knowledge, this is probably the first report of expression of full length human tissue transglutaminase (TG2) using the Bac-to-Bac expression system. PMID:27123417

  4. Recombinant human bone morphogenetic protein induces bone formation.

    PubMed Central

    Wang, E A; Rosen, V; D'Alessandro, J S; Bauduy, M; Cordes, P; Harada, T; Israel, D I; Hewick, R M; Kerns, K M; LaPan, P

    1990-01-01

    We have purified and characterized active recombinant human bone morphogenetic protein (BMP) 2A. Implantation of the recombinant protein in rats showed that a single BMP can induce bone formation in vivo. A dose-response and time-course study using the rat ectopic bone formation assay revealed that implantation of 0.5-115 micrograms of partially purified recombinant human BMP-2A resulted in cartilage by day 7 and bone formation by day 14. The time at which bone formation occurred was dependent on the amount of BMP-2A implanted; at high doses bone formation could be observed at 5 days. The cartilage- and bone-inductive activity of the recombinant BMP-2A is histologically indistinguishable from that of bone extracts. Thus, recombinant BMP-2A has therapeutic potential to promote de novo bone formation in humans. Images PMID:2315314

  5. The structure of full-length LysR-type transcriptional regulators. Modeling of the full-length OxyR transcription factor dimer

    PubMed Central

    Zaim, Jolanta; Kierzek, Andrzej M.

    2003-01-01

    The LysR-type transcriptional regulators (LTTRs) comprise the largest family of prokaryotic transcription factors. These proteins are composed of an N-terminal DNA binding domain (DBD) and a C-terminal cofactor binding domain. To date, no structure of the DBD has been solved. According to the SUPERFAMILY and MODBASE databases, a reliable homology model of LTTR DBDs may be built using the structure of the Escherichia coli ModE transcription factor, containing a winged helix– turn–helix (HTH) motif, as a template. The remote, but statistically significant, sequence similarity between ModE and LTTR DBDs and an alignment generated using SUPERFAMILY and MODBASE methods was independently confirmed by alignment of sequence profiles representing ModE and LTTR family DBDs. Using the crystal structure of the E.coli OxyR C-terminal domain and the DBD alignments we constructed a structural model of the full-length dimer of this LTTR family member and used it to investigate the mode of protein–DNA interaction. We also applied the model to interpret, in a structural context, the results of numerous biochemical studies of mutated LTTRs. A comparison of the LTTR DBD model with the structures of other HTH proteins also provides insights into the interaction of LTTRs with the C-terminal domain of the RNA polymerase α subunit. PMID:12595552

  6. Full-Length GB Virus C (Hepatitis G Virus) RNA Transcripts Are Infectious in Primary CD4-Positive T Cells

    PubMed Central

    Xiang, Jinhua; Wünschmann, Sabina; Schmidt, Warren; Shao, Jianqiang; Stapleton, Jack T.

    2000-01-01

    GB virus C (GBV-C or hepatitis G virus) is a recently described flavivirus which frequently leads to chronic viremia in humans. Although GBV-C is associated with acute posttransfusion hepatitis, it is not clear if the virus is pathogenic for humans. We constructed a full-length cDNA from the plasma of a person with chronic GBV-C viremia. Peripheral blood mononuclear cells (PBMCs) transfected with full-length RNA transcripts from this GBV-C clone resulted in viral replication. This was demonstrated by serial passage of virus from cell culture supernatants, detection of increasing concentrations of positive- and negative-sense GBV-C RNA over time, and the detection of the GBV-C E2 antigen by confocal microscopy. In addition, two types of GBV-C particles were identified in cell lysates; these particles had buoyant densities of 1.06 and 1.12 to 1.17 g/ml in sucrose gradients. PBMCs sorted for expression of CD4 contained 100-fold-more GBV-C RNA than CD4-negative cells. Taken together, these data demonstrate that RNA transcripts from GBV-C full-length cDNA are infectious in primary CD4-positive T cells. In contrast, RNA transcripts from an infectious hepatitis C virus clone did not replicate in the same cell culture system. Infectious RNA transcripts from GBV-C cDNA should prove useful for studying viral replication and may allow identification of differences between GBV-C and hepatitis C virus cultivation in vitro. PMID:10982359

  7. Loss of GATA-1 full length as a cause of Diamond-Blackfan anemia phenotype.

    PubMed

    Parrella, Sara; Aspesi, Anna; Quarello, Paola; Garelli, Emanuela; Pavesi, Elisa; Carando, Adriana; Nardi, Margherita; Ellis, Steven R; Ramenghi, Ugo; Dianzani, Irma

    2014-07-01

    Mutations in the hematopoietic transcription factor GATA-1 alter the proliferation/differentiation of hemopoietic progenitors. Mutations in exon 2 interfere with the synthesis of the full-length isoform of GATA-1 and lead to the production of a shortened isoform, GATA-1s. These mutations have been found in patients with Diamond-Blackfan anemia (DBA), a congenital erythroid aplasia typically caused by mutations in genes encoding ribosomal proteins. We sequenced GATA-1 in 23 patients that were negative for mutations in the most frequently mutated DBA genes. One patient showed a c.2T > C mutation in the initiation codon leading to the loss of the full-length GATA-1 isoform. PMID:24453067

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

    NASA Astrophysics Data System (ADS)

    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.

  9. [Isolation, identification and full-length genome sequence analysis of encephalomyocarditis virus from local aardvarks].

    PubMed

    Chang, Hong-Tao; Liu, Hui-Min; He, Xiu-Yuan; Zhao, Jun; Chen, Lu; Wang, Xin-Wei; Yang, Xia; Yao, Hui-Xia; Wang, Chuan-Qing

    2014-07-01

    Encephalomyocarditis virus (EMCV) is a natural epidemic zoonotic pathogen. However, no reports have been published regarding the isolation, identification and full-length genome of EMCV from a local aardvark population. In present study, an EMCV isolate HNXX13 was isolated from aardvarks named Huainan-pig in Henan Province. The systematic identification, full-length genome sequencing and molecular characteristic analysis of the isolate HNXX13 were conducted. The result showed that the isolate was spherical with a diameter of 24-30 nm, neither heat- nor acid-resistant, sensitive to trypsin, insensitive to chloroform, not protected by bivalent cationic, and the specific fluorescence was observed in the cytoplasm of BHK-21 cells infected with the isolate by using indirect fluorescence assay. The full-length genome of EMCV HNXX13 generated a 7 725bp sequence (GenBank: F771002), with 81.0%-99.9% nucleotide identity to reference strains from different animals, and 99.5% with a Chinese reference strain isolated earlier from a commercial pig herd. The phylogenetic tree based on the full-length genome and ORF sequences identified that all EMCV strains were divided into three groups G1, G2 and G3, and strain HNXX13 belonging to the G1 group with other Chinese reference strains. The result also identified that this EMCV infection could cause severe clinical signs in a local aardvark population, and enriches the molecular epidemiological data of EMCV in China. Regional differences exist in EMCV genome and transmission is limited within a certain area. However, the cross-infection and transmission of EMCV between aardvark and mice appears most likely. Mutations have occurred in some amino acids of EMCV strain HNXX13 during the transmission in local aardvark herd and these mutations might make the virus easier to infect the aardvark. PMID:25272589

  10. Full-length high-temperature severe fuel damage test No. 2. Final safety analysis

    SciTech Connect

    Hesson, G.M.; Lombardo, N.J.; Pilger, J.P.; Rausch, W.N.; King, L.L.; Hurley, D.E.; Parchen, L.J.; Panisko, F.E.

    1993-09-01

    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.

  11. Detection and Full-Length Genome Characterization of Novel Canine Vesiviruses

    PubMed Central

    Pinto, Pierfrancesco; Lorusso, Eleonora; Di Martino, Barbara; Wang, Qiuhong; Larocca, Vittorio; Cavalli, Alessandra; Camero, Michele; Decaro, Nicola; Bányai, Krisztián; Saif, Linda J.; Buonavoglia, Canio

    2015-01-01

    Vesiviruses have been detected in several animal species and as accidental contaminants of cells. We detected vesiviruses in asymptomatic kennel dogs (64.8%) and symptomatic (1.1%) and asymptomatic (3.5%) household dogs in Italy. The full-length genome of 1 strain, Bari/212/07/ITA, shared 89%–90% nt identity with vesiviruses previously detected in contaminated cells. PMID:26196075

  12. Hormone Binding to Recombinant Estrogen Receptors from Human, Alligator, Quail, Salamander, and Fathead Minnow

    EPA Science Inventory

    In this work, a 96-well plate estrogen receptor binding assay was developed to facilitate the direct comparison of chemical binding to full-length recombinant estrogen receptors across vertebrate classes. Receptors were generated in a baculovirus expression system. This approach ...

