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Sample records for asv integrase proteins

  1. Integrase protein of murine leukemia viruses

    Energy Technology Data Exchange (ETDEWEB)

    Yang, Wen K.; Ch ang, Lan-Yang; Sundseth, R.; Myer, F.E.; Yang, Den-Mei (Oak Ridge National Lab., TN (USA); Tennessee Univ., Knoxville, TN (USA); Oak Ridge National Lab., TN (USA))

    1989-01-01

    The defined mapping of the MuLV integrase in previous work (10,11) has made it possible for us to design and construct molecular clones for expression of this protein in bacteria and thus easily obtain specific antisera. Our repeated inability to isolate the antigenic MuLV virion p46 protein without the use of a denaturing reagent, although preventing us from carrying out the contemplated enzymological studies, would imply that the integration function of MuLV is more stringently controlled at the virion structure level than is that of avian retroviruses. Thus, gene transfer and therapy procedures would be safer to the recipient by using the MuLV retroviral vector than the avian retroviral vector, since p46 is less likely than p32 to be aberrantly released to cause genomic damage to the cell. The apparent cytotoxity of transfection with mammalian expression vectors carrying the integrase gene is not unexpected in situations where a high level of this protein is expressed and accumulated in the nucleus. The apparently incidental emergence of neoplastic phenotypes in some stably transfected cells would cause genomic instability and gene rearrangement of the oncogenic components. This possibility is particularly noteworthy for endogenous retroviral gene elements, in which the control of integrase gene expression and function may not be as stringent as in the infectious MuLVs. 28 refs., 5 figs.

  2. Coevolutionary Analysis Identifies Protein–Protein Interaction Sites between HIV-1 Reverse Transcriptase and Integrase

    OpenAIRE

    Arachchilage, Madara Hetti; Piontkivska, Helen

    2016-01-01

    The replication of human immunodeficiency virus-1 (HIV-1) requires reverse transcription of the viral RNA genome and integration of newly synthesized pro-viral DNA into the host genome. This is mediated by the viral proteins reverse transcriptase (RT) and integrase (IN). The formation and stabilization of the pre-integration complex (PIC), which is an essential step for reverse transcription, nuclear import, chromatin targeting, and subsequent integration, involves direct and indirect modes o...

  3. The lentiviral integrase binding protein LEDGF/p75 and HIV-1 replication.

    Directory of Open Access Journals (Sweden)

    Alan Engelman

    2008-03-01

    Full Text Available Retroviral replication proceeds through a stable proviral DNA intermediate, and numerous host cell factors have been implicated in its formation. In particular, recent results have highlighted an important role for the integrase-interactor lens epithelium-derived growth factor (LEDGF/p75 in lentiviral integration. Cells engineered to over-express fragments of LEDGF/p75 containing its integrase-binding domain but lacking determinants essential for chromatin association are refractory to HIV-1 infection. Furthermore, both the levels of HIV-1 integration and the genomic distribution of the resultant proviruses are significantly perturbed in cells devoid of endogenous LEDGF/p75 protein. A strong bias towards integration along transcription units is a characteristic feature of lentiviruses. In the absence of LEDGF/p75, HIV-1 in large part loses that preference, displaying concomitant integration surges in the vicinities of CpG islands and gene promoter regions, elements naturally targeted by other types of retroviruses. Together, these findings highlight that LEDGF/p75 is an important albeit not strictly essential cofactor of lentiviral DNA integration, and solidify a role for chromatin-associated LEDGF/p75 as a receptor for lentiviral preintegration complexes. By now one of the best characterized virus-host interactions, the integrase-LEDGF/p75 interface opens a range of opportunities for lentiviral vector targeting for gene therapy applications as well as for the development of novel classes of antiretroviral drugs.

  4. B′-protein phosphatase 2A is a functional binding partner of delta-retroviral integrase

    Science.gov (United States)

    Maertens, Goedele N.

    2016-01-01

    To establish infection, a retrovirus must insert a DNA copy of its RNA genome into host chromatin. This reaction is catalysed by the virally encoded enzyme integrase (IN) and is facilitated by viral genus-specific host factors. Herein, cellular serine/threonine protein phosphatase 2A (PP2A) is identified as a functional IN binding partner exclusive to δ-retroviruses, including human T cell lymphotropic virus type 1 and 2 (HTLV-1 and HTLV-2) and bovine leukaemia virus (BLV). PP2A is a heterotrimer composed of a scaffold, catalytic and one of any of four families of regulatory subunits, and the interaction is specific to the B′ family of the regulatory subunits. B′-PP2A and HTLV-1 IN display nuclear co-localization, and the B′ subunit stimulates concerted strand transfer activity of δ-retroviral INs in vitro. The protein–protein interaction interface maps to a patch of highly conserved residues on B′, which when mutated render B′ incapable of binding to and stimulating HTLV-1 and -2 IN strand transfer activity. PMID:26657642

  5. HIV-2 integrase variation in integrase inhibitor-naive adults in Senegal, West Africa.

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    Geoffrey S Gottlieb

    Full Text Available BACKGROUND: Antiretroviral therapy for HIV-2 infection is hampered by intrinsic resistance to many of the drugs used to treat HIV-1. Limited studies suggest that the integrase inhibitors (INIs raltegravir and elvitegravir have potent activity against HIV-2 in culture and in infected patients. There is a paucity of data on genotypic variation in HIV-2 integrase that might confer intrinsic or transmitted INI resistance. METHODS: We PCR amplified and analyzed 122 HIV-2 integrase consensus sequences from 39 HIV-2-infected, INI-naive adults in Senegal, West Africa. We assessed genetic variation and canonical mutations known to confer INI-resistance in HIV-1. RESULTS: No amino acid-altering mutations were detected at sites known to be pivotal for INI resistance in HIV-1 (integrase positions 143, 148 and 155. Polymorphisms at several other HIV-1 INI resistance-associated sites were detected at positions 72, 95, 125, 154, 165, 201, 203, and 263 of the HIV-2 integrase protein. CONCLUSION: Emerging genotypic and phenotypic data suggest that HIV-2 is susceptible to the new class of HIV integrase inhibitors. We hypothesize that intrinsic HIV-2 integrase variation at "secondary" HIV-1 INI-resistance sites may affect the genetic barrier to HIV-2 INI resistance. Further studies will be needed to assess INI efficacy as part of combination antiretroviral therapy in HIV-2-infected patients.

  6. Hamming distance geometry of a protein conformational space. Application to the clustering of a 4 ns molecular dynamics trajectory of the HIV-1 integrase catalytic core

    CERN Document Server

    Laboulais, C; Le Bret, M; Gabarro-Arpa, J; Laboulais, Cyril; Ouali, Mohammed; Bret, Marc Le; Gabarro-Arpa, Jacques

    2001-01-01

    Protein structures can be encoded into binary sequences, these are used to define a Hamming distance in conformational space: the distance between two different molecular conformations is the number of different bits in their sequences. Each bit in the sequence arises from a partition of conformational space in two halves. Thus, the information encoded in the binary sequences is also used to characterize the regions of conformational space visited by the system. We apply this distance and their associated geometric structures, to the clustering and analysis of conformations sampled during a 4 ns molecular dynamics simulation of the HIV-1 integrase catalytic core. The cluster analysis of the simulation shows a division of the trajectory into two segments of 2.6 and 1.4 ns length, which are qualitatively different: the data points to the fact that equilibration is only reached at the end of the first segment. Some length of the paper is devoted to compare the Hamming distance to the r.m.s. deviation measure. Th...

  7. Multifunctional facets of retrovirus integrase

    Institute of Scientific and Technical Information of China (English)

    Duane; P; Grandgenett; Krishan; K; Pandey; Sibes; Bera; Hideki; Aihara

    2015-01-01

    The retrovirus integrase(IN) is responsible for integration of the reverse transcribed linear c DNA into the host DNA genome. First, IN cleaves a dinucleotide from the 3’ OH blunt ends of the viral DNA exposing the highly conserved CA sequence in the recessed ends. IN utilizes the 3’ OH ends to catalyze the concerted integration of the two ends into opposite strands of the cellular DNA producing 4 to 6 bp staggered insertions, depending on the retrovirus species. The staggered ends are repaired by host cell machinery that results in a permanent copy of the viral DNA in the cellular genome. Besides integration, IN performs other functions in the replication cycle of several studied retroviruses. The proper organization of IN within the viral internal core is essential for the correct maturation of the virus. IN plays a major role in reverse transcription by interacting directly with the reverse transcriptase and by binding to the viral capsid protein and a cellular protein. Recruitment of several other host proteins into the viral particle are also promoted by IN. IN assists with the nuclear transport of the preintegration complex across the nuclear membrane. With several retroviruses, IN specifically interacts with different host protein factors that guide the preintegration complex to preferentially integrate the viral genome into specific regions of the host chromosomal target. Human gene therapy using retrovirus vectors is directly affected by the interactions of IN with these host factors. Inhibitors directed against the human immunodeficiency virus(HIV) IN bind within the active site of IN containing viral DNA ends thus preventing integration and subsequent HIV/AIDS.

  8. Human immunodeficiency virus type 1 integrase: effect on viral replication of mutations at highly conserved residues.

    OpenAIRE

    Cannon, P M; Wilson, W; Byles, E; Kingsman, S M; Kingsman, A J

    1994-01-01

    Sequence comparisons of the integrase (IN) proteins from different retroviruses have identified several highly conserved residues. We have introduced mutations at 16 of these sites into the integrase gene of human immunodeficiency virus type 1 and analyzed the phenotypes of the resulting viruses. The viruses were all normal for p24 content and reverse transcriptase activity. In addition, all of the mutants could infect T-cell lines and undergo reverse transcription, as assessed by PCR analysi...

  9. Developing a dynamic pharmacophore model for HIV-1 integrase.

    Science.gov (United States)

    Carlson, H A; Masukawa, K M; Rubins, K; Bushman, F D; Jorgensen, W L; Lins, R D; Briggs, J M; McCammon, J A

    2000-06-01

    We present the first receptor-based pharmacophore model for HIV-1 integrase. The development of "dynamic" pharmacophore models is a new method that accounts for the inherent flexibility of the active site and aims to reduce the entropic penalties associated with binding a ligand. Furthermore, this new drug discovery method overcomes the limitation of an incomplete crystal structure of the target protein. A molecular dynamics (MD) simulation describes the flexibility of the uncomplexed protein. Many conformational models of the protein are saved from the MD simulations and used in a series of multi-unit search for interacting conformers (MUSIC) simulations. MUSIC is a multiple-copy minimization method, available in the BOSS program; it is used to determine binding regions for probe molecules containing functional groups that complement the active site. All protein conformations from the MD are overlaid, and conserved binding regions for the probe molecules are identified. Those conserved binding regions define the dynamic pharmacophore model. Here, the dynamic model is compared to known inhibitors of the integrase as well as a three-point, ligand-based pharmacophore model from the literature. Also, a "static" pharmacophore model was determined in the standard fashion, using a single crystal structure. Inhibitors thought to bind in the active site of HIV-1 integrase fit the dynamic model but not the static model. Finally, we have identified a set of compounds from the Available Chemicals Directory that fit the dynamic pharmacophore model, and experimental testing of the compounds has confirmed several new inhibitors. PMID:10841789

  10. The A128T resistance mutation reveals aberrant protein multimerization as the primary mechanism of action of allosteric HIV-1 integrase inhibitors.

    Science.gov (United States)

    Feng, Lei; Sharma, Amit; Slaughter, Alison; Jena, Nivedita; Koh, Yasuhiro; Shkriabai, Nikolozi; Larue, Ross C; Patel, Pratiq A; Mitsuya, Hiroaki; Kessl, Jacques J; Engelman, Alan; Fuchs, James R; Kvaratskhelia, Mamuka

    2013-05-31

    Allosteric HIV-1 integrase (IN) inhibitors (ALLINIs) are a very promising new class of anti-HIV-1 agents that exhibit a multimodal mechanism of action by allosterically modulating IN multimerization and interfering with IN-lens epithelium-derived growth factor (LEDGF)/p75 binding. Selection of viral strains under ALLINI pressure has revealed an A128T substitution in HIV-1 IN as a primary mechanism of resistance. Here, we elucidated the structural and mechanistic basis for this resistance. The A128T substitution did not affect the hydrogen bonding between ALLINI and IN that mimics the IN-LEDGF/p75 interaction but instead altered the positioning of the inhibitor at the IN dimer interface. Consequently, the A128T substitution had only a minor effect on the ALLINI IC50 values for IN-LEDGF/p75 binding. Instead, ALLINIs markedly altered the multimerization of IN by promoting aberrant higher order WT (but not A128T) IN oligomers. Accordingly, WT IN catalytic activities and HIV-1 replication were potently inhibited by ALLINIs, whereas the A128T substitution in IN resulted in significant resistance to the inhibitors both in vitro and in cell culture assays. The differential multimerization of WT and A128T INs induced by ALLINIs correlated with the differences in infectivity of HIV-1 progeny virions. We conclude that ALLINIs primarily target IN multimerization rather than IN-LEDGF/p75 binding. Our findings provide the structural foundations for developing improved ALLINIs with increased potency and decreased potential to select for drug resistance. PMID:23615903

  11. HIV-2 Integrase Variation in Integrase Inhibitor-Naïve Adults in Senegal, West Africa

    OpenAIRE

    Geoffrey S Gottlieb; Smith, Robert A.; Dia Badiane, Ndeye Mery; Ba, Selly; Stephen E Hawes; Toure, Macoumba; Starling, Alison K.; Traore, Fatou; Sall, Fatima; Cherne, Stephen L.; Stern, Joshua; Kim G Wong; Lu, Paul; Kim, Moon; Dana N Raugi

    2011-01-01

    Background Antiretroviral therapy for HIV-2 infection is hampered by intrinsic resistance to many of the drugs used to treat HIV-1. Limited studies suggest that the integrase inhibitors (INIs) raltegravir and elvitegravir have potent activity against HIV-2 in culture and in infected patients. There is a paucity of data on genotypic variation in HIV-2 integrase that might confer intrinsic or transmitted INI resistance. Methods We PCR amplified and analyzed 122 HIV-2 integrase consensus sequenc...

  12. In vitro selection of G-rich RNA aptamers that target HIV-1 integrase

    Institute of Scientific and Technical Information of China (English)

    2008-01-01

    Aptamers that interact with various HIV-1 proteins,such as reverse transcriptase,Rev,Tat protein,and nuclear capsule protein,have been prepared through SELEX (systematic evolution of ligands by ex-ponential enrichment) technique. However,there are few reports about the DNA or RNA aptamers that target HIV-1 integrase. In this investigation,we selected alternative RNA aptamers specific for the HIV-1 integrase by using a different binding buffer containing 10 mmol·L-1 MgCl2 and 100 mmol·L-1 KCl. Aptamer IN1,IN2,IN3 had similar and the highest Kd values from 145 to 239 nmol·L-1. Structural studies showed that they formed similar stem-loop structure. Deletion of any stem structure resulted in diminished affinity. In addition,structure probing study with antisense DNA indicated that the stem-loop structure in the random region was critical for integrase binding. Although aptamer IN1 failed to form G-quartet structure,it might directly interact with the DDE motif of integrase,which is the virus DNA-binding site,because G-quadruplex T40214 competitively inhibited the interaction between IN1 and integrase. Together,this study generated a novel RNA aptamer IN1,which could be useful in basic research and anti-HIV drug screening.

  13. Dolutegravir (S/GSK1349572) Exhibits Significantly Slower Dissociation than Raltegravir and Elvitegravir from Wild-Type and Integrase Inhibitor-Resistant HIV-1 Integrase-DNA Complexes▿†

    OpenAIRE

    Hightower, Kendra E.; Wang, RuoLan; DeAnda, Felix; Johns, Brian A.; Weaver, Kurt; Shen, Yingnian; Tomberlin, Ginger H.; Carter, H. Luke; Broderick, Timothy; Sigethy, Scott; Seki, Takahiro; KOBAYASHI, Masanori; Underwood, Mark R.

    2011-01-01

    The integrase inhibitor (INI) dolutegravir (DTG; S/GSK1349572) has significant activity against HIV-1 isolates with raltegravir (RAL)- and elvitegravir (ELV)-associated resistance mutations. As an initial step in characterizing the different resistance profiles of DTG, RAL, and ELV, we determined the dissociation rates of these INIs with integrase (IN)-DNA complexes containing a broad panel of IN proteins, including IN substitutions corresponding to signature RAL and ELV resistance mutations....

  14. Inhibition of HIV-1 Integrase gene expression by 10-23 DNAzyme

    Indian Academy of Sciences (India)

    Nirpendra Singh; Atul Ranjan; Souvik Sur; Ramesh Chandra; Vibha Tandon

    2012-07-01

    HIV Integrase (IN) is an enzyme that is responsible for the integration of the proviral genome into the human genome, and this integration step is the first step of the virus hijacking the human cell machinery for its propagation and replication. 10-23 DNAzyme has the potential to suppress gene expressions through sequence-specific mRNA cleavage. We have designed three novel DNAzymes, DIN54, DIN116, and DIN152, against HIV-1 Integrase gene using Mfold software and evaluated them for target site cleavage activity on the in vitro transcribed mRNA. All DNAzymes were tested for its inhibition of expression of HIV Integrase protein in the transiently transfected cell lines. DIN116 and DIN152 inhibited IN-EGFP expression by 80% and 70% respectively.

  15. In vitro selection of G-rich RNA aptamers that target HIV-1 integrase

    Institute of Scientific and Technical Information of China (English)

    LIU YingChun; ZHANG Yan; YE GuoZhu; YANG ZhenJun; ZHANG LiangRen; ZHANG LiHe

    2008-01-01

    Aptamers that interact with various HIV-1 proteins, such as reverse transcriptase, Rev, Tat protein, and nuclear capsule protein, have been prepared through SELEX (systematic evolution of ligands by ex-ponential enrichment) technique. However, there are few reports about the DNA or RNA aptamers that target HIV-1 integrase. In this investigation, we selected alternative RNA aptamers specific for the HIV-1 Aptamer IN1, IN2, IN3 had similar and the highest Kd values from 145 to 239 nmol. L-1. Structural studies showed that they formed similar stem-loop structure. Deletion of any stem structure resulted in diminished affinity. In addition, structure probing study with antisense DNA indicated that the stem-loop structure in the random region was critical for integrase binding. Although aptamer IN1 failed to form G-quartet structure, it might directly interact with the DDE motif of integrase, which is the virus DNA-binding site, because G-quadruplex T40214 competitively inhibited the interaction between IN1 and integrase. Together, this study generated a novel RNA aptamer IN1, which could be useful in basic research and anti-HIV drug screening.

  16. Use of Integrase-Minus Lentiviral Vector for Transient Expression

    Directory of Open Access Journals (Sweden)

    Hossein Azadeh

    2012-01-01

    Full Text Available Objective: Lentivirus-derived vectors are among the most promising viral vectors for gene therapy which is currently available, but their use in clinical practice is limited due to associated risk of insertional mutagenesis. Gene targeting is an ideal method for gene therapy, but it has low efficiency in comparison to viral vector methods. In this study, we are going to design and construct an integrase-minus lentiviral vector. This vector is suitable for transient expression of gene and gene targeting with viral vector.Materials and Methods: In this experimental study, three missense mutations were induced in the catalytic domain of Integrase gene in the pLP1 plasmid and resulted D64V, D116A and E152G changes in the amino acid sequence through site directed mutagenesis. The pLenti6.2-GW/EmGFP transfer vector, associated with native and mutated packaging mix, was transfected into 293T cell line. In order to titer the lentivirus stock, the viruses were harvested. Finally, the viruses transduced into COS-7 cell line to assess green fluorescent protein (GFP gene expression by a fluorescence microscopy.Results: Recombinant and wild lentiviruses titer was about 5~8×106 transducing units/ml in COS-7 cell line. The number of GFP-positive cells transduced with native viruses was decreased slightly during two weeks after viral transduction. In contrast, in the case of integrase-minus viruses, a dramatic decrease in the number of GFP positive cells was observed.Conclusion: This study was conducted to overcome the integration of lentiviral genome into a host genome. Nonintegrating lentiviral vectors can be used for transient gene expression and gene targeting if a Target gene cassette is placed in the lentivirus gene structure. This combination method decreases disadvantages of both processes, such as random integration of lentiviruses and low efficiency of gene targeting.

  17. Virtual screening with AutoDock Vina and the common pharmacophore engine of a low diversity library of fragments and hits against the three allosteric sites of HIV integrase: participation in the SAMPL4 protein-ligand binding challenge.

    Science.gov (United States)

    Perryman, Alexander L; Santiago, Daniel N; Forli, Stefano; Santos-Martins, Diogo; Olson, Arthur J

    2014-04-01

    To rigorously assess the tools and protocols that can be used to understand and predict macromolecular recognition, and to gain more structural insight into three newly discovered allosteric binding sites on a critical drug target involved in the treatment of HIV infections, the Olson and Levy labs collaborated on the SAMPL4 challenge. This computational blind challenge involved predicting protein-ligand binding against the three allosteric sites of HIV integrase (IN), a viral enzyme for which two drugs (that target the active site) have been approved by the FDA. Positive control cross-docking experiments were utilized to select 13 receptor models out of an initial ensemble of 41 different crystal structures of HIV IN. These 13 models of the targets were selected using our new "Rank Difference Ratio" metric. The first stage of SAMPL4 involved using virtual screens to identify 62 active, allosteric IN inhibitors out of a set of 321 compounds. The second stage involved predicting the binding site(s) and crystallographic binding mode(s) for 57 of these inhibitors. Our team submitted four entries for the first stage that utilized: (1) AutoDock Vina (AD Vina) plus visual inspection; (2) a new common pharmacophore engine; (3) BEDAM replica exchange free energy simulations, and a Consensus approach that combined the predictions of all three strategies. Even with the SAMPL4's very challenging compound library that displayed a significantly lower amount of structural diversity than most libraries that are conventionally employed in prospective virtual screens, these approaches produced hit rates of 24, 25, 34, and 27 %, respectively, on a set with 19 % declared binders. Our only entry for the second stage challenge was based on the results of AD Vina plus visual inspection, and it ranked third place overall according to several different metrics provided by the SAMPL4 organizers. The successful results displayed by these approaches highlight the utility of the computational

  18. Peptides derived from HIV-1 integrase that bind Rev stimulate viral genome integration.

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    Aviad Levin

    Full Text Available BACKGROUND: The human immunodeficiency virus type 1 (HIV-1 integrase protein (IN, catalyzes the integration of viral DNA into the host cell genome. IN catalyzes the first step of the integration process, namely the 3'-end processing in which IN removes a pGT dinucleotide from the 3' end of each viral long terminal repeat (LTR. Following nuclear import of the viral preintegration complex, the host chromosomal DNA becomes accessible to the viral cDNA and the second step of the integration process, namely the strand-transfer step takes place. This ordered sequence of events, centered on integration, is mandatory for HIV replication. METHODOLOGY/PRINCIPAL FINDINGS: Using an integrase peptide library, we selected two peptides, designated INr-1 and INr-2, which interact with the Rev protein and probably mediate the Rev-integrase interaction. Using an in-vitro assay system, we show that INr-1 and INr-2 are able to abrogate the inhibitory effects exerted by Rev and Rev-derived peptides on integrase activity. Both INr-1 and INr-2 were found to be cell-permeable and nontoxic, allowing a study of their effect in HIV-1-infected cultured cells. Interestingly, both INr peptides stimulated virus infectivity as estimated by production of the viral P24 protein, as well as by determination of the appearance of newly formed virus particles. Furthermore, kinetics studies revealed that the cell-permeable INr peptides enhance the integration process, as was indeed confirmed by direct determination of viral DNA integration by real-time PCR. CONCLUSIONS/SIGNIFICANCE: The results of the present study raise the possibility that in HIV-infected cells, the Rev protein may be involved in the integration of proviral DNA by controlling/regulating the activity of the integrase. Release from such inhibition leads to stimulation of IN activity and multiple viral DNA integration events.

  19. Cloning, Expression, and In Vitro Functional Activity Assay of phiC31 Integrase cDNA in Escherichia coli

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    Mohammad Hadi Sekhavati

    2013-01-01

    Full Text Available Objective: The aim of present study was cloning and expression of phiC31 integrase cDNA in a bacterial expression vector. Thus, an intra molecular assay vector was applied to show in vitro activity of recombinant protein.Materials and Methods: In this experimental study, phiC31 cDNA was subcloned into a prokaryotic expression vector and transformed into E.coli Bl21 (DE3. Recombinant phiC31 integrase was purified form the bacterial cell lysates and its activity was verified by an in vitro functional assessment.Results: Sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE of the purifi ed phiC31 integrase confirmed the size of protein (70 kDa. Finally, the functionality of purified phiC31 integrase was verified.Conclusion: The results of this study indicated that the purified integrase has a great potential application for in vitro site-specific integration.

  20. Raltegravir: first in class HIV integrase inhibitor

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    Zelalem Temesgen

    2008-06-01

    Full Text Available Zelalem Temesgen1, Dawd S Siraj21Mayo Clinic, Rochester, MN, USA; 2East Carolina University Greenville, NC, USAAbstract: On October 16, 2007, the US Food and Drug Administration (FDA approved raltegravir for treatment of human immunodeficiency virus (HIV-1 infection in combination with other antiretroviral agents in treatment-experienced adult patients who have evidence of viral replication and HIV-1 strains resistant to multiple antiretroviral agents. Raltegravir is first in a novel class of antiretroviral drugs known as integrase inhibitors. It has demonstrated potent anti HIV activity in both antiretroviral treatment-naïve and experienced patients. The most common adverse events reported with raltegravir during phase 2 and 3 clinical trials were diarrhea, nausea, and headache. Laboratory abnormalities include mild elevations in liver transaminases and creatine phosphokinase.Keywords: raltegravir, HIV, antiretroviral agents, integrase inhibitors

  1. HIV-1 integrase inhibitors that compete with the target DNA substrate define a unique strand transfer conformation for integrase

    OpenAIRE

    Espeseth, Amy S.; Felock, Peter; Wolfe, Abigail; Witmer, Marc; Grobler, Jay; Anthony, Neville; Egbertson, Melissa; Melamed, Jeffrey Y.; Young, Steve; Hamill, Terence; Cole, James L.; Hazuda, Daria J.

    2000-01-01

    Diketo acids such as L-731,988 are potent inhibitors of HIV-1 integrase that inhibit integration and viral replication in cells. These compounds exhibit the unique ability to inhibit the strand transfer activity of integrase in the absence of an effect on 3′ end processing. To understand the reasons for this distinct inhibitory profile, we developed a scintillation proximity assay that permits analysis of radiolabeled inhibitor binding and integrase function. High-...

  2. Cloning, Expression, and in vitro Functional Activity Assay of phiC31 Integrase cDNA in Escherichia coli

    OpenAIRE

    Mohammad Hadi Sekhavati; Mojtaba Tahmoorespur; Kamran Ghaedi; Kianoush Dormiani, Pharm; Mohammad Reza Nassiri; Yahya Khazaie; Mahboubeh Foruzanfar; Morteza Hosseini; Mohammad Hossein Nasr Esfahani

    2013-01-01

    Objective: The aim of present study was cloning and expression of phiC31 integrase cDNA in a bacterial expression vector. Thus, an intra molecular assay vector was applied to show in vitro activity of recombinant protein. Materials and Methods: In this experimental study, phiC31 cDNA was subcloned into a prokaryotic expression vector and transformed into E.coli Bl21 (DE3). Recombinant phiC31 integrase was purified form the bacterial cell lysates and its activity was verified by an in vitro fu...

  3. Functional characteristics of a highly specific integrase encoded by an LTR-retrotransposon

    OpenAIRE

    Babacar Faye; Frederick Arnaud; Eric Peyretaillade; Emilie Brasset; Bernard Dastugue; Chantal Vaury

    2008-01-01

    International audience BACKGROUND: The retroviral Integrase protein catalyzes the insertion of linear viral DNA into host cell DNA. Although different retroviruses have been shown to target distinctive chromosomal regions, few of them display a site-specific integration. ZAM, a retroelement from Drosophila melanogaster very similar in structure and replication cycle to mammalian retroviruses is highly site-specific. Indeed, ZAM copies target the genomic 5'-CGCGCg-3' consensus-sequences. To...

  4. Primary resistance to integrase strand-transfer inhibitors in Europe

    DEFF Research Database (Denmark)

    Casadellà, M; van Ham, P M; Noguera-Julian, M;

    2015-01-01

    OBJECTIVES: The objective of this study was to define the natural genotypic variation of the HIV-1 integrase gene across Europe for epidemiological surveillance of integrase strand-transfer inhibitor (InSTI) resistance. METHODS: This was a multicentre, cross-sectional study within the European...

  5. Tight regulation of the intS gene of the KplE1 prophage: a new paradigm for integrase gene regulation.

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    Gaël Panis

    2010-10-01

    Full Text Available Temperate phages have the ability to maintain their genome in their host, a process called lysogeny. For most, passive replication of the phage genome relies on integration into the host's chromosome and becoming a prophage. Prophages remain silent in the absence of stress and replicate passively within their host genome. However, when stressful conditions occur, a prophage excises itself and resumes the viral cycle. Integration and excision of phage genomes are mediated by regulated site-specific recombination catalyzed by tyrosine and serine recombinases. In the KplE1 prophage, site-specific recombination is mediated by the IntS integrase and the TorI recombination directionality factor (RDF. We previously described a sub-family of temperate phages that is characterized by an unusual organization of the recombination module. Consequently, the attL recombination region overlaps with the integrase promoter, and the integrase and RDF genes do not share a common activated promoter upon lytic induction as in the lambda prophage. In this study, we show that the intS gene is tightly regulated by its own product as well as by the TorI RDF protein. In silico analysis revealed that overlap of the attL region with the integrase promoter is widely encountered in prophages present in prokaryotic genomes, suggesting a general occurrence of negatively autoregulated integrase genes. The prediction that these integrase genes are negatively autoregulated was biologically assessed by studying the regulation of several integrase genes from two different Escherichia coli strains. Our results suggest that the majority of tRNA-associated integrase genes in prokaryotic genomes could be autoregulated and that this might be correlated with the recombination efficiency as in KplE1. The consequences of this unprecedented regulation for excessive recombination are discussed.

  6. The hunt for HIV-1 integrase inhibitors.

    Science.gov (United States)

    Lataillade, Max; Kozal, Michael J

    2006-07-01

    Currently, there are three distinct mechanistic classes of antiretrovirals: inhibitors of the HIV- 1 reverse transcriptase and protease enzymes and inhibitors of HIV entry, including receptor and coreceptor binding and cell fusion. A new drug class that inhibits the HIV-1 integrase enzyme (IN) is in development and may soon be available in the clinic. IN is an attractive drug target because it is essential for a stable and productive HIV-1 infection and there is no mammalian homologue of IN. Inhibitors of integrase enzyme (INI) block the integration of viral double-stranded DNA into the host cell's chromosomal DNA. HIV-1 integration has many potential steps that can be inhibited and several new compounds that target specific integration steps have been identified by drug developers. Recently, two INIs, GS-9137 and MK-0518, demonstrated promising early clinical trial results and have been advanced into later stage trials. In this review, we describe how IN facilitates HIV-1 integration, the needed enzyme cofactors, and the resultant byproducts created during integration. Furthermore, we review the different INIs under development, their mechanism of actions, site of IN inhibition, potency, resistance patterns, and discuss the early clinical trial results. PMID:16839248

  7. The hunt for HIV-1 integrase inhibitors.

    Science.gov (United States)

    Lataillade, Max; Kozal, Michael J

    2006-07-01

    Currently, there are three distinct mechanistic classes of antiretrovirals: inhibitors of the HIV- 1 reverse transcriptase and protease enzymes and inhibitors of HIV entry, including receptor and coreceptor binding and cell fusion. A new drug class that inhibits the HIV-1 integrase enzyme (IN) is in development and may soon be available in the clinic. IN is an attractive drug target because it is essential for a stable and productive HIV-1 infection and there is no mammalian homologue of IN. Inhibitors of integrase enzyme (INI) block the integration of viral double-stranded DNA into the host cell's chromosomal DNA. HIV-1 integration has many potential steps that can be inhibited and several new compounds that target specific integration steps have been identified by drug developers. Recently, two INIs, GS-9137 and MK-0518, demonstrated promising early clinical trial results and have been advanced into later stage trials. In this review, we describe how IN facilitates HIV-1 integration, the needed enzyme cofactors, and the resultant byproducts created during integration. Furthermore, we review the different INIs under development, their mechanism of actions, site of IN inhibition, potency, resistance patterns, and discuss the early clinical trial results.

  8. phiC31 integrase-mediated site-specific recombination in barley.

    Directory of Open Access Journals (Sweden)

    Eszter Kapusi

    Full Text Available The Streptomyces phage phiC31 integrase was tested for its feasibility in excising transgenes from the barley genome through site-specific recombination. We produced transgenic barley plants expressing an active phiC31 integrase and crossed them with transgenic barley plants carrying a target locus for recombination. The target sequence involves a reporter gene encoding green fluorescent protein (GFP, which is flanked by the attB and attP recognition sites for the phiC31 integrase. This sequence disruptively separates a gusA coding sequence from an upstream rice actin promoter. We succeeded in producing site-specific recombination events in the hybrid progeny of 11 independent barley plants carrying the above target sequence after crossing with plants carrying a phiC31 expression cassette. Some of the hybrids displayed fully executed recombination. Excision of the GFP gene fostered activation of the gusA gene, as visualized in tissue of hybrid plants by histochemical staining. The recombinant loci were detected in progeny of selfed F(1, even in individuals lacking the phiC31 transgene, which provides evidence of stability and generative transmission of the recombination events. In several plants that displayed incomplete recombination, extrachromosomal excision circles were identified. Besides the technical advance achieved in this study, the generated phiC31 integrase-expressing barley plants provide foundational stock material for use in future approaches to barley genetic improvement, such as the production of marker-free transgenic plants or switching transgene activity.

  9. Detection of integron integrase genes on King George Island, Antarctica

    Institute of Scientific and Technical Information of China (English)

    Vernica Antelo; Hctor Romero; Silvia Batista

    2015-01-01

    The presence and diversity of class 1 integrase gene (intI) sequences were evaluated by PCR using previously designed primers. Two clone libraries were constructed from DNA in sediment and microbial mat samples collected on Fildes Peninsula, King George Island, Antarctica.The libraries constructed from samples collected at Halfthree Point (HP) and Norma Cove (NC) contained 62 and 36 partial intI sequences, respectively. These sequences clustered into 10 different groups with <95% amino acid identity. Alignment of the deduced amino acid sequences with those from recognized integron-encoded integrases demonstrated the presence of highly conserved motifs characteristic of intI integrases. The HP library contained 42 nucleotide sequences identical to the class 1 intI gene found in a collection of trimethoprim-resistant (Tmpr) Antarctic Enterobacter sp. isolates, previously collected in the same area. These integrons, located on plasmids, had a genetic organization similar to that of pKOX105 from Klebsiella oxytoca. The 20 remaining HP and NC library sequences were similar to integrase sequences previously determined in a metagenomic analysis of environmental samples. We have demonstrated the presence of integron integrase genes in Antarctic sediment samples. About half these genes were very similar to the class 1 integrons found in human-associated microbiota, suggesting that they originated from human-dominated ecosystems. The remaining integrase genes were probably associated with endemic bacteria.