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

    PubMed

    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

  14. cDNA Library Enrichment of Full Length Transcripts for SMRT Long Read Sequencing

    PubMed Central

    Hartwig, Benjamin; Reinhardt, Richard; Schneeberger, Korbinian

    2016-01-01

    The utility of genome assemblies does not only rely on the quality of the assembled genome sequence, but also on the quality of the gene annotations. The Pacific Biosciences Iso-Seq technology is a powerful support for accurate eukaryotic gene model annotation as it allows for direct readout of full-length cDNA sequences without the need for noisy short read-based transcript assembly. We propose the implementation of the TeloPrime Full Length cDNA Amplification kit to the Pacific Biosciences Iso-Seq technology in order to enrich for genuine full-length transcripts in the cDNA libraries. We provide evidence that TeloPrime outperforms the commonly used SMARTer PCR cDNA Synthesis Kit in identifying transcription start and end sites in Arabidopsis thaliana. Furthermore, we show that TeloPrime-based Pacific Biosciences Iso-Seq can be successfully applied to the polyploid genome of bread wheat (Triticum aestivum) not only to efficiently annotate gene models, but also to identify novel transcription sites, gene homeologs, splicing isoforms and previously unidentified gene loci. PMID:27327613

  15. Cognitive flexibility deficits in a mouse model for the absence of full-length dystrophin.

    PubMed

    Remmelink, E; Aartsma-Rus, A; Smit, A B; Verhage, M; Loos, M; van Putten, M

    2016-07-01

    Duchenne muscular dystrophy (DMD) is a progressive muscle-wasting disorder, caused by mutations in the DMD gene and the resulting lack of dystrophin. The DMD gene has seven promoters, giving rise to multiple full-length and shorter isoforms. Besides the expression of dystrophin in muscles, the majority of dystrophin isoforms is expressed in brain and dystrophinopathy can lead to cognitive deficits, including intellectual impairments and deficits in executive function. In contrast to the muscle pathology, the impact of the lack of dystrophin on the brain is not very well studied. Here, we study the behavioral consequences of a lack of full-length dystrophin isoforms in mdx mice, particularly with regard to domains of executive functions and anxiety. We observed a deficit in cognitive flexibility in mdx mice in the absence of motor dysfunction or general learning impairments using two independent behavioral tests. In addition, increased anxiety was observed, but its expression depended on the context. Overall, these results suggest that the absence of full-length dystrophin in mice has specific behavioral effects that compare well to deficits observed in DMD patients. PMID:27220066

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

    PubMed

    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

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

    PubMed

    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

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

    PubMed

    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

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

    PubMed

    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

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

    PubMed

    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

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

    PubMed Central

    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

  2. Mesenchymal stromal cell delivery of full-length tumor necrosis factor–related apoptosis-inducing ligand is superior to soluble type for cancer therapy

    PubMed Central

    Yuan, ZhengQiang; Kolluri, Krishna K.; Sage, Elizabeth K.; Gowers, Kate H.C.; Janes, Sam M.

    2015-01-01

    Background aims Mesenchymal stromal cell (MSC) delivery of pro-apoptotic tumor necrosis factor (TNF)-related apoptosis-inducing ligand (TRAIL) is an attractive strategy for anticancer therapy. MSCs expressing full-length human TRAIL (flT) or its soluble form (sT) have previously been shown to be effective for cancer killing. However, a comparison between the two forms has never been performed, leaving it unclear which approach is most effective. This study addresses the issue for the possible clinical application of TRAIL-expressing MSCs in the future. Methods MSCs were transduced with lentiviruses expressing flT or an isoleucine zipper-fused sT. TRAIL expression was examined and cancer cell apoptosis was measured after treatment with transduced MSCs or with MSC-derived soluble TRAIL. Results The transduction does not adversely affect cell phenotype. The sT-transduced MSCs (MSC-sT) secrete abundant levels of soluble TRAIL but do not present the protein on the cell surface. Interestingly, the flT-transduced MSCs (MSC-flT) not only express cell-surface TRAIL but also release flT into medium. These cells were examined for inducing apoptosis in 20 cancer cell lines. MSC-sT cells showed very limited effects. By contrast, MSC-flT cells demonstrated high cancer cell-killing efficiency. More importantly, MSC-flT cells can overcome some cancer cell resistance to recombinant TRAIL. In addition, both cell surface flT and secreted flT are functional for inducing apoptosis. The secreted flT was found to have higher cancer cell-killing capacity than either recombinant TRAIL or MSC-secreted sT. Conclusions These observations demonstrate that MSC delivery of flT is superior to MSC delivery of sT for cancer therapy. PMID:25888191

  3. The Human Transcript Database: A Catalogue of Full Length cDNA Inserts

    SciTech Connect

    Bouckk John; Michael McLeod; Kim Worley; Richard Gibbs

    1999-09-10

    The BCM Search Launcher provided improved access to web-based sequence analysis services during the granting period and beyond. The Search Launcher web site grouped analysis procedures by function and provided default parameters that provided reasonable search results for most applications. For instance, most queries were automatically masked for repeat sequences prior to sequence database searches to avoid spurious matches. In addition to the web-based access and arrangements that were made using the functions easier, the BCM Search Launcher provided unique value-added applications like the BEAUTY sequence database search tool that combined information about protein domains and sequence database search results to give an enhanced, more complete picture of the reliability and relative value of the information reported. This enhanced search tool made evaluating search results more straight-forward and consistent. Some of the favorite features of the web site are the sequence utilities and the batch client functionality that allows processing of multiple samples from the command line interface. One measure of the success of the BCM Search Launcher is the number of sites that have adopted the models first developed on the site. The graphic display on the BLAST search from the NCBI web site is one such outgrowth, as is the display of protein domain search results within BLAST search results, and the design of the Biology Workbench application. The logs of usage and comments from users confirm the great utility of this resource.

  4. The full-length form of the Drosophila amyloid precursor protein is involved in memory formation.

    PubMed

    Bourdet, Isabelle; Preat, Thomas; Goguel, Valérie

    2015-01-21

    The APP plays a central role in AD, a pathology that first manifests as a memory decline. Understanding the role of APP in normal cognition is fundamental in understanding the progression of AD, and mammalian studies have pointed to a role of secreted APPα in memory. In Drosophila, we recently showed that APPL, the fly APP ortholog, is required for associative memory. In the present study, we aimed to characterize which form of APPL is involved in this process. We show that expression of a secreted-APPL form in the mushroom bodies, the center for olfactory memory, is able to rescue the memory deficit caused by APPL partial loss of function. We next assessed the impact on memory of the Drosophila α-secretase kuzbanian (KUZ), the enzyme initiating the nonamyloidogenic pathway that produces secreted APPLα. Strikingly, KUZ overexpression not only failed to rescue the memory deficit caused by APPL loss of function, it exacerbated this deficit. We further show that in addition to an increase in secreted-APPL forms, KUZ overexpression caused a decrease of membrane-bound full-length species that could explain the memory deficit. Indeed, we observed that transient expression of a constitutive membrane-bound mutant APPL form is sufficient to rescue the memory deficit caused by APPL reduction, revealing for the first time a role of full-length APPL in memory formation. Our data demonstrate that, in addition to secreted APPL, the noncleaved form is involved in memory, raising the possibility that secreted and full-length APPL act together in memory processes. PMID:25609621

  5. Performance of initial full-length RHIC (Relativistic Heavy Ion Collider) dipoles

    SciTech Connect

    Dahl, P.; Cottingham, J.; Garber, M.; Ghosh, A.; Goodzeit, C.; Greene, A.; Herrera, J.; Kahn, S.; Kelly, E.; Morgan, G.

    1987-01-01

    The first four full-length (9.7 m) R and D dipoles for the proposed Relativistic Heavy Ion Collider (RHIC) have been successfully tested. The magnets reached a quench plateau of approximately 4.5 T with very reasonable training - a field level comfortably above the design field of 3.45 T required for operation with beams of 100 GeV/amu gold nuclei. Measured field multipoles are considered to be quite acceptable for this series of R and D magnets.

  6. Purification, crystallization and preliminary crystallographic analysis of the full-length cystathionine β-synthase from Apis mellifera

    PubMed Central

    Oyenarte, Iker; Majtan, Tomas; Ereño, June; Corral-Rodríguez, María Angeles; Klaudiny, Jaroslav; Majtan, Juraj; Kraus, Jan P.; Martínez-Cruz, Luis Alfonso

    2012-01-01

    Cystathionine β-synthase (CBS) is a pyridoxal-5′-phosphate-dependent enzyme that catalyzes the first step of the transsulfuration pathway, namely the condensation of serine with homocysteine to form cystathionine. Mutations in the CBS gene are the single most common cause of hereditary homocystinuria, a multisystemic disease affecting to various extents the vasculature, connective tissues and central nervous system. At present, the crystal structure of CBS from Drosophila melanogaster is the only available structure of the full-length enzyme. Here we describe a cloning, overexpression, purification and preliminary crystallographic analysis of a full-length CBS from Apis mellifera (AmCBS) which maintains 51 and 46% sequence identity with its Drosophila and human homologs, respectively. The AmCBS yielded crystals belonging to space group P212121, with unit-cell parameters a = 85.90, b = 95.87, c = 180.33 Å. Diffraction data were collected to a resolution of 3.0 Å. The crystal structure contained two molecules in the asymmetric unit which presumably correspond to the dimeric species observed in solution. PMID:23143241

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

    PubMed

    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

  8. The influence of recombination on human genetic diversity.

    PubMed

    Spencer, Chris C A; Deloukas, Panos; Hunt, Sarah; Mullikin, Jim; Myers, Simon; Silverman, Bernard; Donnelly, Peter; Bentley, David; McVean, Gil

    2006-09-22

    In humans, the rate of recombination, as measured on the megabase scale, is positively associated with the level of genetic variation, as measured at the genic scale. Despite considerable debate, it is not clear whether these factors are causally linked or, if they are, whether this is driven by the repeated action of adaptive evolution or molecular processes such as double-strand break formation and mismatch repair. We introduce three innovations to the analysis of recombination and diversity: fine-scale genetic maps estimated from genotype experiments that identify recombination hotspots at the kilobase scale, analysis of an entire human chromosome, and the use of wavelet techniques to identify correlations acting at different scales. We show that recombination influences genetic diversity only at the level of recombination hotspots. Hotspots are also associated with local increases in GC content and the relative frequency of GC-increasing mutations but have no effect on substitution rates. Broad-scale association between recombination and diversity is explained through covariance of both factors with base composition. To our knowledge, these results are the first evidence of a direct and local influence of recombination hotspots on genetic variation and the fate of individual mutations. However, that hotspots have no influence on substitution rates suggests that they are too ephemeral on an evolutionary time scale to have a strong influence on broader scale patterns of base composition and long-term molecular evolution. PMID:17044736

  9. Effects of Full-Length Kisspeptin Administration on Follicular Development in Japanese Black Beef Cows

    PubMed Central

    NANIWA, Yousuke; NAKATSUKASA, Keisuke; SETSUDA, Shohei; OISHI, Shinya; FUJII, Nobutaka; MATSUDA, Fuko; UENOYAMA, Yoshihisa; TSUKAMURA, Hiroko; MAEDA, Kei-ichiro; OHKURA, Satoshi

    2013-01-01

    Abstract Kisspeptin is a key molecule that stimulates gonadotropin secretion via release of gonadotropin-releasing hormone (GnRH). In the present study, our aim was to investigate whether kisspeptin has stimulatory effects on follicular development via GnRH/gonadotropin secretion in cows. Japanese Black beef cows were intravenously injected with full-length bovine kisspeptin [Kp-53 (0.2 or 2 nmol/kg)] or vehicle 5 days after they exhibited standing estrus (Day 0). In cows injected with Kp-53 at 2 nmol/kg, the follicular sizes of the first dominant follicles increased on Day 6 and thereafter. Ovulation of the first dominant follicle occurred in 1 out of 4 cows treated with Kp-53 at 2 nmol/kg. Injection of Kp-53 at 2 nmol/kg increased the concentration of plasma luteinizing hormone (LH) but not follicle-stimulating hormone, over a 4-h period following injection in all cows. The present study suggests that administration of full-length kisspeptin causes LH secretion, which is sustained for a few hours, and it is capable of stimulating follicular development and/or ovulation. PMID:24107742

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

    PubMed Central

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

    2016-01-01

    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. PMID:27021073

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

    SciTech Connect

    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.

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

    PubMed

    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

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

    SciTech Connect

    Lanning, D.D.; Lombardo, N.J.; Hensley, W.K.; Fitzsimmons, D.E.; Panisko, F.E.; Hartwell, J.K.

    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.

  14. Design, fabrication, and testing of an external fuel (UO2), full-length thermionic converter

    NASA Technical Reports Server (NTRS)

    Schock, A.; Raab, B.

    1971-01-01

    The development of a full-length external-fuel thermionic converter for in-pile testing is described. The development program includes out-of-pile performance testing of the fully fueled-converter, using RF-induction heating, before its installation in the in-pile test capsule. The external-fuel converter is cylindrical in shape, and consists of an inner, centrally cooled collector, and an outer emitter surrounded by nuclear fuel. The term full-length denotes that the converter is long enough to extend over the full height of the reactor core. Thus, the converter is not a scaled-down test device, but a full-scale fuel element of the thermionic reactor. The external-fuel converter concept permits a number of different design options, particularly with respect to the fuel composition and shape, and the collector cooling arrangement. The converter described was developed for the Jet Propulsion Laboratory, and is based on their concept for a thermionic reactor with uninsulated collector cooling as previously described. The converter is double-ended, with through-flow cooling, and with ceramic seals and emitter and collector power take-offs at both ends. The design uses a revolver-shaped tungsten emitter body, with the central emitter hole surrounded by six peripheral fuel holes loaded with cylindrical UO2 pellets.

  15. Two methods for full-length RNA sequencing for low quantities of cells and single cells

    PubMed Central

    Pan, Xinghua; Durrett, Russell E.; Zhu, Haiying; Tanaka, Yoshiaki; Li, Yumei; Zi, Xiaoyuan; Marjani, Sadie L.; Euskirchen, Ghia; Ma, Chao; LaMotte, Robert H.; Park, In-Hyun; Snyder, Michael P.; Mason, Christopher E.; Weissman, Sherman M.

    2013-01-01

    The ability to determine the gene expression pattern in low quantities of cells or single cells is important for resolving a variety of problems in many biological disciplines. A robust description of the expression signature of a single cell requires determination of the full-length sequence of the expressed mRNAs in the cell, yet existing methods have either 3′ biased or variable transcript representation. Here, we report our protocols for the amplification and high-throughput sequencing of very small amounts of RNA for sequencing using procedures of either semirandom primed PCR or phi29 DNA polymerase-based DNA amplification, for the cDNA generated with oligo-dT and/or random oligonucleotide primers. Unlike existing methods, these protocols produce relatively uniformly distributed sequences covering the full length of almost all transcripts independent of their sizes, from 1,000 to 10 cells, and even with single cells. Both protocols produced satisfactory detection/coverage of the abundant mRNAs from a single K562 erythroleukemic cell or a single dorsal root ganglion neuron. The phi29-based method produces long products with less noise, uses an isothermal reaction, and is simple to practice. The semirandom primed PCR procedure is more sensitive and reproducible at low transcript levels or with low quantities of cells. These methods provide tools for mRNA sequencing or RNA sequencing when only low quantities of cells, a single cell, or even degraded RNA are available for profiling. PMID:23267071

  16. EFFECTS OF PHOSPHORYLATION ON THE SELF-ASSEMBLY OF NATIVE FULL-LENGTH PORCINE AMELOGENIN AND ITS REGULATION OF CALCIUM PHOSPHATE FORMATION IN VITRO

    PubMed Central

    Wiedemann-Bidlack, Felicitas B.; Kwak, Seo-Young; Beniash, Elia; Yamakoshi, Yasuo; Simmer, James P.; Margolis, Henry C.

    2010-01-01

    The self-assembly of the predominant extracellular enamel matrix protein amelogenin plays an essential role in regulating the growth and organization of enamel mineral during early stages of dental enamel formation. The present study describes the effect of the phosphorylation of a single site on the full-length native porcine amelogenin P173 on self-assembly and on the regulation of spontaneous calcium phosphate formation in vitro. Studies were also conducted using recombinant non-phosphorylated (rP172) porcine amelogenin, along with the most abundant amelogenin cleavage product (P148) and its recombinant form (rP147). Amelogenin self-assembly was assessed using dynamic light scattering (DLS) and transmission electron microscopy (TEM). Using these approaches, we have shown that self-assembly of each amelogenin is very sensitive to pH and appears to be affected by both hydrophilic and hydrophobic interactions. Furthermore, our results suggest that the phosphorylation of the full-length porcine amelogenin P173 has a small but potentially important effect on its higher-order self-assembly into chain-like structures under physiological conditions of pH, temperature, and ionic strength. Although phosphorylation has a subtle effect on the higher-order assembly of full-length amelogenin, native phosphorylated P173 was found to stabilize amorphous calcium phosphate for extended periods of time, in sharp contrast to previous findings using non-phosphorylated rP172. The biological relevance of these findings is discussed. PMID:21074619

  17. Characterisation of a new infectious full-length cDNA clone of BVDV genotype 2 and generation of virus mutants.

    PubMed

    Mischkale, Katrin; Reimann, Ilona; Zemke, J; König, P; Beer, Martin

    2010-04-21

    Based on their genomic sequences, two genotypes of Bovine viral diarrhea virus (BVDV) can be differentiated, BVDV type 1 (BVDV-1) and BVDV type 2 (BVDV-2). The complete genomic sequence of the highly virulent BVDV-2 strain 890 was cloned as cDNA to establish the infectious cDNA clone p890FL. In vitro-synthesised full-length RNA of p890FL was transfected into bovine cells and infectious virus could be recovered (v890FL). In vitro, recombinant v890FL showed similar growth characteristics as wild type virus v890WT. However, infection experiments in calves revealed an attenuation of recombinant v890FL in comparison to the parental isolate. Both leukocytopenia and fever were less pronounced in v890FL-infected calves. Nevertheless, viremia and virus shedding were comparable between recombinant and parental BVDV 890. Furthermore, mutants with partial deletions of the genomic region encoding for the autoprotease N(pro) (p890DeltaN(pro)) or the capsid protein (p890DeltaC) were constructed and characterised. In order to generate pseudovirions, replicon p890DeltaC was efficiently trans-complemented on a helper cell line. In summary, the newly developed construct p890FL represents the first infectious full-length cDNA clone for the BVDV-2 strain 890 and offers a useful tool for further studies on the pathogenesis of BVDV-2 and the development of novel recombinant BVDV-2 specific vaccine candidates. PMID:19875251

  18. Infectious foot-and-mouth disease virus derived from a cloned full-length cDNA.

    PubMed Central

    Zibert, A; Maass, G; Strebel, K; Falk, M M; Beck, E

    1990-01-01

    A full-length cDNA plasmid of foot-and-mouth disease virus has been constructed. RNA synthesized in vitro by means of a bacteriophage SP6 promoter inserted in front of the cDNA led to the production of infectious particles upon transfection of BHK-21 cells. These particles were also found to be highly infectious for primary bovine kidney cells as well as for baby mice. The difficulty in cloning the foot-and-mouth disease virus cytidyl tract in Escherichia coli was circumvented by joining two separate cloned parts, representing the S and L fragments of the genome, and, in a second step, inserting a dC-dG homopolymer. Homopolymeric sequences of up to 25 cytidyl residues did not lead to the production of virus. Replicons containing poly(C) tracts long enough to permit virus replication were first established in yeast cells. One of these constructs could also be maintained in E. coli and was used to produce infectious RNA in vitro. The length of the poly(C) sequence in this cDNA plasmid was 32 nucleotides. However, the poly(C) tracts of two recombinant viruses found in transfected BHK-21 cells were 60 and 80 nucleotides long, respectively. Possible mechanisms leading to the enlargement of the poly(C) tract during virus replication are discussed. Images PMID:2159523

  19. Production of biologically active recombinant human lactoferrin in Aspergillus oryzae.

    PubMed

    Ward, P P; Lo, J Y; Duke, M; May, G S; Headon, D R; Conneely, O M

    1992-07-01

    We report the production of recombinant human lactoferrin in Aspergillus oryzae. Expression of human lactoferrin (hLF), a 78 kD glycoprotein, was achieved by placing the cDNA under the control of the A. oryzae alpha-amylase promoter and the 3' flanking region of the A. niger glucoamylase gene. Using this system, hLF is expressed and secreted into the growth medium at levels up to 25 mg/l. The recombinant lactoferrin is indistinguishable from human milk lactoferrin with respect to its size, immunoreactivity, and iron-binding capacity. The recombinant protein appears to be appropriately N-linked glycosylated and correctly processed at the N-terminus by the A. oryzae secretory apparatus. Lactoferrin is the largest heterologous protein and the first mammalian glycoprotein expressed in the Aspergillus system to date. Hence, this expression system appears suitable for the large-scale production and secretion of biologically active mammalian glycoproteins. PMID:1368268