  10. Crystal structure of the HIV-1 integrase core domain in complex with sucrose reveals details of an allosteric inhibitory binding site

    Energy Technology Data Exchange (ETDEWEB)

    Wielens, Jerome; Headey, Stephen J.; Jeevarajah, Dharshini; Rhodes, David I.; Deadman, John; Chalmers, David K.; Scanlon, Martin J.; Parker, Michael W. (SVIMR-A); (Avea); (Monash IPS)

    2010-04-19

    HIV integrase (IN) is an essential enzyme in HIV replication and an important target for drug design. IN has been shown to interact with a number of cellular and viral proteins during the integration process. Disruption of these important interactions could provide a mechanism for allosteric inhibition of IN. We present the highest resolution crystal structure of the IN core domain to date. We also present a crystal structure of the IN core domain in complex with sucrose which is bound at the dimer interface in a region that has previously been reported to bind integrase inhibitors.

  11. Monoclonal antibodies against human immunodeficiency virus type 1 integrase: epitope mapping and differential effects on integrase activities in vitro.

    Science.gov (United States)

    Nilsen, B M; Haugan, I R; Berg, K; Olsen, L; Brown, P O; Helland, D E

    1996-01-01

    Human immunodeficiency virus type 1 (HIV-1) integrase (IN) catalyzes the integration of viral DNA into the host chromosome, an essential step in retroviral replication. As a tool to study the structure and function of this enzyme, monoclonal antibodies (MAbs) against HIV-1 IN were produced. Epitope mapping demonstrated that the 17 MAbs obtained could be divided into seven different groups, and the selection of MAbs representing these groups were tested for their effect on in vitro activities of IN. Four groups of MAbs recognized epitopes within the region of amino acids (aa) 1 to 16, 17 to 38, or 42 to 55 in and around the conserved HHCC motif near the N terminus of IN. MAbs binding to these epitopes inhibited end processing and DNA joining and either stimulated or had little effect on disintegration and reintegration activities of IN. Two MAbs binding to epitopes within the region of aa 56 to 102 in the central core or aa 186 to 250 in the C-terminal half of the protein showed only minor effects on the in vitro activities of IN. Three Mabs which recognized on epitope within the region of aa262 to 271 of HIV-1 IN cross-reacted with HIV-2 IN. MAbs binding to this epitope clearly inhibited end processing and DNA joining and stimulated or had little effect on disintegration. In contrast to the N-terminal-specific MAbs, these C-terminal-specific MAbs abolished reintegration activity of IN. PMID:8627677

  12. Allosteric Inhibition of Human Immunodeficiency Virus Integrase

    Science.gov (United States)

    Gupta, Kushol; Brady, Troy; Dyer, Benjamin M.; Malani, Nirav; Hwang, Young; Male, Frances; Nolte, Robert T.; Wang, Liping; Velthuisen, Emile; Jeffrey, Jerry; Van Duyne, Gregory D.; Bushman, Frederic D.

    2014-01-01

    HIV-1 replication in the presence of antiviral agents results in evolution of drug-resistant variants, motivating the search for additional drug classes. Here we report studies of GSK1264, which was identified as a compound that disrupts the interaction between HIV-1 integrase (IN) and the cellular factor lens epithelium-derived growth factor (LEDGF)/p75. GSK1264 displayed potent antiviral activity and was found to bind at the site occupied by LEDGF/p75 on IN by x-ray crystallography. Assays of HIV replication in the presence of GSK1264 showed only modest inhibition of the early infection steps and little effect on integration targeting, which is guided by the LEDGF/p75·IN interaction. In contrast, inhibition of late replication steps was more potent. Particle production was normal, but particles showed reduced infectivity. GSK1264 promoted aggregation of IN and preformed LEDGF/p75·IN complexes, suggesting a mechanism of inhibition. LEDGF/p75 was not displaced from IN during aggregation, indicating trapping of LEDGF/p75 in aggregates. Aggregation assays with truncated IN variants revealed that a construct with catalytic and C-terminal domains of IN only formed an open polymer associated with efficient drug-induced aggregation. These data suggest that the allosteric inhibitors of IN are promising antiviral agents and provide new information on their mechanism of action. PMID:24904063

  13. Anodic stripping voltammetry – ASV for determination of heavy metals

    International Nuclear Information System (INIS)

    Although voltammetric methods presented a number of difficulties in its early stages, nowadays ''ASV'' anodic stripping voltammetry is considered one of the most sensitive electro-analytical and suitable for trace-level determination of many metals and compounds in environmental samples, clinical and industrial. Its sensitivity is attributed to the combination of a step of pre-concentration effective together with an electrochemical advanced measurement of accumulated analyte. This paper presents an overview of the voltammetry, which includes a group of electro-analytical methods, in them the information about analyte is obtained from measurements of the current flowing in an electrochemical cell when applied a potential difference to an suitable electrode system

  14. Intraarterial calcium stimulation (ASVS) for pancreatic insulinoma: comparison of preoperative localization procedures

    International Nuclear Information System (INIS)

    Evaluation of clinical relevance of the arterial stimulation procedure with venous sampling (ASVS) in the preoperative localization of insulinoma. Thirteen patients with endogenous hyperinsulinism underwent preoperative transabdominal ultrasound (US), helical CT (CT), MRI, endoscopic ultrasound (EUS), and angiography (DSA) in conjunction with the ASVS-test for the detection of insulinoma. The results were compared with intraoperative findings, intraoperative ultrasound (IOUS) and histology. Sensitivity was as follows: US 8%, MRI 27%, CT 46%, EUS 50%,DSA 69%,and ASVS 92%.Intraoperative palpation and IOUS yielded a sensitivity of 77%. In 3 patients the tumors were neither palpable nor detectable by IOUS, the mode of resection was based on preoperative diagnostics.The ASVS procedure as a functional test was superior to all other modalities for the preoperative tumor detection. The ASVS was the most sensitive diagnostic modality. It should especially be considered in terms of health economical aspects when CT or MRI do not yield conclusive results. (orig.)

  15. Rapid Optimization of Engineered Metabolic Pathways with Serine Integrase Recombinational Assembly (SIRA).

    Science.gov (United States)

    Merrick, C A; Wardrope, C; Paget, J E; Colloms, S D; Rosser, S J

    2016-01-01

    Metabolic pathway engineering in microbial hosts for heterologous biosynthesis of commodity compounds and fine chemicals offers a cheaper, greener, and more reliable method of production than does chemical synthesis. However, engineering metabolic pathways within a microbe is a complicated process: levels of gene expression, protein stability, enzyme activity, and metabolic flux must be balanced for high productivity without compromising host cell viability. A major rate-limiting step in engineering microbes for optimum biosynthesis of a target compound is DNA assembly, as current methods can be cumbersome and costly. Serine integrase recombinational assembly (SIRA) is a rapid DNA assembly method that utilizes serine integrases, and is particularly applicable to rapid optimization of engineered metabolic pathways. Using six pairs of orthogonal attP and attB sites with different central dinucleotide sequences that follow SIRA design principles, we have demonstrated that ΦC31 integrase can be used to (1) insert a single piece of DNA into a substrate plasmid; (2) assemble three, four, and five DNA parts encoding the enzymes for functional metabolic pathways in a one-pot reaction; (3) generate combinatorial libraries of metabolic pathway constructs with varied ribosome binding site strengths or gene orders in a one-pot reaction; and (4) replace and add DNA parts within a construct through targeted postassembly modification. We explain the mechanism of SIRA and the principles behind designing a SIRA reaction. We also provide protocols for making SIRA reaction components and practical methods for applying SIRA to rapid optimization of metabolic pathways. PMID:27417934

  16. Human immunodeficiency virus type 1 integrase: effect on viral replication of mutations at highly conserved residues.

    Science.gov (United States)

    Cannon, P M; Wilson, W; Byles, E; Kingsman, S M; Kingsman, A J

    1994-08-01

    Sequence comparisons of the integrase (IN) proteins from different retroviruses have identified several highly conserved residues. We have introduced mutations at 16 of these sites into the integrase gene of human immunodeficiency virus type 1 and analyzed the phenotypes of the resulting viruses. The viruses were all normal for p24 content and reverse transcriptase activity. In addition, all of the mutants could infect T-cell lines and undergo reverse transcription, as assessed by PCR analysis. Most of the mutant viruses also had normal Western blot (immunoblot) profiles, although three of the mutations resulted in reduced signals for IN relative to the wild type on the immunoblots and mutation of residue W235 completely abolished recognition of the protein by pooled sera from human immunodeficiency virus type 1-positive patients. Mutations that have previously been shown to abolish activity in in vitro studies produced noninfectious viruses. The substitution of W235 was notable in producing a noninfectious virus, despite previous reports of this residue being nonessential for IN activity in vitro (A.D. Leavitt, L. Shiue, and H.E. Varmus, J. Biol. Chem. 268:2113-2119, 1993). In addition, we have identified four highly conserved residues that can be mutated without any affect on viral replication in T-cell lines. PMID:8035478

  17. The role of lysine 186 in HIV-1 integrase multimerization

    International Nuclear Information System (INIS)

    HIV-1 integrase (IN) catalyzes biochemical reactions required for viral cDNA insertion into host cell chromosomal DNA, an essential step in the HIV-1 replication cycle. In one of these reactions, the two ends of the linear viral cDNA are believed to be simultaneously ligated to chromosomal DNA by a tetrameric form of IN. The structure of the full-length IN tetramer is not known but a model consisting of the N-terminal domain and the catalytic core revealed basic residues 186 to 188 at the interface between the two IN dimers. We found that alteration of these residues, in particular changing IN lysine residue 186 to glutamate (K186Q), impairs IN oligomerization in the yeast two-hybrid system and decreases oligomeric forms of IN within virions. When expressed independently of other viral proteins in human cells, IN-K186Q did not concentrate in the nucleus as did wild-type IN. Co-expression of wild-type IN restored the multimerization defects of IN-K186Q, in both the two-hybrid system and in virions, and also rescued the nuclear targeting defects. Virions bearing IN-K186Q were not infectious in a single cycle of replication but when mixed virions containing two different IN mutants were produced, IN-K186Q was capable of complementing the catalytically inactive mutant IN-D116A. Our biochemical and functional data support the crystallographic model in which IN residue K186 lies at the interface between IN dimers and suggest that tetramerization is important, not only for concerted integration, but also for IN nuclear targeting

  18. Lead Screening for HIV-1 Integrase (IN Inhibited by Traditional Chinese Medicine

    Directory of Open Access Journals (Sweden)

    Tzu-Chieh Hung

    2014-01-01

    Full Text Available Human immunodeficiency virus causes the acquired immunodeficiency syndrome (AIDS and becomes a serious world-wide problem because of this disease's rapid propagation and incurability. Integrase strand transfer inhibitors (INSTIs supports HIV have rapid drug resistance for antitreatment. Screening the traditional Chinese medicine (TCM database by simulating molecular docking and molecular dynamics may select molecular compounds to inhibit INSTIs against HIV drug resistance. (S-cathinone and (1S,2S-norpseudoephedrine are selected based on structure and ligand-based drugs are designed and then get higher bioactivity predicted score from SVM than Raltegravir and other TCM compounds. The molecular dynamics are helpful in the analysis and detection of protein-ligand interactions. According to the docking poses, hydrophobic interactions and hydrogen bond variations define the main regions of important amino acids in integrase. In addition to the detection of TCM compound efficacy, we suggest (1S,2S-norpseudoephedrine is better than the others based on the analysis of interaction and the effect on the structural variation.

  19. Health hazards by lead exposure: evaluation using ASV and XRF.

    Science.gov (United States)

    Herman, D'Souza Sunil; Geraldine, Menezes; Scott, Clark C; Venkatesh, Thuppil

    2006-07-01

    Globally, of many toxic heavy metals, lead is the most widely used for various purposes, resulting in a variety of health hazards due to environmental contamination. Lead in the workplace enters the workers through inhalation of lead-contaminated air, by ingestion, and sometimes through dermal exposure. Furthermore, exposure outside the workplace can occur from inhalation of lead-contaminated air, ingestion of lead-contaminated dust and soil, consumption of lead polluted water, lead adulterated food and lead supplemented medicine. In the present study, an evaluation of blood lead was carried out with the aid of a 3010 B lead analyser, based on the principle of anodic stripping voltametry (ASV), and environmental lead in paint, soil and dust samples by a field portable X-ray fluorescence (XRF) analyser. This revealed a high incidence of lead toxicity in most of the lead-based industrial workers in the four facilities tested in India and high levels of lead in the environmental samples. Developed countries have complied with the global standards for regulating environmental lead poisoning in the workplace, eliminating to some degree excessive exposure to lead. A developing country, such as India, can tackle this problem by implementing national and international policies. The US Occupational Safety and Health Administration (OSHA) and Environmental Protection Agency (EPA) regulations, which are of prime importance, or similar regulations, can be adapted for use in India and implemented to minimize lead exposure and to reduce the resultant health hazards.

  20. Dolutegravir (S/GSK1349572) exhibits significantly slower dissociation than raltegravir and elvitegravir from wild-type and integrase inhibitor-resistant HIV-1 integrase-DNA complexes.

    Science.gov (United States)

    Hightower, Kendra E; Wang, Ruolan; Deanda, Felix; Johns, Brian A; Weaver, Kurt; Shen, Yingnian; Tomberlin, Ginger H; Carter, H Luke; Broderick, Timothy; Sigethy, Scott; Seki, Takahiro; Kobayashi, Masanori; Underwood, Mark R

    2011-10-01

    The integrase inhibitor (INI) dolutegravir (DTG; S/GSK1349572) has significant activity against HIV-1 isolates with raltegravir (RAL)- and elvitegravir (ELV)-associated resistance mutations. As an initial step in characterizing the different resistance profiles of DTG, RAL, and ELV, we determined the dissociation rates of these INIs with integrase (IN)-DNA complexes containing a broad panel of IN proteins, including IN substitutions corresponding to signature RAL and ELV resistance mutations. DTG dissociates slowly from a wild-type IN-DNA complex at 37°C with an off-rate of 2.7 × 10(-6) s(-1) and a dissociative half-life (t(1/2)) of 71 h, significantly longer than the half-lives for RAL (8.8 h) and ELV (2.7 h). Prolonged binding (t(1/2), at least 5 h) was observed for DTG with IN-DNA complexes containing E92, Y143, Q148, and N155 substitutions. The addition of a second substitution to either Q148 or N155 typically resulted in an increase in the off-rate compared to that with the single substitution. For all of the IN substitutions tested, the off-rate of DTG from IN-DNA complexes was significantly slower (from 5 to 40 times slower) than the off-rate of RAL or ELV. These data are consistent with the potential for DTG to have a higher genetic barrier to resistance, provide evidence that the INI off-rate may be an important component of the mechanism of INI resistance, and suggest that the slow dissociation of DTG may contribute to its distinctive resistance profile. PMID:21807982

  1. Dolutegravir (S/GSK1349572) exhibits significantly slower dissociation than raltegravir and elvitegravir from wild-type and integrase inhibitor-resistant HIV-1 integrase-DNA complexes.

    Science.gov (United States)

    Hightower, Kendra E; Wang, Ruolan; Deanda, Felix; Johns, Brian A; Weaver, Kurt; Shen, Yingnian; Tomberlin, Ginger H; Carter, H Luke; Broderick, Timothy; Sigethy, Scott; Seki, Takahiro; Kobayashi, Masanori; Underwood, Mark R

    2011-10-01

    The integrase inhibitor (INI) dolutegravir (DTG; S/GSK1349572) has significant activity against HIV-1 isolates with raltegravir (RAL)- and elvitegravir (ELV)-associated resistance mutations. As an initial step in characterizing the different resistance profiles of DTG, RAL, and ELV, we determined the dissociation rates of these INIs with integrase (IN)-DNA complexes containing a broad panel of IN proteins, including IN substitutions corresponding to signature RAL and ELV resistance mutations. DTG dissociates slowly from a wild-type IN-DNA complex at 37°C with an off-rate of 2.7 × 10(-6) s(-1) and a dissociative half-life (t(1/2)) of 71 h, significantly longer than the half-lives for RAL (8.8 h) and ELV (2.7 h). Prolonged binding (t(1/2), at least 5 h) was observed for DTG with IN-DNA complexes containing E92, Y143, Q148, and N155 substitutions. The addition of a second substitution to either Q148 or N155 typically resulted in an increase in the off-rate compared to that with the single substitution. For all of the IN substitutions tested, the off-rate of DTG from IN-DNA complexes was significantly slower (from 5 to 40 times slower) than the off-rate of RAL or ELV. These data are consistent with the potential for DTG to have a higher genetic barrier to resistance, provide evidence that the INI off-rate may be an important component of the mechanism of INI resistance, and suggest that the slow dissociation of DTG may contribute to its distinctive resistance profile.

  2. Lack of integrase inhibitors associated resistance mutations among HIV-1C isolates

    OpenAIRE

    Mulu, Andargachew; Maier, Melanie; Liebert, Uwe Gerd

    2015-01-01

    Background Although biochemical analysis of HIV-1 integrase enzyme suggested the use of integrase inhibitors (INIs) against HIV-1C, different viral subtypes may favor different mutational pathways potentially leading to varying levels of drug resistance. Thus, the aim of this study was to search for the occurrence and natural evolution of integrase polymorphisms and/or resistance mutations in HIV-1C Ethiopian clinical isolates prior to the introduction of INIs. Methods Plasma samples from chr...

  3. The mechanism of φC31 integrase directionality: experimental analysis and computational modelling

    OpenAIRE

    Pokhilko, Alexandra; Zhao, Jia; Ebenhöh, Oliver; Smith, Margaret C M; Stark, W. Marshall; Colloms, Sean D.

    2016-01-01

    Serine integrases, DNA site-specific recombinases used by bacteriophages for integration and excision of their DNA to and from their host genomes, are increasingly being used as tools for programmed rearrangements of DNA molecules for biotechnology and synthetic biology. A useful feature of serine integrases is the simple regulation and unidirectionality of their reactions. Recombination between the phage attP and host attB sites is promoted by the serine integrase alone, giving recombinant a...

  4. Cell type differences in activity of the Streptomyces bacteriophage phiC31 integrase.

    Science.gov (United States)

    Maucksch, Christof; Aneja, Manish Kumar; Hennen, Elisabeth; Bohla, Alexander; Hoffmann, Florian; Elfinger, Markus; Rosenecker, Joseph; Rudolph, Carsten

    2008-10-01

    Genomic integration by the Streptomyces bacteriophage C31 integrase is a promising tool for non-viral gene therapy of various genetic disorders. We investigated the C31 integrase recombination activity in T cell derived cell lines, primary T lymphocytes and CD34(+) haematopoietic stem cells in comparison to mesenchymal stem cells and cell lines derived from lung-, liver- and cervix-tissue. In T cell lines, enhanced long-term expression above control was observed only with high amounts of integrase mRNA. Transfections of C31 integrase plasmids were not capable of mediating enhanced long-term transgene expression in T cell lines. In contrast, moderate to high efficiency could be detected in human mesenchymal stem cells, human lung, liver and cervix carcinoma cell lines. Up to 100-fold higher levels of recombination product was found in C31 integrase transfected A549 lung than Jurkat T cells. When the C31 integrase activity was normalized to the intracellular integrase mRNA levels, a 16-fold difference was found. As one possible inhibitor of the C31 integrase, we found 3- to 5-fold higher DAXX levels in Jurkat than in A549 cells, which could in addition to other yet unknown factors explain the observed discrepancy of C31 integrase activity.

  5. Next-generation site-directed transgenesis in the malaria vector mosquito Anopheles gambiae: self-docking strains expressing germline-specific phiC31 integrase.

    Directory of Open Access Journals (Sweden)

    Janet M Meredith

    Full Text Available Diseases transmitted by mosquitoes have a devastating impact on global health and the situation is complicated due to difficulties with both existing control measures and the impact of climate change. Genetically modified mosquitoes that are refractory to disease transmission are seen as having great potential in the delivery of novel control strategies. The Streptomyces phage phiC31 integrase system has been successfully adapted for site-directed transgene integration in a range of insects, thus overcoming many limitations due to size constraints and random integration associated with transposon-mediated transformation. Using this technology, we previously published the first site-directed transformation of Anopheles gambiae, the principal vector of human malaria. Mosquitoes were initially engineered to incorporate the phiC31 docking site at a defined genomic location. A second phase of genetic modification then achieved site-directed integration of an anti-malarial effector gene. In the current publication we report improved efficiency and utility of the phiC31 integrase system following the generation of Anopheles gambiae self-docking strains. Four independent strains, with docking sites at known locations on three different chromosome arms, were engineered to express integrase under control of the regulatory regions of the nanos gene from Anopheles gambiae. The resulting protein accumulates in the posterior oocyte to provide integrase activity at the site of germline development. Two self-docking strains, exhibiting significantly different levels of integrase expression, were assessed for site-directed transgene integration and found to demonstrate greatly improved survival and efficiency of transformation. In the fight against malaria, it is imperative to establish a broad repertoire of both anti-malarial effector genes and tissue-specific promoters to regulate their expression, enabling those offering maximum effect with minimum fitness

  6. HIV-1 Integrase Inhibitor Resistance and Its Clinical Implications

    OpenAIRE

    Blanco, Jose-Luis; Varghese, Vici; Rhee, Soo-Yon; Gatell, Jose M.; Shafer, Robert W.

    2011-01-01

    With the approval in 2007 of the first integrase inhibitor (INI), raltegravir, clinicians became better able to suppress virus replication in patients infected with human immunodeficiency virus type 1 (HIV-1) who were harboring many of the most highly drug-resistant viruses. Raltegravir also provided clinicians with additional options for first-line therapy and for the simplification of regimens in patients with stable virological suppression. Two additional INIs in advanced clinical developm...

  7. Development of a high-throughput assay for the HIV-1 integrase disintegration reaction

    Institute of Scientific and Technical Information of China (English)

    2010-01-01

    Both HIV-1 integrase (IN) and the central catalytic domain of IN (IN-CCD) catalyze the disintegration reaction in vitro.In this study,IN and IN-CCD proteins were expressed and purified,and a high-throughput format enzyme-linked immunosorbent assay (ELISA) was developed for the disintegration reaction.IN exhibited a marked preference for Mn2+ over Mg2+ as the divalent cation cofactor in disintegration.Baicalein,a known IN inhibitor,was found to be an IN-CCD inhibitor.The assay is sensitive and specific for the study of disintegration reaction as well as for the in vitro identification of antiviral drugs targeting IN,especially targeting IN-CCD.

  8. Dialysis purification of integrase-DNA complexes provides high-resolution atomic force microscopy images: dimeric recombinant HIV-1 integrase binding and specific looping on DNA.

    Directory of Open Access Journals (Sweden)

    Tatsuaki Tsuruyama

    Full Text Available It remains difficult to obtain high-resolution atomic force microscopy images of HIV-1 integrase bound to DNA in a dimeric or tetrameric fashion. We therefore constructed specific target DNAs to assess HIV-1 integrase binding and purified the complex by dialysis prior to analysis. Our resulting atomic force microscopy analyses indicated precise size of binding human immunodeficiency virus type 1 (HIV-1 recombinant integrase in a tetrameric manner, inducing formation of a loop-like or figure-eight-like secondary structure in the target DNA. Our findings regarding the target DNA secondary structure provide new insights into the intermediate states of retroviral integration.

  9. Dialysis purification of integrase-DNA complexes provides high-resolution atomic force microscopy images: dimeric recombinant HIV-1 integrase binding and specific looping on DNA.

    Science.gov (United States)

    Tsuruyama, Tatsuaki; Nakai, Tonau; Ohmori, Rei; Ozeki, Munetaka; Tamaki, Keiji; Yoshikawa, Kenichi

    2013-01-01

    It remains difficult to obtain high-resolution atomic force microscopy images of HIV-1 integrase bound to DNA in a dimeric or tetrameric fashion. We therefore constructed specific target DNAs to assess HIV-1 integrase binding and purified the complex by dialysis prior to analysis. Our resulting atomic force microscopy analyses indicated precise size of binding human immunodeficiency virus type 1 (HIV-1) recombinant integrase in a tetrameric manner, inducing formation of a loop-like or figure-eight-like secondary structure in the target DNA. Our findings regarding the target DNA secondary structure provide new insights into the intermediate states of retroviral integration.

  10. Arsenic response of AtPCS1- and CePCS-expressing plants - effects of external As(V) concentration on As-accumulation pattern and NPT metabolism.

    Science.gov (United States)

    Wojas, Sylwia; Clemens, Stephan; Skłodowska, Aleksandra; Maria Antosiewicz, Danuta

    2010-02-15

    Phytochelatins (PCs) are small, cysteine-rich peptides, known to play a major role in detoxification of both cadmium and arsenic. The aim of this study was to determine whether overexpression of either of two PC synthase (PCS) genes, AtPCS1 and CePCS in Nicotiana tabacum (previously shown to cause decrease and increase, respectively, of cadmium tolerance of tobacco - Wojas et al., 2008) also contributes to such contrasting phenotypes with respect to arsenic (As) tolerance and accumulation, and how observed responses relate to non-protein thiol (NPT) metabolism. The expression of both genes resulted in an increase of As-tolerance, with CePCS plants most tolerant. We showed for the first time that the response of PCS overexpressing plants to As qualitatively depends on the external As(V) concentration. At the less toxic 50muM As(V), AtPCS1 and CePCS transformants accumulated more As in roots and leaves than WT. An increase in PC production and the level of PC2 species was detected in leaves of AtPCS1 and CePCS plants, which might explain their enhanced As-accumulation and tolerance. In contrast, at the highly toxic 200muM As(V), several disturbances in thiol metabolism of PCS overexpressing plants were found, surprisingly, including decrease of PC levels both in roots and leaves of transgenic plants relative to WT. The increase in As-tolerance and accumulation due to AtPCS1 and CePCS overexpression, observed at the As(V) concentrations similar to those found in As-contaminated soils, makes these genes promising candidates for plant engineering for phytoremediation.

  11. Effect of fulvic acid on adsorptive removal of Cr(VI) and As(V) from groundwater by iron oxide-based adsorbents

    KAUST Repository

    Uwamariya, V.

    2015-05-15

    Abstract Natural contamination has become a challenging problem in drinking water production due to metal contamination of groundwater throughout the world, and arsenic and chromium are well-known toxic elements. In this study, iron oxide-coated sand (IOCS) and granular ferric hydroxide (GFH) were used to study the effects of fulvic acid (FA) on the adsorptive removal of Cr(VI) and As(V) from synthetic groundwater. IOCS and GFH were characterized by SEM/EDS, and experiments were performed at different pH levels (6, 7, and 8). The surface of IOCS and GFH showed a high content of Fe and O (75 and 60 % of the atomic composition, respectively), suggesting that they can highly effectively adsorb Cr(VI) and As(V). Adsorption tests with the simultaneous presence of As(V) and FA, on the one hand, and Cr(VI) with FA, on the other hand, revealed that the role of FA on chromate and arsenate adsorption was insignificant at almost all pH values investigated with both adsorbents. A small influence as a result of FA was only observed for the removal of As(V) by IOCS at pH 6 with a decrease of 13 and 23 % when 2 and 5 mg/l were added to the synthetic water, respectively. It was also found that organic matter (OM) was leached from the IOCS during batch adsorption experiments. The use of FEEM revealed that humic-like, fulvic-like, and protein-like organic matter fractions are present on the IOCS surface. © 2015 Springer International Publishing Switzerland.

  12. HIV-1 Protease, Reverse Transcriptase, and Integrase Variation

    Science.gov (United States)

    Sankaran, Kris; Varghese, Vici; Winters, Mark A.; Hurt, Christopher B.; Eron, Joseph J.; Parkin, Neil; Holmes, Susan P.; Holodniy, Mark; Shafer, Robert W.

    2016-01-01

    ABSTRACT HIV-1 protease (PR), reverse transcriptase (RT), and integrase (IN) variability presents a challenge to laboratories performing genotypic resistance testing. This challenge will grow with increased sequencing of samples enriched for proviral DNA such as dried blood spots and increased use of next-generation sequencing (NGS) to detect low-abundance HIV-1 variants. We analyzed PR and RT sequences from >100,000 individuals and IN sequences from >10,000 individuals to characterize variation at each amino acid position, identify mutations indicating APOBEC-mediated G-to-A editing, and identify mutations resulting from selective drug pressure. Forty-seven percent of PR, 37% of RT, and 34% of IN positions had one or more amino acid variants with a prevalence of ≥1%. Seventy percent of PR, 60% of RT, and 60% of IN positions had one or more variants with a prevalence of ≥0.1%. Overall 201 PR, 636 RT, and 346 IN variants had a prevalence of ≥0.1%. The median intersubtype prevalence ratios were 2.9-, 2.1-, and 1.9-fold for these PR, RT, and IN variants, respectively. Only 5.0% of PR, 3.7% of RT, and 2.0% of IN variants had a median intersubtype prevalence ratio of ≥10-fold. Variants at lower prevalences were more likely to differ biochemically and to be part of an electrophoretic mixture compared to high-prevalence variants. There were 209 mutations indicative of APOBEC-mediated G-to-A editing and 326 mutations nonpolymorphic treatment selected. Identification of viruses with a high number of APOBEC-associated mutations will facilitate the quality control of dried blood spot sequencing. Identifying sequences with a high proportion of rare mutations will facilitate the quality control of NGS. IMPORTANCE Most antiretroviral drugs target three HIV-1 proteins: PR, RT, and IN. These proteins are highly variable: many different amino acids can be present at the same position in viruses from different individuals. Some of the amino acid variants cause drug

  13. As(V) adsorption onto nanoporous titania adsorbents (NTAs): effects of solution composition.

    Science.gov (United States)

    Han, Dong Suk; Batchelor, Bill; Park, Sung Hyuk; Abdel-Wahab, Ahmed

    2012-08-30

    This study has focused on developing two nanoporous titania adsorbents (NTA) to enhance removal efficiency of adsorption process for As(V) by characterizing the effects of pH and phosphate concentration on their sorption capacities and behaviors. One type of adsorbent is a mesoporous titania (MT) solid phase and the other is group of a highly ordered mesoporous silica solids (SBA-15) that can incorporate different levels of reactive titania sorption sites. Microscopic analysis showed that Ti((25))-SBA-15 (Ti/SBA=0.25 g/g) had titania nanostructured mesopores that do not rupture the highly ordered hexagonal silica framework. However, MT has disordered, wormhole-like mesopores that are caused by interparticle porosity. Adsorption experiments showed that Ti((25))-SBA-15 had a greater sorption capacity for As(V) than did Ti((15))-SBA-15 or Ti((35))-SBA-15 and the amount of As(V) adsorbed generally decreased as pH increased. Higher removal of As(V) was observed with Ti((25))-SBA-15 than with MT at pH 4, but MT had higher removals at higher pH (7, 9.5), even though MT has a lower specific surface area. However, in the presence of phosphate, MT showed higher removal of As(V) at low pH rather than did Ti((25))-SBA-15. As expected, the NTAs showed very fast sorption kinetics, but they followed a bi-phasic sorption pattern. PMID:22727482

  14. The discovery of S/GSK1265744: a carbamoyl pyridone HIV-1 integrase inhibitor

    Directory of Open Access Journals (Sweden)

    H Murai

    2012-11-01

    Full Text Available Background: HIV-1 integrase is a virally encoded enzyme essential for lentiviral replication. Assiduous medicinal chemistry efforts culminated in the discovery of raltegravir, the first marketed HIV-1 integrase inhibitor (INI. However, there is significant opportunity for improvement including overall dose burden, dosing interval and potency against resistant viruses. Our molecular design approach used a two-metal binding pharmacophore strategy and succeeded in identification of carbamoyl pyridone HIV-1 INIs. This enriched core scaffold has abundant structural features expanding the opportunity to control drug properties, leading to the discovery of S/GSK1265744. Methods: The carbamoyl pyridone scaffold was derivatized and evaluated for antiviral activity against wild-type virus (±HSA along with key INI-resistant mutants. Animal pharmacokinetic profiles including a key measure of the trough drug concentration over protein-adjusted antiviral potency (C24/PAIC50 along with in vitro DMPK properties, were used along with the virological data for compound selection. Results: The carbamoyl pyridone series inhibitors exhibited potent antiviral profiles with promising DMPK properties. S/GSK1265744 demonstrated good coverage of C24 over PAIC50 predicting low mg unboosted once daily dosing, now validated in phase 2 clinical studies. These preclinical data along with a long human T1/2 of ~30 hours in oral tablet study supports S/GSK1265744 as a long acting parenteral agent for once-monthly or less frequent dosing. Conclusions: A medicinal chemistry approach utilizing key viral mutants in combination with C24/PAIC50 has allowed for discovery of S/GSK1265744. This agent is currently in phase 2 development evaluating a novel, long-acting parenteral route of administration and may enable new approaches to HIV therapy and prevention.

  15. Application of Liquid Emulsion Membrane Technique for the Removal of As(V) from Aqueous Solutions

    Science.gov (United States)

    Binnal, Prakash; Hiremath, Poornima G.

    2012-08-01

    Liquid emulsion membrane technique was used to remove As(V) from synthetic aqueous solutions. The emulsion was composed of Aliquat 336 as an extractant, commercial kerosene as a diluent and Span 80 (Sorbiton monooleate) as an emulsifying agent. Different types of internal phases were used, namely, sodium hydroxide, sodium carbonate, ammonium bicarbonate, sodium sulphate and sodium chloride. The effect of process parameters affecting extraction efficiency, such as, initial concentration of As(V) in feed solution, pH of feed solution, concentrations of Aliquat 336 and Span 80 in membrane phase, volume ratio of stripping phase to membrane phase, concentration of internal phase, type of internal phase, volume ratio of emulsion to feed, agitation speed during extraction and time of extraction was investigated. The optimum conditions for the extraction were determined. A maximum As(V) removal rate of 97.8 was observed under optimum conditions.

  16. As(V) and P Competitive Sorption on Soils, By-Products and Waste Materials

    Science.gov (United States)

    Rivas-Pérez, Ivana María; Paradelo-Núñez, Remigio; Nóvoa-Muñoz, Juan Carlos; Arias-Estévez, Manuel; Fernández-Sanjurjo, María José; Álvarez-Rodríguez, Esperanza; Núñez-Delgado, Avelino

    2015-01-01

    Batch-type experiments were used to study competitive As(V) and P sorption on various soils and sorbent materials. The materials assayed were a forest soil, a vineyard soil, pyritic material, granitic material, coarsely and finely ground mussel shell, calcinated mussel shell ash, pine sawdust and slate processing fines. Competition between As(V) and P was pronounced in the case of both soils, granitic material, slate fines, both shells and pine sawdust, showing more affinity for P. Contrary, the pyritic material and mussel shell ash showed high and similar affinity for As(V) and P. These results could be useful to make a correct use of the soils and materials assayed when focusing on As and P removal in solid or liquid media, in circumstances where both pollutants may compete for sorption sites. PMID:26690456

  17. As(V and P Competitive Sorption on Soils, By-Products and Waste Materials

    Directory of Open Access Journals (Sweden)

    Ivana María Rivas-Pérez

    2015-12-01

    Full Text Available Batch-type experiments were used to study competitive As(V and P sorption on various soils and sorbent materials. The materials assayed were a forest soil, a vineyard soil, pyritic material, granitic material, coarsely and finely ground mussel shell, calcinated mussel shell ash, pine sawdust and slate processing fines. Competition between As(V and P was pronounced in the case of both soils, granitic material, slate fines, both shells and pine sawdust, showing more affinity for P. Contrary, the pyritic material and mussel shell ash showed high and similar affinity for As(V and P. These results could be useful to make a correct use of the soils and materials assayed when focusing on As and P removal in solid or liquid media, in circumstances where both pollutants may compete for sorption sites.

  18. Structural and Functional Insights into Foamy Viral Integrase

    Directory of Open Access Journals (Sweden)

    Cha-Gyun Shin

    2013-07-01

    Full Text Available Successful integration of retroviral DNA into the host chromosome is an essential step for viral replication. The process is mediated by virally encoded integrase (IN and orchestrated by 3'-end processing and the strand transfer reaction. In vitro reaction conditions, such as substrate specificity, cofactor usage, and cellular binding partners for such reactions by the three distinct domains of prototype foamy viral integrase (PFV-IN have been described well in several reports. Recent studies on the three‑dimensional structure of the interacting complexes between PFV-IN and DNA, cofactors, binding partners, or inhibitors have explored the mechanistic details of such interactions and shown its utilization as an important target to develop anti-retroviral drugs. The presence of a potent, non-transferable nuclear localization signal in the PFV C-terminal domain extends its use as a model for investigating cellular trafficking of large molecular complexes through the nuclear pore complex and also to identify novel cellular targets for such trafficking. This review focuses on recent advancements in the structural analysis and in vitro functional aspects of PFV-IN.

  19. The successive polarographic determination of As(III) and As(V)

    NARCIS (Netherlands)

    Cosijn, A.H.M.; Molen, H.J. van deer

    1958-01-01

    A method has been described for the successive polarographic determination of As(III) and As(V)in a sulphuric acid solution. A directly recorded polarogram shows a limiting current corresponding to the As(III) concentration, another polarogram, recorded with a second sample after reduction of As(V)

  20. Transgenic Xenopus laevis embryos can be generated using ϕC31 integrase

    OpenAIRE

    Allen, Bryan G.; Weeks, Daniel L.

    2005-01-01

    Phage ϕC31 encodes an integrase that can mediate the insertion of extrachromosomal DNA into genomic DNA1. Here we show ϕC31 integrase can be used to generate transgenic Xenopus laevis embryos. mRNA encoding integrase was co-injected with a reporter plasmid containing a CMV promoter driven GFP into one cell embryos. The reporter plasmid was integrated into the genome. GFP expression, though robust, was in a limited number of tissues and varied among the embryos analyzed. We attributed this res...

  1. HIV Integrase Inhibitors with Nucleobase Scaffolds: Discovery of a Highly Potent anti-HIV Agent

    OpenAIRE

    Nair, Vasu; Chi, Guochen; Ptak, Roger; Neamati, Nouri

    2006-01-01

    HIV integrase is essential for HIV replication. However, there are currently no integrase inhibitors in clinical use for AIDS. We have discovered a conceptually new β-diketo acid that is a powerful inhibitor of both the 3′-processing and strand transfer steps of HIV-1 integrase. The in vitro anti-HIV data of this inhibitor were remarkable as exemplified by its highly potent antiviral therapeutic efficacy against HIVTEKI and HIV-1NL4-3 replication in PBMC (TI >4,000 and >10,000, respectively).

  2. Structural Basis for the Recognition Between HIV-1 Integrase and Transcriptional Coactivator p75

    Energy Technology Data Exchange (ETDEWEB)

    Cherepanov,P.; Ambrosio, A.; Rahman, S.; Ellenberger, T.; Engelman, A.

    2005-01-01

    Integrase (IN) is an essential retroviral enzyme, and human transcriptional coactivator p75, which is also referred to as lens epithelium-derived growth factor (LEDGF), is the dominant cellular binding partner of HIV-1 IN. Here, we report the crystal structure of the dimeric catalytic core domain of HIV-1 IN complexed to the IN-binding domain of LEDGF. Previously identified LEDGF hotspot residues anchor the protein to both monomers at the IN dimer interface. The principal structural features of IN that are recognized by the host factor are the backbone conformation of residues 168-171 from one monomer and a hydrophobic patch that is primarily comprised of {alpha}-helices 1 and 3 of the second IN monomer. Inspection of diverse retroviral primary and secondary sequence elements helps to explain the apparent lentiviral tropism of the LEDGF-IN interaction. Because the lethal phenotypes of HIV-1 mutant viruses unable to interact with LEDGF indicate that IN function is highly sensitive to perturbations of the structure around the LEDGF-binding site, we propose that small molecule inhibitors of the protein-protein interaction might similarly disrupt HIV-1 replication.

  3. HIV-1 group O integrase displays lower susceptibility to raltegravir and has a different mutational pathway for resistance than HIV-1 group M

    Directory of Open Access Journals (Sweden)

    Agnès Depatureaux

    2014-11-01

    Full Text Available Introduction: HIV-1 group O (HIV-O is a rare HIV-1 variant characterized by a high number of polymorphisms, especially in the integrase gene, e.g. positions L74I, S153A, G163Q and T206S. As HIV-O integrase enzymes have not previously been studied, our aim was to assess the impact of HIV-O integrase polymorphisms on susceptibility to integrase inhibitors and emergence of resistance associated mutations. Viruses and Methods: We cloned and purified integrase proteins from each of HIV-1 Group O clades A (HIV-O/A and B (HIV-O/B, a HIV-O divergent strain (HIV-O/Div, and HIV-1 group M (subtype B, HIV-M/B and characterized these enzymes for susceptibility to integrase strand transfer inhibitors (INSTIs in cell-free assays and in tissue culture, in the absence or presence of varying concentrations of several INSTIs. The inhibition constant (Ki and IC50 were calculated and compared for HIV-M and HIV-O integrases. Selections for resistance-related mutations were performed using cord blood mononuclear cells and increasing concentration of INSTIs. Results: HIV-O integrase and viruses were more susceptible to raltegravir (RAL in competitive inhibition assays and in tissue culture than were HIV-M enzymes and viruses, respectively. During selection, we observed different pathways of resistance depending on the drug and clade. Mutations selected in HIV-O can be classified as follows: (1 mutations described for HIV-M such as T97A, Q148R, V151A/I (RAL, T66I, E92Q, E157Q (EVG and M50I, R263K (DTG and (2 signature mutations for HIV-O (i.e. not described in HIV-M F121C (HIV-O/B for RAL, V75I (HIV-O/A for RAL and S153V (HIV-O/A for DTG. Only the HIV-O/Div selected the Q148R mutation for RAL and R263K+M50I for DTG, as previously described for HIV-M. None of the HIV-O viruses selected either N155H or Y143C. The selection of the specific S153V mutation could be explained at the nucleotide level: HIV-O at this position contains an alanine and substitution of alanine to

  4. Removal of arsenic from water using manganese (III) oxide: Adsorption of As(III) and As(V).

    Science.gov (United States)

    Babaeivelni, Kamel; Khodadoust, Amid P

    2016-01-01

    Removal of arsenic from water was evaluated with manganese (III) oxide (Mn2O3) as adsorbent. Adsorption of As(III) and As(V) onto Mn2O3 was favorable according to the Langmuir and Freundlich adsorption equilibrium equations, while chemisorption of arsenic occurred according to the Dubinin-Radushkevich equation. Adsorption parameters from the Langmuir, Freundlich, and Temkin equations showed a greater adsorption and removal of As(III) than As(V) by Mn2O3. Maximum removal of As(III) and As(V) occurred at pH 3-9 and at pH 2, respectively, while removal of As(V) in the pH range of 6-9 was 93% (pH 6) to 61% (pH 9) of the maximum removal. Zeta potential measurements for Mn2O3 in As(III) was likely converted to As(V) solutions indicated that As(III) was likely converted to As(V) on the Mn2O3 surface at pH 3-9. Overall, the effective Mn2O3 sorbent rapidly removed As(III) and As(V) from water in the pH range of 6-9 for natural waters.

  5. Chemistry and Structure-Activity Relationship of the Styrylquinoline-Type HIV Integrase Inhibitors

    OpenAIRE

    Didier Desmaële; Jean-François Mouscadet

    2010-01-01

    In spite of significant progress in anti-HIV-1 therapy, current antiviral chemo-therapy still suffers from deleterious side effects and emerging drug resistance. Therefore, the development of novel antiviral drugs remains a crucial issue for the fight against AIDS. HIV-1 integrase is a key enzyme in the replication cycle of the retrovirus since it catalyzes the integration of the reverse transcribed viral DNA into the chromosomal DNA. Efforts to develop anti-integrase drugs started during the...

  6. Effects of Etravirine on the Pharmacokinetics of the Integrase Inhibitor S/GSK1265744

    OpenAIRE

    Ford, Susan L.; Gould, Elizabeth; Chen, Shuguang; Lou, Yu; Dumont, Etienne; Spreen, William; Piscitelli, Stephen

    2013-01-01

    HIV integrase inhibitors such as raltegravir and elvitegravir halt HIV progression, but treatment-emergent resistance and cross-resistance have been observed. The nonnucleoside reverse transcriptase inhibitor etravirine (ETR) may be used in combination with integrase inhibitors in patients with drug resistance. This single-center, open-label, two-period, single-sequence crossover study evaluated the effects of ETR coadministration on the pharmacokinetic profile of S/GSK1265744, an investigati...

  7. Serine integrase chimeras with activity in E. coli and HeLa cells

    OpenAIRE

    Farruggio, Alfonso P.; Calos, Michele P.

    2014-01-01

    In recent years, application of serine integrases for genomic engineering has increased in popularity. The factor-independence and unidirectionality of these large serine recombinases makes them well suited for reactions such as site-directed vector integration and cassette exchange in a wide variety of organisms. In order to generate information that might be useful for altering the specificity of serine integrases and to improve their efficiency, we tested a hybridization strategy that has ...

  8. Serine integrase chimeras with activity in E. coli and HeLa cells

    OpenAIRE

    Farruggio, Alfonso P.; Calos, Michele P.

    2014-01-01

    ABSTRACT In recent years, application of serine integrases for genomic engineering has increased in popularity. The factor-independence and unidirectionality of these large serine recombinases makes them well suited for reactions such as site-directed vector integration and cassette exchange in a wide variety of organisms. In order to generate information that might be useful for altering the specificity of serine integrases and to improve their efficiency, we tested a hybridization strategy ...

  9. HIV-1 integrase inhibitor resistance and its clinical implications.

    Science.gov (United States)

    Blanco, Jose-Luis; Varghese, Vici; Rhee, Soo-Yon; Gatell, Jose M; Shafer, Robert W

    2011-05-01

    With the approval in 2007 of the first integrase inhibitor (INI), raltegravir, clinicians became better able to suppress virus replication in patients infected with human immunodeficiency virus type 1 (HIV-1) who were harboring many of the most highly drug-resistant viruses. Raltegravir also provided clinicians with additional options for first-line therapy and for the simplification of regimens in patients with stable virological suppression. Two additional INIs in advanced clinical development-elvitegravir and S/GSK1349572-may prove equally versatile. However, the INIs have a relatively low genetic barrier to resistance in that 1 or 2 mutations are capable of causing marked reductions in susceptibility to raltegravir and elvitegravir, the most well-studied INIs. This perspective reviews the genetic mechanisms of INI resistance and their implications for initial INI therapy, the treatment of antiretroviral-experienced patients, and regimen simplification. PMID:21459813

  10. QSAR study of curcumine derivatives as HIV-1 integrase inhibitors.

    Science.gov (United States)

    Gupta, Pawan; Sharma, Anju; Garg, Prabha; Roy, Nilanjan

    2013-03-01

    A QSAR study was performed on curcumine derivatives as HIV-1 integrase inhibitors using multiple linear regression. The statistically significant model was developed with squared correlation coefficients (r(2)) 0.891 and cross validated r(2) (r(2) cv) 0.825. The developed model revealed that electronic, shape, size, geometry, substitution's information and hydrophilicity were important atomic properties for determining the inhibitory activity of these molecules. The model was also tested successfully for external validation (r(2) pred = 0.849) as well as Tropsha's test for model predictability. Furthermore, the domain analysis was carried out to evaluate the prediction reliability of external set molecules. The model was statistically robust and had good predictive power which can be successfully utilized for screening of new molecules. PMID:23286784

  11. HIV-1 integrase inhibitor resistance and its clinical implications.

    Science.gov (United States)

    Blanco, Jose-Luis; Varghese, Vici; Rhee, Soo-Yon; Gatell, Jose M; Shafer, Robert W

    2011-05-01

    With the approval in 2007 of the first integrase inhibitor (INI), raltegravir, clinicians became better able to suppress virus replication in patients infected with human immunodeficiency virus type 1 (HIV-1) who were harboring many of the most highly drug-resistant viruses. Raltegravir also provided clinicians with additional options for first-line therapy and for the simplification of regimens in patients with stable virological suppression. Two additional INIs in advanced clinical development-elvitegravir and S/GSK1349572-may prove equally versatile. However, the INIs have a relatively low genetic barrier to resistance in that 1 or 2 mutations are capable of causing marked reductions in susceptibility to raltegravir and elvitegravir, the most well-studied INIs. This perspective reviews the genetic mechanisms of INI resistance and their implications for initial INI therapy, the treatment of antiretroviral-experienced patients, and regimen simplification.

  12. Anti - HIV-1 integrase activity of Thai Medicinal Plants

    Directory of Open Access Journals (Sweden)

    Kingkan Bunluepuech

    2009-08-01

    Full Text Available For the purpose of discovering anti-HIV-1 agents from natural sources, the aqueous and EtOH extracts of eight Thaiplants including Clerodendron indicum (whole plant, Tiliacora triandra (stem, Capparis micracantha (wood, Harrissoniaperforata (wood, Ficus glomerata (wood, Diospyros decandra (wood, Dracaena loureiri (heartwood, and Tinospora crispa (stem were screened for their inhibitory activities against HIV-1 integrase (IN using the multiplate integration assay(MIA. Of the EtOH extracts, Ficus glomerata (wood was the most potent with an IC50 value of 7.8 g/ml; whereas the water extract of Harrisonia perforata (wood was the most potent aqueous extract with an IC50 value of 2.3 g/ml. The isolation of active principles against HIV-1 IN from Ficus glomerata is now actively pursued.

  13. Development of a receptor model for efficient in silico screening of HIV-1 integrase inhibitors.

    Science.gov (United States)

    Quevedo, Mario A; Ribone, Sergio R; Briñón, Margarita C; Dehaen, Wim

    2014-07-01

    Integrase (IN) is a key viral enzyme for the replication of the type-1 human immunodeficiency virus (HIV-1), and as such constitutes a relevant therapeutic target for the development of anti-HIV agents. However, the lack of crystallographic data of HIV IN complexed with the corresponding viral DNA has historically hindered the application of modern structure-based drug design techniques to the discovery of new potent IN inhibitors (INIs). Consequently, the development and validation of reliable HIV IN structural models that may be useful for the screening of large databases of chemical compounds is of particular interest. In this study, four HIV-1 IN homology models were evaluated respect to their capability to predict the inhibition potency of a training set comprising 36 previously reported INIs with IC50 values in the low nanomolar to the high micromolar range. Also, 9 inactive structurally related compounds were included in this training set. In addition, a crystallographic structure of the IN-DNA complex corresponding to the prototype foamy virus (PFV) was also evaluated as structural model for the screening of inhibitors. The applicability of high throughput screening techniques, such as blind and ligand-guided exhaustive rigid docking was assessed. The receptor models were also refined by molecular dynamics and clustering techniques to assess protein sidechain flexibility and solvent effect on inhibitor binding. Among the studied models, we conclude that the one derived from the X-ray structure of the PFV integrase exhibited the best performance to rank the potencies of the compounds in the training set, with the predictive power being further improved by explicitly modeling five water molecules within the catalytic side of IN. Also, accounting for protein sidechain flexibility enhanced the prediction of inhibition potencies among the studied compounds. Finally, an interaction fingerprint pattern was established for the fast identification of potent IN

  14. Development of a receptor model for efficient in silico screening of HIV-1 integrase inhibitors.

    Science.gov (United States)

    Quevedo, Mario A; Ribone, Sergio R; Briñón, Margarita C; Dehaen, Wim

    2014-07-01

    Integrase (IN) is a key viral enzyme for the replication of the type-1 human immunodeficiency virus (HIV-1), and as such constitutes a relevant therapeutic target for the development of anti-HIV agents. However, the lack of crystallographic data of HIV IN complexed with the corresponding viral DNA has historically hindered the application of modern structure-based drug design techniques to the discovery of new potent IN inhibitors (INIs). Consequently, the development and validation of reliable HIV IN structural models that may be useful for the screening of large databases of chemical compounds is of particular interest. In this study, four HIV-1 IN homology models were evaluated respect to their capability to predict the inhibition potency of a training set comprising 36 previously reported INIs with IC50 values in the low nanomolar to the high micromolar range. Also, 9 inactive structurally related compounds were included in this training set. In addition, a crystallographic structure of the IN-DNA complex corresponding to the prototype foamy virus (PFV) was also evaluated as structural model for the screening of inhibitors. The applicability of high throughput screening techniques, such as blind and ligand-guided exhaustive rigid docking was assessed. The receptor models were also refined by molecular dynamics and clustering techniques to assess protein sidechain flexibility and solvent effect on inhibitor binding. Among the studied models, we conclude that the one derived from the X-ray structure of the PFV integrase exhibited the best performance to rank the potencies of the compounds in the training set, with the predictive power being further improved by explicitly modeling five water molecules within the catalytic side of IN. Also, accounting for protein sidechain flexibility enhanced the prediction of inhibition potencies among the studied compounds. Finally, an interaction fingerprint pattern was established for the fast identification of potent IN

  15. Mechanism of action of the HIV-1 integrase inhibitory peptide LEDGF 361-370.

    Science.gov (United States)

    Hayouka, Zvi; Levin, Aviad; Maes, Michal; Hadas, Eran; Shalev, Deborah E; Volsky, David J; Loyter, Abraham; Friedler, Assaf

    2010-04-01

    The HIV-1 integrase protein (IN) mediates integration of the viral cDNA into the host genome and is a target for anti-HIV drugs. We have recently described a peptide derived from residues 361-370 of the IN cellular partner protein LEDGF/p75, which inhibited IN catalytic activity in vitro and HIV-1 replication in cells. Here we performed a comprehensive study of the LEDGF 361-370 mechanism of action in vitro, in cells and in vivo. Alanine scan, fluorescence anisotropy binding studies, homology modeling and NMR studies demonstrated that all residues in LEDGF 361-370 contribute to IN binding and inhibition. Kinetic studies in cells showed that LEDGF 361-370 specifically inhibited integration of viral cDNA. Thus, the full peptide was chosen for in vivo studies, in which it inhibited the production of HIV-1 RNA in mouse model. We conclude that the full LEDGF 361-370 peptide is a potent HIV-1 inhibitor and may be used for further development as an anti-HIV lead compound.

  16. Small molecule inhibitors of the LEDGF site of human immunodeficiency virus integrase identified by fragment screening and structure based design.

    Directory of Open Access Journals (Sweden)

    Thomas S Peat

    Full Text Available A fragment-based screen against human immunodeficiency virus type 1 (HIV integrase led to a number of compounds that bound to the lens epithelium derived growth factor (LEDGF binding site of the integrase catalytic core domain. We determined the crystallographic structures of complexes of the HIV integrase catalytic core domain for 10 of these compounds and quantitated the binding by surface plasmon resonance. We demonstrate that the compounds inhibit the interaction of LEDGF with HIV integrase in a proximity AlphaScreen assay, an assay for the LEDGF enhancement of HIV integrase strand transfer and in a cell based assay. The compounds identified represent a potential framework for the development of a new series of HIV integrase inhibitors that do not bind to the catalytic site of the enzyme.

  17. [Mechanism of groundwater As(V) removal with ferric flocculation and direct filtration].

    Science.gov (United States)

    Kang, Ying; Duan, Jin-Ming; Jing, Chuan-Yong

    2015-02-01

    The As removal process and mechanism from groundwater using ferric flocculation-direct filtration system was investigated using batch, field pilot tests, extended X-ray absorption fine structure ( EXAFS) spectroscopy, and charge-distribution multisite complexation (CD-MUSIC) model. The results showed that arsenate [As(V)] was the dominant As species in the groundwater with a concentration of 40 μg x L(-1). The treatment system could supply 64 984 L As-safe drinking water (concentration (5 mg x L(-1)). EXAFS and CD-MUSIC model indicated that As(V) was adsorbed onto ferric hydroxide via bidentate binuclear complexes in the pH range of 3 to 9.5, while formation of precipitate with Ca or Mg dominated the As removal at pH > 9.5. PMID:26031078

  18. Electromembrane extraction and spectrophotometric determination of As(V) in water samples.

    Science.gov (United States)

    Kamyabi, Mohammad Ali; Aghaei, Ali

    2016-12-01

    In this study, for the first time electromembrane extraction (EME) was used as a highly efficient sample pre-treatment method for the UV-VIS spectrophotometric determination of As(V) in water samples. The influences of experimental parameters during EME were investigated and optimized using one-variable-at-a-time methodology as follows: organic solvent: 1-octanol+2.5% (V/V) di-(2-ethylhexyl) phosphate, applied voltage: 70V, extraction time: 15min, pH of acceptor: 13, stirring rate: 750rpm. The method allowed the determination of As(V) in the range of 5-300ngmL(-1). The relative standard deviation was found to be within the range of 3.4-7.6%. The limit of detection, corresponding to a signal to noise ratio of three, was 1.5ngmL(-1). The proposed method was finally applied to the determination of As(V) in water samples and relative recoveries ranging from 95 to 102% were obtained.

  19. Electromembrane extraction and spectrophotometric determination of As(V) in water samples.

    Science.gov (United States)

    Kamyabi, Mohammad Ali; Aghaei, Ali

    2016-12-01

    In this study, for the first time electromembrane extraction (EME) was used as a highly efficient sample pre-treatment method for the UV-VIS spectrophotometric determination of As(V) in water samples. The influences of experimental parameters during EME were investigated and optimized using one-variable-at-a-time methodology as follows: organic solvent: 1-octanol+2.5% (V/V) di-(2-ethylhexyl) phosphate, applied voltage: 70V, extraction time: 15min, pH of acceptor: 13, stirring rate: 750rpm. The method allowed the determination of As(V) in the range of 5-300ngmL(-1). The relative standard deviation was found to be within the range of 3.4-7.6%. The limit of detection, corresponding to a signal to noise ratio of three, was 1.5ngmL(-1). The proposed method was finally applied to the determination of As(V) in water samples and relative recoveries ranging from 95 to 102% were obtained. PMID:27374507

  20. Design of inhibitors of the HIV-1 integrase core domain using virtual screening.

    Science.gov (United States)

    Regon, Preetom; Gogoi, Dhrubajyoti; Rai, Ashok Kumar; Bordoloi, Manabjyoti; Bezbaruah, Rajib Lochan

    2014-01-01

    Acquired immunodeficiency syndrome (AIDS) is a disease of the human immune system caused by the human immunodeficiency virus (HIV). The integrase (IN) enzyme of HIV interacts with several cellular and viral proteins during the integration process. Thus, it represents an appropriate target for antiretroviral drugs (ARVs). We performed virtual screening of database compounds and designed analogues using Elvitegravir (EVG) as a standard compound. The 378 screened compounds were retrieved from ZINC, ChemSpider, PubChem, and ChemBank Chemical Databases based on chemical similarity and literature searches related to the structure of EVG. The Physiochemical properties, Bioactivity, Toxicity and Absorption, Distribution, Metabolism and Excretion of Molecules (ADME) of these compounds were predicted and docking Experiments were conducted using Molegro Virtual Docker software. The docking and ADME suggested very significant results in regard to EVG. The MolDock and Rerank scores were used to analyze the results. The compounds ZINC26507991 (-84.22), Analogue 9 (-68.49), ZINC20731658 (-66.79), ZINC00210363 (-43.44) showed better binding orientation with IN receptor model with respect to EVG (182.52). The ZINC26507991 has showed significant ADME result.

  1. Design of inhibitors of the HIV-1 integrase core domain using virtual screening.

    Science.gov (United States)

    Regon, Preetom; Gogoi, Dhrubajyoti; Rai, Ashok Kumar; Bordoloi, Manabjyoti; Bezbaruah, Rajib Lochan

    2014-01-01

    Acquired immunodeficiency syndrome (AIDS) is a disease of the human immune system caused by the human immunodeficiency virus (HIV). The integrase (IN) enzyme of HIV interacts with several cellular and viral proteins during the integration process. Thus, it represents an appropriate target for antiretroviral drugs (ARVs). We performed virtual screening of database compounds and designed analogues using Elvitegravir (EVG) as a standard compound. The 378 screened compounds were retrieved from ZINC, ChemSpider, PubChem, and ChemBank Chemical Databases based on chemical similarity and literature searches related to the structure of EVG. The Physiochemical properties, Bioactivity, Toxicity and Absorption, Distribution, Metabolism and Excretion of Molecules (ADME) of these compounds were predicted and docking Experiments were conducted using Molegro Virtual Docker software. The docking and ADME suggested very significant results in regard to EVG. The MolDock and Rerank scores were used to analyze the results. The compounds ZINC26507991 (-84.22), Analogue 9 (-68.49), ZINC20731658 (-66.79), ZINC00210363 (-43.44) showed better binding orientation with IN receptor model with respect to EVG (182.52). The ZINC26507991 has showed significant ADME result. PMID:24616558

  2. Identification of small peptides inhibiting the integrase-LEDGF/p75 interaction through targeting the cellular co-factor.

    Science.gov (United States)

    Cavalluzzo, Claudia; Christ, Frauke; Voet, Arnout; Sharma, Ajendra; Singh, Brajendra Kumar; Zhang, Kam Y J; Lescrinier, Eveline; De Maeyer, Marc; Debyser, Zeger; Van der Eycken, Erik

    2013-10-01

    The integration of the viral DNA into the host genome is one of the essential steps in the HIV replication cycle. This process is mediated by the viral enzyme integrase (IN) and lens epithelium-derived growth factor (LEDGF/p75). LEDGF/p75 has been identified as a crucial cellular co-factor of integration that acts by tethering IN to the cellular chromatin. Recently, circular peptides were identified that bind to the C-terminal domain of IN and disrupt the interaction with LEDGF/p75. Starting from the circular peptides, we identified a short peptidic sequence able to inhibit the LEDGF/p75-IN interaction at low μM concentration through its binding to the IN binding site of LEDGF/p75. This discovery can lead to the synthesis of peptidomimetics with high anti-HIV activity targeting the cellular co-factor LEDGF/p75 and not the viral protein IN.

  3. Transient gene expression mediated by integrase-defective retroviral vectors.

    Science.gov (United States)

    Yu, Seung Shin; Dan, Kazuyuki; Chono, Hideto; Chatani, Emi; Mineno, Junichi; Kato, Ikunoshin

    2008-04-18

    Nonintegrating retroviral vectors were produced from a Moloney murine leukemia virus (MoMLV)-based retroviral vector system by introducing a point mutation into the integrase (IN) gene of the packaging plasmid. The efficacy of IN-defective retroviral vectors was measured through the transient expression of ZsGreen or luciferase in human cell lines. The IN-defective retroviral vectors could transduce target cells efficiently, but their gene expression was transient and lower than that seen with the integrating vectors. IN-defective retroviral vector gene expression decreased to background levels in fewer than 10 days. Southern blot analysis of transduced K562 cells confirmed the loss of a detectable vector sequence by 15 days. The residual integration activity of the IN-defective vector was 1000- to 10,000-fold lower than that of the integrating vector. These results demonstrate that the IN-defective retroviral vectors can provide a useful tool for efficient transient gene expression targeting of primary hematopoietic stem cells and lymphoid cells.

  4. A method for producing transgenic cells using a multi-integrase system on a human artificial chromosome vector.

    Directory of Open Access Journals (Sweden)

    Shigeyuki Yamaguchi

    Full Text Available The production of cells capable of expressing gene(s of interest is important for a variety of applications in biomedicine and biotechnology, including gene therapy and animal transgenesis. The ability to insert transgenes at a precise location in the genome, using site-specific recombinases such as Cre, FLP, and ΦC31, has major benefits for the efficiency of transgenesis. Recent work on integrases from ΦC31, R4, TP901-1 and Bxb1 phages demonstrated that these recombinases catalyze site-specific recombination in mammalian cells. In the present study, we examined the activities of integrases on site-specific recombination and gene expression in mammalian cells. We designed a human artificial chromosome (HAC vector containing five recombination sites (ΦC31 attP, R4 attP, TP901-1 attP, Bxb1 attP and FRT; multi-integrase HAC vector and de novo mammalian codon-optimized integrases. The multi-integrase HAC vector has several functions, including gene integration in a precise locus and avoiding genomic position effects; therefore, it was used as a platform to investigate integrase activities. Integrases carried out site-specific recombination at frequencies ranging from 39.3-96.8%. Additionally, we observed homogenous gene expression in 77.3-87.5% of colonies obtained using the multi-integrase HAC vector. This vector is also transferable to another cell line, and is capable of accepting genes of interest in this environment. These data suggest that integrases have high DNA recombination efficiencies in mammalian cells. The multi-integrase HAC vector enables us to produce transgene-expressing cells efficiently and create platform cell lines for gene expression.

  5. Pharmacovirological impact of an integrase inhibitor on human immunodeficiency virus type 1 cDNA species in vivo.

    Science.gov (United States)

    Goffinet, Christine; Allespach, Ina; Oberbremer, Lena; Golden, Pamela L; Foster, Scott A; Johns, Brian A; Weatherhead, Jason G; Novick, Steven J; Chiswell, Karen E; Garvey, Edward P; Keppler, Oliver T

    2009-08-01

    Clinical trials of the first approved integrase inhibitor (INI), raltegravir, have demonstrated a drop in the human immunodeficiency virus type 1 (HIV-1) RNA loads of infected patients that was unexpectedly more rapid than that with a potent reverse transcriptase inhibitor, and apparently dose independent. These clinical outcomes are not understood. In tissue culture, although their inhibition of integration is well documented, the effects of INIs on levels of unintegrated HIV-1 cDNAs have been variable. Furthermore, there has been no report to date on an INI's effect on these episomal species in vivo. Here, we show that prophylactic treatment of transgenic rats with the strand transfer INI GSK501015 reduced levels of viral integrants in the spleen by up to 99.7%. Episomal two-long-terminal-repeat (LTR) circles accumulated up to sevenfold in this secondary lymphoid organ, and this inversely correlated with the impact on the proviral burden. Contrasting raltegravir's dose-ranging study with HIV patients, titration of GSK501015 in HIV-infected animals demonstrated dependence of the INI's antiviral effect on its serum concentration. Furthermore, the in vivo 50% effective concentration calculated from these data best matched GSK501015's in vitro potency when serum protein binding was accounted for. Collectively, this study demonstrates a titratable, antipodal impact of an INI on integrated and episomal HIV-1 cDNAs in vivo. Based on these findings and known biological characteristics of viral episomes, we discuss how integrase inhibition may result in additional indirect antiviral effects that contribute to more rapid HIV-1 decay in HIV/AIDS patients. PMID:19458008

  6. Pharmacovirological impact of an integrase inhibitor on human immunodeficiency virus type 1 cDNA species in vivo.

    Science.gov (United States)

    Goffinet, Christine; Allespach, Ina; Oberbremer, Lena; Golden, Pamela L; Foster, Scott A; Johns, Brian A; Weatherhead, Jason G; Novick, Steven J; Chiswell, Karen E; Garvey, Edward P; Keppler, Oliver T

    2009-08-01

    Clinical trials of the first approved integrase inhibitor (INI), raltegravir, have demonstrated a drop in the human immunodeficiency virus type 1 (HIV-1) RNA loads of infected patients that was unexpectedly more rapid than that with a potent reverse transcriptase inhibitor, and apparently dose independent. These clinical outcomes are not understood. In tissue culture, although their inhibition of integration is well documented, the effects of INIs on levels of unintegrated HIV-1 cDNAs have been variable. Furthermore, there has been no report to date on an INI's effect on these episomal species in vivo. Here, we show that prophylactic treatment of transgenic rats with the strand transfer INI GSK501015 reduced levels of viral integrants in the spleen by up to 99.7%. Episomal two-long-terminal-repeat (LTR) circles accumulated up to sevenfold in this secondary lymphoid organ, and this inversely correlated with the impact on the proviral burden. Contrasting raltegravir's dose-ranging study with HIV patients, titration of GSK501015 in HIV-infected animals demonstrated dependence of the INI's antiviral effect on its serum concentration. Furthermore, the in vivo 50% effective concentration calculated from these data best matched GSK501015's in vitro potency when serum protein binding was accounted for. Collectively, this study demonstrates a titratable, antipodal impact of an INI on integrated and episomal HIV-1 cDNAs in vivo. Based on these findings and known biological characteristics of viral episomes, we discuss how integrase inhibition may result in additional indirect antiviral effects that contribute to more rapid HIV-1 decay in HIV/AIDS patients.

  7. Self-assessment test on concepts related to Anodic Stripping Voltammetry (ASV)

    OpenAIRE

    Milla González, Miguel; Espada Bellido, Estrella

    2014-01-01

    This exercise is intended to reinforce and assess knowledge on Anodic Stripping Voltammetry (ASV) as an electroanalytical technique. It is designed as a multiple entry test in which several options for a concept related to this analysis technique is proposed. The exercise can be resolved page to page (answers are ticked and the truth or falsity of the chosen option is obtained) or globally. In the latter, the user ticks the "correct" answers and obtains a grade at the end of the test. Going ...