  20. Modular structure of the full-length DNA gyrase B subunit revealed by small-angle X-ray scattering.

    PubMed

    Costenaro, Lionel; Grossmann, J Günter; Ebel, Christine; Maxwell, Anthony

    2007-03-01

    DNA gyrase, the only topoisomerase able to introduce negative supercoils into DNA, is essential for bacterial transcription and replication; absent from humans, it is a successful target for antibacterials. From biophysical experiments in solution, we report a structural model at approximately 12-15 A resolution of the full-length B subunit (GyrB). Analytical ultracentrifugation shows that GyrB is mainly a nonglobular monomer. Ab initio modeling of small-angle X-ray scattering data for GyrB consistently yields a "tadpole"-like envelope. It allows us to propose an organization of GyrB into three domains-ATPase, Toprim, and Tail-based on their crystallographic and modeled structures. Our study reveals the modular organization of GyrB and points out its potential flexibility, needed during the gyrase catalytic cycle. It provides important insights into the supercoiling mechanism by gyrase and suggests new lines of research. PMID:17355868

  1. Mechanism of activation gating in the full-length KcsA K[superscript +] channel

    SciTech Connect

    Uysal, Serdar; Cuello, Luis G.; Cortes, D. Marien; Koide, Shohei; Kossiakoff, Anthony A.; Perozo, Eduardo

    2012-10-25

    Using a constitutively active channel mutant, we solved the structure of full-length KcsA in the open conformation at 3.9 {angstrom}. The structure reveals that the activation gate expands about 20 {angstrom}, exerting a strain on the bulge helices in the C-terminal domain and generating side windows large enough to accommodate hydrated K{sup +} ions. Functional and spectroscopic analysis of the gating transition provides direct insight into the allosteric coupling between the activation gate and the selectivity filter. We show that the movement of the inner gate helix is transmitted to the C-terminus as a straightforward expansion, leading to an upward movement and the insertion of the top third of the bulge helix into the membrane. We suggest that by limiting the extent to which the inner gate can open, the cytoplasmic domain also modulates the level of inactivation occurring at the selectivity filter.

  2. Full-length apolipoprotein E protects against the neurotoxicity of an apoE-related peptide

    PubMed Central

    Crutcher, K.A.; Lilley, H.N.; Anthony, S. R.; Zhou, W.; Narayanaswami, V.

    2009-01-01

    Apolipoprotein E was found to protect against the neurotoxic effects of a dimeric peptide derived from the receptor-binding region of this protein (residues 141–149). Both apoE3 and apoE4 conferred protection but the major N-terminal fragment of each isoform did not. Nor was significant protection provided by bovine serum albumin or apoA-I. Full-length apoE3 and apoE4 also inhibited the uptake of a fluorescent-labeled derivative of the peptide, suggesting that the mechanism of inhibition might involve competition for cell surface receptors/proteoglycans that mediate endocytosis and/or signaling pathways. These results might bear on the question of the role of apoE in neuronal degeneration, such as occurs in Alzheimer’s disease where apoE4 confers a significantly greater risk of pathology. PMID:19836363

  3. On the Minimum Weight of Simple Full-Length Array LDPC Codes

    NASA Astrophysics Data System (ADS)

    Sugiyama, Kenji; Kaji, Yuichi

    We investigate the minimum weights of simple full-length array LDPC codes (SFA-LDPC codes). The SFA-LDPC codes are a subclass of LDPC codes, and constructed algebraically according to two integer parameters p and j. Mittelholzer and Yang et al. have studied the minimum weights of SFA-LDPC codes, but the exact minimum weights of the codes are not known except for some small p and j. In this paper, we show that the minimum weights of the SFA-LDPC codes with j=4 and j=5 are upper-bounded by 10 and 12, respectively, independent from the prime number p. By combining the results with Yang's lower-bound limits, we can conclude that the minimum weights of the SFA-LDPC codes with j=4 and p>7 are exactly 10 and those of the SFA-LDPC codes with j=5 are 10 or 12.

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

    PubMed

    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

  5. Efficient expression of full-length antibodies in the cytoplasm of engineered bacteria

    PubMed Central

    Robinson, Michael-Paul; Ke, Na; Lobstein, Julie; Peterson, Cristen; Szkodny, Alana; Mansell, Thomas J.; Tuckey, Corinna; Riggs, Paul D.; Colussi, Paul A.; Noren, Christopher J.; Taron, Christopher H.; DeLisa, Matthew P.; Berkmen, Mehmet

    2015-01-01

    Current methods for producing immunoglobulin G (IgG) antibodies in engineered cells often require refolding steps or secretion across one or more biological membranes. Here, we describe a robust expression platform for biosynthesis of full-length IgG antibodies in the Escherichia coli cytoplasm. Synthetic heavy and light chains, both lacking canonical export signals, are expressed in specially engineered E. coli strains that permit formation of stable disulfide bonds within the cytoplasm. IgGs with clinically relevant antigen- and effector-binding activities are readily produced in the E. coli cytoplasm by grafting antigen-specific variable heavy and light domains into a cytoplasmically stable framework and remodelling the fragment crystallizable domain with amino-acid substitutions that promote binding to Fcγ receptors. The resulting cytoplasmic IgGs—named ‘cyclonals'—effectively bypass the potentially rate-limiting steps of membrane translocation and glycosylation. PMID:26311203

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

    PubMed

    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

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

    NASA Astrophysics Data System (ADS)

    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.

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

    PubMed

    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

  9. Fabrication and Testing of Full-Length Single-Cell Externally Fueled Converters for Thermionic Reactors

    SciTech Connect

    Schock, Alfred

    1994-06-01

    The preceding paper described designs and analyses of thermionic reactors employing full-core-length single-cell converters with their heated emitters located on the outside of their internally cooled collectors, and it presented results of detailed parametric analyses which illustrate the benefits of this unconventional design. The present paper describes the fabrication and testing of full-length prototypical converters, both unfueled and fueled, and presents parametric results of electrically heated tests. The unfueled converter tests demonstrated the practicality of operating such long converters without shorting across a 0.3-mm interelectrode gap. They produced a measured peak output of 751 watts(e) from a single diode and a peak efficiency of 15.4%. The fueled converter tests measured the parametric performance of prototypic UO(subscript 2)-fueled converters designed for subsequent in-pile testing. They employed revolver-shaped tungsten elements with a central emitter hole surrounded by six fuel chambers. The full-length converters were heated by a water-cooled RF-induction coil inside an ion-pumped vacuum chamber. This required development of high-vacuum coaxial RF feedthroughs. In-pile test rules required multiple containment of the UO (subscript 2)-fuel, which complicated the fabrication of the test article and required successful development of techniques for welding tungsten and other refractory components. The test measured a peak power output of 530 watts(e) or 7.1 watts/cm (superscript 2) at an efficiency of 11.5%. There are three copies in the file. Cross-Reference a copy FSC-ESD-217-94-529 in the ESD files with a CID #8574.

  10. The feline oral microbiome: a provisional 16S rRNA gene based taxonomy with full-length reference sequences.

    PubMed

    Dewhirst, Floyd E; Klein, Erin A; Bennett, Marie-Louise; Croft, Julie M; Harris, Stephen J; Marshall-Jones, Zoe V

    2015-02-25

    The human oral microbiome is known to play a significant role in human health and disease. While less well studied, the feline oral microbiome is thought to play a similarly important role. To determine roles oral bacteria play in health and disease, one first has to be able to accurately identify bacterial species present. 16S rRNA gene sequence information is widely used for molecular identification of bacteria and is also useful for establishing the taxonomy of novel species. The objective of this research was to obtain full 16S rRNA gene reference sequences for feline oral bacteria, place the sequences in species-level phylotypes, and create a curated 16S rRNA based taxonomy for common feline oral bacteria. Clone libraries were produced using "universal" and phylum-selective PCR primers and DNA from pooled subgingival plaque from healthy and periodontally diseased cats. Bacteria in subgingival samples were also cultivated to obtain isolates. Full-length 16S rDNA sequences were determined for clones and isolates that represent 171 feline oral taxa. A provisional curated taxonomy was developed based on the position of each taxon in 16S rRNA phylogenetic trees. The feline oral microbiome curated taxonomy and 16S rRNA gene reference set will allow investigators to refer to precisely defined bacterial taxa. A provisional name such as "Propionibacterium sp. feline oral taxon FOT-327" is an anchor to which clone, strain or GenBank names or accession numbers can point. Future next-generation-sequencing studies of feline oral bacteria will be able to map reads to taxonomically curated full-length 16S rRNA gene sequences. PMID:25523504

  11. Expression of Recombinant Human Amelogenin in Iranian Lizard Leishmania and Its Biological Function Assay

    PubMed Central

    YADEGARI, Zahra; BANDEHPOUR, Mojgan; KAZEMI, Bahram; SHARIFI-SARASIABI, Khojasteh

    2015-01-01

    Background: Amelogenins are the major components of enamel matrix proteins. Enamel matrix derivatives (EMD) can be used in periodontal diseases to regenerate periodontal tissues. The main aim of this study was to evaluate expression of full-length functional recombinant human amelogenin (rhAm) in Iranian lizard Leishmania (I.L.L.) as an alternative eukaryotic expression system. Methods: Human cDNA encoding a 175-amino acid amelogenin expression cassette was sub cloned into a pLEXSY vector. The construct was transferred into Leishmania cells by electroporation. The protein production was surveyed in the transcription and the translation levels. The expressed protein was purified and some of its biological properties were investigated in comparison to EMD and negative control. Results: Expression of rhAm was confirmed by RT-PCR and western blot test in Leishmania cells. Purified rhAm significantly inhibited the formation of tartrate-resistant acid phosphatase positive (TRAP+) multinuclear cells in calcitriol stimulated mouse marrow cultures. Moreover, it significantly promoted proliferation and DNA synthesis in L929 mouse fibroblast cells. Conclusion: Functional rhAm was successfully expressed in I.L.L. Easy handling and post translation modification were the main advantages of this expression system. It is suggested to investigate molecular properties of this rhAm in the future. PMID:26576377

  12. How to obtain a non-digital Full-length lower limb Radiographs using regular X-ray plates

    PubMed Central

    Parihar, Mangal; Singh, Tarandeep; Shah, Lalitkumar

    2011-01-01

    Full-length hip-to-ankle radiographs are an important part of planning and execution of any realignment procedure of the lower limb. This technical note describes a simple and inexpensive technique to obtain plain, standing, full-length hip-to-ankle radiograph using regular x-ray plates.

  13. Factors Influencing the Production of MFSV Full-Length Clone: Maize Fine Streak Virus Proteins in Drosophila S2 Cells

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Maize fine streak virus (MFSV) is negative-sense RNA virus member of the genus Nucleorhabdovirus. Our goal is to determine whether Drosophila S2 cells can support the production of a full-length clone of MFSV. We have previously demonstrated that the full-length MFSV nucleoprotein (N) and phosphopro...

  14. Structure and Dynamics of Full Length HIV-1 Capsid Protein in Solution

    PubMed Central

    Deshmukh, Lalit; Schwieters, Charles D.; Grishaev, Alexander; Ghirlando, Rodolfo; Baber, James L.; Clore, G. Marius

    2013-01-01

    The HIV-1 capsid protein plays a crucial role in viral infectivity, assembling into a cone that encloses the viral RNA. In the mature virion, the N-terminal domain of the capsid protein forms hexameric and pentameric rings, while C-terminal domain homodimers connect adjacent N-terminal domain rings to one another. Structures of disulfide-linked hexamer and pentamer assemblies, as well as structures of the isolated domains have been solved previously. The dimer configuration in C-terminal domain constructs differs in solution (residues 144–231) and crystal (residues 146–231) structures by ~30°, and it has been postulated that the former connects the hexamers while the latter links pentamers to hexamers. Here we study the structure and dynamics of full-length capsid protein in solution, comprising a mixture of monomeric and dimeric forms in dynamic equilibrium, using ensemble simulated annealing driven by experimental NMR residual dipolar couplings and X-ray scattering data. The complexity of the system necessitated the development of a novel computational framework that should be generally applicable to many other challenging systems that currently escape structural characterization by standard application of mainstream techniques of structural biology. We show that the orientation of the C-terminal domains in dimeric full-length capsid and isolated C-terminal domain constructs is the same in solution, and obtain a quantitative description of the conformational space sampled by the N-terminal domain relative to the C-terminal domain on the nano- to millisecond time-scale. The positional distribution of the N-terminal domain relative to the C-terminal domain is large and modulated by the oligomerization state of the C-terminal domain. We also show that a model of the hexamer/pentamer assembly can be readily generated with a single configuration of the C-terminal domain dimer, and that capsid assembly likely proceeds via conformational selection of sparsely

  15. Recombination in the Human Pseudoautosomal Region PAR1

    PubMed Central

    Hinch, Anjali G.; Altemose, Nicolas; Noor, Nudrat; Donnelly, Peter; Myers, Simon R.

    2014-01-01

    The pseudoautosomal region (PAR) is a short region of homology between the mammalian X and Y chromosomes, which has undergone rapid evolution. A crossover in the PAR is essential for the proper disjunction of X and Y chromosomes in male meiosis, and PAR deletion results in male sterility. This leads the human PAR with the obligatory crossover, PAR1, to having an exceptionally high male crossover rate, which is 17-fold higher than the genome-wide average. However, the mechanism by which this obligatory crossover occurs remains unknown, as does the fine-scale positioning of crossovers across this region. Recent research in mice has suggested that crossovers in PAR may be mediated independently of the protein PRDM9, which localises virtually all crossovers in the autosomes. To investigate recombination in this region, we construct the most fine-scale genetic map containing directly observed crossovers to date using African-American pedigrees. We leverage recombination rates inferred from the breakdown of linkage disequilibrium in human populations and investigate the signatures of DNA evolution due to recombination. Further, we identify direct PRDM9 binding sites using ChIP-seq in human cells. Using these independent lines of evidence, we show that, in contrast with mouse, PRDM9 does localise peaks of recombination in the human PAR1. We find that recombination is a far more rapid and intense driver of sequence evolution in PAR1 than it is on the autosomes. We also show that PAR1 hotspot activities differ significantly among human populations. Finally, we find evidence that PAR1 hotspot positions have changed between human and chimpanzee, with no evidence of sharing among the hottest hotspots. We anticipate that the genetic maps built and validated in this work will aid research on this vital and fascinating region of the genome. PMID:25033397

  16. Recombination in the human Pseudoautosomal region PAR1.

    PubMed

    Hinch, Anjali G; Altemose, Nicolas; Noor, Nudrat; Donnelly, Peter; Myers, Simon R

    2014-07-01

    The pseudoautosomal region (PAR) is a short region of homology between the mammalian X and Y chromosomes, which has undergone rapid evolution. A crossover in the PAR is essential for the proper disjunction of X and Y chromosomes in male meiosis, and PAR deletion results in male sterility. This leads the human PAR with the obligatory crossover, PAR1, to having an exceptionally high male crossover rate, which is 17-fold higher than the genome-wide average. However, the mechanism by which this obligatory crossover occurs remains unknown, as does the fine-scale positioning of crossovers across this region. Recent research in mice has suggested that crossovers in PAR may be mediated independently of the protein PRDM9, which localises virtually all crossovers in the autosomes. To investigate recombination in this region, we construct the most fine-scale genetic map containing directly observed crossovers to date using African-American pedigrees. We leverage recombination rates inferred from the breakdown of linkage disequilibrium in human populations and investigate the signatures of DNA evolution due to recombination. Further, we identify direct PRDM9 binding sites using ChIP-seq in human cells. Using these independent lines of evidence, we show that, in contrast with mouse, PRDM9 does localise peaks of recombination in the human PAR1. We find that recombination is a far more rapid and intense driver of sequence evolution in PAR1 than it is on the autosomes. We also show that PAR1 hotspot activities differ significantly among human populations. Finally, we find evidence that PAR1 hotspot positions have changed between human and chimpanzee, with no evidence of sharing among the hottest hotspots. We anticipate that the genetic maps built and validated in this work will aid research on this vital and fascinating region of the genome. PMID:25033397

  17. Genetically encoded optical activation of DNA recombination in human cells.

    PubMed

    Luo, J; Arbely, E; Zhang, J; Chou, C; Uprety, R; Chin, J W; Deiters, A

    2016-06-30

    We developed two tightly regulated, light-activated Cre recombinase enzymes through site-specific incorporation of two genetically-encoded photocaged amino acids in human cells. Excellent optical off to on switching of DNA recombination was achieved. Furthermore, we demonstrated precise spatial control of Cre recombinase through patterned illumination. PMID:27277957

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

    PubMed

    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

  19. Molecular cloning and properties of a full-length putative thyroid hormone receptor coactivator.

    PubMed

    Takeshita, A; Yen, P M; Misiti, S; Cardona, G R; Liu, Y; Chin, W W

    1996-08-01

    Thyroid hormone receptors (TRs) are ligand-dependent transcription factors that regulate target gene transcription. The conserved carboxy-terminal region of the ligand-binding domain (AF-2) has been thought to play a critical role in mediating ligand-dependent transactivation by the interaction with coactivator(s). Using bacterially-expressed TR as a probe, far-Western-based expression cDNA library screening identified cDNAs that encode, in part, the recently reported partial steroid receptor coactivator-1 (SRC-1) sequence. Additional work, including 5' RACE, has characterized a full-length cDNA that encodes a approximately 160 kD protein as a putative thyroid hormone receptor coactivator (F-SRC-1). In vitro binding studies show that F-SRC-1 binds to a variety of nuclear hormone receptors in a ligand-dependent manner, along with TBP and TFIIB, suggesting that F-SRC-1 may play a role as a bridging molecule between nuclear hormone receptors and general transcription factors. Interestingly, AF-2 mutants also retain ligand-dependent interaction with F-SRC-1. Although F-SRC-1 recognizes the ligand-induced conformational changes of nuclear hormone receptors, our observations suggest that F-SRC-1 may bind directly with subregion(s) in nuclear hormone receptors other than the AF-2 region. PMID:8754792

  20. Comprehensive Analysis of the Green-to-Blue Photoconversion of Full-Length Cyanobacteriochrome Tlr0924

    PubMed Central

    Hardman, Samantha J.O.; Hauck, Anna F.E.; Clark, Ian P.; Heyes, Derren J.; Scrutton, Nigel S.

    2014-01-01

    Cyanobacteriochromes are members of the phytochrome superfamily of photoreceptors and are of central importance in biological light-activated signaling mechanisms. These photoreceptors are known to reversibly convert between two states in a photoinitiated process that involves a basic E/Z isomerization of the bilin chromophore and, in certain cases, the breakage of a thioether linkage to a conserved cysteine residue in the bulk protein structure. The exact details and timescales of the reactions involved in these photoconversions have not been conclusively shown. The cyanobacteriochrome Tlr0924 contains phycocyanobilin and phycoviolobilin chromophores, both of which photoconvert between two species: blue-absorbing and green-absorbing, and blue-absorbing and red-absorbing, respectively. Here, we followed the complete green-to-blue photoconversion process of the phycoviolobilin chromophore in the full-length form of Tlr0924 over timescales ranging from femtoseconds to seconds. Using a combination of time-resolved visible and mid-infrared transient absorption spectroscopy and cryotrapping techniques, we showed that after photoisomerization, which occurs with a lifetime of 3.6 ps, the phycoviolobilin twists or distorts slightly with a lifetime of 5.3 μs. The final step, the formation of the thioether linkage with the protein, occurs with a lifetime of 23.6 ms. PMID:25418104

  1. The full-length transcripts and promoter analysis of intergenic microRNAs in Drosophila melanogaster.

    PubMed

    Qian, Jinjun; Zhang, Zan; Liang, Jingdong; Ge, Qiongqiong; Duan, Xuchu; Ma, Fei; Li, Fei

    2011-05-01

    MicroRNA (miRNA) transcription is still not well understood until now. To increase the miRNA abundance, we stimulated miRNA transcription with CuSO(4) and knocked down Drosha enzyme using dsRNA in Drosophila S2 cells. The full length transcripts of bantam, miR-276a and miR-277, the 5'-end of miR-8, the 3'-end of miR-2b and miR-10 were obtained. We also conducted a series of miRNA promoter analysis to prove the reliability of RACE results. Luciferase-reporter assays proved that both bantam and miR-276a promoters successfully drove the expressions of downstream luciferase genes. The promoter activities were impaired by introducing one or multiple mutations at predicted transcription factor binding sites. Chromatin immunoprecipitation analysis confirmed that hypophosphorylated RNA polymerase II and transcription factor c-Myc physically bind at miRNA promoter. RNA interference of transcription factors Mad and Prd led to down-expression of bantam, miR-277 and miR-2b but not miR-276a, whereas RNAi of Dorsal had the opposite effect. PMID:21333734