  8. As(V), Cr(III) and Cr(VI) sorption on biochars and soil

    Science.gov (United States)

    Diamadopoulos, Evan; Agrafioti, Evita; Kalderis, Dimitrios

    2014-05-01

    The use of biochar, as a cost effective sorbent for heavy metal removal from contaminated water and soils is becoming a very promising practice. In this study, rice husk, the organic fraction of solid wastes, as well as sewage sludge were used as precursors for biochar production. The first was chosen as one of the most abundant types of biomass worldwide and the other two in order to find alternative innovative uses of these wastes. A series of batch kinetic and equilibrium (sorption and desorption) experiments was conducted using As(V), Cr(III) and Cr(VI) as adsorbates. The specific heavy metals were chosen in order to assess biochars removal capacity towards both anionic and cationic metals. Apart from biochars, a sandy loam soil was also used as adsorbent for metal removal. Knowing the separate behavior of biochars and soil towards metal sorption, it could be the first step in explaining the fate of heavy metals in a biochar amended soil. The kinetic study showed that, for all adsorbents and metals examined, sorption can be well described by the pseudo-second order kinetic model. What is more, simulation of sorption isotherms gave a better fit for the Freundlich model, possibly due to the heterogeneous surface of the initial biomasses and the fine aggregates that soil consists of. Based on the equilibrium study, the materials examined removed more than 95% of the initial Cr(III). This is possibly related to the electrostatic interactions between adsorbents negative surface charge and Cr(III) cations. However, removal rates for As(V) and Cr(VI) anions were significantly lower. Biochar derived from sewage sludge was efficient in removing 89% of Cr(VI) and 53% of As(V). Its ash high Fe2O3 content may have enhanced metal adsorption via precipitation. Soil was the most effective material for the removal of As(V), yet it could not strongly retain metal anions compared to biochars, as a significant amount of the adsorbed metal was released during desorption

  9. Quantitative prediction of integrase inhibitor resistance from genotype through consensus linear regression modeling

    Science.gov (United States)

    2013-01-01

    Background Integrase inhibitors (INI) form a new drug class in the treatment of HIV-1 patients. We developed a linear regression modeling approach to make a quantitative raltegravir (RAL) resistance phenotype prediction, as Fold Change in IC50 against a wild type virus, from mutations in the integrase genotype. Methods We developed a clonal genotype-phenotype database with 991 clones from 153 clinical isolates of INI naïve and RAL treated patients, and 28 site-directed mutants. We did the development of the RAL linear regression model in two stages, employing a genetic algorithm (GA) to select integrase mutations by consensus. First, we ran multiple GAs to generate first order linear regression models (GA models) that were stochastically optimized to reach a goal R2 accuracy, and consisted of a fixed-length subset of integrase mutations to estimate INI resistance. Secondly, we derived a consensus linear regression model in a forward stepwise regression procedure, considering integrase mutations or mutation pairs by descending prevalence in the GA models. Results The most frequently occurring mutations in the GA models were 92Q, 97A, 143R and 155H (all 100%), 143G (90%), 148H/R (89%), 148K (88%), 151I (81%), 121Y (75%), 143C (72%), and 74M (69%). The RAL second order model contained 30 single mutations and five mutation pairs (p INI naïve patients. Conclusions We describe a systematic approach to derive a model for predicting INI resistance from a limited amount of clonal samples. Our RAL second order model is made available as an Additional file for calculating a resistance phenotype as the sum of integrase mutations and mutation pairs. PMID:23282253

  10. Subtype diversity associated with the development of HIV-1 resistance to integrase inhibitors.

    Science.gov (United States)

    Brenner, Bluma G; Lowe, Matthew; Moisi, Daniela; Hardy, Isabelle; Gagnon, Simon; Charest, Hugues; Baril, Jean Guy; Wainberg, Mark A; Roger, Michel

    2011-05-01

    We used genotypic and phylogenetic analysis to determine integrase diversity among subtypes, and studied natural polymorphisms and mutations implicated in resistance to integrase inhibitors (INI) in treatment-naïve persons (n = 220) and -experienced individuals (n = 24). Phylogenetics revealed 7 and 10% inter-subtype diversity in the integrase and reverse transcriptase (RT)/protease regions, respectively. Integrase sequencing identified a novel A/B recombinant in which all viruses in a male-sex-male (MSM) transmission cluster (n = 12) appeared to possess subtype B in integrase and subtype A in the remainder of the pol region. Natural variations and signature polymorphisms were observed at codon positions 140, 148, 151, 157, and 160 among HIV subtypes. These variations predicted higher genetic barriers to G140S and G140C in subtypes C, CRF02_AG, and A/CRF01_AE, as well as higher genetic barriers toward acquisition of V151I in subtypes CRF02_AG and A/CRF01_AE. The E157Q and E160Q mutational motif was observed in 35% of INI-naïve patients harboring subtype C infections, indicating intra-subtype variations. Thirteen patients failed raltegravir (RAL)-containing regimens within 8 ± 1 months, in association with the major Q148K/R/H and G140A/S (n = 8/24) or N155H (n = 5/24) mutational pathways. Of note, the remaining patients on RAL regimens for 14 ± 3 months harbored no or only minor integrase mutations/polymorphisms (T66I, T97A, H114P, S119P, A124S, G163R, I203M, R263K). These results demonstrate the importance of understanding subtype variability in the development of resistance to INIs. PMID:21360548

  11. Subtype diversity associated with the development of HIV-1 resistance to integrase inhibitors.

    Science.gov (United States)

    Brenner, Bluma G; Lowe, Matthew; Moisi, Daniela; Hardy, Isabelle; Gagnon, Simon; Charest, Hugues; Baril, Jean Guy; Wainberg, Mark A; Roger, Michel

    2011-05-01

    We used genotypic and phylogenetic analysis to determine integrase diversity among subtypes, and studied natural polymorphisms and mutations implicated in resistance to integrase inhibitors (INI) in treatment-naïve persons (n = 220) and -experienced individuals (n = 24). Phylogenetics revealed 7 and 10% inter-subtype diversity in the integrase and reverse transcriptase (RT)/protease regions, respectively. Integrase sequencing identified a novel A/B recombinant in which all viruses in a male-sex-male (MSM) transmission cluster (n = 12) appeared to possess subtype B in integrase and subtype A in the remainder of the pol region. Natural variations and signature polymorphisms were observed at codon positions 140, 148, 151, 157, and 160 among HIV subtypes. These variations predicted higher genetic barriers to G140S and G140C in subtypes C, CRF02_AG, and A/CRF01_AE, as well as higher genetic barriers toward acquisition of V151I in subtypes CRF02_AG and A/CRF01_AE. The E157Q and E160Q mutational motif was observed in 35% of INI-naïve patients harboring subtype C infections, indicating intra-subtype variations. Thirteen patients failed raltegravir (RAL)-containing regimens within 8 ± 1 months, in association with the major Q148K/R/H and G140A/S (n = 8/24) or N155H (n = 5/24) mutational pathways. Of note, the remaining patients on RAL regimens for 14 ± 3 months harbored no or only minor integrase mutations/polymorphisms (T66I, T97A, H114P, S119P, A124S, G163R, I203M, R263K). These results demonstrate the importance of understanding subtype variability in the development of resistance to INIs.

  12. Radiation induced emulsion graft polymerization of 4-vinylpyridine onto PE/PP nonwoven fabric for As(V) adsorption

    Science.gov (United States)

    Akkaş Kavaklı, Pınar; Kavaklı, Cengiz; Seko, Noriaki; Tamada, Masao; Güven, Olgun

    2016-10-01

    A novel nonwoven fabric adsorbent having 4-vinylpyridine functional groups was prepared by using radiation-induced emulsion graft polymerization method and grafting 4-vinylpyridine monomer onto a polyethylene-coated polypropylene nonwoven fabric (NWF) in aqueous emulsion solution. The grafting conditions of the 4-vinylpyridine monomer onto the NWF were optimised and 150% Dg VP-g-NWF was prepared using 30 kGy pre-irradiation dose, 5% VP monomer concentration and 0.5% (w/w) Tween 20 in aqueous emulsion. Grafted 4-vinylpyridine chains on the NWF were then quaternized for the preparation of QVP-g-NWF adsorbent. All fabric structures were characterized by using Fourier-transform infrared spectrometer, x-ray photoelectron spectrometer and scanning electron microscope. QVP-g-NWF adsorbent was used in batch adsorption experiments for As(V) ions by studying the pH, contact time, and initial As(V) ion concentration parameters. Results showed that QVP-g-NWF adsorbent has significant As(V) adsorption and experimental As(V) adsorption capacity was 98.04 mg As(V)/g polymer from 500 mg/L initial As(V) concentration at pH 7.00.

  13. Effect of calcium on adsorptive removal of As(III) and As(V) by iron oxide-based adsorbents

    KAUST Repository

    Uwamariya, V.

    2014-06-25

    The effects of calcium on the equilibrium adsorption capacity of As(III) and As(V) onto iron oxide-coated sand (IOCS) and granular ferric hydroxide (GFH) were investigated through batch experiments, rapid small-scale column tests (RSSCT) and kinetics modelling. Batch experiments showed that at calcium concentrations≤20 mg/L, high As(III) and As(V) removal efficiencies by IOCS and GFH are achieved at pH 6. An increase of the calcium concentration to 40 and 80 mg/L reversed this trend, giving higher removal efficiencies at higher pH (8). The adsorption capacities of IOCS and GFH at an equilibrium arsenic concentration of 10 g/L were found to be between 2.0 and 3.1 mg/g for synthetic water without calcium and between 2.8 and 5.3 mg/g when 80 mg/L of calcium was present at the studied pH values. After 10 hours of filter run in RSSCT, approximately 1000 empty bed volumes, the ratios of C/Co for As(V) were 26% and 18% for calcium-free model water; and only 1% and 0.2% after addition of 80 mg/L of Ca for filter columns with IOCS and GFH, respectively. The adsorption of As(III) and As(V) onto GFH follows a second-order reaction, with and without addition of calcium. The adsorption of As(III) and As(V) onto IOCS follows a first-order reaction without calcium addition, and moves to the second-reaction-order kinetics when calcium is added. Based on the intraparticle diffusion model, the main controlling mechanism for As(III) adsorption is intraparticle diffusion, while surface diffusion contributes greatly to the adsorption of As(V).

  14. The mechanisms of detoxification of As(III), dimethylarsinic acid (DMA) and As(V) in the microalga Chlorella vulgaris.

    Science.gov (United States)

    Pantoja Munoz, L; Purchase, D; Jones, H; Raab, A; Urgast, D; Feldmann, J; Garelick, H

    2016-06-01

    The response of Chlorella vulgaris when challenged by As(III), As(V) and dimethylarsinic acid (DMA) was assessed through experiments on adsorption, efflux and speciation of arsenic (reduction, oxidation, methylation and chelation with glutathione/phytochelatin [GSH/PC]). Our study indicates that at high concentrations of phosphate (1.62mM of HPO4(2-)), upon exposure to As(V), cells are able to shift towards methylation of As(V) rather than PC formation. Treatment with As(V) caused a moderate decrease in intracellular pH and a strong increase in the concentration of free thiols (GSH). Passive surface adsorption was found to be negligible for living cells exposed to DMA and As(V). However, adsorption of As(III) was observed to be an active process in C. vulgaris, because it did not show saturation at any of the exposure periods. Chelation of As(III) with GS/PC and to a lesser extent hGS/hPC is a major detoxification mechanism employed by C. vulgaris cells when exposed to As(III). The increase of bound As-GS/PC complexes was found to be strongly related to an increase in concentration of As(III) in media. C. vulgaris cells did not produce any As-GS/PC complex when exposed to As(V). This may indicate that a reduction step is needed for As(V) complexation with GSH/PC. C. vulgaris cells formed DMAS(V)-GS upon exposure to DMA independent of the exposure period. As(III) triggers the formation of arsenic complexes with PC and homophytochelatins (hPC) and their compartmentalisation to vacuoles. A conceptual model was devised to explain the mechanisms involving ABCC1/2 transport. The potential of C. vulgaris to bio-remediate arsenic from water appeared to be highly selective and effective without the potential hazard of reducing As(V) to As(III), which is more toxic to humans.

  15. Gated rotation mechanism of site-specific recombination by ϕC31 integrase.

    Science.gov (United States)

    Olorunniji, Femi J; Buck, Dorothy E; Colloms, Sean D; McEwan, Andrew R; Smith, Margaret C M; Stark, W Marshall; Rosser, Susan J

    2012-11-27

    Integrases, such as that of the Streptomyces temperate bacteriophage ϕC31, promote site-specific recombination between DNA sequences in the bacteriophage and bacterial genomes to integrate or excise the phage DNA. ϕC31 integrase belongs to the serine recombinase family, a large group of structurally related enzymes with diverse biological functions. It has been proposed that serine integrases use a "subunit rotation" mechanism to exchange DNA strands after double-strand DNA cleavage at the two recombining att sites, and that many rounds of subunit rotation can occur before the strands are religated. We have analyzed the mechanism of ϕC31 integrase-mediated recombination in a topologically constrained experimental system using hybrid "phes" recombination sites, each of which comprises a ϕC31 att site positioned adjacent to a regulatory sequence recognized by Tn3 resolvase. The topologies of reaction products from circular substrates containing two phes sites support a right-handed subunit rotation mechanism for catalysis of both integrative and excisive recombination. Strand exchange usually terminates after a single round of 180° rotation. However, multiple processive "360° rotation" rounds of strand exchange can be observed, if the recombining sites have nonidentical base pairs at their centers. We propose that a regulatory "gating" mechanism normally blocks multiple rounds of strand exchange and triggers product release after a single round.

  16. Identification of minority resistance mutations in the HIV-1 integrase coding region using next generation sequencing

    DEFF Research Database (Denmark)

    Fonager, Jannik; Larsson, Jonas T; Hussing, Christian;

    2015-01-01

    : raltegravir (RAL), elvitegravir (EVG) and dolutegravir (DTG). STUDY DESIGN: NGS and Sseq were used to analyze RT-PCR products of the HIV-1 integrase coding region from six patients and in serial samples from two patients. NGS sequences were assembled and analyzed using the low frequency variant detection...

  17. Determination of As(III) and As(V) by Flow Injection-Hydride Generation-Atomic Absorption Spectrometry via On-line Reduction of As(V) by KI

    DEFF Research Database (Denmark)

    Nielsen, Steffen; Hansen, Elo Harald

    1997-01-01

    interferents. Thus, without impairing the assay, interferents such as Cu, Co, Ni and Se could, at a As(V) level of 5 mu g l(-1), be tolerated at a weight excess of 2000, 30000, 200 and 200 times, respectively. The assay of a certified drinking water sample by means of multiple standard addition (five levels......A volume-based flow injection (FI) procedure is described for the determination and speciation of trace inorganic arsenic, As(III) and As(V), via hydride generation-atomic absorption spectrometry (HG-AAS) of As(III). The determination of total arsenic is obtained by on-line reduction of As(V) to As...... reactor immersed in a water bath at 10 degrees C. By using the very same volume-based FI-HG-AAS system without the heating and cooling reactors, and employing mild hydrochloric acid conditions, As(V) is not converted to arsine, thereby allowing the selective determination of As(III). The injected sample...

  18. Determination of As(III and As(V in waters by chronopotentiometric stripping analysis

    Directory of Open Access Journals (Sweden)

    Švarc-Gajić Jaroslava V.

    2006-01-01

    Full Text Available Arsenic is a naturally occurring toxic and carcinogenic element. The degree of the toxicity depends on its chemical form and the concentration. Application of a sensitive, selective, simple and rapid method for detection and monitoring of different oxidation states of arsenic in waters is of great importance because main route of population exposure is through drinking water. In this work chronopotentiometric stripping analysis (CSA was used for the determination of As(III and As(V in tap, well, river and rain waters from Vojvodina (Serbia. Gold film electrode on the glassy carbon support was used as the working electrode. The experimental parameters of the technique were investigated and optimized. Detection limit of the method for the electrolysis time of 600 s was 2 μg/dm3 of As(III.

  19. TISSUE DISTRIBUTION OF INORGANIC ARSENIC (AS) AND ITS METHYLATED METABOLITES IN MICE FOLLOWING ORAL ADMINISTRATION OF ARSENATE (ASV)

    Science.gov (United States)

    TISSUE DISTRIBUTION OF INORGANIC ARSENIC (iAs) AND ITS METHYLATED METABOLITES IN MICE FOLLOWING ORAL ADMINISTRATION OF ARSENATE (AsV). E M Kenyon1, L M Del Razo2, and M F Hughes1. 1NHEERL, ORD, US EPA, RTP, NC, USA; 2CINVESTAV-IPN, Mexico City, Mexico.The relationship o...

  20. The first examples of 1-D organic hybrid lanthanoid thioarsenates based on two [As(V)S4](3-) linkage modes.

    Science.gov (United States)

    Zhao, Rong-Qing; Liu, Xing; Zhou, Jian; Xiao, Hong-Ping; Zou, Hua-Hong; Fu, Lianshe; Tang, Qiuling; Tan, Xiao-Feng

    2016-04-14

    A series of new 1-D organic hybrid lanthanoid thioarsenates [Ln(dap)2]2(μ-η(1):η(1):η(1):η(1)-AsS4)(μ-η(1):η(1)-As(V)S4)]n {Ln = Ce (Ia), Pr (Ib), Nd (Ic), and Sm (Id); dap = diaminopropane} have been prepared under solvothermal conditions and structurally characterized. Compounds Ia-d contain two [As(V)S4](3-) linkage modes, namely μ-η(1):η(1):η(1):η(1)-As(V)S4 and μ-η(1):η(1)-As(V)S4, which are linked alternately with [Ln(dap)2](3+) groups into 1-D neutral chains [Ln(dap)2]2(μ-η(1):η(1):η(1):η(1)-As(V)S4)(μ-η(1):η(1)-As(V)S4)]n, which represent the first examples of 1-D organic hybrid lanthanoid thioarsenates based on two [As(V)S4](3-) linkage modes. To learn more about the influence of lanthanide contraction on the formation of lanthanoid thioarsenates, five organic hybrid lanthanoid thioarsenates [Ln(dap)3As(V)S4] [Ln = Tb (IIa), Dy (IIb), Ho (IIIc), and Er (IIId)] and [Er(dien)2As(V)S4] (III, dien = diethylenetriamine) are also provided. Both II and III contain neutral lanthanide-centred complexes, where the tetrahedral anion [As(V)S4](3-) acts as a chelating ligand to the complex [Ln(dap)3](3+)/[Er(dien)2](3+) cation. Their optical properties have been characterized by UV-vis spectra, and the density functional theory calculation of Ia has been performed.

  1. Synthesis of 6-sulfamoyl-4-oxoquinoline-3-carboxylic acid derivatives as integrase antagonists with anti-HIV activity

    Institute of Scientific and Technical Information of China (English)

    Zai Gang Luo; Cheng Chu Zeng; Lei Fu Yang; Hong Qiu He; Cun Xin Wang; Li Ming Hu

    2009-01-01

    A series of novel 6-sulfamoyl-4-oxoquinoline-3-earboxylic acids derivatives have been synthesized and screened for HIV integrase inhibition activity. Their structures were confirmed by ESI-MS, 1H NMR and 13C NMR.

  2. Optimization of Streptomyces bacteriophage phi C31 integrase system to prevent post integrative gene silencing in pulmonary type II cells.

    Science.gov (United States)

    Aneja, Manish Kumar; Geiger, Johannes; Imker, Rabea; Uzgun, Senta; Kormann, Michael; Hasenpusch, Guenther; Maucksch, Christof; Rudolph, Carsten

    2009-12-31

    phi C31 integrase has emerged as a potent tool for achieving long-term gene expression in different tissues. The present study aimed at optimizing elements of phi C31 integrase system for alveolar type II cells. Luciferase and beta-galactosidase activities were measured at different time points post transfection. 5-Aza-2'deoxycytidine (AZA) and trichostatin A (TSA) were used to inhibit DNA methyltransferase and histone deacetylase complex (HDAC) respectively. In A549 cells, expression of the integrase using a CMV promoter resulted in highest integrase activity, whereas in MLE12 cells, both CAG and CMV promoter were equally effective. Effect of polyA site was observed only in A549 cells, where replacement of SV40 polyA by bovine growth hormone (BGH) polyA site resulted in an enhancement of integrase activity. Addition of a C-terminal SV40 nuclear localization signal (NLS) did not result in any significant increase in integrase activity. Long-term expression studies with AZA and TSA, provided evidence for post-integrative gene silencing. In MLE12 cells, both DNA methylases and HDACs played a significant role in silencing, whereas in A549 cells, it could be attributed majorly to HDAC activity. Donor plasmids comprising cellular promoters ubiquitin B (UBB), ubiquitin C (UCC) and elongation factor 1 alpha (EF1 alpha) in an improved backbone prevented post-integrative gene silencing. In contrast to A549 and MLE12 cells, no silencing could be observed in human bronchial epithelial cells, BEAS-2B. Donor plasmid coding for murine erythropoietin under the EF1 alpha promoter when combined with phi C31 integrase resulted in higher long-term erythropoietin expression and subsequently higher hematocrit levels in mice after intravenous delivery to the lungs. These results provide evidence for cell specific post integrative gene silencing with C31 integrase and demonstrate the pivotal role of donor plasmid in long-term expression attained with this system.

  3. Biosorption of Cr(VI) and As(V) at high concentrations by organic and inorganic wastes

    Science.gov (United States)

    María Rivas Pérez, Ivana; Paradelo Núñez, Remigio; Nóvoa Muñoz, Juan Carlos; Arias Estévez, Manuel; José Fernández Sanjurjo, María; Álvarez Rodríguez, Esperanza; Núñez Delgado, Avelino

    2016-04-01

    The potential reutilization of several wastes as biosorbents for As(V) and Cr(VI) has been assessed in batch-type experiments. The materials studied were one inorganic: mussel shell, and three organic: pine bark, oak ash and hemp waste. Batch experiments were performed in order to determine the removal capacity of the wastes under conditions of high As(V) and Cr(VI) loads. For this, 3 g of each waste material were added with 30 mL NaNO3 0.01 M dissolutions containing 0, 0.5, 1.5, 3 and 6 mmol As(V) L-1 or Cr(VI) L-1, prepared from analytical grade Na2HAsO4 or K2Cr2O7. The resulting suspensions were shaken for 24 h, centrifuged and filtered. Once each batch experiment corresponding to the sorption trials ended, each individual sample was added with 30 mL of NaNO3 0.01 M to desorb As(V) or Cr(VI), shaken for 24 h, centrifuged and filtered as in the sorption trials. Oak ash showed high sorption (>76%) and low desorption (hemp waste (98%) with very low desorption (hemp waste and mussel shell, that presented very low Cr(VI) sorption (<10%). Sorption data for both elements were better described by the Freundlich than by the Langmuir model. The variable results obtained for the removal of the two anionic contaminants for a given sorbent suggest that different mechanisms govern removal from the solution in each case. In summary, oak ash would be an efficient sorbent material for As(V), but not for Cr(VI), while pine bark would be the best sorbent for Cr(VI) removal.

  4. Actinophage R4 integrase-based site-specific chromosomal integration of non-replicative closed circular DNA.

    Science.gov (United States)

    Miura, Takamasa; Nishizawa, Akito; Nishizawa, Tomoyasu; Asayama, Munehiko; Shirai, Makoto

    2016-06-01

    The actinophage R4 integrase (Sre)-based molecular genetic engineering system was developed for the chromosomal integration of multiple genes in Escherichia coli. A cloned DNA fragment containing two attP sites, green fluorescent protein (gfp) as a first transgene, and an antibiotic resistance gene as a selection marker was self-ligated to generate non-replicative closed circular DNA (nrccDNA) for integration. nrccDNA was introduced into attB-inserted E. coli cells harboring the plasmid expressing Sre by electroporation. The expressed Sre catalyzed site-specific integration between one of the two attP sites on nrccDNA and the attB site on the E. coli chromosome. The integration frequency was affected by the chromosomal location of the target site. A second nrccDNA containing two attB sites, lacZα encoding the alpha fragment of β-galactosidase as a transgene, and another antibiotic resistance gene was integrated into the residual attP site on the gfp-integrated E. coli chromosome via one of the two attB sites according to reiterating site-specific recombination. The integrants clearly exhibited β-galactosidase activity and green fluorescence, suggesting the simultaneous expression of multiple recombinant proteins in E. coli. The results of the present study showed that a step-by-step integration procedure using nrccDNA achieved the chromosomal integration of multiple genes. PMID:26870903

  5. Optimization of Streptomyces bacteriophage φC31 integrase system to prevent post integrative gene silencing in pulmonary type II cells

    OpenAIRE

    Aneja, Manish Kumar; Geiger, Johannes; Imker, Rabea; Üzgün, Senta; Kormann, Michael; Hasenpusch, Guenther; Maucksch, Christof; Rudolph, Carsten

    2009-01-01

    φC31 integrase has emerged as a potent tool for achieving long-term gene expression in different tissues. The present study aimed at optimizing elements of φC31 integrase system for alveolar type II cells. Luciferase and β-galactosidase activities were measured at different time points post transfection. 5-Aza-2'deoxycytidine (AZA) and trichostatin A (TSA) were used to inhibit DNA methyltransferase and histone deacetylase complex (HDAC) respectively. In A549 cells, expression of the integrase...

  6. Novel 3′-Processing Integrase Activity Assay by Real-Time PCR for Screening and Identification of HIV-1 Integrase Inhibitors

    OpenAIRE

    Supachai Sakkhachornphop; Weeraya Thongkum; Chatchai Tayapiwatana

    2015-01-01

    The 3′-end processing (3′P) of each viral long terminal repeat (LTR) during human immunodeficiency virus type-1 (HIV-1) integration is a vital step in the HIV life cycle. Blocking the 3′P using 3′P inhibitor has recently become an attractive strategy for HIV-1 therapeutic intervention. Recently, we have developed a novel real-time PCR based assay for the detection of 3′P activity in vitro. The methodology usually involves biotinylated HIV-1 LTR, HIV-1 integrase (IN), and specific primers and ...

  7. Cancer-specific binary expression system activated in mice by bacteriophage HK022 Integrase.

    Science.gov (United States)

    Elias, Amer; Spector, Itay; Sogolovsky-Bard, Ilana; Gritsenko, Natalia; Rask, Lene; Mainbakh, Yuli; Zilberstein, Yael; Yagil, Ezra; Kolot, Mikhail

    2016-01-01

    Binary systems based on site-specific recombination have been used for tumor specific transcription targeting of suicide genes in animal models. In these binary systems a site specific recombinase or integrase that is expressed from a tumor specific promoter drives tumor specific expression of a cytotoxic gene. In the present study we developed a new cancer specific binary expression system activated by the Integrase (Int) of the lambdoid phage HK022. We demonstrate the validity of this system by the specific expression of a luciferase (luc) reporter in human embryonic kidney 293T (HEK293T) cells and in a lung cancer mouse model. Due to the absence viral vectors and of cytotoxicity the Int based binary system offers advantages over previously described counterparts and may therefore be developed into a safer cancer cell killing system. PMID:27117628

  8. Molecular modeling study on the allosteric inhibition mechanism of HIV-1 integrase by LEDGF/p75 binding site inhibitors.

    Directory of Open Access Journals (Sweden)

    Weiwei Xue

    Full Text Available HIV-1 integrase (IN is essential for the integration of viral DNA into the host genome and an attractive therapeutic target for developing antiretroviral inhibitors. LEDGINs are a class of allosteric inhibitors targeting LEDGF/p75 binding site of HIV-1 IN. Yet, the detailed binding mode and allosteric inhibition mechanism of LEDGINs to HIV-1 IN is only partially understood, which hinders the structure-based design of more potent anti-HIV agents. A molecular modeling study combining molecular docking, molecular dynamics simulation, and binding free energy calculation were performed to investigate the interaction details of HIV-1 IN catalytic core domain (CCD with two recently discovered LEDGINs BI-1001 and CX14442, as well as the LEDGF/p75 protein. Simulation results demonstrated the hydrophobic domain of BI-1001 and CX14442 engages one subunit of HIV-1 IN CCD dimer through hydrophobic interactions, and the hydrophilic group forms hydrogen bonds with HIV-1 IN CCD residues from other subunit. CX14442 has a larger tert-butyl group than the methyl of BI-1001, and forms better interactions with the highly hydrophobic binding pocket of HIV-1 IN CCD dimer interface, which can explain the stronger affinity of CX14442 than BI-1001. Analysis of the binding mode of LEDGF/p75 with HIV-1 IN CCD reveals that the LEDGF/p75 integrase binding domain residues Ile365, Asp366, Phe406 and Val408 have significant contributions to the binding of the LEDGF/p75 to HIV1-IN. Remarkably, we found that binding of BI-1001 and CX14442 to HIV-1 IN CCD induced the structural rearrangements of the 140 s loop and oration displacements of the side chains of the three conserved catalytic residues Asp64, Asp116, and Glu152 located at the active site. These results we obtained will be valuable not only for understanding the allosteric inhibition mechanism of LEDGINs but also for the rational design of allosteric inhibitors of HIV-1 IN targeting LEDGF/p75 binding site.

  9. 3-Hydroxypyrimidine-2,4-diones as an Inhibitor Scaffold of HIV Integrase

    OpenAIRE

    Tang, Jing; Maddali, Kasthuraiah; Sham, Yuk Y.; Vince, Robert; Pommier, Yves; Wang, Zhengqiang

    2011-01-01

    Integrase (IN) represents a clinically validated target for the development of antivirals against human immunodeficiency virus (HIV). Inhibitors with a novel structure core are essential for combating resistance associated with known IN inhibitors (INIs). We have previously disclosed a novel dual inhibitor scaffold of HIV IN and reverse transcriptase (RT). Here we report the complete structure-activity relationship (SAR), molecular modeling and resistance profile of this inhibitor type on IN ...

  10. The systemic effects of sclerostin overexpression using ΦC31 integrase in mice.

    Science.gov (United States)

    Zhang, Dongdong; Park, Bo Mi; Kang, Myengmo; Nam, HeeJin; Kim, Eun Jin; Bae, ChuHyun; Lim, Sung Kil

    2016-04-01

    Sclerostin, encoded by the Sost gene, is mainly produced by osteocytes in bone and antagonizes the Wnt/β-catenin signaling pathway, which is a requisite for bone formation. Currently, human anti-sclerostin antibodies are being tested in phase III clinical trials. In addition, serum sclerostin levels are reported to be associated with bone mineral density and fracture risk in normal individuals; however, the correlation between serum sclerostin and bone mass remains controversial. To study the effects of the continuous exposure of exogenous sclerostin on bone, a ΦC31 integrase system, which has the characteristics of site-specificity and efficiency, was applied for the delivery of the Sost gene in this study. We injected Sost-attB plasmid with or without ΦC31 integrase plasmid into the mouse tail vein using a hydrodynamic-based method. The site-specific integration of the Sost gene into the mouse genome was confirmed by examining a pseudo-attP site on the hepatic genomic DNA. Sclerostin was expressed in the hepatocytes, secreted into the blood flow, and maintained at high concentrations in the mice with both Sost-attB plasmid and ΦC31 integrase plasmid injections, which was observed by serial measurement. Moreover, the mice with long-term high levels of serum sclerostin showed trabecular bone loss on micro-CT analysis. Peripheral B cell populations were not affected. Our results suggested that sclerostin could be expressed in the liver and sustained successfully at high levels in the blood by using the ΦC31 integrase system, leading to trabecular bone loss. These findings may help to further ascertain the effects of sclerostin introduced exogenously on the skeleton.

  11. Lack of Pharmacokinetic Interaction between Rilpivirine and Integrase Inhibitors Dolutegravir and GSK1265744

    OpenAIRE

    Ford, Susan L.; Gould, Elizabeth; Chen, Shuguang; Margolis, David; Spreen, William; Crauwels, Herta; Piscitelli, Stephen

    2013-01-01

    Dolutegravir (DTG) and GSK1265744 are HIV integrase inhibitors (INIs) in clinical development. The oral formulation of rilpivirine (RPV), a nonnucleoside reverse transcriptase inhibitor (NNRTI), has been approved for treatment-naive HIV infection. Long-acting depot injections of GSK1265744 and RPV are also being developed. This study evaluated the potential for drug interactions between RPV and these INIs. This phase 1, open-label, two-cohort, three-period, single-sequence crossover study eva...

  12. Identifying and Characterizing a Functional HIV-1 Reverse Transcriptase-binding Site on Integrase*

    OpenAIRE

    Wilkinson, Thomas A.; Januszyk, Kurt; Phillips, Martin L.; Tekeste, Shewit S.; Zhang, Min; Miller, Jennifer T.; Stuart F. J. Le Grice; Clubb, Robert T.; Chow, Samson A

    2009-01-01

    Integrase (IN) from human immunodeficiency virus, type 1 (HIV-1) exerts pleiotropic effects in the viral replication cycle. Besides integration, IN mutations can impact nuclear import, viral maturation, and reverse transcription. IN and reverse transcriptase (RT) interact in vitro, and the IN C-terminal domain (CTD) is both necessary and sufficient for binding RT. We used nuclear magnetic resonance spectroscopy to identify a putative RT-binding surface on the IN CTD, a...

  13. Structural Properties of HIV Integrase. Lens Epithelium-derived Growth Factor Oligomers

    Energy Technology Data Exchange (ETDEWEB)

    Gupta, K.; Diamond, T; Hwang, Y; Bushman, F; Van Duyne, G

    2010-01-01

    Integrase (IN) is the catalytic component of the preintegration complex, a large nucleoprotein assembly critical for the integration of the retroviral genome into a host chromosome. Although partial crystal structures of human immunodeficiency virus IN alone and its complex with the integrase binding domain of the host factor PSIP1/lens epithelium-derived growth factor (LEDGF)/p75 are available, many questions remain regarding the properties and structures of LEDGF-bound IN oligomers. Using analytical ultracentrifugation, multiangle light scattering, and small angle x-ray scattering, we have established the oligomeric state, stoichiometry, and molecular shapes of IN {center_dot} LEDGF complexes in solution. Analyses of intact IN tetramers bound to two different LEDGF truncations allow for placement of the integrase binding domain by difference analysis. Modeling of the small angle x-ray scattering envelopes using existing structural data suggests domain arrangements in the IN oligomers that support and extend existing biochemical data for IN {center_dot} LEDGF complexes and lend new insights into the quaternary structure of LEDGF-bound IN tetramers. These IN oligomers may be involved in stages of the viral life cycle other than integration, including assembly, budding, and early replication.

  14. Increasing Deaths Due to Malignancy in HIV+ Patients is Associated with Integrase Inhibitor Therapy

    Institute of Scientific and Technical Information of China (English)

    Daniel O. Griffin[1,2,3; Arif Dharsee[1; Juan Carlos Rico[1; Joseph McGowan[1

    2014-01-01

    Despite reductions in AIDS-related deaths, registries show HIV+ patients are still dying at a younger age than HIV-peers. Although overall mortality has declined in this population, a growing percent of deaths are due to malignancy. Since the data demonstrating the growing percentage of deaths due to malignancy in the HIV+ population is derived from registries, the study explores whether the subset dying from malignancy has particular characteristics that can be seen in a well-characterized cohort. In the well-characterized HIV+ cohort, the percentage of deaths due to cancer was seen to increase over four years (2010-2013) from 21% to 24% to 38% to 40%. The mean CD4-count of those who died from malignancy was 252+/-42 and 333+/-36 in patients with death from other causes. The viral load was not suppressed in 26% of patients dying from malignancy. Of patients on integrase inhibitor therapy, 48% of deaths were due to malignancy while in patients not on this therapy, 10% of deaths were due to malignancy (relative risk = 4.8). In HIV+ patients, a low CD4-count, failure to achieve viral suppression, and use of integrase inhibitors were associated with malignancy as the cause of death. The association of a specific therapy, integrase inhibition, with malignancy is seen in the study.

  15. The Promise of Dolutegravir: A Novel Second Generation Integrase Strand Transfer Inhibitor.

    Science.gov (United States)

    Singh, Harmanjit; Kaur, Mandeep; Kakkar, Ashish K; Kumar, Harish

    2016-01-01

    Dolutegravir (DTG), a novel integrase strand transfer inhibitor (INSTI), is one of the newest addition to the arsenal of anti-human immunodeficiency virus (HIV) therapeutics. Dolutegravir is the first member of the class of second generation integrase strand transfer inhibitors aimed primarily to address the current unmet need for novel unboosted integrase inhibitors with convenient once-daily dosing and a superior resistance profile. During its clinical development, DTG has demonstrated noninferior or superior efficacy in both treatment-naive as well as treatmentexperienced individuals including those who have previously failed first generation INSTIs. Other potential advantages include a favorable safety profile, low propensity for drug-drug interactions, and prolonged serum half-life permitting once-daily administration in treatment-naive or treatment-experienced INSTI naive HIV patients. Twice-daily administration is recommended in individuals with established or suspected resistance to first-generation INSTIs. This review outlines the need for new HIV therapeutics and summarizes the efficacy, safety, and pharmacokinetic profile of dolutegravir. PMID:27157040

  16. Molecular features related to HIV integrase inhibition obtained from structure- and ligand-based approaches.

    Directory of Open Access Journals (Sweden)

    Luciana L de Carvalho

    Full Text Available Among several biological targets to treat AIDS, HIV integrase is a promising enzyme that can be employed to develop new anti-HIV agents. The aim of this work is to propose a mechanistic interpretation of HIV-1 integrase inhibition and to rationalize the molecular features related to the binding affinity of studied ligands. A set of 79 HIV-1 integrase inhibitors and its relationship with biological activity are investigated employing 2D and 3D QSAR models, docking analysis and DFT studies. Analyses of docking poses and frontier molecular orbitals revealed important features on the main ligand-receptor interactions. 2D and 3D models presenting good internal consistency, predictive power and stability were obtained in all cases. Significant correlation coefficients (r(2 = 0.908 and q(2= 0.643 for 2D model; r(2= 0.904 and q(2= 0.719 for 3D model were obtained, indicating the potential of these models for untested compounds. The generated holograms and contribution maps revealed important molecular requirements to HIV-1 IN inhibition and several evidences for molecular modifications. The final models along with information resulting from molecular orbitals, 2D contribution and 3D contour maps should be useful in the design of new inhibitors with increased potency and selectivity within the chemical diversity of the data.

  17. Integrase-independent HIV-1 infection is augmented under conditions of DNA damage and produces a viral reservoir

    Energy Technology Data Exchange (ETDEWEB)

    Ebina, Hirotaka, E-mail: hebina@virus.kyoto-u.ac.jp; Kanemura, Yuka; Suzuki, Yasutsugu; Urata, Kozue; Misawa, Naoko; Koyanagi, Yoshio

    2012-05-25

    HIV-1 possesses a viral protein, integrase (IN), which is necessary for its efficient integration in target cells. However, it has been reported that an IN-defective HIV strain is still capable of integration. Here, we assessed the ability of wild type (WT) HIV-1 to establish infection in the presence of IN inhibitors. We observed a low, yet clear infection of inhibitor-incubated cells infected with WT HIV which was identical to cells infected with IN-deficient HIV, D64A. Furthermore, the IN-independent integration could be enhanced by the pretreatment of cells with DNA-damaging agents suggesting that integration is mediated by a DNA repair system. Moreover, significantly faster viral replication kinetics with augmented viral DNA integration was observed after infection in irradiated cells treated with IN inhibitor compared to nonirradiated cells. Altogether, our results suggest that HIV DNA has integration potential in the presence of an IN inhibitor and may serve as a virus reservoir.

  18. Huwe1, a novel cellular interactor of Gag-Pol through integrase binding, negatively influences HIV-1 infectivity.

    Science.gov (United States)

    Yamamoto, Seiji P; Okawa, Katsuya; Nakano, Takashi; Sano, Kouichi; Ogawa, Kanako; Masuda, Takao; Morikawa, Yuko; Koyanagi, Yoshio; Suzuki, Youichi

    2011-04-01

    Integration, an indispensable step for retrovirus replication, is executed by integrase (IN), which is expressed as a part of a Gag-Pol precursor. Although mechanistic detail of the IN-catalyzed integration reaction is well defined, numerous evidence have demonstrated that IN is involved in multiple steps of retrovirus replication other than integration. In this study, Huwe1, a HECT-type E3 ubiquitin ligase, was identified as a new cellular interactor of human immunodeficiency virus type 1 (HIV-1) IN. The interaction was mediated through the catalytic core domain of IN and a wide-range region of Huwe1. Interestingly, although depletion of Huwe1 in target cells did not affect the early phase of HIV-1 infection in a human T cell line, we found that infectivity of HIV-1 released from the Huwe1 knockdown cells was significantly augmented more than that of virus produced from control cells. The increase in infectivity occurred in proviral DNA synthesis. Further analysis revealed that Huwe1 interacted with HIV-1 Gag-Pol precursor protein through an IN domain. Our results suggest that Huwe1 in HIV-1 producer cells has a negative impact on early post-entry events during the next round of virus infection via association with an IN region of Gag-Pol. PMID:21167302

  19. A new large-DNA-fragment delivery system based on integrase activity from an integrative and conjugative element.

    Science.gov (United States)

    Miyazaki, Ryo; van der Meer, Jan Roelof

    2013-07-01

    During the past few decades, numerous plasmid vectors have been developed for cloning, gene expression analysis, and genetic engineering. Cloning procedures typically rely on PCR amplification, DNA fragment restriction digestion, recovery, and ligation, but increasingly, procedures are being developed to assemble large synthetic DNAs. In this study, we developed a new gene delivery system using the integrase activity of an integrative and conjugative element (ICE). The advantage of the integrase-based delivery is that it can stably introduce a large DNA fragment (at least 75 kb) into one or more specific sites (the gene for glycine-accepting tRNA) on a target chromosome. Integrase recombination activity in Escherichia coli is kept low by using a synthetic hybrid promoter, which, however, is unleashed in the final target host, forcing the integration of the construct. Upon integration, the system is again silenced. Two variants with different genetic features were produced, one in the form of a cloning vector in E. coli and the other as a mini-transposable element by which large DNA constructs assembled in E. coli can be tagged with the integrase gene. We confirmed that the system could successfully introduce cosmid and bacterial artificial chromosome (BAC) DNAs from E. coli into the chromosome of Pseudomonas putida in a site-specific manner. The integrase delivery system works in concert with existing vector systems and could thus be a powerful tool for synthetic constructions of new metabolic pathways in a variety of host bacteria. PMID:23686268

  20. DNA cleavage is independent of synapsis during Streptomyces phage phiBT1 integrase-mediated site-specific recombination.

    Science.gov (United States)

    Zhang, Lin; Wang, Lu; Wang, Jin; Ou, Xijun; Zhao, Guoping; Ding, Xiaoming

    2010-10-01

    Bacteriophage-encoded serine recombinases have great potential in genetic engineering but their catalytic mechanisms have not been adequately studied. Integration of ϕBT1 and ϕC31 via their attachment (att) sites is catalyzed by integrases of the large serine recombinase subtype. Both ϕBT1 and ϕC31 integrases were found to cleave single-substrate att sites without synaptic complex formation, and ϕBT1 integrase relaxed supercoiled DNA containing a single integration site. Systematic mutation of the central att site dinucleotide revealed that cleavage was independent of nucleotide sequence, but rejoining was crucially dependent upon complementarity of the cleavage products. Recombination between att sites containing dinucleotides with antiparallel complementarity led to antiparallel recombination. Integrase-substrate pre-incubation experiments revealed that the enzyme can form an attP-integrase tetramer complex that then captures naked attB DNA, and suggested that two alternative assembly pathways can lead to synaptic complex formation.

  1. Exploring binding mode for styrylquinoline HIV-1 integrase inhibitors using comparative molecular field analysis and docking studies

    Institute of Scientific and Technical Information of China (English)

    Xiao-hui MA; Xiao-yi ZHANG; Jian-jun TAN; Wei-zu CHEN; Cun-xin WANG

    2004-01-01

    AIM: To understand pharmacophore properties of styrylquinoline derivatives and to design inhibitors of HIV-1integrase. METHODS: Comparative molecular field analysis (CoMFA) was performed to analyze three-dimensional quantitative structure-activity relationship (3D-QSAR) of styrylquinoline derivatives. Thirty-eight compounds were randomly divided into a training set of 28 compounds and a test set of 10 compounds. The stability of 3DQSAR models was proved by the analysis of cross-validated and non-cross-validated methods. Moreover, the binding mode of these compounds and integrase was constructed by AutoDock program. RESULTS: The CoMFA model of the training compounds was reasonably predicted with cross-validated coefficient (q2) and conventional (r2) values (up to 0.696 and 0.754). Then the model was validated by the test set. The resulting CoMFA maps visualized structural requirements for the biological activity of these inhibitors. Docking results showed that a carboxyl group at C-7 and a hydroxyl group at C-8 in the quinoline subunit, bound closely to the divalent metal cofactor (Mg2+) around the integrase catalytic site. Moreover, there is a linear correlation between the binding energy of the inhibitors with integrase and their inhibitory effect. CONCLUSIONS: The present study indicated that the CoMFA model together with docking results could give us helpful hints for drug design as well as interpretation of the binding affinity between these inhibitors and integrase.

  2. Synthesis, characterization and adsorptive properties of carbon with iron nanoparticles and iron carbide for the removal of As(V) from water.

    Science.gov (United States)

    Gutierrez-Muñiz, O E; García-Rosales, G; Ordoñez-Regil, E; Olguin, M T; Cabral-Prieto, A

    2013-01-15

    This manuscript presents the synthesis of carbon modified with iron nanoparticles (CFe) and iron carbide (CarFe) from the pyrolyzed crown leaves of pineapple (Ananas comosus) treated with iron salts. The materials that were obtained were used for the removal of As(V) from aqueous media. The carbonaceous materials were characterized by Scanning Electron Microscopy (SEM), Transmission electron microscopy (TEM), X-ray diffraction (XRD), X-Ray Photoelectron Spectroscopy (XPS) and Mossbauer Spectroscopy. The specific area (BET), number site density and point of zero charge (pH(pzc)) were also determined. The kinetic parameters were obtained by fitting the experimental data to the pseudo-first-order and pseudo-second-order models. Different isotherm models were applied to describe the As(V) adsorption behavior. The kinetics of As(V) sorption by CFe and CarFe was well defined for the pseudo-second-order model (R(2) = 0.9994 and 0.999, respectively). The maximum As(V) uptake was 1.8 mg g(-1) for CFe and 1.4 mg g(-1) for CarFe. The results obtained indicated that both materials are equally useful for As(V) sorption. The As(V) experimental isotherm data were described by the Freundlich model for CFe and CarFe.

  3. Genetic diversity on the integrase region of the pol gene among HIV type 1-infected patients naive for integrase inhibitors in São Paulo City, Brazil.

    Science.gov (United States)

    Arruda, Liã Bárbara; Fonseca, Luiz Augusto M; Duarte, Alberto J S; Casseb, Jorge

    2010-01-01

    The presence of mutations associated with integrase inhibitor (INI) resistance among INI-naive patients may play an important clinical role in the use of those drugs Samples from 76 HIV-1-infected subjects naive to INIs were submitted to direct sequencing. No differences were found between naive (25%) subjects and subjects on HAART (75%). No primary mutation associated with raltegravir or elvitegravir resistance was found. However, 78% of sequences showed at least one accessory mutation associated with resistance. The analysis of the 76 IN sequences showed a high polymorphic level on this region among Brazilian HIV-1-infected subjects, including a high prevalence of aa substitutions related to INI resistance. The impact of these findings remains unclear and further studies are necessary to address these questions. PMID:20055590

  4. Genetic diversity on the integrase region of the pol gene among HIV type 1-infected patients naive for integrase inhibitors in São Paulo City, Brazil.

    Science.gov (United States)

    Arruda, Liã Bárbara; Fonseca, Luiz Augusto M; Duarte, Alberto J S; Casseb, Jorge

    2010-01-01

    The presence of mutations associated with integrase inhibitor (INI) resistance among INI-naive patients may play an important clinical role in the use of those drugs Samples from 76 HIV-1-infected subjects naive to INIs were submitted to direct sequencing. No differences were found between naive (25%) subjects and subjects on HAART (75%). No primary mutation associated with raltegravir or elvitegravir resistance was found. However, 78% of sequences showed at least one accessory mutation associated with resistance. The analysis of the 76 IN sequences showed a high polymorphic level on this region among Brazilian HIV-1-infected subjects, including a high prevalence of aa substitutions related to INI resistance. The impact of these findings remains unclear and further studies are necessary to address these questions.

  5. Screening of potential pseudo att sites of Streptomyces phage ΦC31 integrase in the human genome

    Institute of Scientific and Technical Information of China (English)

    Zhi-peng HU; Lu-sheng CHEN; Cai-yan JIA; Huan-zhang ZHU; Wei WANG; Jiang ZHONG

    2013-01-01

    Aim:ΦC31 integrase mediates site-specific recombination between two short sequences,attP and attB,in phage and bacterial genomes,which is a promising tool in gene regulation-based therapy since the zinc finger structure is probably the DNA recognizing domain that can further be engineered.The aim of this study was to screen potential pseudo att sites of ΦC31 integrase in the human genome,and evaluate the risks of its application in human gene therapy.Methods:TFBS (transcription factor binding sites) were found on the basis of reported pseudo att sites using multiple motif-finding tools,including AlignACE,BioProspector,Consensus,MEME,and Weeder.The human genome with the proposed motif was scanned tc find the potential pseudo att sites of ΦC31 integrase.Results:The possible recognition motif of ΦC31 integrase was identified,which was composed of two co-occurrence conserved elements that were reverse complement to each other flanking the core sequence TTG.In the human genome,a total of 27924 potential pseudo att sites of ΦC31 integrase were found,which were distributed in each human chromosome with high-risk specificity values in the chromosomes 16,17,and 19.When the risks of the sites were evaluate more rigorously,53 hits were discovered,and some of them were just the vital functional genes or regulatory regions,such as ACYP2,AKR1B1,DUSP4,etc.Conclusion:The results provide clues for more comprehensive evaluation of the risks of using ΦC31 integrase in human gene therapy and for drug discovery.

  6. Effect of attC structure on cassette excision by integron integrases

    Directory of Open Access Journals (Sweden)

    Larouche André

    2011-02-01

    Full Text Available Abstract Background Integrons are genetic elements able to integrate and disseminate genes as cassettes by a site-specific recombination mechanism. These elements contain a gene coding for an integrase that carries out recombination by interacting with two different target sites; the attI site in cis with the integrase and the palindromic attC site of a gene cassette. Integron integrases (IntIs bind specifically to the bottom strand of attC sites. The extrahelical bases resulting from folding of attC bottom strands are important for the recognition by integrases. These enzymes are directly involved in the accumulation and formation of new cassette arrangements in the variable region of integrons. Thus, it is important to better understand interactions between IntIs and their substrates. Results We compared the ability of five IntIs to carry out excision of several cassettes flanked by different attC sites. The results showed that for most cassettes, IntI1 was the most active integrase. However, IntI2*179E and SonIntIA could easily excise cassettes containing the attCdfrA1 site located upstream, whereas IntI1 and IntI3 had only a weak excision activity for these cassettes. Analysis of the secondary structure adopted by the bottom strand of attCdfrA1 has shown that the identity of the extrahelical bases and the distance between them (A-N7-8-C differ from those of attCs contained in the cassettes most easily excisable by IntI1 (T-N6-G. We used the attCdfrA1 site upstream of the sat2 gene cassette as a template and varied the identity and spacing between the extrahelical bases in order to determine how these modifications influence the ability of IntI1, IntI2*179E, IntI3 and SonIntIA to excise cassettes. Our results show that IntI1 is more efficient in cassette excision using T-N6-G or T-N6-C attCs while IntI3 recognizes only a limited range of attCs. IntI2*179E and SonIntIA are more tolerant of changes to the identity and spacing of extrahelical

  7. Treating chemical diversity in QSAR analysis: modeling diverse HIV-1 integrase inhibitors using 4D fingerprints.

    Science.gov (United States)

    Iyer, Manisha; Hopfinger, A J

    2007-01-01

    A set of 213 compounds across 12 structurally diverse classes of HIV-1 integrase inhibitors was used to develop and evaluate a combined clustering and QSAR modeling methodology to construct significant, reliable, and robust models for structurally diverse data sets. The trial-descriptor pool for both clustering- and QSAR-model building consisted of 4D fingerprints and classic QSAR descriptors. Clustering was carried out using a combination of the partitioning around medoids method and divisive hierarchical clustering. QSAR models were constructed for members of each cluster by linear-regression fitting and model optimization using the genetic function approximation. The 12 structurally diverse classes of integrase inhbitors were partitioned into five clusters from which corresponding QSAR models, overwhelmingly composed of 4D fingerprint descriptors, were constructed. Analysis of the five QSAR models suggests that three models correspond to structurally diverse inhibitors that likely bind at a common site on integrase characterized by a common inhibitor hydrogen-bond donor, but involving somewhat different alignments and/or poses for the inhibitors of each of the three clusters. The particular alignments for the inhibitors of each of the three QSAR models involve specific distributions of nonpolar groups over the inhibitors. The two other clusters, one for coumarins and the other for depsides and depsidones, lead to QSAR models with less-defined pharmacophores, likely representing an inhibitor binding to a site(s) different from that of the other nine classes of inhibitors. Overall, the clustering and QSAR methodology employed in this study suggests that it can meaningfully partition structurally diverse compounds expressing a common endpoint in such a manner that leads to statistically significant and pharmacologically insightful composite QSAR models. PMID:17661457

  8. Effects of etravirine on the pharmacokinetics of the integrase inhibitor S/GSK1265744.

    Science.gov (United States)

    Ford, Susan L; Gould, Elizabeth; Chen, Shuguang; Lou, Yu; Dumont, Etienne; Spreen, William; Piscitelli, Stephen

    2013-01-01

    HIV integrase inhibitors such as raltegravir and elvitegravir halt HIV progression, but treatment-emergent resistance and cross-resistance have been observed. The nonnucleoside reverse transcriptase inhibitor etravirine (ETR) may be used in combination with integrase inhibitors in patients with drug resistance. This single-center, open-label, two-period, single-sequence crossover study evaluated the effects of ETR coadministration on the pharmacokinetic profile of S/GSK1265744, an investigational integrase inhibitor in phase 2 studies. Healthy subjects received 30 mg of S/GSK1265744 alone once daily for 10 days (period 1) and in combination with 200 mg of ETR twice daily for 14 days (period 2). Serial plasma samples for pharmacokinetic analyses were collected on day 10 during period 1 and on day 14 during period 2. All treatments were well tolerated. Etravirine had no effects on S/GSK1265744 geometric mean ratios of the area under the curve from time zero until the end of the dosing interval (1.01; 90% confidence interval [CI], 0.956 to 1.06), of the maximum observed plasma concentration (1.04; 90% CI, 0.987 to 1.09), or of the plasma concentration at the end of the dosing interval (0.999; 90% CI, 0.942 to 1.06). Etravirine pharmacokinetics (PK) parameters observed following coadministration with S/GSK1265744 were in the range of historical values reported for ETR alone in healthy subjects. These results indicate that 30 mg of S/GSK1265744 for 10 days as monotherapy followed by an additional 14 days in combination with ETR was well tolerated in healthy subjects and that no dose adjustment of S/GSK1265744 is required when it is coadministered with ETR. PMID:23114768

  9. Conserved sequences in the carboxyl terminus of integrase that are essential for human immunodeficiency virus type 1 replication.

    OpenAIRE

    Cannon, P M; Byles, E D; Kingsman, S M; Kingsman, A J

    1996-01-01

    We have previously identified a residue in the carboxyl terminus of human immunodeficiency virus type 1 integrase (HIV-1 IN), W-235, the requirement for which is only revealed in viral assays for integrase function (P. M. Cannon, W. Wilson, E. Byles, S. M. Kingsman, and A. J. Kingsman, J. Virol. 68:4768-4775, 1994). Our further analysis of this region of retroviral IN has now identified several sequence motifs which are conserved in all the retroviruses we examined, apart from human spumaretr...

  10. 3-Hydroxypyrimidine-2,4-diones as an inhibitor scaffold of HIV integrase.

    Science.gov (United States)

    Tang, Jing; Maddali, Kasthuraiah; Metifiot, Mathieu; Sham, Yuk Y; Vince, Robert; Pommier, Yves; Wang, Zhengqiang

    2011-04-14

    Integrase (IN) represents a clinically validated target for the development of antivirals against human immunodeficiency virus (HIV). Inhibitors with a novel structure core are essential for combating resistance associated with known IN inhibitors (INIs). We have previously disclosed a novel dual inhibitor scaffold of HIV IN and reverse transcriptase (RT). Here we report the complete structure-activity relationship (SAR), molecular modeling, and resistance profile of this inhibitor type on IN inhibition. These studies support an antiviral mechanism of dual inhibition against both IN and RT and validate 3-hydroxypyrimidine-2,4-diones as an IN inhibitor scaffold. PMID:21381765

  11. 3-Hydroxypyrimidine-2,4-diones as an inhibitor scaffold of HIV integrase.

    Science.gov (United States)

    Tang, Jing; Maddali, Kasthuraiah; Metifiot, Mathieu; Sham, Yuk Y; Vince, Robert; Pommier, Yves; Wang, Zhengqiang

    2011-04-14

    Integrase (IN) represents a clinically validated target for the development of antivirals against human immunodeficiency virus (HIV). Inhibitors with a novel structure core are essential for combating resistance associated with known IN inhibitors (INIs). We have previously disclosed a novel dual inhibitor scaffold of HIV IN and reverse transcriptase (RT). Here we report the complete structure-activity relationship (SAR), molecular modeling, and resistance profile of this inhibitor type on IN inhibition. These studies support an antiviral mechanism of dual inhibition against both IN and RT and validate 3-hydroxypyrimidine-2,4-diones as an IN inhibitor scaffold.

  12. Biosorption of As(III) and As(V) from aqueous solution by macrofungus (Inonotus hispidus) biomass: Equilibrium and kinetic studies

    International Nuclear Information System (INIS)

    The biosorption characteristics of As(III) and As(V) from aqueous solution using the macrofungus (Inonotus hispidus) biomass were investigated as a function of pH, biomass dosage, contact time, and temperature. Langmuir, Freundlich and Dubinin-Radushkevich (D-R) models were applied to describe the biosorption isotherm of the metal ions by I. hispidus biomass. Langmuir model fitted the equilibrium data better than the Freundlich isotherm. The biosorption capacity of I. hispidus for As(III) and As(V) was found to be 51.9 mg/g and 59.6 mg/g, respectively at optimum conditions of pH 6 for As(III) and pH 2 for As(V), contact time of 30 min and temperature of 20 deg. C. The metal ions were desorbed from I. hispidus using both 1 M HCl and 1 M HNO3. The high stability of I. hispidus permitted 10 times of adsorption-elution process along the studies with a decrease about 11-28% in recovery of As(III) and 10-25% for As(V). The mean free energy values evaluated from the D-R model indicated that the biosorption of As(III) and As(V) onto I. hispidus biomass was taken place by chemical ion-exchange. The calculated thermodynamic parameters showed that the biosorption of As(III) and As(V) ions onto I. hispidus biomass was feasible, spontaneous and exothermic under examined conditions. Kinetic examination of the experimental data were showed that the biosorption processes of both As(III) and As(V) followed well pseudo-second-order kinetics.

  13. Construction and evaluation of As(V) selective electrodes based on iron oxyhydroxide embedded in silica gel membrane

    Energy Technology Data Exchange (ETDEWEB)

    Rodriguez, J.A. [Departamento de Quimica Analitica, Facultad de Ciencias, Universidad de Valladolid, Prado de la Magdalena s/n, 47005 Valladolid (Spain); Centro de Investigaciones Quimicas, Universidad Autonoma del Estado de Hidalgo, Crta. Pachuca-Tulancingo, Km 4.5, 42076 Pachuca, Hidalgo (Mexico); Barrado, E. [Departamento de Quimica Analitica, Facultad de Ciencias, Universidad de Valladolid, Prado de la Magdalena s/n, 47005 Valladolid (Spain)]. E-mail: ebarrado@qa.uva.es; Vega, M. [Departamento de Quimica Analitica, Facultad de Ciencias, Universidad de Valladolid, Prado de la Magdalena s/n, 47005 Valladolid (Spain); Prieto, F. [Centro de Investigaciones Quimicas, Universidad Autonoma del Estado de Hidalgo, Crta. Pachuca-Tulancingo, Km 4.5, 42076 Pachuca, Hidalgo (Mexico); Lima, J.L.F.C. [REQUIMTE/Departamento de Quimica-Fisica, Faculdade de Farmacia, Universidade do Porto, Rua Anibal Cunha, 164, 4099-030 Porto (Portugal)

    2005-05-10

    Two As(V) selective electrodes (with and without inner reference solution) have been developed using selective membranes based on iron oxyhydroxide embedded on silica gel mixed with ultrapure graphite at a 2/98 (w/w) ratio. The active component of the membrane was synthesised by means of the sol-gel technique and characterized by X-ray and FTIR spectroscopy. This compound shows a great affinity towards As(V) ions adsorbing 408 mg g{sup -1}. Using 1 mol l{sup -1} phosphate buffer (at a 1/1, v/v ratio) to adjust the pH and the ionic strength to 7 and 0.5 mol l{sup -1}, respectively, the calibration curve is linear from 1.0 x 10{sup -1} to 1.0 x 10{sup -6} mol l{sup -1} As(V), with a practical detection limit of 4 x 10{sup -7} mol l{sup -1} (0.03 mg l{sup -1}) and a slope close to 30 mV decade{sup -1}. The effect of potentially interfering ions was investigated. The accuracy and precision of the procedure have been tested on arsenic-free drinking water samples spiked with known amounts of arsenic and on groundwater samples containing high levels of arsenic. Total arsenic in these samples was determined after oxidation of As(III) with iodine at pH 7. The results of total As were comparable to those generated by ET-AAS.

  14. VizieR Online Data Catalog: Photometry of stars at NAOR and ASV in 2013-2014 (Cvetkovic+, 2016)

    Science.gov (United States)

    Cvetkovic, Z.; Pavlovic, R.; Boeva, S.

    2016-10-01

    The previous seven series of CCD observations of double and multiple stars performed by the Belgrade team at the Bulgarian National Astronomical Observatory at Rozhen (NAOR) with a CCD camera attached to the 2m telescope took place in 2004, 2005, 2006, 2009, 2010, 2011, and 2012, respectively. The results have been published in Pavlovic et al. (2005SerAJ.171...49P; 2013, Cat. J/AJ/146/52) and Cvetkovic et al. (2006SerAJ.172...53C; 2007SerAJ.174...83C; 2010SerAJ.180..103C; 2011, Cat. J/AJ/142/73; 2015, Cat. J/AJ/149/150). The eighth series comprising observations of 271 double or multiple stars took place during seven nights: five nights in 2013 and two nights in 2014. The telescope is of the Ritchey-Chretien-Coude-type with a focal length of 16m as given by the manufacturer. The frames were obtained by using the CCD camera VersArray 1300B. The basic characteristics of this camera and its orientation error are given in Table1. For each double or multiple star, 10 frames were obtained: 5 in the Johnson B filter and 5 in the Johnson V filter. During the summer of 2011, the first CCD observations of double and multiple stars from the Astronomical Station on the mountain of Vidojevica (ASV) took place. The results for the previous two series have been published in Pavlovic et al. 2013 (Cat. J/AJ/146/52) and Cvetkovic et al. 2015 (Cat. J/AJ/149/150). This station belongs to the Astronomical Observatory of Belgrade. The 60cm telescope has an equatorial mounting and a Cassegrain optical system. The telescope focal length is 600cm as given by the manufacturer. We carried out CCD observations of 343 visual double or multiple stars at ASV during 21 nights: 12 nights in 2013 and 9 nights in 2014. This is the third series of observations at ASV. For these nights, we used either SBIG ST-10ME or Apogee Alta U42 CCD cameras. Its basic characteristics are summarized in Table1. For each star pair 10 frames were obtained: 5 in the Cousins/Bessel B filter and 5 in the Cousins/Bessel V

  15. An unusual helix turn helix motif in the catalytic core of HIV-1 integrase binds viral DNA and LEDGF.

    Directory of Open Access Journals (Sweden)

    Hayate Merad

    Full Text Available BACKGROUND: Integrase (IN of the type 1 human immunodeficiency virus (HIV-1 catalyzes the integration of viral DNA into host cellular DNA. We identified a bi-helix motif (residues 149-186 in the crystal structure of the catalytic core (CC of the IN-Phe185Lys variant that consists of the alpha(4 and alpha(5 helices connected by a 3 to 5-residue turn. The motif is embedded in a large array of interactions that stabilize the monomer and the dimer. PRINCIPAL FINDINGS: We describe the conformational and binding properties of the corresponding synthetic peptide. This displays features of the protein motif structure thanks to the mutual intramolecular interactions of the alpha(4 and alpha(5 helices that maintain the fold. The main properties are the binding to: 1- the processing-attachment site at the LTR (long terminal repeat ends of virus DNA with a K(d (dissociation constant in the sub-micromolar range; 2- the whole IN enzyme; and 3- the IN binding domain (IBD but not the IBD-Asp366Asn variant of LEDGF (lens epidermal derived growth factor lacking the essential Asp366 residue. In our motif, in contrast to the conventional HTH (helix-turn-helix, it is the N terminal helix (alpha(4 which has the role of DNA recognition helix, while the C terminal helix (alpha(5 would rather contribute to the motif stabilization by interactions with the alpha(4 helix. CONCLUSION: The motif, termed HTHi (i, for inverted emerges as a central piece of the IN structure and function. It could therefore represent an attractive target in the search for inhibitors working at the DNA-IN, IN-IN and IN-LEDGF interfaces.

  16. HIV-1 integrase strand-transfer inhibitors: design, synthesis and molecular modeling investigation.

    Science.gov (United States)

    De Luca, Laura; De Grazia, Sara; Ferro, Stefania; Gitto, Rosaria; Christ, Frauke; Debyser, Zeger; Chimirri, Alba

    2011-02-01

    This study is focused on a new series of benzylindole derivatives with various substituents at the benzene-fused ring, suggested by our 3D pharmacophore model developed for HIV-1 integrase inhibitors (INIs). All synthesized compounds proved to be active in the nanomolar range (6-35 nM) on the strand-transfer step (ST). In particular, derivative 4-[1-(4-fluorobenzyl)-5,7-dimethoxy-1H-indol-3-yl]-2-hydroxy-4-oxobut-2-enoic acid (8e), presenting the highest best-fit value on pharmacophore model, showed a potency comparable to that of clinical INSTIs GS 9137 (1) and MK-0518 (2). The binding mode of our molecules has been investigated using the recently published crystal structure of the complex of full-length integrase from the prototype foamy virus in complex with its cognate DNA (PFV-IN/DNA). The results highlighted the ability of derivative 8e to assume the same binding mode of MK-0518 and GS 9137. PMID:21227550

  17. Three-dimensional Quantitative Structure-activity Relationship Models of HIV-1 Integrase Inhibitors of DKAs

    Institute of Scientific and Technical Information of China (English)

    ZHANG Mei-Qing; ZHAO Wen-Na; LU Shao-Yong

    2012-01-01

    As one of the three viral encoded enzymes of HIV-1 infection, HIV-1 integrase has become an attractive drug target for the treatment. Diketoacid compounds (DKAs) are one kind of potent and selective inhibitors of HIV-1 IN. In the present work, two three-dimensional QSAR techniques (CoMFA and CoMSIA) were employed to correlate the molecular structure with the activity of inhibiting the strand transfer for 147 DKAs. The all-oritation search (AOS) and all-placement search (APS) were used to optimize the CoMFA model. The diketo and keto-enol tautomers of DKAs were also used to establish the CoMFA models. The results indicated that the enol was the dominant conformation in the HIV-1 IN and DKAs complexes. It can provide a new method and reference to identify the bioactive conformation of drugs by using QSAR analysis. The best CoMSIA model, with five fields combined, implied that the hydrophobic field is very important as well as the steric and electrostatic fields. All models indicated favorable internal validation. A comparative analysis with the three models demonstrated that the CoMFA model seems to be more predictive. The contour maps could afford steric, electrostatic, hydrophobic and H-bond information about the interaction of ligand-receptor complex visually. The models would give some useful guidelines for designing novel and potent HIV-1 integrase inhibitors.

  18. HIV-1 integrase strand-transfer inhibitors: design, synthesis and molecular modeling investigation.

    Science.gov (United States)

    De Luca, Laura; De Grazia, Sara; Ferro, Stefania; Gitto, Rosaria; Christ, Frauke; Debyser, Zeger; Chimirri, Alba

    2011-02-01

    This study is focused on a new series of benzylindole derivatives with various substituents at the benzene-fused ring, suggested by our 3D pharmacophore model developed for HIV-1 integrase inhibitors (INIs). All synthesized compounds proved to be active in the nanomolar range (6-35 nM) on the strand-transfer step (ST). In particular, derivative 4-[1-(4-fluorobenzyl)-5,7-dimethoxy-1H-indol-3-yl]-2-hydroxy-4-oxobut-2-enoic acid (8e), presenting the highest best-fit value on pharmacophore model, showed a potency comparable to that of clinical INSTIs GS 9137 (1) and MK-0518 (2). The binding mode of our molecules has been investigated using the recently published crystal structure of the complex of full-length integrase from the prototype foamy virus in complex with its cognate DNA (PFV-IN/DNA). The results highlighted the ability of derivative 8e to assume the same binding mode of MK-0518 and GS 9137.

  19. Expanding the diversity of oenococcal bacteriophages: insights into a novel group based on the integrase sequence.

    Science.gov (United States)

    Jaomanjaka, Fety; Ballestra, Patricia; Dols-lafargue, Marguerite; Le Marrec, Claire

    2013-09-01

    Temperate bacteriophages are a contributor of the genetic diversity in the lactic acid bacterium Oenococcus oeni. We used a classification scheme for oenococcal prophages based on integrase gene polymorphism, to analyze a collection of Oenococcus strains mostly isolated in the area of Bordeaux, which represented the major lineages identified through MLST schemes in the species. Genome sequences of oenococcal prophages were clustered into four integrase groups (A to D) which were related to the chromosomal integration site. The prevalence of each group was determined and we could show that members of the intB- and intC-prophage groups were rare in our panel of strains. Our study focused on the so far uncharacterized members of the intD-group. Various intD viruses could be easily isolated from wine samples, while intD lysogens could be induced to produce phages active against two permissive O. oeni isolates. These data support the role of this prophage group in the biology of O. oeni. Global alignment of three relevant intD-prophages revealed significant conservation and highlighted a number of unique ORFs that may contribute to phage and lysogen fitness.

  20. Determination of As(III) and As(V) in soils using sequential extraction combined with flow injection hydride generation atomic fluorescence detection

    International Nuclear Information System (INIS)

    An analytical procedure for determination of As(III) and As(V) in soils using sequential extraction combined with flow injection (FI) hydride generation atomic fluorescence spectrometry (HG-AFS) was presented. The soils were sequentially extracted by water, 0.6 mol l-1 KH2PO4 solution, 1% (v/v) HCl solution and 1% (w/v) NaOH solution. The arsenite (As(III)) in extract was analyzed by HG-AFS in the medium of 0.1 mol l-1 citric acid solution, then the total arsenic in extract was determined by HG-AFS using on-line reduction of arsenate with L-cysteine. The concentration of arsenate (As(V)) was calculated by the difference. The optimum conditions of extraction and determination were studied in detail. The detection limit (3σ) for As(III) and As(V) were 0.11 and 0.07 μg l-1, respectively. The relative standard deviation (R.S.D.) was 1.43% (n=11) at the 10 μg l-1 As level. The method was applied in the determination of As(III) and As(V) of real soils and the recoveries of As(III) and As(V) were in the range of 89.3-118 and 80.4-111%, respectively

  1. Design, Synthesis and Anti-HIV Integrase Evaluation of 4-Oxo-4H-quinolizine-3-carboxylic Acid Derivatives

    Directory of Open Access Journals (Sweden)

    Li-Ming Hu

    2009-02-01

    Full Text Available 4-Oxo-4H-quinolizine-3-carboxylic acid derivatives bearing sulfamido, carboxylamido, benzimidazole and benzothiazole substituents have been designed and synthesized. The structures of these new compounds were confirmed by 1H-NMR, 13C- NMR, IR and ESI (or HRMS spectra. Compounds were screened for possible HIV integrase inhibitory activity.

  2. Nanobody-based chimeric receptor gene integration in Jurkat cells mediated by PhiC31 integrase

    International Nuclear Information System (INIS)

    The crucial role of T lymphocytes in anti-tumor immunity has led to the development of novel strategies that can target and activate T cells against tumor cells. Recombinant DNA technology has been used to generate non-MHC-restricted chimeric antigen receptors (CARs). Here, we constructed a panel of recombinant CAR that harbors the anti-MUC1 nanobody and the signaling and co-signaling moieties (CD3ζ/CD28) with different spacer regions derived from human IgG3 with one or two repeats of the hinge sequence or the hinge region of FcγRII. The PhiC31 integrase system was employed to investigate if the recombination efficiency could be recruited for high and stable expression of T cell chimeric receptor genes. The effect of nuclear localization signal (NLS) and two different promoters (CMV and CAG) on efficacy of PhiC31 integrase in human T cell lines was evaluated. The presence of integrase in combination with NLS, mediated up to 7.6 and 8.5 fold increases in CAR expression in ZCHN-attB and ZCHHN-attB cassette integrated T cells, respectively. Our results showed that highly efficient and stable transduction of the Jurkat cell line by PhiC31 integrase is a feasible modality for generating anti-cancer chimeric T cells for use in cancer immunotherapy.