  2. Efficiency of recombinant human TNF in human cancer therapy.

    PubMed

    Lejeune, Ferdy J; Liénard, Danielle; Matter, Maurice; Rüegg, Curzio

    2006-01-01

    Recombinant human tumour necrosis factor (TNF) has a selective effect on angiogenic vessels in tumours. Given that it induces vasoplegia, its clinical use has been limited to administration through isolated limb perfusion (ILP) for regionally advanced melanomas and soft tissue sarcomas of the limbs. When combined with the alkylating agent melphalan, a single ILP produces a very high objective response rate. In melanoma, the complete response (CR) rate is around 80% and the overall objective response rate greater than 90%. In soft tissue sarcomas that are inextirpable, ILP is a neoadjuvant treatment resulting in limb salvage in 80% of the cases. The CR rate averages 20% and the objective response rate is around 80%. The mode of action of TNF-based ILP involves two distinct and successive effects on the tumour-associated vasculature: first, an increase in endothelium permeability leading to improved chemotherapy penetration within the tumour tissue, and second, a selective killing of angiogenic endothelial cells resulting in tumour vessel destruction. The mechanism whereby these events occur involves rapid (of the order of minutes) perturbation of cell-cell adhesive junctions and inhibition of alphavbeta3 integrin signalling in tumour-associated vessels, followed by massive death of endothelial cells and tumour vascular collapse 24 hours later. New, promising approaches for the systemic use of TNF in cancer therapy include TNF targeting by means of single chain antibodies or endothelial cell ligands, or combined administration with drugs perturbing integrin-dependent signalling and sensitizing angiogenic endothelial cells to TNF-induced death. PMID:16551058

  3. Applications of recombinant DNA technology in the production of glycosylated recombinant human granulocyte colony stimulating factor.

    PubMed

    Holloway, C J

    1994-01-01

    Lenograstim has been developed by recombinant DNA technology and is expressed in large-scale mammalian cell culture. It has been shown that lenograstim is indistinguishable in its physicochemical, structural and biological properties with respect to native granulocyte colony stimulating factor isolated from a human cell line. In particular, both the recombinant and natural proteins have identical amino acid sequences, contain the same intra-polypeptide chain disulphide bridges and exhibit the same posttranslational carbohydrate structures. Lenograstim is manufactured by expanding inoculum from vials of the Manufacturer's Working Cell Bank (from molecular cloning) followed by culture in a large bioreactor. Purification of lenograstim involves a four-step chromatographic process. The active ingredient is monitored by in-process controls at all stages of manufacture and routinely as purified bulk. The finished product is formulated into excipients reflecting conditions close to the natural environment of the protein with respect to pH, osmolarity and the presence of human serum albumin. PMID:7535067

  4. Insulin allergy treated with human insulin (recombinant DNA).

    PubMed

    De Leeuw, I; Delvigne, C; Bekaert, J

    1982-01-01

    Two insulin-dependent diabetic subjects treated with pork and beef insulin during a period of 6 mo developed severe local reactions. Both patients had an important allergic history (asthma, urticaria, drug reactions, rhinitis). Skin-testing revealed type I allergy to beef and pork insulin. Specific IgE-insulin binding was demonstrated with both insulins. After negative skin testing with NPH Lilly human insulin (recombinant DNA), treatment was started with this compound and remained successful during a period of 6-9 mo. In one patient a local reaction occurred when regular human insulin (recombinant DNA) was added to NPH in order to obtain better control. Skin testing with regular human insulin was positive, but not with NPH human insulin alone. The mechanism of this phenomenon remains unsolved. PMID:6765530

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

    PubMed Central

    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

  6. [Cloning of full-length coding sequence of tree shrew CD4 and prediction of its molecular characteristics].

    PubMed

    Tian, Wei-Wei; Gao, Yue-Dong; Guo, Yan; Huang, Jing-Fei; Xiao, Chang; Li, Zuo-Sheng; Zhang, Hua-Tang

    2012-02-01

    The tree shrews, as an ideal animal model receiving extensive attentions to human disease research, demands essential research tools, in particular cellular markers and monoclonal antibodies for immunological studies. In this paper, a 1 365 bp of the full-length CD4 cDNA encoding sequence was cloned from total RNA in peripheral blood of tree shrews, the sequence completes two unknown fragment gaps of tree shrews predicted CD4 cDNA in the GenBank database, and its molecular characteristics were analyzed compared with other mammals by using biology software such as Clustal W2.0 and so forth. The results showed that the extracellular and intracellular domains of tree shrews CD4 amino acid sequence are conserved. The tree shrews CD4 amino acid sequence showed a close genetic relationship with Homo sapiens and Macaca mulatta. Most regions of the tree shrews CD4 molecule surface showed positive charges as humans. However, compared with CD4 extracellular domain D1 of human, CD4 D1 surface of tree shrews showed more negative charges, and more two N-glycosylation sites, which may affect antibody binding. This study provides a theoretical basis for the preparation and functional studies of CD4 monoclonal antibody. PMID:22345010

  7. Limited human infection due to recombinant raccoon pox virus

    USGS Publications Warehouse

    Rocke, T.E.; Dein, F.J.; Fuchsberger, M.; Fox, B.C.; Stinchcomb, D.T.; Osorio, J.G.

    2004-01-01

    A laboratory accident resulted in human exposure to a recombinant raccoon poxvirus (RCN) developed as a vaccine vector for antigens of Yersinia pestis for protection of wild rodents (and other animals) against plague. Within 9 days, the patient developed a small blister that healed within 4 weeks. Raccoon poxvirus was cultured from the lesion, and the patient developed antibody to plague antigen (F1) and RCN. This is the first documented case of human exposure to RCN.

  8. Construction and characterization of bacterial artificial chromosomes (BACs) containing herpes simplex virus full-length genomes.

    PubMed

    Nagel, Claus-Henning; Pohlmann, Anja; Sodeik, Beate

    2014-01-01

    Bacterial artificial chromosomes (BACs) are suitable vectors not only to maintain the large genomes of herpesviruses in Escherichia coli but also to enable the traceless introduction of any mutation using modern tools of bacterial genetics. To clone a herpes simplex virus genome, a BAC replication origin is first introduced into the viral genome by homologous recombination in eukaryotic host cells. As part of their nuclear replication cycle, genomes of herpesviruses circularize and these replication intermediates are then used to transform bacteria. After cloning, the integrity of the recombinant viral genomes is confirmed by restriction length polymorphism analysis and sequencing. The BACs may then be used to design virus mutants. Upon transfection into eukaryotic cells new herpesvirus strains harboring the desired mutations can be recovered and used for experiments in cultured cells as well as in animal infection models. PMID:24671676

  9. Expression and purification of recombinant human c-Fos/c-Jun that is highly active in DNA binding and transcriptional activation in vitro

    PubMed Central

    Ferguson, Heather A.; Goodrich, James A.

    2001-01-01

    c-Fos and c-Jun are members of the AP-1 family of transcriptional activators that regulate the expression of genes during cell proliferation. To facilitate in vitro studies of mechanisms of transcriptional activation by c-Jun and c-Fos we developed a method for obtaining recombinant c-Fos/c-Jun that is highly active in DNA binding and transcriptional activation in vitro. Full-length human c-Fos and c-Jun were expressed in Escherichia coli. The expression of c-Fos was dependent on a helper plasmid that encodes rare ArgtRNAs. Both over-expressed c-Fos and c-Jun were recovered from inclusion bodies. A c-Fos/c-Jun complex was generated by co-renaturation and purified via a His-tag on the full-length human c-Fos. The resulting c-Fos/c-Jun bound DNA with high affinity and specificity, and activated transcription in a reconstituted human RNA polymerase II transcription system. The availability of active recombinant human c-Fos/c-Jun will allow future biochemical studies of these important transcriptional activators. PMID:11600717

  10. Fate of prions in soil: trapped conformation of full-length ovine prion protein induced by adsorption on clays.

    PubMed

    Revault, M; Quiquampoix, H; Baron, M H; Noinville, S

    2005-08-01

    Studying the mechanism of retention of ovine prion protein in soils will tackle the environmental aspect of potential dissemination of scrapie infectious agent. We consider the surface-induced conformational changes that the recombinant ovine prion protein (ovPrP) may undergo under different pH conditions when interacting with soil minerals of highly adsorptive capacities such as montmorillonite. The conformational states of the full-length ovine prion protein adsorbed on the electronegative clay surface are compared to its solvated state in deuterated buffer in the pD range 3.5-9, using FTIR spectroscopy. The in vitro pH-induced conversion of the alpha-helical monomer of ovPrP into oligomers of beta-like structure prone to self-aggregation does not occur when the protein is adsorbed on the clay surface. The conformation of the trapped ovPrP molecules on montmorillonite is pH-independent and looks like that of the ovPrP solvated state at pD higher than 7, suggesting the major role of Arg and Lys residues in the electrostatic origin of adsorption. The uneven distribution of positively and negatively charged residues of the ovPrP protein would promote a favored orientation of the protein towards the clay, so that not only the basic residues embedded in the N-terminal flexible part but also external basic residues in the globular part of the protein might participate to the attractive interaction. From these results, it appears unlikely that the interaction of normal prions (PrP(C)) with soil clay surfaces could induce a change of conformation leading to the pathogenic form of prions (PrP(Sc)). PMID:15950385

  11. 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

    SciTech Connect

    Deymier, Martin J.; Claiborne, Daniel T.; Ende, Zachary; Ratner, Hannah K.; Kilembe, William; Hunter, Eric

    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.

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

    PubMed Central

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

    2015-01-01

    Selenocysteine (Sec), the 21st amino acid in translation, uses its specific tRNA (tRNASec) to recognize the UGA codon. The Sec-specific elongation factor SelB brings the selenocysteinyl-tRNASec (Sec-tRNASec) 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-tRNASec 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-tRNASec, SECIS RNA, and the 70S ribosome suggest that the unique secondary structure of tRNASec allows SelB to specifically recognize tRNASec and characteristically place it at the ribosomal A-site. PMID:26304550

  13. Purification and Activity Testing of the Full-Length YycFGHI Proteins of Staphylococcus aureus

    PubMed Central

    Türck, Michael; Bierbaum, Gabriele

    2012-01-01

    Background The YycFG two-component regulatory system (TCS) of Staphylococcus aureus represents the only essential TCS that is almost ubiquitously distributed in Gram-positive bacteria with a low G+C-content. YycG (WalK/VicK) is a sensor histidine-kinase and YycF (WalR/VicR) is the cognate response regulator. Both proteins play an important role in the biosynthesis of the cell envelope and mutations in these proteins have been involved in development of vancomycin and daptomycin resistance. Methodology/Principal Findings Here we present high yield expression and purification of the full-length YycG and YycF proteins as well as of the auxiliary proteins YycH and YycI of Staphylococcus aureus. Activity tests of the YycG kinase and a mutated version, that harbours an Y306N exchange in its cytoplasmic PAS domain, in a detergent-micelle-model and a phosholipid-liposome-model showed kinase activity (autophosphorylation and phosphoryl group transfer to YycF) only in the presence of elevated concentrations of alkali salts. A direct comparison of the activity of the kinases in the liposome-model indicated a higher activity of the mutated YycG kinase. Further experiments indicated that YycG responds to fluidity changes in its microenvironment. Conclusions/Significance The combination of high yield expression, purification and activity testing of membrane and membrane-associated proteins provides an excellent experimental basis for further protein-protein interaction studies and for identification of all signals received by the YycFGHI system. PMID:22276191

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

    PubMed

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

    PubMed

    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

  16. Identification and quantification of full-length BK channel variants in the developing mouse cochlea.

    PubMed

    Sakai, Yoshihisa; Harvey, Margaret; Sokolowski, Bernd

    2011-11-01

    Maxi-K(+) (BK) channel diversity is attributed to alternative splicing in the kcnma1 gene. The resultant variants manifest themselves in different cell types, tissues, and functions, such as excitation, metabolism, and signaling. Immunoelectron microscopy revealed immunogold particle labeling of BK in apical and basal regions of inner and outer hair cells, respectively. Additional labeling occurs in Deiters' cells and the inner mitochondrial membrane. Identification of full-length sequences reveals 27 BK variants from embryonic and postnatal mouse inner ear, per classification by tail motif, VYR, DEC, and ERL, and by exon usage. Three predicted start codons are found encoding MAN, MSS, and MDA, of which MDA shows the greatest expression through all stages in development, whereas MAN is undetectable. Complex splice sites occur between exons 9 and 10 and between 21 and 23. Spliced-in/out exons between 8 and 10 reveal a short fragment composed of exons 8 + 10, detectable on postnatal day (PD) 14 and PD30, and a longer fragment composed of exons 8 + 9 + 10 that is upregulated on embryonic day (ED) 14. Spliced-in exons 22 or 23 are expressed on ED14 but decrease over time; however, exon 22 increases again on PD34. Using tail-specific primers, qRT-PCR from ED14, PD4, -14, and -30 shows that BK-VYR and -ERL dominate expression on ED14, whereas DEC dominates after birth in all cochlear regions. The localization of BK and the changes in expression of its exons and tail types, by alternative splicing during development, may contribute to cochlear organization, acquisition of hearing, and intracellular signaling. PMID:21800349

  17. Mapping Full-Length Porcine Endogenous Retroviruses in a Large White Pig

    PubMed Central

    Herring, C.; Quinn, G.; Bower, R.; Parsons, N.; Logan, N. A.; Brawley, A.; Elsome, K.; Whittam, A.; Fernandez-Suarez, X. M.; Cunningham, D.; Onions, D.; Langford, G.; Scobie, L.

    2001-01-01

    Xenotransplantation may bridge the widening gap between the shortage of donor organs and the increasing number of patients waiting for transplantation. However, a major safety issue is the potential cross-species transmission of porcine endogenous retroviruses (PERV). This problem could be resolved if it is possible to produce pigs that do not contain replication-competent copies of this virus. In order to determine the feasibility of this, we have determined the number of potentially replication-competent full-length PERV proviruses and obtained data on their integration sites within the porcine genome. We have screened genomic DNA libraries from a Large White pig for potentially intact proviruses. We identified six unique PERV B proviruses that were apparently intact in all three genes, while the majority of isolated proviruses were defective in one or more genes. No intact PERV A proviruses were found in this pig, despite the identification of multiple defective A proviruses. Genotyping of 30 unrelated pigs for these unique proviruses showed a heterogeneous distribution. Two proviruses were uncommon, present in 7 of 30 and 3 of 30 pigs, while three were each present in 24 of 30 pigs, and one was present in 30 of 30 animals examined. Our data indicate that few PERV proviruses in Large White pigs are capable of productive infection and suggest that many could be removed by selective breeding. Further studies are required to determine if all potentially functional proviruses could be removed by breeding or whether gene knockout techniques will be required to remove the residuum. PMID:11711616

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

    PubMed

    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

  19. Chemical Polysialylation of Recombinant Human Proteins.

    PubMed

    Smirnov, Ivan V; Vorobiev, Ivan I; Belogurov, Alexey A; Genkin, Dmitry D; Deyev, Sergey M; Gabibov, Alexander G

    2015-01-01

    Design of drug with prolonged therapeutic action is one of the rapid developing fields of modern medical science and required implementation of different methods of protein chemistry and molecular biology. There are several therapeutic proteins needing increasing of their stability, pharmacokinetic, and pharmacodynamics parameters. To make long-live DNA-encoded drug PEGylation was proposed. Alternatively polysialic (colominic) acid, extracted from the cell wall of E. coli, fractionated to the desired size by anion-exchange chromatography and chemically activated to the amine-reactive aldehyde form, may be chemically attached to the polypeptide chain. Conjugates of proteins and polysialic acid generally resemble properties of protein-PEG conjugates, but possess significant negative net charge and are thought to be fully degradable after endocytosis due to the presence of intracellular enzymes, hydrolyzing the polysialic acid. Complete biodegradation of the polysialic acid moiety makes this kind of conjugates preferable for creation of drugs, intended for chronic use. Here, we describe two different protocols of chemical polysialylation. First protocol was employed for the CHO-derived human butyrylcholinesterase with optimized for recovery of specific enzyme activity. Polysialic acid moieties are attached at various lysine residues. Another protocol was developed for high-yield conjugation of human insulin; major conjugation point is the N-terminal residue of the insulin's light chain. These methods may allow to produce polysialylated conjugates of various proteins or polypeptides with reasonable yield and without significant loss of functional activity. PMID:26082236

  20. Immunization of malignant melanoma patients with full-length NY-ESO-1 protein using Toll-like receptor 7 agonist imiquimod as vaccine adjuvant

    PubMed Central

    Adams, Sylvia; O'Neill, David W.; Nonaka, Daisuke; Hardin, Elizabeth; Chiriboga, Luis; Siu, Kimberly; Cruz, Crystal M.; Angiulli, Angelica; Angiulli, Francesca; Ritter, Erika; Holman, Rose Marie; Shapiro, Richard L.; Berman, Russell S.; Berner, Natalie; Shao, Yongzhao; Manches, Olivier; Pan, Linda; Venhaus, Ralph R.; Hoffman, Eric W.; Jungbluth, Achim; Gnjatic, Sacha; Old, Lloyd; Pavlick, Anna C.; Bhardwaj, Nina

    2008-01-01

    T cell-mediated immunity to microbes and to cancer can be enhanced by the activation of dendritic cells (DCs) via Toll-like receptors (TLRs). In this study, we evaluated the safety and feasibility of topical imiquimod, a TLR7 agonist, in a series of vaccinations (26) proteins,(27) and DNA, (28, 29) as well as in vaccines using recombinant Listeria(30) or DCs.(31) In humans, it was shown that topical imiquimod treatment may enhance the immunogenicity of a melanoma peptide vaccine when given with systemic FLT3 ligand. (32) In addition, injection of immature DCs into imiquimod pretreated skin lead to DC activation in situ and enhanced migratory capacity to draining lymph nodes in cancer patients. (33) In this study, we test the safety and feasibility of imiquimod in a vaccine against the cancer/testis antigen NY-ESO-1, and evaluate the immunogenicity of the combination. NY-ESO-1 is detectable in approximately 30% of metastatic melanomas. (34-36) It is against the cancer/testis antigen NY-ESO-1 in patients with malignant melanoma. Recombinant, full-length NY-ESO-1 protein was administered intradermally into imiquimod pre-conditioned sites followed by additional topical applications of imiquimod. The regimen was very well-tolerated with only mild and transient local reactions and constitutional symptoms. Secondarily, we examined the systemic immune response induced by the imiquimod/NY-ESO-1 combination, and show that it elicited both humoral and cellular responses in a significant fraction of patients. Skin biopsies were assessed for imiquimod's in situ immunomodulatory effects. Compared with untreated skin, topical imiquimod induced dermal mononuclear cell infiltrates in all patients composed primarily of T cells, monocytes, macrophages, myeloid DCs and natural killer (NK) cells, and to a lesser extent plasmacytoid DCs. DC activation was evident. This study demonstrates the feasibility and excellent safety profile of a topically applied TLR7 agonist utilized as a

  1. Multiple biological activities of human recombinant interleukin 1.

    PubMed Central

    Dinarello, C A; Cannon, J G; Mier, J W; Bernheim, H A; LoPreste, G; Lynn, D L; Love, R N; Webb, A C; Auron, P E; Reuben, R C