  3. Nanobody-based chimeric receptor gene integration in Jurkat cells mediated by PhiC31 integrase

    Energy Technology Data Exchange (ETDEWEB)

    Iri-Sofla, Farnoush Jafari [Department of Medical Biotechnology, Faculty of Medical Sciences, Tarbiat Modares University, Tehran (Iran, Islamic Republic of); Rahbarizadeh, Fatemeh, E-mail: rahbarif@modares.ac.ir [Department of Medical Biotechnology, Faculty of Medical Sciences, Tarbiat Modares University, Tehran (Iran, Islamic Republic of); Ahmadvand, Davoud [Center of Pharmaceutical Nanotechnology and Nanotoxicology, Department of Pharmaceutics and Analytical Chemistry, University of Copenhagen, Universitetsparken 2, DK-2100 Copenhagen O (Denmark); Rasaee, Mohammad J. [Department of Medical Biotechnology, Faculty of Medical Sciences, Tarbiat Modares University, Tehran (Iran, Islamic Republic of)

    2011-11-01

    The crucial role of T lymphocytes in anti-tumor immunity has led to the development of novel strategies that can target and activate T cells against tumor cells. Recombinant DNA technology has been used to generate non-MHC-restricted chimeric antigen receptors (CARs). Here, we constructed a panel of recombinant CAR that harbors the anti-MUC1 nanobody and the signaling and co-signaling moieties (CD3{zeta}/CD28) with different spacer regions derived from human IgG3 with one or two repeats of the hinge sequence or the hinge region of Fc{gamma}RII. The PhiC31 integrase system was employed to investigate if the recombination efficiency could be recruited for high and stable expression of T cell chimeric receptor genes. The effect of nuclear localization signal (NLS) and two different promoters (CMV and CAG) on efficacy of PhiC31 integrase in human T cell lines was evaluated. The presence of integrase in combination with NLS, mediated up to 7.6 and 8.5 fold increases in CAR expression in ZCHN-attB and ZCHHN-attB cassette integrated T cells, respectively. Our results showed that highly efficient and stable transduction of the Jurkat cell line by PhiC31 integrase is a feasible modality for generating anti-cancer chimeric T cells for use in cancer immunotherapy.

  4. Mode of inhibition of HIV-1 Integrase by a C-terminal domain-specific monoclonal antibody*

    Directory of Open Access Journals (Sweden)

    Merkel George

    2006-06-01

    Full Text Available Abstract Background To further our understanding of the structure and function of HIV-1 integrase (IN we developed and characterized a library of monoclonal antibodies (mAbs directed against this protein. One of these antibodies, mAb33, which is specific for the C-terminal domain, was found to inhibit HIV-1 IN processing activity in vitro; a corresponding Fv fragment was able to inhibit HIV-1 integration in vivo. Our subsequent studies, using heteronuclear nuclear magnetic resonance spectroscopy, identified six solvent accessible residues on the surface of the C-terminal domain that were immobilized upon binding of the antibody, which were proposed to comprise the epitope. Here we test this hypothesis by measuring the affinity of mAb33 to HIV-1 proteins that contain Ala substitutions in each of these positions. To gain additional insight into the mode of inhibition we also measured the DNA binding capacity and enzymatic activities of the Ala substituted proteins. Results We found that Ala substitution of any one of five of the putative epitope residues, F223, R224, Y226, I267, and I268, caused a decrease in the affinity of the mAb33 for HIV-1 IN, confirming the prediction from NMR data. Although IN derivatives with Ala substitutions in or near the mAb33 epitope exhibited decreased enzymatic activity, none of the epitope substitutions compromised DNA binding to full length HIV-1 IN, as measured by surface plasmon resonance spectroscopy. Two of these derivatives, IN (I276A and IN (I267A/I268A, exhibited both increased DNA binding affinity and uncharacteristic dissociation kinetics; these proteins also exhibited non-specific nuclease activity. Results from these investigations are discussed in the context of current models for how the C-terminal domain interacts with substrate DNA. Conclusion It is unlikely that inhibition of HIV-1 IN activity by mAb33 is caused by direct interaction with residues that are essential for substrate binding. Rather

  5. Cross-Resistance Profile Determination of Two Second-Generation HIV-1 Integrase Inhibitors Using a Panel of Recombinant Viruses Derived from Raltegravir-Treated Clinical Isolates ▿

    OpenAIRE

    Van Wesenbeeck, L.; Rondelez, E.; Feyaerts, M.; Verheyen, A.; Van der Borght, K; Smits, V.; Cleybergh, C.; Wolf, H; Van Baelen, K.; Stuyver, L. J.

    2010-01-01

    The integrase inhibitor raltegravir (RAL) is currently used for the treatment of both treatment-naïve and treatment-experienced HIV-1-infected patients. Elvitegravir (EVG) is in late phases of clinical development. Since significant cross-resistance between RAL and EVG is observed, there is a need for second-generation integrase inhibitors (INIs) with a higher genetic barrier and limited cross-resistance to RAL/EVG. A panel of HIV-1 integrase recombinants, derived from plasma samples from ral...

  6. The use of artificial neural network for modelling of phycoremediation of toxic elements As(III) and As(V) from wastewater using Botryococcus braunii.

    Science.gov (United States)

    Podder, M S; Majumder, C B

    2016-02-15

    In the present study, a thorough investigation has been done on the removal efficiency of both As(III) and As (V) from synthetic wastewater by phycoremediation of Botryococcus braunii algal biomass. Artificial neural networks (ANNs) are practised for predicting % phycoremediation efficiency of both As(III) and As(V) ions. The influence of several parameters for example initial pH, inoculum size, contact time and initial arsenic concentration (either As(III) or As(V)) was examined systematically. The maximum phycoremediation of As(III) and As(V) was found to be 85.22% and 88.15% at pH9.0, equilibrium time of 144h by using algal inoculum size of 10% (v/v) and initial arsenic concentration of 50mg/L. The data acquired from laboratory scale experimental set up was utilized for training a three-layer feed-forward back propagation (BP) with Levenberg-Marquardt (LM) training algorithm having 4:5:1 architecture. A comparison between the experimental data and model outputs provided a high correlation coefficient (R(2)all_ANN equal to 0.9998) and exhibited that the model was capable for predicting the phycoremediation of both As(III) and As(V) from wastewater. The network topology was optimized by changing number of neurons in hidden layers. ANNs are efficient to model and simulate highly non-liner multivariable relationships. Absolute error and Standard deviation (SD) with respect to experimental output were calculated for ANN model outputs. The comparison of phycoremediation efficiencies of both As(III) and As(V) between experimental results and ANN model outputs exhibited that ANN model can determine the behaviour of As(III) and As(V) elimination process under various circumstances.

  7. Development of magnetic graphene oxide adsorbent for the removal and preconcentration of As(III) and As(V) species from environmental water samples.

    Science.gov (United States)

    Rashidi Nodeh, Hamid; Wan Ibrahim, Wan Aini; Ali, Imran; Sanagi, Mohd Marsin

    2016-05-01

    New-generation adsorbent, Fe3O4@SiO2/GO, was developed by modification of graphene oxide (GO) with silica-coated (SiO2) magnetic nanoparticles (Fe3O4). The synthesized adsorbent was characterized using Fourier transform infrared spectroscopy, X-ray diffractometry, energy-dispersive X-ray spectroscopy, and field emission scanning electron microscopy. The developed adsorbent was used for the removal and simultaneous preconcentration of As(III) and As(V) from environmental waters prior to ICP-MS analysis. Fe3O4@SiO2/GO provided high adsorption capacities, i.e., 7.51 and 11.46 mg g(-1) for As(III) and As(V), respectively, at pH 4.0. Adsorption isotherm, kinetic, and thermodynamic were investigated for As(III) and As(V) adsorption. Preconcentration of As(III) and As(V) were studied using magnetic solid-phase extraction (MSPE) method at pH 9.0 as the adsorbent showed selective adsorption for As(III) only in pH range 7-10. MSPE using Fe3O4@SiO2/GO was developed with good linearities (0.05-2.0 ng mL(-1)) and high coefficient of determination (R (2) = 0.9992 and 0.9985) for As(III) and As(V), respectively. The limits of detection (LODs) (3× SD/m, n = 3) obtained were 7.9 pg mL(-1) for As(III) and 28.0 pg mL(-1) for As(V). The LOD obtained is 357-1265× lower than the WHO maximum permissible limit of 10.0 ng mL(-1). The developed MSPE method showed good relative recoveries (72.55-109.71 %) and good RSDs (0.1-4.3 %, n = 3) for spring water, lake, river, and tap water samples. The new-generation adsorbent can be used for the removal and simultaneous preconcentration of As(III) and As(V) from water samples successfully. The adsorbent removal for As(III) is better than As(V).

  8. Novel 3′-Processing Integrase Activity Assay by Real-Time PCR for Screening and Identification of HIV-1 Integrase Inhibitors

    Directory of Open Access Journals (Sweden)

    Supachai Sakkhachornphop

    2015-01-01

    Full Text Available The 3′-end processing (3′P of each viral long terminal repeat (LTR during human immunodeficiency virus type-1 (HIV-1 integration is a vital step in the HIV life cycle. Blocking the 3′P using 3′P inhibitor has recently become an attractive strategy for HIV-1 therapeutic intervention. Recently, we have developed a novel real-time PCR based assay for the detection of 3′P activity in vitro. The methodology usually involves biotinylated HIV-1 LTR, HIV-1 integrase (IN, and specific primers and probe. In this novel assay, we designed the HIV-1 LTR substrate based on a sequence with a homology to HIV-1 LTR labeled at its 3′ end with biotin on the sense strand. Two nucleotides at the 3′ end were subsequently removed by IN activity. Only two nucleotides labeled biotin were captured on an avidin-coated tube; therefore, inhibiting the binding of primers and probe results in late signals in the real-time PCR. This novel assay has successfully detected both the 3′P activity of HIV-1 IN and the anti-IN activity by Raltegravir and sodium azide agent. This real-time PCR assay has been shown to be effective and inexpensive for a high-throughput screening of novel IN inhibitors.

  9. Daxx Interacts With Phage ΦBT1 Integrase and Inhibits Its Recombination%Daxx与ΦBT1相互作用并抑制其重组活性

    Institute of Scientific and Technical Information of China (English)

    王臻臻; 王然; 李文娟; 薛京伦; 陈金中

    2013-01-01

    The bacterial phage ΦBT1 integrase is a promising tool due to its site-specific transgene character.It enriches the site-specific transgenic tools and provides the possibility for multiple site-specific transgenic manipulations.To improve its safety as a vector of gene therapy,it is necessary to investigate the potential interactions between ΦBT1 and proteins in mammalian host cells.Yeast mating and co-immunoprecipitation assay indicated that a tetrapeptide 433RFAL436 in ΦBT1 integrase was responsible for ΦBT1 and Daxx interaction.It was also demonstrated that over-expression of Daxx could reduce ΦBT1 mediated recombination rate in 293T cells by using ΦBT1 report system.It is the first time to identify a cellular protein interacting with ΦBT1 integrase and inhibiting its recombination efficiency.This result might be useful for improving the ΦBT1 integrase mediated transgene methods and directing the selection of target cells for ΦBT1 integrase.%噬菌体整合酶BT1因其具有可介导转基因位点特异性整合的能力而成为了基因治疗中一种有效的工具,它丰富了转基因载体的选择,并使得多位点特异性整合成为可能.为了能够更加安全有效地利用ΦBT1整合酶作为基因治疗载体,有必要了解ΦBT1整合酶与宿主细胞内蛋白质相互作用的情况.酵母配对实验与免疫共沉淀实验揭示了ΦBT1整合酶中4个氨基酸433RFAL436对整合酶与PML-NBs蛋白Daxx的结合起到了关键作用.通过进一步构建并利用ΦBT1整合酶哺乳动物细胞报告系统,证实过表达Daxx会抑制ΦBT1整合酶在293T细胞中的重组效率.以上结果表明,细胞内的蛋白质可以与ΦBT1整合酶发生相互作用并抑制其重组活性,对于改善ΦBT1整合酶介导的转基因操作以及选择ΦBT1整合酶靶细胞方面具有重要的参考意义.

  10. Genetic Innovation in Vertebrates: Gypsy Integrase Genes and Other Genes Derived from Transposable Elements

    Directory of Open Access Journals (Sweden)

    Domitille Chalopin

    2012-01-01

    Full Text Available Due to their ability to drive DNA rearrangements and to serve as a source of new coding and regulatory sequences, transposable elements (TEs are considered as powerful evolutionary agents within genomes. In this paper, we review the mechanism of molecular domestication, which corresponds to the formation of new genes derived from TE sequences. Many genes derived from retroelements and DNA transposons have been identified in mammals and other vertebrates, some of them fulfilling essential functions for the development and survival of their host organisms. We will particularly focus on the evolution and expression of Gypsy integrase (GIN genes, which have been formed from ancient event(s of molecular domestication and have evolved differentially in some vertebrate sublineages. What we describe here is probably only the tip of the evolutionary iceberg, and future genome analyses will certainly uncover new TE-derived genes and biological functions driving genetic innovation in vertebrates and other organisms.

  11. Comparative docking and CoMFA analysis of curcumine derivatives as HIV-1 integrase inhibitors.

    Science.gov (United States)

    Gupta, Pawan; Garg, Prabha; Roy, Nilanjan

    2011-08-01

    The docking studies and comparative molecular field analysis (CoMFA) were performed on highly active molecules of curcumine derivatives against 3' processing activity of HIV-1 integrase (IN) enzyme. The optimum CoMFA model was selected with statistically significant cross-validated r(2) value of 0.815 and non-cross validated r (2) value of 0.99. The common pharmacophore of highly active molecules was used for screening of HIV-1 IN inhibitors. The high contribution of polar interactions in pharmacophore mapping is well supported by docking and CoMFA results. The results of docking, CoMFA, and pharmacophore mapping give structural insights as well as important binding features of curcumine derivatives as HIV-1 IN inhibitors which can provide guidance for the rational design of novel HIV-1 IN inhibitors. PMID:21327540

  12. Investigating the role of metal chelation in HIV-1 integrase strand transfer inhibitors.

    Science.gov (United States)

    Bacchi, Alessia; Carcelli, Mauro; Compari, Carlotta; Fisicaro, Emilia; Pala, Nicolino; Rispoli, Gabriele; Rogolino, Dominga; Sanchez, Tino W; Sechi, Mario; Sinisi, Valentina; Neamati, Nouri

    2011-12-22

    HIV-1 integrase (IN) has been validated as an attractive target for the treatment of HIV/AIDS. Several studies have confirmed that the metal binding function is a crucial feature in many of the reported IN inhibitors. To provide new insights on the metal chelating mechanism of IN inhibitors, we prepared a series of metal complexes of two ligands (HL1 and HL2), designed as representative models of the clinically used compounds raltegravir and elvitegravir. Potentiometric measurements were conducted for HL2 in the presence of Mg(II), Mn(II), Co(II), and Zn(II) in order to delineate a metal speciation model. We also determined the X-ray structures of both of the ligands and of three representative metal complexes. Our results support the hypothesis that several selective strand transfer inhibitors preferentially chelate one cation in solution and that the metal complexes can interact with the active site of the enzyme.

  13. A novel co-crystal structure affords the design of gain-of-function lentiviral integrase mutants in the presence of modified PSIP1/LEDGF/p75.

    Directory of Open Access Journals (Sweden)

    Stephen Hare

    2009-01-01

    Full Text Available Lens epithelium derived growth factor (LEDGF, also known as PC4 and SFRS1 interacting protein 1 (PSIP1 and transcriptional co-activator p75, is the cellular binding partner of lentiviral integrase (IN proteins. LEDGF accounts for the characteristic propensity of Lentivirus to integrate within active transcription units and is required for efficient viral replication. We now present a crystal structure containing the N-terminal and catalytic core domains (NTD and CCD of HIV-2 IN in complex with the IN binding domain (IBD of LEDGF. The structure extends the known IN-LEDGF interface, elucidating primarily charge-charge interactions between the NTD of IN and the IBD. A constellation of acidic residues on the NTD is characteristic of lentiviral INs, and mutations of the positively charged residues on the IBD severely affect interaction with all lentiviral INs tested. We show that the novel NTD-IBD contacts are critical for stimulation of concerted lentiviral DNA integration by LEDGF in vitro and for its function during the early steps of HIV-1 replication. Furthermore, the new structural details enabled us to engineer a mutant of HIV-1 IN that primarily functions only when presented with a complementary LEDGF mutant. These findings provide structural basis for the high affinity lentiviral IN-LEDGF interaction and pave the way for development of LEDGF-based targeting technologies for gene therapy.

  14. Development and identification of a novel anti-HIV-1 peptide derived by modification of the N-terminal domain of HIV-1 integrase

    Directory of Open Access Journals (Sweden)

    Marina eSala

    2016-06-01

    Full Text Available The viral enzyme integrase (IN is essential for the replication of human immunodeficiency virus type 1 (HIV-1 and represents an important target for the development of new antiretroviral drugs. In this study, we focused on the N-terminal domain (NTD, which is mainly involved into protein oligomerization process, for the development and synthesis of a library of overlapping peptide sequences, with specific length and specific offset covering the entire native protein sequence NTD IN 1-50. The most potent fragment, VVAKEIVAH (peptide 18, which includes a His residue instead of the natural Ser at position 39, inhibits the HIV-1 IN activity with an IC50 value of 4.5 M. Amino acid substitution analysis on this peptide revealed essential residues for activity and allowed us to identify two nonapeptides (peptides 24 and 25, that show a potency of inhibition similar to the one of peptide 18. Interestingly, peptide 18 does not interfere with the dynamic interplay between IN subunits, while peptides 24 and 25 modulated these interactions in different manners. In fact, peptide 24 inhibited the IN-IN dimerization, while peptide 25 promoted IN multimerization, with IC50 values of 32 and 4.8 µM, respectively. In addition, peptide 25 has shown to have selective anti-infective cell activity for HIV-1. These results confirmed peptide 25 as a hit for further development of new chemotherapeutic agents against HIV-1.

  15. Rapid activity prediction of HIV-1 integrase inhibitors: harnessing docking energetic components for empirical scoring by chemometric and artificial neural network approaches.

    Science.gov (United States)

    Thangsunan, Patcharapong; Kittiwachana, Sila; Meepowpan, Puttinan; Kungwan, Nawee; Prangkio, Panchika; Hannongbua, Supa; Suree, Nuttee

    2016-06-01

    Improving performance of scoring functions for drug docking simulations is a challenging task in the modern discovery pipeline. Among various ways to enhance the efficiency of scoring function, tuning of energetic component approach is an attractive option that provides better predictions. Herein we present the first development of rapid and simple tuning models for predicting and scoring inhibitory activity of investigated ligands docked into catalytic core domain structures of HIV-1 integrase (IN) enzyme. We developed the models using all energetic terms obtained from flexible ligand-rigid receptor dockings by AutoDock4, followed by a data analysis using either partial least squares (PLS) or self-organizing maps (SOMs). The models were established using 66 and 64 ligands of mercaptobenzenesulfonamides for the PLS-based and the SOMs-based inhibitory activity predictions, respectively. The models were then evaluated for their predictability quality using closely related test compounds, as well as five different unrelated inhibitor test sets. Weighting constants for each energy term were also optimized, thus customizing the scoring function for this specific target protein. Root-mean-square error (RMSE) values between the predicted and the experimental inhibitory activities were determined to be rapid weighted energy tuning methods via PLS and SOMs analyses is a viable approach to predict the potential inhibitory activity and to discriminate among small drug-like molecules to target a specific protein of interest. PMID:27314501

  16. Development and Identification of a Novel Anti-HIV-1 Peptide Derived by Modification of the N-Terminal Domain of HIV-1 Integrase

    Science.gov (United States)

    Sala, Marina; Spensiero, Antonia; Esposito, Francesca; Scala, Maria C.; Vernieri, Ermelinda; Bertamino, Alessia; Manfra, Michele; Carotenuto, Alfonso; Grieco, Paolo; Novellino, Ettore; Cadeddu, Marta; Tramontano, Enzo; Schols, Dominique; Campiglia, Pietro; Gomez-Monterrey, Isabel M.

    2016-01-01

    The viral enzyme integrase (IN) is essential for the replication of human immunodeficiency virus type 1 (HIV-1) and represents an important target for the development of new antiretroviral drugs. In this study, we focused on the N-terminal domain (NTD), which is mainly involved into protein oligomerization process, for the development and synthesis of a library of overlapping peptide sequences, with specific length and specific offset covering the entire native protein sequence NTD IN 1–50. The most potent fragment, VVAKEIVAH (peptide 18), which includes a His residue instead of the natural Ser at position 39, inhibits the HIV-1 IN activity with an IC50 value of 4.5 μM. Amino acid substitution analysis on this peptide revealed essential residues for activity and allowed us to identify two nonapeptides (peptides 24 and 25), that show a potency of inhibition similar to the one of peptide 18. Interestingly, peptide 18 does not interfere with the dynamic interplay between IN subunits, while peptides 24 and 25 modulated these interactions in different manners. In fact, peptide 24 inhibited the IN-IN dimerization, while peptide 25 promoted IN multimerization, with IC50 values of 32 and 4.8 μM, respectively. In addition, peptide 25 has shown to have selective anti-infective cell activity for HIV-1. These results confirmed peptide 25 as a hit for further development of new chemotherapeutic agents against HIV-1. PMID:27375570

  17. Impact of carbon nanotubes on the toxicity of inorganic arsenic [AS(III) and AS(V)] to Daphnia magna: The role of certain arsenic species.

    Science.gov (United States)

    Wang, Xinghao; Qu, Ruijuan; Allam, Ahmed A; Ajarem, Jamaan; Wei, Zhongbo; Wang, Zuoyao

    2016-07-01

    As a type of emerging nanomaterial, hydroxylated multiwalled carbon nanotubes (OH-MWCNTs) may interact with other pollutants in the aquatic environments and further influence their toxicity, transport, and fate. Thus, evaluation of toxicity to arsenic in the presence of CNTs needs to receive much more attention. The present study was conducted to explore the underlying mechanisms of OH-MWCNT-induced arsenic (As[III] and As[V]) toxicity changes in the aquatic organism Daphnia magna at different pH levels. The most toxic species for As(III) and As(V) to D. magna were found to be H2 AsO3 (-) and H2 AsO4 (-) . It appeared that the pH values were of greatest importance when the biological toxicity of As(III) and As(V) was compared. Furthermore, the effects of OH-MWCNTs on arsenic toxicity to D. magna indicated that the presence of OH-MWCNTs could enhance the toxicity of arsenic. The interactions of arsenic with OH-MWCNTs were further investigated by conducting adsorption experiments. The adsorption capacity of As(V) by OH-MWCNTs was found to be higher than that of As(III). To conclude, adsorption of certain arsenic species onto OH-MWCNTs is crucial for a reliable interpretation of enhanced toxicity. Environ Toxicol Chem 2016;35:1852-1859. © 2016 SETAC. PMID:26681408

  18. Nature and reactivity of layered double hydroxides formed by coprecipitating Mg, Al and As(V): Effect of arsenic concentration, pH, and aging.

    Science.gov (United States)

    Sommella, Alessia; Caporale, Antonio G; Denecke, Melissa A; Mangold, Stefan; Pigna, Massimo; Santoro, Anna; Terzano, Roberto; Violante, Antonio

    2015-12-30

    Arsenic (As) co-precipitation is one of the major processes controlling As solubility in soils and waters. When As is co-precipitated with Al and Mg, the possible formation of layered double hydroxides (LDHs) and other nanocomposites can stabilize As in their structures thus making this toxic element less available. We investigated the nature and reactivity of Mg-Al-arsenate [As(V)] co-precipitated LDHs formed in solution affected by As concentration, pH, and aging. At the beginning of the co-precipitation process, poorly crystalline LDH and non-crystalline Al(Mg)-oxides form. Prolonged aging of the samples promotes crystallization of LDHs, evidenced by an increase in As K XANES intensities and XRD peak intensities. During aging Al- and/or Mg-oxides are likely transformed by dissolution/re-precipitation processes into more crystalline but still defective LDHs. Surface area, chemical composition, reactivity of the precipitates, and anion exchange properties of As(V) in the co-precipitates are influenced by pH, aging, and As concentration. This study demonstrates that (i) As(V) retards or inhibits the formation and transformation of LDHs and (ii) more As(V) is removed from solution if co-precipitated with Mg and Al than by sorption onto well crystallized LDHs. PMID:26241870

  19. Toxicity of As(III and As(V on morphological traits and pigments of Gram Seed (Cicer arietinum during germination and early seedling growth

    Directory of Open Access Journals (Sweden)

    Naba Kumar Mondal

    2016-03-01

    Full Text Available Arsenic (As contamination is an important environmental consequence in some part of India and other countries. In this study, we investigated that individual phytotoxicity of both As(III and As(V on Cicer arietinum. Total five different concentrations (0, 2, 4, 6, 8, and 10 mg/L of both As(III and As(V have been considered for this study. Results indicate that percent germination significantly (p < 0.05 reduced with increasing concentration of both As(III and As(V. However, As(III showed pronounced effects than As(V. The variation of root and shoot length was equally affected by arsenite, where as arsenate showed higher negative impact on shoot length than root length up to the concentration 6 mg/L. However, arsenate concentration greater than 6 mg/L showed opposite trend of variation in root and shoot length. The pigment (Chlorophyll ‘a’, ‘b’ and total chlorophylls and carotenoids level also gradually decrease with increasing concentration of both forms of arsenic.

  20. Speciation of AsIII and AsV in fruit juices by dispersive liquid–liquid microextraction and hydride generation-atomic fluorescence spectrometry

    Science.gov (United States)

    A new procedure was developed to speciate and quantify As(III) and As(V) in fruit juices. At pH 3.0, As(III) and ammonium pyrrolidine dithiocarbamate (APDC) formed a complex, which was extracted into carbon tetrachloride by dispersive liquid–liquid microextraction (DLLME) and subsequently quantified...

  1. Stability study of As(III), As(V), MMA and DMA by anion exchange chromatography and HG-AFS in wastewater samples

    Energy Technology Data Exchange (ETDEWEB)

    Segura, Marta; Munoz, Juan; Madrid, Yolanda; Camara, Carmen [Departamento de Quimica Analitica, Facultad de Ciencias Quimicas, Universidad Complutense, 28040 Madrid (Spain)

    2002-10-01

    The stability of arsenic species {l_brace}arsenate [As(V)], monomethylarsonate [MMA], dimethylarsinate [DMA] and arsenite [As(III)]{r_brace} in two types of urban wastewater samples (raw and treated) was evaluated. Water samples containing a mixture of the different arsenic species were stored in the absence of light at three different temperatures: +4 C, +20 C and +40 C. At regular time intervals, arsenic species were determined by high performance liquid chromatography (HPLC)-hydride generation (HG)-atomic fluorescence spectrometry (AFS). The experimental conditions for the separation of arsenic species by HPLC and their determination by AFS were directly optimised from wastewater samples. As(III), As(V), MMA and DMA were separated on an anion exchange column using phosphate buffer (pH 6.0) as the mobile phase. Under these conditions the four arsenic species were separated in less than 10 min. The detection limits were 0.6, 0.9, 0.9 and 1.8 {mu}g L{sup -1} for As(III), DMA, MMA and As(V), respectively. As(V), MMA and DMA were found stable in the two types of urban wastewater samples over the 4-month period at the three different temperatures tested, while the concentration of As(III) in raw wastewater sample decreased after 2 weeks of storage. A greater stability of As(III) was found in the treated urban wastewater sample. As(III) remained unaltered in this matrix at pH 7.27 over the period studied, while at lower pH (1.6) losses of As(III) were detected after 1 month of storage. The results show that the decrease in As(III) concentration with time was accompanied by an increase in As(V) concentration. (orig.)

  2. Development, characterization and evaluation of iron-coated honeycomb briquette cinders for the removal of As(V from aqueous solutions

    Directory of Open Access Journals (Sweden)

    Tiantian Sheng

    2014-01-01

    Full Text Available The adsorptive removal of As(V from aqueous solutions using iron-coated honeycomb briquette cinder (Fe-HBC is presented. Low cost mechanical granulation process was integrated with surface amendment technology to prepare iron-oxide modified granular adsorbent for clean water production. Detailed characterizations were performed using FTIR, XRD, EDS and SEM techniques. Operating parameters including initial As(V concentration, pH, contact time, adsorbent dose, iron leaching and the effects of competing ions on As(V removal were evaluated. Results demonstrated that high amount of arsenate (961.5 μg g−1 was adsorbed at pH 7.5 in 14 h contact time. Langmuir, Freundlich and Temkin isotherm models were used to analyze the adsorption data, whereas Langmuir model was found to best represent the data with a correlation co-efficient (R2 = 0.999. Thus, As(V sorption on Fe-HBC surface suggested monolayer adsorption and indicated surface homogeneity. Moreover, the dimensionless parameter (RL value calculated to be about 0.118 that reiterated the process is favorable and spontaneous. The influences of competing ions on As(V removal decreased in the following order:PO43−>HCO3−>F−>Cl−. The profound inhibition effects ofPO43− revealed a high affinity toward iron(oxy hydroxide. Life-cycle assessment confirmed that spent HBC is non-hazardous and can be used as a promising sorbent for arsenic removal.

  3. A New Large-DNA-Fragment Delivery System Based on Integrase Activity from an Integrative and Conjugative Element

    OpenAIRE

    Miyazaki, Ryo; van der Meer, Jan Roelof

    2013-01-01

    During the past few decades, numerous plasmid vectors have been developed for cloning, gene expression analysis, and genetic engineering. Cloning procedures typically rely on PCR amplification, DNA fragment restriction digestion, recovery, and ligation, but increasingly, procedures are being developed to assemble large synthetic DNAs. In this study, we developed a new gene delivery system using the integrase activity of an integrative and conjugative element (ICE). The advantage of the integr...

  4. Pharmacovirological Impact of an Integrase Inhibitor on Human Immunodeficiency Virus Type 1 cDNA Species In Vivo ▿

    OpenAIRE

    Goffinet, Christine; Allespach, Ina; Oberbremer, Lena; Golden, Pamela L.; Foster, Scott A.; Johns, Brian A.; Weatherhead, Jason G.; Novick, Steven J.; Chiswell, Karen E.; Garvey, Edward P.; Keppler, Oliver T.

    2009-01-01

    Clinical trials of the first approved integrase inhibitor (INI), raltegravir, have demonstrated a drop in the human immunodeficiency virus type 1 (HIV-1) RNA loads of infected patients that was unexpectedly more rapid than that with a potent reverse transcriptase inhibitor, and apparently dose independent. These clinical outcomes are not understood. In tissue culture, although their inhibition of integration is well documented, the effects of INIs on levels of unintegrated HIV-1 cDNAs have be...

  5. A Novel Strategy to Assemble the β-Diketo Acid Pharmacophore of HIV Integrase Inhibitors on Purine Nucleobase Scaffolds

    OpenAIRE

    Uchil, Vinod; Seo, Byung; Nair, Vasu

    2007-01-01

    Claisen condensation, the key step in constructing the pharmacophore of aryl β-diketo acids (DKA) as integrase inhibitors, fails in certain cases of highly electron-deficient heterocycles such as purines. A general synthetic strategy to assemble the DKA motif on the purine scaffold has been accomplished. The synthetic sequence entails a palladium-catalyzed cross-coupling, a C-acylation involving a tandem addition/elimination reaction and a novel ferric ion-catalyzed selective hydrolysis of an...

  6. Diketo acid inhibitor mechanism and HIV-1 integrase: Implications for metal binding in the active site of phosphotransferase enzymes

    OpenAIRE

    Grobler, Jay A.; Stillmock, Kara; Hu, Binghua; Witmer, Marc; Felock, Peter; Espeseth, Amy S.; Wolfe, Abigail; Egbertson, Melissa; Bourgeois, Michele; Melamed, Jeffrey; Wai, John S.; Young, Steve; Vacca, Joseph; Hazuda, Daria J.

    2002-01-01

    The process of integrating the reverse-transcribed HIV-1 DNA into the host chromosomal DNA is catalyzed by the virally encoded enzyme integrase (IN). Integration requires two metal-dependent reactions, 3′ end processing and strand transfer. Compounds that contain a diketo acid moiety have been shown to selectively inhibit the strand transfer reaction of IN in vitro and in infected cells and are effective as inhibitors of HIV-1 replication. To characterize the molecular basis of inhibition, we...

  7. A Profile of Native Integration Sites Used by φC31 Integrase in the Bovine Genome

    Institute of Scientific and Technical Information of China (English)

    Lijuan Qu; Qingwen Ma; Zaiwei Zhou; Haiyan Ma; Ying Huang; Shuzhen Huang; Fanyi Zeng; Yitao Zeng

    2012-01-01

    The Streptomyces phage φC31 integrase can efficiently target attB-beadng transgenes to endogenous pseudo attP sites within mammalian genomes.To better understand the activity of φC31 integrase in the bovine genome,DNA sequences of 44 integration events were analyzed,and 32 pseudo attP sites were identified.The majority of these sites share a sequence motif that contains inverted repeats and has similarities to wild-type attP site.Genomic DNA flanking these sites typically contained repetitive sequence elements,such as short and long interspersed repetitive elements.These sequence features indicate that DNA sequence recognition plays an important role in guiding φC31-mediated site-specific integration.In addition,BF27 integration hotspot sites were identified in the bovine genome,which accounted for 13.6% of all isolated integration events and mapped to an intron of the deleted in liver cancer 1 (DLC1) gene.Also we found that the pseudo attP sites in the bovine genome had other features in common with those in the human genome.This study represents the first time that the sequence features of pseudo attP sites in the bovine genome were analyzed.We conclude that this sitespecific integrase system has great potential for applied modifications of the bovine genome.

  8. Secondary mutations in viruses resistant to HIV-1 integrase inhibitors that restore viral infectivity and replication kinetics.

    Science.gov (United States)

    Nakahara, Koichiro; Wakasa-Morimoto, Chiaki; Kobayashi, Masanori; Miki, Shigeru; Noshi, Takeshi; Seki, Takahiro; Kanamori-Koyama, Mikiko; Kawauchi, Shinobu; Suyama, Akemi; Fujishita, Toshio; Yoshinaga, Tomokazu; Garvey, Edward P; Johns, Brian A; Foster, Scott A; Underwood, Mark R; Sato, Akihiko; Fujiwara, Tamio

    2009-02-01

    Passage of HIV-1 in the presence of integrase inhibitors (INIs) generates resistant viruses that have mutations in the integrase region. Integrase-resistant mutations Q148K and Q148R were identified as primary mutations with the passage of HIV-1 IIIB in the presence of INIs S-1360 or S/GSK-364735, respectively. Secondary amino acid substitutions E138K or G140S were observed when passage with INI was continued. The role of these mutations was investigated with molecular clones. Relative to Q148K alone, Q148K/E138K had 2- and >6-fold increases in resistance to S-1360 and S/GSK-364735, respectively, and the double mutant had slightly better infectivity and replication kinetics. In contrast, Q148K/G140S and Q148R/E138K had nearly equivalent or slightly reduced fold resistance to the INI compared with their respective Q148 primary mutants, and had increases in infectivity and replication kinetics. Recovery of these surrogates of viral fitness coincided with the recovery of integration efficiency of viral DNA into the host cell chromosome for these double mutants. These data show that recovery of viral integration efficiency can be an important factor for the emergence and maintenance of INI-resistant mutations. PMID:19027039

  9. Secondary mutations in viruses resistant to HIV-1 integrase inhibitors that restore viral infectivity and replication kinetics.