    1986-01-01

    Complementary DNA coding for human monocyte interleukin 1 (IL-1), pI 7 form, was expressed in Escherichia coli. During purification, IL-1 activity on murine T cells was associated with the recombinant protein. Homogeneous human recombinant IL-1 (hrIL-1) was tested in several assays to demonstrate the immunological and inflammatory properties attributed to this molecule. hrIL-1 induced proliferative responses in a cloned murine T cell in the presence of suboptimal concentrations of mitogen, whereas no effect was observed with hrIL-1 alone. At concentrations of 0.05 ng/ml, hrIL-1 doubled the response to mitogen (5 X 10(6) half maximal units/mg). Human peripheral blood T cells depleted of adherent cells underwent a blastogenic response and released interleukin 2 in the presence of hrIL-1 and mitogen. hrIL-1 was a potent inflammatory agent by its ability to induce human dermal fibroblast prostaglandin E2 production in vitro and to produce monophasic (endogenous pyrogen) fever when injected into rabbits or endotoxin-resistant mice. These studies establish that the dominant pI 7 form of recombinant human IL-1 possesses immunological and inflammatory properties and acts on the central nervous system to produce fever. Images PMID:3519678

  2. Recombinant human erythrocyte cytochrome b5.

    PubMed

    Lloyd, E; Ferrer, J C; Funk, W D; Mauk, M R; Mauk, A G

    1994-09-27

    The gene encoding the human erythrocyte form of cytochrome b5 (97 residues in length) has been prepared by mutagenesis of an expression vector encoding lipase-solubilized bovine liver microsomal cytochrome b5 (93 residues in length) (Funk et al., 1990). Efficient expression of this gene in Escherichia coli has provided the first opportunity to obtain this protein in quantities sufficient for physical and functional characterization. Comparison of the erythrocytic cytochrome with the trypsin-solubilized bovine liver cytochrome b5 by potentiometric titration indicates that the principal electrostatic difference between the two proteins results from two additional His residues present in the human erythrocytic protein. The midpoint reduction potential of this protein determined by direct electrochemistry is -9 +/- 2 mV vs SHE at pH 7.0 (mu = 0.10 M, 25.0 degrees C), and this value varies with pH in a fashion that is consistent with the presence of a single ionizable group that changes pKa from 6.0 +/- 0.1 in the ferricytochrome to 6.3 +/- 0.1 in the ferrocytochrome with delta H degrees = -3.2 +/- 0.1 kcal/mol and delta S degrees = -11.5 +/- 0.3 eu (pH 7.0, mu = 0.10). The 1D 1H NMR spectrum of the erythrocytic ferricytochrome indicates that 90% of the protein binds heme in the "major" orientation and 10% of the protein binds heme in the "minor" orientation (pH 7.0, 25 degrees C) with delta H degrees = -2.9 +/- 0.3 kcal/mol and delta S degrees = -5.4 +/- 0.9 eu for this equilibrium. PMID:7918357

  3. Recombinant expression, in vitro refolding, and biophysical characterization of the human glucagon-like peptide-1 receptor.

    PubMed

    Schröder-Tittmann, Kathrin; Bosse-Doenecke, Eva; Reedtz-Runge, Steffen; Ihling, Christian; Sinz, Andrea; Tittmann, Kai; Rudolph, Rainer

    2010-09-14

    Activation of the glucagon-like peptide-1 receptor (GLP-1R) upon ligand binding leads to the release of insulin from pancreatic cells. This strictly glucose-dependent process renders the receptor and its ligands useful in the treatment of type II diabetes mellitus. To enable a biophysical characterization in vitro, we expressed the human full-length GLP-1R in the cytosol of Escherichia coli as inclusion bodies. After purification, refolding of the SDS-solubilized receptor was achieved by the exchange of SDS against the detergent Brij78 using an artificial chaperone system. Far-UV circular dichroism spectroscopic studies revealed that the receptor adopts a characteristic alpha-helical structure in Brij78 micelles. Ligand binding of the renatured protein was quantified by fluorescence quenching and surface plasmon resonance spectroscopy. In the presence of Brij micelles, the refolded receptor binds the agonist exendin-4 with an apparent dissociation constant of approximately 100 nM in a reversible one-step mechanism. To demonstrate that the detected ligand binding activity is not only due to an autonomously functional N-terminal domain (nGLP-1R) but also due to additional contacts with the juxtamembrane part, we separately expressed and refolded the extracellular domain relying on identical protocols established for the full-length GLP-1R. In support of the suggested multidomain binding mode, the nGLP-1R binds exendin-4 with a lower affinity (K(app) in the micromolar range) and a different kinetic mechanism. The lower ligand affinity of the nGLP-1R results entirely from a decreased kinetic stability of the receptor-ligand complex, dissociation of which is approximately 40-fold faster in the case of the nGLP-1R compared to the full-length GLP-1R. In summary, a framework was developed to produce functional human full-length GLP-1R by recombinant expression in E. coli as a prerequisite for eventual structure determination and a rigorous biophysical characterization

  4. Crystallization of recombinant human interleukin 1β

    NASA Astrophysics Data System (ADS)

    Einspahr, Howard; Clancy, L. L.; Muchmore, S. W.; Watenpaugh, K. D.; Harris, P. K. W.; Carter, D. B.; Curry, K. A.; Tomich, C.-S. C.; Yem, A. W.; Deibel, M. R.; Tracey, D. E.; Paslay, J. W.; Staite, N. D.; Carter, J. B.; Theriault, N. Y.; Reardon, I. M.; Zurcher-Neely, H. A.; Heinrikson, R. L.

    1988-07-01

    The gene for the fully processed form of human interleukin 1β was cloned from SK-hep-1 hepatoma cellular RNA and expressed at high levels in E. coli. The protein produced in E. coli. was purified to homogeneity by standard chromatographic methods, including adsorption and desorption from Procion Red Sepharose, sizing on a Superose 12 FPLC column, and anion exchange chromatography on QAE Sepharose. The result is a biologically active protein, rIL-1β, that migrates on two-dimensional gels as a single spot with a pI of 6.5 ± 0.2 and a molecular mass of 17, 500 daltons. Crystals of rIL-1β have been produced from concentrated solutions of the protein by ammonium sulfate precipitation. The crystals are tetragonal, have space group P41 or its enantiomer, have lattice constants of a = 58.46(1) Å and c = 77.02(3) Å, and scatter to at least 2 Å resolution. A structure determination ba these crystals is underway.

  5. Human Recombinant Insulin 1g - ug

    NASA Technical Reports Server (NTRS)

    2004-01-01

    Proteins are the building blocks of our bodies and the living world around us. Within our bodies proteins make it possible for red blood cells to carry oxygen throughout the body. Others help transmit nerve impulses so we can hear, smell and feel the world around us. While others play a crucial role in preventing or causing disease. If the structure of a protein is known, then companies can develop new or improved drugs to fight the disease of which the protein is a part. To determine protein structure, researchers must grow near-perfect crystals of the protein. On Earth convection currents, sedimentation and other gravity-induced phenomena hamper crystal growth efforts. In microgravity researchers can grow near-perfect crystals in an environment free of these effects. Because of the enormous potential for new pharmaceutical products the Center for Macromolecular Crystallography--the NASA Commercial Space Center responsible for commercial protein crystal growth efforts has more than fifty major industry and academic partners. Research on crystals of human insulin could lead to improved treatments for diabetes.

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

    SciTech Connect

    Watanabe, Yasuko; Hiraoka, Wakako; Igarashi, Manabu; Ito, Kimihito; Shimoyama, Yuhei; Horiuchi, Motohiro; Yamamori, Tohru; Yasui, Hironobu; Kuwabara, Mikinori; Inagaki, Fuyuhiko; Inanami, Osamu

    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}.

  7. Soluble expression, purification and characterization of the full length IS2 Transposase

    PubMed Central

    2011-01-01

    Background The two-step transposition pathway of insertion sequences of the IS3 family, and several other families, involves first the formation of a branched figure-of-eight (F-8) structure by an asymmetric single strand cleavage at one optional donor end and joining to the flanking host DNA near the target end. Its conversion to a double stranded minicircle precedes the second insertional step, where both ends function as donors. In IS2, the left end which lacks donor function in Step I acquires it in Step II. The assembly of two intrinsically different protein-DNA complexes in these F-8 generating elements has been intuitively proposed, but a barrier to testing this hypothesis has been the difficulty of isolating a full length, soluble and active transposase that creates fully formed synaptic complexes in vitro with protein bound to both binding and catalytic domains of the ends. We address here a solution to expressing, purifying and structurally analyzing such a protein. Results A soluble and active IS2 transposase derivative with GFP fused to its C-terminus functions as efficiently as the native protein in in vivo transposition assays. In vitro electrophoretic mobility shift assay data show that the partially purified protein prepared under native conditions binds very efficiently to cognate DNA, utilizing both N- and C-terminal residues. As a precursor to biophysical analyses of these complexes, a fluorescence-based random mutagenesis protocol was developed that enabled a structure-function analysis of the protein with good resolution at the secondary structure level. The results extend previous structure-function work on IS3 family transposases, identifying the binding domain as a three helix H + HTH bundle and explaining the function of an atypical leucine zipper-like motif in IS2. In addition gain- and loss-of-function mutations in the catalytic active site define its role in regional and global binding and identify functional signatures that are common

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

    PubMed

    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

  9. Bioinformatic Analysis of the Human Recombinant Iduronate 2-Sulfate Sulfatase

    PubMed Central

    Morales-Álvarez, Edwin D.; Rivera-Hoyos, Claudia M.; Landázuri, Patricia; Poutou-Piñales, Raúl A.; Pedroza-Rodríguez, Aura M.

    2016-01-01

    Mucopolysaccharidosis type II is a human recessive disease linked to the X chromosome caused by deficiency of lysosomal enzyme Iduronate 2-Sulfate Sulfatase (IDS), which leads to accumulation of glycosaminoglycans in tissues and organs. The human enzyme has been expressed in Escherichia coli and Pichia pastoris in attempt to develop more successful expression systems that allow the production of recombinant IDS for Enzyme Replacement Therapy (ERT). However, the preservation of native signal peptide in the sequence has caused conflicts in processing and recognition in the past, which led to problems in expression and enzyme activity. With the main object being the improvement of the expression system, we eliminate the native signal peptide of human recombinant IDS. The resulting sequence showed two modified codons, thus, our study aimed to analyze computationally the nucleotide sequence of the IDSnh without signal peptide in order to determine the 3D structure and other biochemical properties to compare them with the native human IDS (IDSnh). Results showed that there are no significant differences between both molecules in spite of the two-codon modifications detected in the recombinant DNA sequence. PMID:27335624

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

    PubMed

    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