    Science.gov (United States)

    Nakahara, Koichiro; Wakasa-Morimoto, Chiaki; Kobayashi, Masanori; Miki, Shigeru; Noshi, Takeshi; Seki, Takahiro; Kanamori-Koyama, Mikiko; Kawauchi, Shinobu; Suyama, Akemi; Fujishita, Toshio; Yoshinaga, Tomokazu; Garvey, Edward P; Johns, Brian A; Foster, Scott A; Underwood, Mark R; Sato, Akihiko; Fujiwara, Tamio

    2009-02-01

    Passage of HIV-1 in the presence of integrase inhibitors (INIs) generates resistant viruses that have mutations in the integrase region. Integrase-resistant mutations Q148K and Q148R were identified as primary mutations with the passage of HIV-1 IIIB in the presence of INIs S-1360 or S/GSK-364735, respectively. Secondary amino acid substitutions E138K or G140S were observed when passage with INI was continued. The role of these mutations was investigated with molecular clones. Relative to Q148K alone, Q148K/E138K had 2- and >6-fold increases in resistance to S-1360 and S/GSK-364735, respectively, and the double mutant had slightly better infectivity and replication kinetics. In contrast, Q148K/G140S and Q148R/E138K had nearly equivalent or slightly reduced fold resistance to the INI compared with their respective Q148 primary mutants, and had increases in infectivity and replication kinetics. Recovery of these surrogates of viral fitness coincided with the recovery of integration efficiency of viral DNA into the host cell chromosome for these double mutants. These data show that recovery of viral integration efficiency can be an important factor for the emergence and maintenance of INI-resistant mutations.

  10. Creation of Tenecteplase-Producing CHO Cell Line Using Site-Specific Integrase from the Phage φC31

    Directory of Open Access Journals (Sweden)

    Khadijeh Karbalaie

    2010-01-01

    Full Text Available Objective: The aim of this study was to produce a stable CHO cell line expressing tenecteplase.Materials and Methods: In the first step, the tenecteplase coding sequence was clonedin a pDB2 vector containing attB recognition sites for the phage φC31 integrase. Then,using lipofection, the CHO cells were co-transfected with constructed recombinant plasmidencoding tenecteplase and attB recognition sites and the integrase coding sequencecontaining pCMV-Int plasmid. As the recombinant plasmid contained the neomycin resistancegene (neo, stable cells were then selected using G418 as an antibiotic. Stabletransformed cells were assessed using genomic PCR and RT-PCR. Finally, the functionalityof tenecteplase was evaluated on the cell culture media.Results: our results indicated that tenecteplase coding sequence was inserted into theCHO cell genome and was successfully expressed. Moreover, tenecteplase activity assessmentindicated the presence of our functional tenecteplase in the cell culture medium.Conclusion: Considering the data obtained from this study, φC31 integrase can be usedfor the production of a stable cell line and it be used to introduce ectopic genes into mammaliancells.

  11. Impact of hydrodynamic injection and phiC31 integrase on tumor latency in a mouse model of MYC-induced hepatocellular carcinoma.

    Directory of Open Access Journals (Sweden)

    Lauren E Woodard

    Full Text Available BACKGROUND: Hydrodynamic injection is an effective method for DNA delivery in mouse liver and is being translated to larger animals for possible clinical use. Similarly, phiC31 integrase has proven effective in mediating long-term gene therapy in mice when delivered by hydrodynamic injection and is being considered for clinical gene therapy applications. However, chromosomal aberrations have been associated with phiC31 integrase expression in tissue culture, leading to questions about safety. METHODOLOGY/PRINCIPAL FINDINGS: To study whether hydrodynamic delivery alone, or in conjunction with delivery of phiC31 integrase for long-term transgene expression, could facilitate tumor formation, we used a transgenic mouse model in which sustained induction of the human C-MYC oncogene in the liver was followed by hydrodynamic injection. Without injection, mice had a median tumor latency of 154 days. With hydrodynamic injection of saline alone, the median tumor latency was significantly reduced, to 105 days. The median tumor latency was similar, 106 days, when a luciferase donor plasmid and backbone plasmid without integrase were administered. In contrast, when active or inactive phiC31 integrase and donor plasmid were supplied to the mouse liver, the median tumor latency was 153 days, similar to mice receiving no injection. CONCLUSIONS/SIGNIFICANCE: Our data suggest that phiC31 integrase does not facilitate tumor formation in this C-MYC transgenic mouse model. However, in groups lacking phiC31 integrase, hydrodynamic injection appeared to contribute to C-MYC-induced hepatocellular carcinoma in adult mice. Although it remains to be seen to what extent these findings may be extrapolated to catheter-mediated hydrodynamic delivery in larger species, they suggest that caution should be used during translation of hydrodynamic injection to clinical applications.

  12. Removal and co-transport of Zn, As(V), and Cd during leachate seepage through downgradient mine soils: A batch sorption and column study.

    Science.gov (United States)

    Kim, Juhee; Nam, Seung Mo; Hyun, Seunghun

    2016-05-01

    The removal of Zn, As(V), and Cd during the leachate seepage process was measured in single, binary, and ternary solute systems by batch sorption and 1-D column flow experiments, followed by a sequential extraction procedure (SEP). In single-solute systems, sorption (Kd(⁎)) occurred in the order of As(V)>Zn≫Cd, and this sequence did not change in the presence of other solutes. In multi-solute systems, the sorption of Zn (~20%) and Cd (~27%) was enhanced by As(V), while Zn and Cd suppressed the sorption of each other. In all cases, As(V) sorption was not affected by the cations, indicating that As(V) is prioritized by sorption sites to a much greater degree than Zn and Cd. Element retention by column soils was strongly correlated (r(2)=0.77) with Kd(⁎). Across column segments, mass retention was in the order of inlet (36-54%)>middle (26-35%)>outlet (20-31%), except for Cd in the Zn-Cd binary system. The result of SEP revealed that most of the retained Cd (98-99%) and Zn (56-71%) was in the labile fraction (e.g., the sum of F1 and F2) while only 9-12% of As(V) was labile and most (>55%) was specifically adsorbed to Fe/Al oxides. Plots of the labile fraction (f(labile)) and the fast sorption fraction (f(fast)) suggested that the kinetics of specific As(V) sorption occur rapidly (f(fast)>f(labile)), whereas labile Zn and Cd sorption occurs slowly (f(labile)>f(fast)), indicating the occurrence of kinetically limited labile sorption sites, probably due to Zn-Cd competition. In conclusion, the element leaching potential of mine leachate can be greatly attenuated during downgradient soil seepage. However, when assessing the soil attenuation process, the impact of sorption competitors and the lability of adsorbed elements should first be considered. PMID:26896586

  13. Removal and co-transport of Zn, As(V), and Cd during leachate seepage through downgradient mine soils: A batch sorption and column study.

    Science.gov (United States)

    Kim, Juhee; Nam, Seung Mo; Hyun, Seunghun

    2016-05-01

    The removal of Zn, As(V), and Cd during the leachate seepage process was measured in single, binary, and ternary solute systems by batch sorption and 1-D column flow experiments, followed by a sequential extraction procedure (SEP). In single-solute systems, sorption (Kd(⁎)) occurred in the order of As(V)>Zn≫Cd, and this sequence did not change in the presence of other solutes. In multi-solute systems, the sorption of Zn (~20%) and Cd (~27%) was enhanced by As(V), while Zn and Cd suppressed the sorption of each other. In all cases, As(V) sorption was not affected by the cations, indicating that As(V) is prioritized by sorption sites to a much greater degree than Zn and Cd. Element retention by column soils was strongly correlated (r(2)=0.77) with Kd(⁎). Across column segments, mass retention was in the order of inlet (36-54%)>middle (26-35%)>outlet (20-31%), except for Cd in the Zn-Cd binary system. The result of SEP revealed that most of the retained Cd (98-99%) and Zn (56-71%) was in the labile fraction (e.g., the sum of F1 and F2) while only 9-12% of As(V) was labile and most (>55%) was specifically adsorbed to Fe/Al oxides. Plots of the labile fraction (f(labile)) and the fast sorption fraction (f(fast)) suggested that the kinetics of specific As(V) sorption occur rapidly (f(fast)>f(labile)), whereas labile Zn and Cd sorption occurs slowly (f(labile)>f(fast)), indicating the occurrence of kinetically limited labile sorption sites, probably due to Zn-Cd competition. In conclusion, the element leaching potential of mine leachate can be greatly attenuated during downgradient soil seepage. However, when assessing the soil attenuation process, the impact of sorption competitors and the lability of adsorbed elements should first be considered.

  14. Determination of traces of Sb(III) using ASV in Sb-rich water samples affected by mining

    Energy Technology Data Exchange (ETDEWEB)

    Cidu, Rosa, E-mail: cidur@unica.it; Biddau, Riccardo; Dore, Elisabetta

    2015-01-07

    Highlights: • Antimony speciation affects the toxicity of this element. • A simple method for Sb(III) analyses in Sb-rich waters was developed. • Sb(III) was determined by ASV in water stabilized with tartaric and nitric acids. • Pre-concentration and/or separation of Sb(III) prior to analysis are not required. - Abstract: Chemical speciation [Sb(V) and Sb(III)] affects the mobility, bioavailability and toxicity of antimony. In oxygenated environments Sb(V) dominates whereas thermodynamically unstable Sb(III) may occur. In this study, a simple method for the determination of Sb(III) in non acidic, oxygenated water contaminated with antimony is proposed. The determination of Sb(III) was performed by anodic stripping voltammetry (ASV, 1–20 μg L{sup −1} working range), the total antimony, Sb(tot), was determined either by inductively coupled plasma mass spectrometry (ICP-MS, 1–100 μg L{sup −1} working range) or inductively coupled plasma optical emission spectrometry (ICP-OES, 100–10,000 μg L{sup −1} working range) depending on concentration. Water samples were filtered on site through 0.45 μm pore size filters. The aliquot for determination of Sb(tot) was acidified with 1% (v/v) HNO{sub 3}. Different preservatives, namely HCl, L(+) ascorbic acid or L(+) tartaric acid plus HNO{sub 3}, were used to assess the stability of Sb(III) in synthetic solutions. The method was tested on groundwater and surface water draining the abandoned mine of Su Suergiu (Sardinia, Italy), an area heavily contaminated with Sb. The waters interacting with Sb-rich mining residues were non acidic, oxygenated, and showed extreme concentrations of Sb(tot) (up to 13,000 μg L{sup −1}), with Sb(III) <10% of total antimony. The stabilization with L(+) tartaric acid plus HNO{sub 3} appears useful for the determination of Sb(III) in oxygenated, Sb-rich waters. Due to the instability of Sb(III), analyses should be carried out within 7 days upon the water collection. The main

  15. Porcine endogenous retrovirus-A/C: biochemical properties of its integrase and susceptibility to raltegravir.

    Science.gov (United States)

    Demange, Antonin; Yajjou-Hamalian, Halima; Gallay, Kathy; Luengo, Catherine; Beven, Véronique; Leroux, Aurélie; Confort, Marie-Pierre; Al Andary, Elsy; Gouet, Patrice; Moreau, Karen; Ronfort, Corinne; Blanchard, Yannick

    2015-10-01

    Porcine endogenous retroviruses (PERVs) are present in the genomes of pig cells. The PERV-A/C recombinant virus can infect human cells and is a major risk of zoonotic disease in the case of xenotransplantation of pig organs to humans. Raltegravir (RAL) is a viral integrase (IN) inhibitor used in highly active antiretroviral treatment. In the present study, we explored the potential use of RAL against PERV-A/C. We report (i) a three-dimensional model of the PERV-A/C intasome complexed with RAL, (ii) the sensitivity of PERV-A/C IN to RAL in vitro and (iii) the sensitivity of a PERV-A/C-IRES-GFP recombinant virus to RAL in cellulo. We demonstrated that RAL is a potent inhibitor against PERV-A/C IN and PERV-A/C replication with IC50s in the nanomolar range. To date, the use of retroviral inhibitors remains the only way to control the risk of zoonotic PERV infection during pig-to-human xenotransplantation. PMID:26296914

  16. Selection of diverse and clinically relevant integrase inhibitor-resistant human immunodeficiency virus type 1 mutants.

    Science.gov (United States)

    Kobayashi, Masanori; Nakahara, Koichiro; Seki, Takahiro; Miki, Shigeru; Kawauchi, Shinobu; Suyama, Akemi; Wakasa-Morimoto, Chiaki; Kodama, Makoto; Endoh, Takeshi; Oosugi, Eiichi; Matsushita, Yoshihiro; Murai, Hitoshi; Fujishita, Toshio; Yoshinaga, Tomokazu; Garvey, Edward; Foster, Scott; Underwood, Mark; Johns, Brian; Sato, Akihiko; Fujiwara, Tamio

    2008-11-01

    Resistance passage studies were conducted with five INIs (integrase inhibitors) that have been tested in clinical trials to date: a new naphthyridinone-type INI S/GSK-364735, raltegravir, elvitegravir, L-870,810 and S-1360. In establishing the passage system and starting from concentrations several fold above the EC(50) value, resistance mutations against S-1360 and related diketoacid-type compounds could be isolated from infected MT-2 cell cultures from day 14 to 28. Q148R and F121Y were the two main pathways of resistance to S/GSK-364735. Q148R/K and N155H, which were found in patients failing raltegravir treatment in Phase IIb studies, were observed during passage with raltegravir with this method. The fold resistance of 40 mutant molecular clones versus wild type virus was compared with these five INIs. The overall resistance pattern of S/GSK-364735 was similar to that of raltegravir and other INIs. However, different fold resistances of particular mutations were noted among different INIs, reflecting a potential to develop INIs with distinctly different resistant profiles. PMID:18625269

  17. Methods for the Analyses of Inhibitor-Induced Aberrant Multimerization of HIV-1 Integrase

    Science.gov (United States)

    Kessl, Jacques J.; Sharma, Amit; Kvaratskhelia, Mamuka

    2016-01-01

    HIV-1 integrase (IN) is an important therapeutic target as its function is essential for the viral lifecycle. The discovery of multifunctional allosteric IN inhibitors or ALLINIs, which potently impair viral replication by promoting aberrant, higher order IN multimerization as well as inhibit IN interactions with its cellular cofactor, LEDGF/p75, has opened new venues to exploit IN multimerization as a therapeutic target. Furthermore, the recent discovery of multimerization selective IN inhibitors or MINIs, has provided new investigational probes to study the direct effects of aberrant IN multimerization in vitro and in infected cells. Here we describe three complementary methods designed to detect and quantify the effects of these new classes of inhibitors on IN multimerization. These methods include a homogenous time-resolved fluorescence-based assay which allows for measuring EC50 values for the inhibitor-induced aberrant IN multimerization, a dynamic light scattering-based assay which allows for monitoring the formation and sizes of oligomeric IN particles in a time-dependent manner, and a chemical cross-linking-based assay of interacting IN subunits which allows for the determination of IN oligomers in viral particles. PMID:26714710

  18. HIV integrase variability and genetic barrier in antiretroviral naïve and experienced patients

    Directory of Open Access Journals (Sweden)

    Comolli Giuditta

    2011-03-01

    Full Text Available Abstract Background HIV-1 integrase (IN variability in treatment naïve patients with different HIV-1 subtypes is a major issue. In fact, the effect of previous exposure to antiretrovirals other than IN inhibitors (INI on IN variability has not been satisfactorily defined. In addition, the genetic barrier for specific INI resistance mutations remains to be calculated. Methods IN variability was analyzed and compared with reverse transcriptase (RT and protease (PR variability in 41 treatment naïve and 54 RT inhibitor (RTI and protease inhibitor (PRI experienced patients from subjects infected with subtype B and non-B strains. In addition, four HIV-2 strains were analyzed in parallel. Frequency and distribution of IN mutations were compared between HAART-naïve and RTI/PI-experienced patients; the genetic barrier for 27 amino acid positions related to INI susceptibility was calculated as well. Results Primary mutations associated with resistance to INI were not detected in patients not previously treated with this class of drug. However, some secondary mutations which have been shown to contribute to INI resistance were found. Only limited differences in codon usage distribution between patient groups were found. HIV-2 strains from INI naïve patients showed the presence of both primary and secondary resistance mutations. Conclusion Exposure to antivirals other than INI does not seem to significantly influence the emergence of mutations implicated in INI resistance. HIV-2 strain might have reduced susceptibility to INI.

  19. Selection of diverse and clinically relevant integrase inhibitor-resistant human immunodeficiency virus type 1 mutants.

    Science.gov (United States)

    Kobayashi, Masanori; Nakahara, Koichiro; Seki, Takahiro; Miki, Shigeru; Kawauchi, Shinobu; Suyama, Akemi; Wakasa-Morimoto, Chiaki; Kodama, Makoto; Endoh, Takeshi; Oosugi, Eiichi; Matsushita, Yoshihiro; Murai, Hitoshi; Fujishita, Toshio; Yoshinaga, Tomokazu; Garvey, Edward; Foster, Scott; Underwood, Mark; Johns, Brian; Sato, Akihiko; Fujiwara, Tamio

    2008-11-01

    Resistance passage studies were conducted with five INIs (integrase inhibitors) that have been tested in clinical trials to date: a new naphthyridinone-type INI S/GSK-364735, raltegravir, elvitegravir, L-870,810 and S-1360. In establishing the passage system and starting from concentrations several fold above the EC(50) value, resistance mutations against S-1360 and related diketoacid-type compounds could be isolated from infected MT-2 cell cultures from day 14 to 28. Q148R and F121Y were the two main pathways of resistance to S/GSK-364735. Q148R/K and N155H, which were found in patients failing raltegravir treatment in Phase IIb studies, were observed during passage with raltegravir with this method. The fold resistance of 40 mutant molecular clones versus wild type virus was compared with these five INIs. The overall resistance pattern of S/GSK-364735 was similar to that of raltegravir and other INIs. However, different fold resistances of particular mutations were noted among different INIs, reflecting a potential to develop INIs with distinctly different resistant profiles.

  20. Class 1 integrase, sulfonamide and tetracycline resistance genes in wastewater treatment plant and surface water.

    Science.gov (United States)

    Makowska, Nicoletta; Koczura, Ryszard; Mokracka, Joanna

    2016-02-01

    Wastewater treatment plants are considered hot spots for multiplication and dissemination of antibiotic-resistant bacteria and resistance genes. In this study, we determined the presence of class 1 integron integrase and genes conferring resistance to tetracyclines and sulfonamides in the genomes of culturable bacteria isolated from a wastewater treatment plant and the river that receives the treated wastewater. Moreover, using PCR-based metagenomic approach, we quantified intI1, tet and sul genes. Wastewater treatment caused the decrease in the total number of culturable heterotrophs and bacteria resistant to tetracycline and sulfonamides, along with the decrease in the number of intI1, sul and tet gene copies per ml, with significant reduction of tet(B). On the other hand, the treatment process increased both the frequency of tetracycline- and sulfonamide-resistant bacteria and intI1-positive strains, and the relative abundance of all quantified antibiotic resistance genes (ARGs) and intI1 gene; in the case of tet(A) and sul2 significantly. The discharge of treated wastewater increased the number of intI1, tet and sul genes in the receiving river water both in terms of copy number per ml and relative abundance. Hence, despite the reduction of the number of ARGs and ARBs, wastewater treatment selects for bacteria with ARGs in effluent.

  1. Study on the drug resistance and the binding mode of HIV-1 integrase with LCA inhibitor

    Institute of Scientific and Technical Information of China (English)

    HU; JianPing; CHANG; Shan; CHEN; WeiZu; WANG; CunXin

    2007-01-01

    Human immunodeficiency virus type 1 (HIV-1) integrase (IN) is an essential enzyme in the lifecycle of this virus and also an important target for the study of anti-HIV drugs. The binding mode of the wild type IN core domain and its G140S mutant with L-Chicoric acid (LCA) inhibitor were investigated by using multiple conformation molecular docking and molecular dynamics (MD) simulation. Based on the binding modes, the drug resistance mechanism was explored for the G140S mutant of IN with LCA. The results indicate that the binding site of the G140S mutant of IN core domain with LCA is different from that of the core domain of the wild type IN, which leads to the partial loss of inhibition potency of LCA. The flexibility of the IN functional loop region and the interactions between Mg2+ ion and the three key residues (i.e., D64, D116, E152) stimulate the biological operation of IN. The drug resistance also lies in several other important effects, such as the repulsion between LCA and E152 in the G140S mutant core domain, the weakening of K159 binding with LCA and Y143 pointing to the pocket of the G140S mutant. All of the above simulation results agree well with experimental data, which provide us with some helpful information for designing the drug of anti-HIV based on the structure of IN.

  2. Porcine endogenous retrovirus-A/C: biochemical properties of its integrase and susceptibility to raltegravir.

    Science.gov (United States)

    Demange, Antonin; Yajjou-Hamalian, Halima; Gallay, Kathy; Luengo, Catherine; Beven, Véronique; Leroux, Aurélie; Confort, Marie-Pierre; Al Andary, Elsy; Gouet, Patrice; Moreau, Karen; Ronfort, Corinne; Blanchard, Yannick

    2015-10-01

    Porcine endogenous retroviruses (PERVs) are present in the genomes of pig cells. The PERV-A/C recombinant virus can infect human cells and is a major risk of zoonotic disease in the case of xenotransplantation of pig organs to humans. Raltegravir (RAL) is a viral integrase (IN) inhibitor used in highly active antiretroviral treatment. In the present study, we explored the potential use of RAL against PERV-A/C. We report (i) a three-dimensional model of the PERV-A/C intasome complexed with RAL, (ii) the sensitivity of PERV-A/C IN to RAL in vitro and (iii) the sensitivity of a PERV-A/C-IRES-GFP recombinant virus to RAL in cellulo. We demonstrated that RAL is a potent inhibitor against PERV-A/C IN and PERV-A/C replication with IC50s in the nanomolar range. To date, the use of retroviral inhibitors remains the only way to control the risk of zoonotic PERV infection during pig-to-human xenotransplantation.

  3. Sequestering of As(III) and As(V) from wastewater using a novel neem leaves/MnFe2O4 composite biosorbent.

    Science.gov (United States)

    Podder, M S; Majumder, C B

    2016-12-01

    An arsenic biosorbent comprising neem leaves (NL) and MnFe2O4 particles was developed and its removal potential was investigated. Physicochemical analysis of the NL/MnFe2O4 composite (MNL) was performed for the Brunauer, Emmett and Teller surface area, Fourier transform infrared spectra (FT-IR), and scanning electron microscopy-Energy-dispersive X-ray (EDX). The following parameters were optimized: pH, biosorbent dose, contact time, temperature, and initial arsenic concentration. The optimum pH values achieved for biosorption of As(III) and As(V) were 7.0 and 4.0, respectively, when the equilibrium time was 110 minutes for both. MNL was found to be efficient with 85.217% and 88.154% biosorption efficiency at a concentration of 50 mg/L of As(III) or As(V) solution, respectively. This was also proved by the FT-IR study of arsenic-loaded biosorbent. For establishing the best suitable correlation for the equilibrium curves exploiting the procedure of the nonlinear regression for curve fitting analysis, isotherm studies were conducted for As(III) and As(V) using 30 isotherm models. The pattern of biosorption fitted well with Brouers-Sotolongo isotherm model for As(III) and Langmuir-Freundlich as well as Sips isotherm models for As(V). Dubinin-Radushkevich (D-R) isotherm studies specified that ion exchange might play a significant role. The influence of various co-existing ions at different concentrations was examined. Desorption study was performed using various concentrations of NaOH solution.

  4. Ukrainas un Sīrijas konfliktu loma ASV Āzijas Klusā okeāna reģiona politikā

    OpenAIRE

    Skladovs, Salvis

    2016-01-01

    Krievijas agresija Ukrainā un Sīrijas pilsoņu kara eskalācija, tajā iesaistoties Daesh, aktualizēja ASV globālo lomu, īpaši Eiropā un Tuvajos Austrumos. ASV tobrīd par stratēģisko prioritāti bija izsludinājusi Āzijas Klusā okeāna reģiona politiku. Atbilstoši ofensīvā reālisma nostādnēm lielvaras tiecas iegrožot potenciālo hegemonu, lai saglabātu savas pozīcijas starptautiskajā sistēmā. Līdz ar to maģistra darbā „Ukrainas un Sīrijas konfliktu loma ASV Āzijas Klusā okeāna reģiona politikā” tiek...

  5. Biochar pyrolytically produced from municipal solid wastes for aqueous As(V) removal: adsorption property and its improvement with KOH activation.

    Science.gov (United States)

    Jin, Hongmei; Capareda, Sergio; Chang, Zhizhou; Gao, Jun; Xu, Yueding; Zhang, Jianying

    2014-10-01

    Biochar converted from waste products is being considered as an alternative adsorbent for removal of aqueous heavy metal(loid)s. In this work, experimental and modeling investigations were conducted to examine the effect of biochars pyrolytically produced from municipal solid wastes on removing aqueous As(V) before and after activated by 2M KOH solution. Results showed that the highest adsorption capacity of pristine biochars was 24.49 mg/g. The pseudo-second-order model and Langmuir adsorption isotherm model can preferably describe the adsorption process. The activated biochar showed enhanced As(V) adsorption ability with an adsorption capacity of 30.98 mg/g, which was more than 1.3 times of pristine biochars, and 2-10 times of modified biochars reported by other literatures. Increase of surface area and changes of porous texture, especially the functional groups on the surface of activated biochars are the major contributors to its more efficient adsorption of As(V). PMID:25103038

  6. Non-catalytic site HIV-1 integrase inhibitors disrupt core maturation and induce a reverse transcription block in target cells.

    Directory of Open Access Journals (Sweden)

    Mini Balakrishnan

    Full Text Available HIV-1 integrase (IN is the target for two classes of antiretrovirals: i the integrase strand-transfer inhibitors (INSTIs and ii the non-catalytic site integrase inhibitors (NCINIs. NCINIs bind at the IN dimer interface and are thought to interfere primarily with viral DNA (vDNA integration in the target cell by blocking IN-vDNA assembly as well as the IN-LEDGF/p75 interaction. Herein we show that treatment of virus-producing cells, but not of mature virions or target cells, drives NCINI antiviral potency. NCINIs target an essential late-stage event in HIV replication that is insensitive to LEDGF levels in the producer cells. Virus particles produced in the presence of NCINIs displayed normal Gag-Pol processing and endogenous reverse transcriptase activity, but were defective at initiating vDNA synthesis following entry into the target cell. NCINI-resistant virus carrying a T174I mutation in the IN dimer interface was less sensitive to the compound-induced late-stage effects, including the reverse transcription block. Wild-type, but not T174I virus, produced in the presence of NCINIs exhibited striking defects in core morphology and an increased level of IN oligomers that was not observed upon treatment of mature cell-free particles. Collectively, these results reveal that NCINIs act through a novel mechanism that is unrelated to the previously observed inhibition of IN activity or IN-LEDGF interaction, and instead involves the disruption of an IN function during HIV-1 core maturation and assembly.

  7. Non-catalytic site HIV-1 integrase inhibitors disrupt core maturation and induce a reverse transcription block in target cells.

    Science.gov (United States)

    Balakrishnan, Mini; Yant, Stephen R; Tsai, Luong; O'Sullivan, Christopher; Bam, Rujuta A; Tsai, Angela; Niedziela-Majka, Anita; Stray, Kirsten M; Sakowicz, Roman; Cihlar, Tomas

    2013-01-01

    HIV-1 integrase (IN) is the target for two classes of antiretrovirals: i) the integrase strand-transfer inhibitors (INSTIs) and ii) the non-catalytic site integrase inhibitors (NCINIs). NCINIs bind at the IN dimer interface and are thought to interfere primarily with viral DNA (vDNA) integration in the target cell by blocking IN-vDNA assembly as well as the IN-LEDGF/p75 interaction. Herein we show that treatment of virus-producing cells, but not of mature virions or target cells, drives NCINI antiviral potency. NCINIs target an essential late-stage event in HIV replication that is insensitive to LEDGF levels in the producer cells. Virus particles produced in the presence of NCINIs displayed normal Gag-Pol processing and endogenous reverse transcriptase activity, but were defective at initiating vDNA synthesis following entry into the target cell. NCINI-resistant virus carrying a T174I mutation in the IN dimer interface was less sensitive to the compound-induced late-stage effects, including the reverse transcription block. Wild-type, but not T174I virus, produced in the presence of NCINIs exhibited striking defects in core morphology and an increased level of IN oligomers that was not observed upon treatment of mature cell-free particles. Collectively, these results reveal that NCINIs act through a novel mechanism that is unrelated to the previously observed inhibition of IN activity or IN-LEDGF interaction, and instead involves the disruption of an IN function during HIV-1 core maturation and assembly. PMID:24040198

  8. Separación electrodialítica de Cu(II y As(V en electrolitos ácidos

    Directory of Open Access Journals (Sweden)

    Ibáñez, J. P.

    2007-02-01

    Full Text Available The separation of copper and arsenic from acidic electrolytes by electrodialysis was investigated at room temperature. The effect of current density and pH was studied in a batch cell during 3 hours. The kinetic parameters showed that Cu(II transport rate was 0.75 mol/m2/h and the As(V transport rate was 0.002 mol/m2/h. An efficient separation between Cu(II and As(V was achieved; generating a concentrated solution of copper with no arsenic, which was obtained independently of the electrolyte acidity and current density used. The effect of the arsenic speciation with pH is discussed as well.

    Se investigó el uso de la electrodiálisis para separar cobre y arsénico desde soluciones ácidas a temperatura ambiente. Se estudió el efecto de la densidad de corriente y grado de
    acidez del electrolito en la separación a través de experimentos en celda batch de 5 compartimientos durante 3 h. Los parámetros cinéticos indican que la velocidad de transporte de Cu(II fue de 0,75 mol/h/m2 y de As(V fue de 0,002 mol/h/m2 a una densidad de corriente de 225 A/m2. Se logró obtener una eficiente separación de Cu(II y As(V, con la generación de una solución concentrada de cobre sin presencia de arsénico, lo cual fue independiente del grado de acidez de la operación y de la densidad de corriente. El efecto de la distribución de las especies iónicas de As(V con el pH también se discute.

  9. Integrase inhibitor (INI) genotypic resistance in treatment-naive and raltegravir-experienced patients infected with diverse HIV-1 clades

    Science.gov (United States)

    Doyle, Tomas; Dunn, David T.; Ceccherini-Silberstein, Francesca; De Mendoza, Carmen; Garcia, Frederico; Smit, Erasmus; Fearnhill, Esther; Marcelin, Anne-Genevieve; Martinez-Picado, Javier; Kaiser, Rolf; Geretti, Anna Maria

    2015-01-01

    Objectives The aim of this study was to characterize the prevalence and patterns of genotypic integrase inhibitor (INI) resistance in relation to HIV-1 clade. Methods The cohort comprised 533 INI-naive subjects and 255 raltegravir recipients with viraemia who underwent integrase sequencing in routine care across Europe, including 134/533 (25.1%) and 46/255 (18.0%), respectively, with non-B clades (A, C, D, F, G, CRF01, CRF02, other CRFs, complex). Results No major INI resistance-associated mutations (RAMs) occurred in INI-naive subjects. Among raltegravir recipients with viraemia (median 3523 HIV-1 RNA copies/mL), 113/255 (44.3%) had one or more major INI RAMs, most commonly N155H (45/255, 17.6%), Q148H/R/K + G140S/A (35/255, 13.7%) and Y143R/C/H (12/255, 4.7%). In addition, four (1.6%) raltegravir recipients showed novel mutations at recognized resistance sites (E92A, S147I, N155D, N155Q) and novel mutations at other integrase positions that were statistically associated with raltegravir exposure (K159Q/R, I161L/M/T/V, E170A/G). Comparing subtype B with non-B clades, Q148H/R/K occurred in 42/209 (20.1%) versus 2/46 (4.3%) subjects (P = 0.009) and G140S/A occurred in 36/209 (17.2%) versus 1/46 (2.2%) subjects (P = 0.005). Intermediate- to high-level cross-resistance to twice-daily dolutegravir was predicted in 40/255 (15.7%) subjects, more commonly in subtype B versus non-B clades (39/209, 18.7% versus 1/46, 2.2%; P = 0.003). A glycine (G) to serine (S) substitution at integrase position 140 required one nucleotide change in subtype B and two nucleotide changes in all non-B clades. Conclusions No major INI resistance mutations occurred in INI-naive subjects. Reduced occurrence of Q148H/R/K + G140S/A was seen in non-B clades versus subtype B, and was explained by the higher genetic barrier to the G140S mutation observed in all non-B clades analysed. PMID:26311843

  10. In vitro DNA tethering of HIV-1 integrase by the transcriptional coactivator LEDGF/p75.

    Science.gov (United States)

    McNeely, Melissa; Hendrix, Jelle; Busschots, Katrien; Boons, Eline; Deleersnijder, Angélique; Gerard, Melanie; Christ, Frauke; Debyser, Zeger

    2011-07-29

    Although LEDGF/p75 is believed to act as a cellular cofactor of lentiviral integration by tethering integrase (IN) to chromatin, there is no good in vitro model to analyze this functionality. We designed an AlphaScreen assay to study how LEDGF/p75 modulates the interaction of human immunodeficiency virus type 1 IN with DNA. IN bound with similar affinity to DNA mimicking the long terminal repeat or to random DNA. While LEDGF/p75 bound DNA strongly, a mutant of LEDGF/p75 with compromised nuclear localization signal (NLS)/AT hook interacted weakly, and the LEDGF/p75 PWWP domain did not interact, corroborating previous reports on the role of NLS and AT hooks in charge-dependent DNA binding. LEDGF/p75 stimulated IN binding to DNA 10-fold to 30-fold. Stimulation of IN-DNA binding required a direct interaction between IN and the C-terminus of LEDGF/p75. Addition of either the C-terminus of LEDGF/p75 (amino acids 325-530) or LEDGF/p75 mutated in the NLS/AT hooks interfered with IN binding to DNA. Our results are consistent with an in vitro model of LEDGF/p75-mediated tethering of IN to DNA. The inhibition of IN-DNA interaction by the LEDGF/p75 C-terminus may provide a novel strategy for the inhibition of HIV IN activity and may explain the potent inhibition of HIV replication observed after the overexpression of C-terminal fragments in cell culture.

  11. A new class of multimerization selective inhibitors of HIV-1 integrase.

    Directory of Open Access Journals (Sweden)

    Amit Sharma

    2014-05-01

    Full Text Available The quinoline-based allosteric HIV-1 integrase (IN inhibitors (ALLINIs are promising candidates for clinically useful antiviral agents. Studies using these compounds have highlighted the role of IN in both early and late stages of virus replication. However, dissecting the exact mechanism of action of the quinoline-based ALLINIs has been complicated by the multifunctional nature of these inhibitors because they both inhibit IN binding with its cofactor LEDGF/p75 and promote aberrant IN multimerization with similar potencies in vitro. Here we report design of small molecules that allowed us to probe the role of HIV-1 IN multimerization independently from IN-LEDGF/p75 interactions in infected cells. We altered the rigid quinoline moiety in ALLINIs and designed pyridine-based molecules with a rotatable single bond to allow these compounds to bridge between interacting IN subunits optimally and promote oligomerization. The most potent pyridine-based inhibitor, KF116, potently (EC50 of 0.024 µM blocked HIV-1 replication by inducing aberrant IN multimerization in virus particles, whereas it was not effective when added to target cells. Furthermore, KF116 inhibited the HIV-1 IN variant with the A128T substitution, which confers resistance to the majority of quinoline-based ALLINIs. A genome-wide HIV-1 integration site analysis demonstrated that addition of KF116 to target or producer cells did not affect LEDGF/p75-dependent HIV-1 integration in host chromosomes, indicating that this compound is not detectably inhibiting IN-LEDGF/p75 binding. These findings delineate the significance of correctly ordered IN structure for HIV-1 particle morphogenesis and demonstrate feasibility of exploiting IN multimerization as a therapeutic target. Furthermore, pyridine-based compounds present a novel class of multimerization selective IN inhibitors as investigational probes for HIV-1 molecular biology.

  12. HIV-1 integrase inhibitors are substrates for the multidrug transporter MDR1-P-glycoprotein

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    Cara Andrea

    2007-03-01

    Full Text Available Abstract Background The discovery of diketoacid-containing derivatives as inhibitors of HIV-1 Integrase (IN (IN inhibitors, IINs has played a major role in validating this enzyme as an important target for antiretroviral therapy. Since the in vivo efficacy depends on access of these drugs to intracellular sites where HIV-1 replicates, we determined whether the IINs are recognized by the multidrug transporter MDR1-P-glycoprotein (P-gp thereby reducing their intracellular accumulation. To address the effect of IINs on drug transport, nine quinolonyl diketo acid (DKA derivatives active on the HIV-1 IN strand transfer (ST step and with EC50 ranging from 1.83 to >50 μm in cell-based assays were tested for their in vitro interaction with P-gp in the CEM-MDR cell system. IINs were investigated for the inhibition and induction of the P-gp function and expression as well as for multidrug resistance (MDR reversing ability. Results The HIV-1 IINs act as genuine P-gp substrates by inhibiting doxorubicin efflux and inducing P-gp functional conformation changes as evaluated by the modulation of UIC2 mAb epitope. Further, IINs chemosensitize MDR cells to vinblastine and induce P-gp expression in drug sensitive revertants of CEM-MDR cells. Conclusion To our knowledge, this is the first demonstration that HIV-1 IINs are P-gp substrates. This biological property may influence the absorption, distribution and elimination of these novels anti HIV-1 compounds.

  13. GCN5-dependent acetylation of HIV-1 integrase enhances viral integration

    Directory of Open Access Journals (Sweden)

    Albanese Alberto

    2010-03-01

    Full Text Available Abstract Background An essential event during the replication cycle of HIV-1 is the integration of the reverse transcribed viral DNA into the host cellular genome. Our former report revealed that HIV-1 integrase (IN, the enzyme that catalyzes the integration reaction, is positively regulated by acetylation mediated by the histone acetyltransferase (HAT p300. Results In this study we demonstrate that another cellular HAT, GCN5, acetylates IN leading to enhanced 3'-end processing and strand transfer activities. GCN5 participates in the integration step of HIV-1 replication cycle as demonstrated by the reduced infectivity, due to inefficient provirus formation, in GCN5 knockdown cells. Within the C-terminal domain of IN, four lysines (K258, K264, K266, and K273 are targeted by GCN5 acetylation, three of which (K264, K266, and K273 are also modified by p300. Replication analysis of HIV-1 clones carrying substitutions at the IN lysines acetylated by both GCN5 and p300, or exclusively by GCN5, demonstrated that these residues are required for efficient viral integration. In addition, a comparative analysis of the replication efficiencies of the IN triple- and quadruple-mutant viruses revealed that even though the lysines targeted by both GCN5 and p300 are required for efficient virus integration, the residue exclusively modified by GCN5 (K258 does not affect this process. Conclusions The results presented here further demonstrate the relevance of IN post-translational modification by acetylation, which results from the catalytic activities of multiple HATs during the viral replication cycle. Finally, this study contributes to clarifying the recent debate raised on the role of IN acetylated lysines during HIV-1 infection.

  14. A new perovskite-type NdFeO3 adsorbent: synthesis, characterization, and As(V) adsorption

    Science.gov (United States)

    Luu, Minh Dai; Nhiem Dao, Ngoc; Van Nguyen, Duc; Chuc Pham, Ngoc; Ninh Vu, The; Dung Doan, Trung

    2016-06-01

    Nanocrystalline NdFeO3 perovskite oxide was prepared by the combustion method using polyvinyl alcohol (PVA) and corresponding metal nitrate precursors under optimum process conditions, using a solution with a pH of 2, a metal/PVA molar ratio of 1:3, and a calcination temperature of 600 °C, and was characterized by x-ray diffraction, scanning electron microscopy, transmission electron microscopy, Brunauer-Emmett-Teller nitrogen adsorption and desorption, energy dispersive x-ray spectroscopy, Fourier transform infrared spectroscopy, laser Raman spectroscopy, and thermogravimetric and differential thermal analysis. The synthesized perovskite NdFeO3, which had an average size of 20 nm and a relatively high surface area of 20 m2 g-1, was investigated for adsorbing hazardous arsenate from aqueous solution. Arsenate adsorption by perovskite NdFeO3 is a pH-dependent process with a high adsorption capacity at pH levels between 4 and 7 and a maximum As(V) adsorption capacity of 126.58 mg g-1, higher than most arsenate adsorbents reported in the literature. Kinetic and equilibrium data of reaction under the experimental conditions are best described by a pseudo-second-order and the Langmuir isotherm equation. The values of enthalpy, Gibbs free energy and entropy changes (ΔH0 = +63.916 kJ mol-1, ΔG0 = -6.551 kJ mol-1 to -14.021 kJ mol-1 at T = 283-313 K, ΔS0 = +0.249 kJ mol-1 K-1) suggested that the reaction was endothermic, spontaneous, and took place with increasing entropy.

  15. A new perovskite-type NdFeO3 adsorbent: synthesis, characterization, and As(V) adsorption

    Science.gov (United States)

    Luu, Minh Dai; Nhiem Dao, Ngoc; Van Nguyen, Duc; Chuc Pham, Ngoc; Ninh Vu, The; Dung Doan, Trung

    2016-06-01

    Nanocrystalline NdFeO3 perovskite oxide was prepared by the combustion method using polyvinyl alcohol (PVA) and corresponding metal nitrate precursors under optimum process conditions, using a solution with a pH of 2, a metal/PVA molar ratio of 1:3, and a calcination temperature of 600 °C, and was characterized by x-ray diffraction, scanning electron microscopy, transmission electron microscopy, Brunauer–Emmett–Teller nitrogen adsorption and desorption, energy dispersive x-ray spectroscopy, Fourier transform infrared spectroscopy, laser Raman spectroscopy, and thermogravimetric and differential thermal analysis. The synthesized perovskite NdFeO3, which had an average size of 20 nm and a relatively high surface area of 20 m2 g‑1, was investigated for adsorbing hazardous arsenate from aqueous solution. Arsenate adsorption by perovskite NdFeO3 is a pH-dependent process with a high adsorption capacity at pH levels between 4 and 7 and a maximum As(V) adsorption capacity of 126.58 mg g‑1, higher than most arsenate adsorbents reported in the literature. Kinetic and equilibrium data of reaction under the experimental conditions are best described by a pseudo-second-order and the Langmuir isotherm equation. The values of enthalpy, Gibbs free energy and entropy changes (ΔH0 = +63.916 kJ mol‑1, ΔG0 = ‑6.551 kJ mol‑1 to ‑14.021 kJ mol‑1 at T = 283‑313 K, ΔS0 = +0.249 kJ mol‑1 K‑1) suggested that the reaction was endothermic, spontaneous, and took place with increasing entropy.

  16. Conserved sequences in the carboxyl terminus of integrase that are essential for human immunodeficiency virus type 1 replication.

    Science.gov (United States)

    Cannon, P M; Byles, E D; Kingsman, S M; Kingsman, A J

    1996-01-01

    We have previously identified a residue in the carboxyl terminus of human immunodeficiency virus type 1 integrase (HIV-1 IN), W-235, the requirement for which is only revealed in viral assays for integrase function (P. M. Cannon, W. Wilson, E. Byles, S. M. Kingsman, and A. J. Kingsman, J. Virol. 68:4768-4775, 1994). Our further analysis of this region of retroviral IN has now identified several sequence motifs which are conserved in all the retroviruses we examined, apart from human spumaretrovirus. We have made mutations within these motifs in HIV-1 IN and examined their phenotypes when reintroduced into an infectious proviral clone. The deleterious effects of several of these mutations demonstrate the importance of these regions for IN function in vivo. We observed a further discrepancy, at a motif that is only conserved in the lentiviruses, in the ability of mutants to function in in vitro and in vivo assays. Substitutions both in this region and at W-235 abolish HIV-1 infectivity but do not affect particle production, morphology, reverse transcription, or nuclear import in T-cell lines. Taken together with the in vitro data suggesting that neither of these residues is directly involved in the catalytic reactions of IN, it seems likely that we have identified regions of IN that are essential for interactions with other components of the integration machinery. PMID:8523588

  17. LEDGIN-mediated Inhibition of Integrase-LEDGF/p75 Interaction Reduces Reactivation of Residual Latent HIV.

    Science.gov (United States)

    Vranckx, Lenard S; Demeulemeester, Jonas; Saleh, Suha; Boll, Annegret; Vansant, Gerlinde; Schrijvers, Rik; Weydert, Caroline; Battivelli, Emilie; Verdin, Eric; Cereseto, Anna; Christ, Frauke; Gijsbers, Rik; Debyser, Zeger

    2016-06-01

    Persistence of latent, replication-competent Human Immunodeficiency Virus type 1 (HIV-1) provirus is the main impediment towards a cure for HIV/AIDS (Acquired Immune Deficiency Syndrome). Therefore, different therapeutic strategies to eliminate the viral reservoirs are currently being explored. We here propose a novel strategy to reduce the replicating HIV reservoir during primary HIV infection by means of drug-induced retargeting of HIV integration. A novel class of integration inhibitors, referred to as LEDGINs, inhibit the interaction between HIV integrase and the LEDGF/p75 host cofactor, the main determinant of lentiviral integration site selection. We show for the first time that LEDGF/p75 depletion hampers HIV-1 reactivation in cell culture. Next we demonstrate that LEDGINs relocate and retarget HIV integration resulting in a HIV reservoir that is refractory to reactivation by different latency-reversing agents. Taken together, these results support the potential of integrase inhibitors that modulate integration site targeting to reduce the likeliness of viral rebound. PMID:27428435

  18. Cross-resistance profile determination of two second-generation HIV-1 integrase inhibitors using a panel of recombinant viruses derived from raltegravir-treated clinical isolates.

    Science.gov (United States)

    Van Wesenbeeck, L; Rondelez, E; Feyaerts, M; Verheyen, A; Van der Borght, K; Smits, V; Cleybergh, C; De Wolf, H; Van Baelen, K; Stuyver, L J

    2011-01-01

    The integrase inhibitor raltegravir (RAL) is currently used for the treatment of both treatment-naïve and treatment-experienced HIV-1-infected patients. Elvitegravir (EVG) is in late phases of clinical development. Since significant cross-resistance between RAL and EVG is observed, there is a need for second-generation integrase inhibitors (INIs) with a higher genetic barrier and limited cross-resistance to RAL/EVG. A panel of HIV-1 integrase recombinants, derived from plasma samples from raltegravir-treated patients (baseline and follow-up samples), were used to study the cross-resistance profile of two second-generation integrase inhibitors, MK-2048 and compound G. Samples with Q148H/R mutations had elevated fold change values with all compounds tested. Although samples with the Y143R/C mutation had reduced susceptibility to RAL, they remained susceptible to MK-2048 and compound G. Samples with the N155H mutation had no reduced susceptibility to compound G. In conclusion, our results allowed ranking of the INIs on the basis of the antiviral activities using recombinant virus stocks from RAL-treated patient viruses. The order according to decreasing susceptibility is compound G, MK-2048, and EVG. PMID:20956600

  19. Cross-resistance profile determination of two second-generation HIV-1 integrase inhibitors using a panel of recombinant viruses derived from raltegravir-treated clinical isolates.

    Science.gov (United States)

    Van Wesenbeeck, L; Rondelez, E; Feyaerts, M; Verheyen, A; Van der Borght, K; Smits, V; Cleybergh, C; De Wolf, H; Van Baelen, K; Stuyver, L J

    2011-01-01

    The integrase inhibitor raltegravir (RAL) is currently used for the treatment of both treatment-naïve and treatment-experienced HIV-1-infected patients. Elvitegravir (EVG) is in late phases of clinical development. Since significant cross-resistance between RAL and EVG is observed, there is a need for second-generation integrase inhibitors (INIs) with a higher genetic barrier and limited cross-resistance to RAL/EVG. A panel of HIV-1 integrase recombinants, derived from plasma samples from raltegravir-treated patients (baseline and follow-up samples), were used to study the cross-resistance profile of two second-generation integrase inhibitors, MK-2048 and compound G. Samples with Q148H/R mutations had elevated fold change values with all compounds tested. Although samples with the Y143R/C mutation had reduced susceptibility to RAL, they remained susceptible to MK-2048 and compound G. Samples with the N155H mutation had no reduced susceptibility to compound G. In conclusion, our results allowed ranking of the INIs on the basis of the antiviral activities using recombinant virus stocks from RAL-treated patient viruses. The order according to decreasing susceptibility is compound G, MK-2048, and EVG.

  20. Lack of pharmacokinetic interaction between rilpivirine and integrase inhibitors dolutegravir and GSK1265744.

    Science.gov (United States)

    Ford, Susan L; Gould, Elizabeth; Chen, Shuguang; Margolis, David; Spreen, William; Crauwels, Herta; Piscitelli, Stephen

    2013-11-01

    Dolutegravir (DTG) and GSK1265744 are HIV integrase inhibitors (INIs) in clinical development. The oral formulation of rilpivirine (RPV), a nonnucleoside reverse transcriptase inhibitor (NNRTI), has been approved for treatment-naive HIV infection. Long-acting depot injections of GSK1265744 and RPV are also being developed. This study evaluated the potential for drug interactions between RPV and these INIs. This phase 1, open-label, two-cohort, three-period, single-sequence crossover study evaluated oral coadministration of RPV with DTG or GSK1265744. Healthy subjects received DTG (50 mg every 24 h for 5 days) or GSK1265744 (30 mg every 24 h for 12 days) in period 1 followed by a washout, RPV (25 mg every 24 h for 11 or 12 days) in period 2, immediately followed by RPV (25 mg every 24 h) plus DTG (50 mg every 24 h) for 5 days or GSK1265744 (30 mg every 24 h) for 12 days in period 3. Steady-state pharmacokinetic (PK) parameters were estimated using noncompartmental analysis of data collected on the last day of each period. The combinations of RPV and DTG (n = 16) and of RPV and GSK1265744 (n = 11) were well tolerated; no grade 3 or 4 adverse events (AEs) or AE-related discontinuations were observed. The 90% confidence intervals for the area under the curve from time zero until the end of the dosage interval [AUC0-τ] and maximum concentration of drug in serum (Cmax) geometric mean ratios were within 0.8 to 1.25. Following administration of DTG + RPV, DTG and RPV Cτ increased by 22% and 21%, respectively. Following administration of GSK1265744 + RPV, RPV Cτ decreased 8%. DTG and GSK1265744 can be administered with RPV without dosage adjustment for either agent. These results support coadministration of RPV with DTG or GSK1265744 as either oral or long-acting depot injection regimens. (This study has been registered at ClinicalTrials.gov under registration no. NCT01467531.). PMID:23979733

  1. Lack of pharmacokinetic interaction between rilpivirine and integrase inhibitors dolutegravir and GSK1265744.

    Science.gov (United States)

    Ford, Susan L; Gould, Elizabeth; Chen, Shuguang; Margolis, David; Spreen, William; Crauwels, Herta; Piscitelli, Stephen

    2013-11-01

    Dolutegravir (DTG) and GSK1265744 are HIV integrase inhibitors (INIs) in clinical development. The oral formulation of rilpivirine (RPV), a nonnucleoside reverse transcriptase inhibitor (NNRTI), has been approved for treatment-naive HIV infection. Long-acting depot injections of GSK1265744 and RPV are also being developed. This study evaluated the potential for drug interactions between RPV and these INIs. This phase 1, open-label, two-cohort, three-period, single-sequence crossover study evaluated oral coadministration of RPV with DTG or GSK1265744. Healthy subjects received DTG (50 mg every 24 h for 5 days) or GSK1265744 (30 mg every 24 h for 12 days) in period 1 followed by a washout, RPV (25 mg every 24 h for 11 or 12 days) in period 2, immediately followed by RPV (25 mg every 24 h) plus DTG (50 mg every 24 h) for 5 days or GSK1265744 (30 mg every 24 h) for 12 days in period 3. Steady-state pharmacokinetic (PK) parameters were estimated using noncompartmental analysis of data collected on the last day of each period. The combinations of RPV and DTG (n = 16) and of RPV and GSK1265744 (n = 11) were well tolerated; no grade 3 or 4 adverse events (AEs) or AE-related discontinuations were observed. The 90% confidence intervals for the area under the curve from time zero until the end of the dosage interval [AUC0-τ] and maximum concentration of drug in serum (Cmax) geometric mean ratios were within 0.8 to 1.25. Following administration of DTG + RPV, DTG and RPV Cτ increased by 22% and 21%, respectively. Following administration of GSK1265744 + RPV, RPV Cτ decreased 8%. DTG and GSK1265744 can be administered with RPV without dosage adjustment for either agent. These results support coadministration of RPV with DTG or GSK1265744 as either oral or long-acting depot injection regimens. (This study has been registered at ClinicalTrials.gov under registration no. NCT01467531.).

  2. Removal of As(V) and simultaneous production of copper powder from a Cu(II)-As(V)-H{sub 2}SO{sub 4} electrolyte by using reactive electrodialysis; Eliminacion de As (V) y produccion simultanea de polvo de cobre de un electrolito de Cu(II) - As(V) - H{sub 2}SO{sub 4} mediante electrodialisis reactiva

    Energy Technology Data Exchange (ETDEWEB)

    Ibanez, J. P.

    2012-11-01

    The removal of As(V) and the simultaneous generation of powder of copper from an electrolyte made of As(V) - Cu(II) - H{sub 2}SO{sub 4} was studied by using electro dialysis at several current densities, temperatures and aeration of the electrolyte. The removal of arsenic was proportional to the current density, temperature and aeration used. The removal of arsenic reached a value of 0.14 mmol/h at 500 A/m2, 25 degree centigrade and without aeration, this value increased to 0.31 mmol/h by increasing the aeration to 6.6 l/h. The Cu(II) was recovered in a 98 % as a fine arsenic free powder of metallic copper with oxides of copper. The arsenic was removed from the electrolyte by adsorption onto the anodic slimes generated from the lead anode oxidation. (Author) 24 refs.

  3. Superparamagnetic nanomaterial Fe3O4-TiO2 for the removal of As(V) and As(III) from aqueous solutions.

    Science.gov (United States)

    Beduk, Fatma

    2016-01-01

    A magnetically separable nanomaterial Fe3O4-TiO2 was synthesized and characterized which was subsequently used for the removal of arsenic (V) from aqueous solutions. The surface morphology, magnetic properties, crystalline structure, thermal stability and Brunauer-Emmet-Teller surface area of the synthesized Fe3O4-TiO2 nanoparticles (NPs) are characterized by scanning electron microscope and high-resolution transmission electron microscope, vibrating sample magnetometry, X-ray diffractometer, thermogravimetric analysis and multi point function surface area analyzer. The saturation magnetization of Fe3O4-TiO2 NPs was determined to be 50.97 emu/g, which makes them superparamagnetic. The surface area of Fe3O4-TiO2 NPs was as much as 94.9 m(2)/g. The main factors affecting adsorption efficiency, such as solution pH, reaction time, initial As(V) concentration and adsorbent concentration are investigated. When the adsorption isotherms were analyzed by the Langmuir, Freundlich and Dubinin-Radushkevich models, equilibrium data were found to be well represented by Freundlich isotherm, and adsorption on Fe3O4-TiO2 NPs fitted well with pseudo-second-order kinetic model. The maximum adsorption capacity of As(V) on Fe3O4-TiO2 NPs, calculated by the Freundlich model was determined at 11.434 µg/g. 1.0 g/L of Fe3O4-TiO2 NPs was efficient for complete removal of 100 µg/L As(V) in 1 h. Fe3O4-TiO2 NPs was also effective for 93% removal of 100 µg/L As(III). Matrix effect was determined using As(V)-contaminated well water. Successfull results were obtained for purification of real well water containing 137.12 µg/L As(V). Results show that Fe3O4-TiO2 NPs are promising adsorbents with an advantage of magnetic separation.

  4. Superparamagnetic nanomaterial Fe3O4-TiO2 for the removal of As(V) and As(III) from aqueous solutions.

    Science.gov (United States)

    Beduk, Fatma

    2016-07-01

    A magnetically separable nanomaterial Fe3O4-TiO2 was synthesized and characterized which was subsequently used for the removal of arsenic (V) from aqueous solutions. The surface morphology, magnetic properties, crystalline structure, thermal stability and Brunauer-Emmet-Teller surface area of the synthesized Fe3O4-TiO2 nanoparticles (NPs) are characterized by scanning electron microscope and high-resolution transmission electron microscope, vibrating sample magnetometry, X-ray diffractometer, thermogravimetric analysis and multi point function surface area analyzer. The saturation magnetization of Fe3O4-TiO2 NPs was determined to be 50.97 emu/g, which makes them superparamagnetic. The surface area of Fe3O4-TiO2 NPs was as much as 94.9 m(2)/g. The main factors affecting adsorption efficiency, such as solution pH, reaction time, initial As(V) concentration and adsorbent concentration are investigated. When the adsorption isotherms were analyzed by the Langmuir, Freundlich and Dubinin-Radushkevich models, equilibrium data were found to be well represented by Freundlich isotherm, and adsorption on Fe3O4-TiO2 NPs fitted well with pseudo-second-order kinetic model. The maximum adsorption capacity of As(V) on Fe3O4-TiO2 NPs, calculated by the Freundlich model was determined at 11.434 µg/g. 1.0 g/L of Fe3O4-TiO2 NPs was efficient for complete removal of 100 µg/L As(V) in 1 h. Fe3O4-TiO2 NPs was also effective for 93% removal of 100 µg/L As(III). Matrix effect was determined using As(V)-contaminated well water. Successfull results were obtained for purification of real well water containing 137.12 µg/L As(V). Results show that Fe3O4-TiO2 NPs are promising adsorbents with an advantage of magnetic separation. PMID:26831455

  5. Corynebacterium glutamicum MTCC 2745 immobilized on granular activated carbon/MnFe2O4 composite: A novel biosorbent for removal of As(III) and As(V) ions.

    Science.gov (United States)

    Podder, M S; Majumder, C B

    2016-11-01

    The optimization of biosorption/bioaccumulation process of both As(III) and As(V) has been investigated by using the biosorbent; biofilm of Corynebacterium glutamicum MTCC 2745 supported on granular activated carbon/MnFe2O4 composite (MGAC). The presence of functional groups on the cell wall surface of the biomass that may interact with the metal ions was proved by FT-IR. To determine the most appropriate correlation for the equilibrium curves employing the procedure of the non-linear regression for curve fitting analysis, isotherm studies were performed for As(III) and As(V) using 30 isotherm models. The pattern of biosorption/bioaccumulation fitted well with Vieth-Sladek isotherm model for As(III) and Brouers-Sotolongo and Fritz-Schlunder-V isotherm models for As(V). The maximum biosorption/bioaccumulation capacity estimated using Langmuir model were 2584.668mg/g for As(III) and 2651.675mg/g for As(V) at 30°C temperature and 220min contact time. The results showed that As(III) and As(V) removal was strongly pH-dependent with an optimum pH value of 7.0. D-R isotherm studies specified that ion exchange might play a prominent role. PMID:27289352

  6. Corynebacterium glutamicum MTCC 2745 immobilized on granular activated carbon/MnFe2O4 composite: A novel biosorbent for removal of As(III) and As(V) ions

    Science.gov (United States)

    Podder, M. S.; Majumder, C. B.

    2016-11-01

    The optimization of biosorption/bioaccumulation process of both As(III) and As(V) has been investigated by using the biosorbent; biofilm of Corynebacterium glutamicum MTCC 2745 supported on granular activated carbon/MnFe2O4 composite (MGAC). The presence of functional groups on the cell wall surface of the biomass that may interact with the metal ions was proved by FT-IR. To determine the most appropriate correlation for the equilibrium curves employing the procedure of the non-linear regression for curve fitting analysis, isotherm studies were performed for As(III) and As(V) using 30 isotherm models. The pattern of biosorption/bioaccumulation fitted well with Vieth-Sladek isotherm model for As(III) and Brouers-Sotolongo and Fritz-Schlunder-V isotherm models for As(V). The maximum biosorption/bioaccumulation capacity estimated using Langmuir model were 2584.668 mg/g for As(III) and 2651.675 mg/g for As(V) at 30 °C temperature and 220 min contact time. The results showed that As(III) and As(V) removal was strongly pH-dependent with an optimum pH value of 7.0. D-R isotherm studies specified that ion exchange might play a prominent role.

  7. Corynebacterium glutamicum MTCC 2745 immobilized on granular activated carbon/MnFe2O4 composite: A novel biosorbent for removal of As(III) and As(V) ions.

    Science.gov (United States)

    Podder, M S; Majumder, C B

    2016-11-01

    The optimization of biosorption/bioaccumulation process of both As(III) and As(V) has been investigated by using the biosorbent; biofilm of Corynebacterium glutamicum MTCC 2745 supported on granular activated carbon/MnFe2O4 composite (MGAC). The presence of functional groups on the cell wall surface of the biomass that may interact with the metal ions was proved by FT-IR. To determine the most appropriate correlation for the equilibrium curves employing the procedure of the non-linear regression for curve fitting analysis, isotherm studies were performed for As(III) and As(V) using 30 isotherm models. The pattern of biosorption/bioaccumulation fitted well with Vieth-Sladek isotherm model for As(III) and Brouers-Sotolongo and Fritz-Schlunder-V isotherm models for As(V). The maximum biosorption/bioaccumulation capacity estimated using Langmuir model were 2584.668mg/g for As(III) and 2651.675mg/g for As(V) at 30°C temperature and 220min contact time. The results showed that As(III) and As(V) removal was strongly pH-dependent with an optimum pH value of 7.0. D-R isotherm studies specified that ion exchange might play a prominent role.

  8. The hepatitis B virus ribonuclease H is sensitive to inhibitors of the human immunodeficiency virus ribonuclease H and integrase enzymes.

    Directory of Open Access Journals (Sweden)

    John E Tavis

    2013-01-01

    Full Text Available Nucleos(tide analog therapy blocks DNA synthesis by the hepatitis B virus (HBV reverse transcriptase and can control the infection, but treatment is life-long and has high costs and unpredictable long-term side effects. The profound suppression of HBV by the nucleos(tide analogs and their ability to cure some patients indicates that they can push HBV to the brink of extinction. Consequently, more patients could be cured by suppressing HBV replication further using a new drug in combination with the nucleos(tide analogs. The HBV ribonuclease H (RNAseH is a logical drug target because it is the second of only two viral enzymes that are essential for viral replication, but it has not been exploited, primarily because it is very difficult to produce active enzyme. To address this difficulty, we expressed HBV genotype D and H RNAseHs in E. coli and enriched the enzymes by nickel-affinity chromatography. HBV RNAseH activity in the enriched lysates was characterized in preparation for drug screening. Twenty-one candidate HBV RNAseH inhibitors were identified using chemical structure-activity analyses based on inhibitors of the HIV RNAseH and integrase. Twelve anti-RNAseH and anti-integrase compounds inhibited the HBV RNAseH at 10 µM, the best compounds had low micromolar IC(50 values against the RNAseH, and one compound inhibited HBV replication in tissue culture at 10 µM. Recombinant HBV genotype D RNAseH was more sensitive to inhibition than genotype H. This study demonstrates that recombinant HBV RNAseH suitable for low-throughput antiviral drug screening has been produced. The high percentage of compounds developed against the HIV RNAseH and integrase that were active against the HBV RNAseH indicates that the extensive drug design efforts against these HIV enzymes can guide anti-HBV RNAseH drug discovery. Finally, differential inhibition of HBV genotype D and H RNAseHs indicates that viral genetic variability will be a factor during drug

  9. A Simple and Highly Efficient Preparation of Structurally Diverse Aryl β-diketoacids as HIV-1 Integrase Inhibitors

    Institute of Scientific and Technical Information of China (English)

    JIANG Xiao-Hua姜晓华; LONG Ya-Qiu龙亚秋

    2004-01-01

    In order to provide a facile and practical access to structurally diverse aryl β-diketoacids, An improved and highly efficient oxalylation method was developed which employed commercially available and cheap reagents.The oxalylation of aryl methyl ketones, the key step to construct the pharmacophore of aryl β-diketoacids, was considerably facilitated by a new combination of dimethyl oxalate as an oxalic source and sodium tert-butoxide as a base. A wide variety of aryl β-diketoacids bearing different functional groups can be prepared rapidly in high yields at room t. emperature with this method, which has significant advantages over the previously reported procedures in a wider application range, much less amount of reagents, pretty higher yields and quite shorter reaction time. The bis-aryldiketoacids 3k and 31, readily prepared by this method, displayed interesting and promising inhibitory activities against HIV-1 integrase and HIV-1 replication in cells.

  10. High-throughput real-time assay based on molecular beacons for HIV-1 integrase 3'-processing reaction

    Institute of Scientific and Technical Information of China (English)

    Hong-qiu HE; Xiao-hui MA; Bin LIU; Xiao-yi ZHANG; Wei-zu CHEN; Cun-xin WANG; Shao-hui CHENG

    2007-01-01

    Aim: To develop a high-throughput real-time assay based on molecular beacons to monitor the integrase 3'-processing reaction in vitro and apply it to inhibitor screening.Methods: The recombinant human immunodeficiency virus (HIV)-1 integrase (IN) is incubated with a 38 mer oligonucleotide substrate, a sequence identical to the U5 end of HIV-1 long terminal repeats (LTR). Based on the fluores-cence properties of molecular beacons, the substrate is designed to form a stem-loop structure labeled with a fluorophore at the 5' end and a quencher at the 3'end.IN cleaves the terminal 3'-dinucleotide containing the quencher, resulting in an increase in fluorescence which can be monitored on a spectrofluorometer. To optimize this assay, tests were performed to investigate the effects of substrates, enzyme and the metal ion concentrations on the IN activity and optimal param-eters were obtained. Moreover, 2 IN inhibitors were employed to test the perfor-mance of this assay in antiviral compound screening.Results: The fluorescent intensity of the reaction mixture varies linearly with time and is proportional to the velocity of the 3'-processing reaction. Tests were performed and the results showed that the optimal rate was obtained for a reaction mixture containing 50 mg/L recom-binant HIV-1 IN, 400 nmol/L substrate, and 10 mmol/L Mn2+. The IN 3'-processing reaction under the optimal conditions showed a more than 18-fold increase in the fluorescence intensity compared to the enzyme-free control. The IC50 values of the IN inhibitors obtained in our assay were similar to the values obtained from a radiolabeled substrate assay.Conclusion: Our results demonstrated that this is a fast, reliable, and sensitive method to monitor HIV IN 3'-processing reaction and that it can be used for inhibitor screening.

  11. Symmetrical 1-pyrrolidineacetamide showing anti-HIV activity through a new binding site on HIV-1 integrase

    Institute of Scientific and Technical Information of China (English)

    Li DU; Ya-xue ZHAO; Liu-meng YANG; Yong-tang ZHENG; Yun TANG; Xu SHEN; Hua-liang JIANG

    2008-01-01

    Aim:To characterize the functional and pharmacological features of a symmetrical 1-pyrrolidineacetamide,N,N'-(methylene-di-4,1-phenylene) bis-1-pyrrolidineacetamide,as a new anti-HIV compound which could competitively inhibit HIV-1 integrase (IN) binding to viral DNA.Methods:A surface plasma resonance (SPR)-based competitive assay was employed to determine the compound's inhibitory activity,and the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide cell assay was used to qualify the antiviral activity.The potential binding sites were predicted by molecular modeling and determined by site-directed mutagenesis and a SPR binding assay.Results:l-pyrrolidineacetamide,N,N'-(methylene-di-4,1-phenylene) bis-1-pyrrolidineacetamide could competitively inhibit IN binding to viral DNA with a 50% inhibitory concentration (IC50) value of 7.29±0.68 μmol/L as investigated by SPR-based investigation.Another antiretroviral activity assay showed that this compound exhibited inhibition against HⅣ-Ⅰ(ⅢB) replication with a 50% effective concentration (EC50) value of 40.54 μmol/L in C8166 cells,and cytotoxicity with a cytotoxic concentration value of 173.84 μmol/L in mock-infected C8166 cells.Molecular docking predicted 3 potential residues as 1-pyrrolidineacetamide,N,N'-(methylene-di-4,1-phenylene)bis-1-pyrrolidineacetamide binding sites.The importance of 3 key amino acid residues (Lys103,Lys173,and Thr174) involved in the binding was further identified by site-directed mutagenesis and a SPR binding assay.Conclusion:This present work identified a new anti-HIV compound through a new IN-binding site which is expected to supply new potential drug-binding site information for HIV-1 integrase inhibitor discovery and development.

  12. Design and Synthesis of Bis-amide and Hydrazide-containing Derivatives of Malonic Acid as Potential HIV-1 Integrase Inhibitors

    Directory of Open Access Journals (Sweden)

    Nouri Neamati

    2008-10-01

    Full Text Available HIV-1 integrase (IN is an attractive and validated target for the development of novel therapeutics against AIDS. In the search for new IN inhibitors, we designed and synthesized three series of bis-amide and hydrazide-containing derivatives of malonic acid. We performed a docking study to investigate the potential interactions of the title compounds with essential amino acids on the IN active site.

  13. Synthesis and evaluation of substituted 4-(N-benzylamino)cinnamate esters as potential anti-cancer agents and HIV-1 integrase inhibitors.

    Science.gov (United States)

    Faridoon; Edkins, Adrienne L; Isaacs, Michelle; Mnkandhla, Dumisani; Hoppe, Heinrich C; Kaye, Perry T

    2016-08-01

    Encouraging selectivity and low micromolar activity against HeLa cervical carcinoma (IC50⩾3.0μM) and the aggressive MDA-MB-231 triple negative breast carcinoma (IC50⩾9.6μM) cell lines has been exhibited by a number of readily accessible 4-(N-benzylamino)cinnamate esters. The potential of the ligands as HIV-1 integrase inhibitors has also been examined. PMID:27317645

  14. A crystal structure of the catalytic core domain of an avian sarcoma and leukemia virus integrase suggests an alternate dimeric assembly.

    Science.gov (United States)

    Ballandras, Allison; Moreau, Karen; Robert, Xavier; Confort, Marie-Pierre; Merceron, Romain; Haser, Richard; Ronfort, Corinne; Gouet, Patrice

    2011-01-01

    Integrase (IN) is an important therapeutic target in the search for anti-Human Immunodeficiency Virus (HIV) inhibitors. This enzyme is composed of three domains and is hard to crystallize in its full form. First structural results on IN were obtained on the catalytic core domain (CCD) of the avian Rous and Sarcoma Virus strain Schmidt-Ruppin A (RSV-A) and on the CCD of HIV-1 IN. A ribonuclease-H like motif was revealed as well as a dimeric interface stabilized by two pairs of α-helices (α1/α5, α5/α1). These structural features have been validated in other structures of IN CCDs. We have determined the crystal structure of the Rous-associated virus type-1 (RAV-1) IN CCD to 1.8 Å resolution. RAV-1 IN shows a standard activity for integration and its CCD differs in sequence from that of RSV-A by a single accessible residue in position 182 (substitution A182T). Surprisingly, the CCD of RAV-1 IN associates itself with an unexpected dimeric interface characterized by three pairs of α-helices (α3/α5, α1/α1, α5/α3). A182 is not involved in this novel interface, which results from a rigid body rearrangement of the protein at its α1, α3, α5 surface. A new basic groove that is suitable for single-stranded nucleic acid binding is observed at the surface of the dimer. We have subsequently determined the structure of the mutant A182T of RAV-1 IN CCD and obtained a RSV-A IN CCD-like structure with two pairs of buried α-helices at the interface. Our results suggest that the CCD of avian INs can dimerize in more than one state. Such flexibility can further explain the multifunctionality of retroviral INs, which beside integration of dsDNA are implicated in different steps of the retroviral cycle in presence of viral ssRNA. PMID:21857987

  15. A crystal structure of the catalytic core domain of an avian sarcoma and leukemia virus integrase suggests an alternate dimeric assembly.

    Directory of Open Access Journals (Sweden)

    Allison Ballandras

    Full Text Available Integrase (IN is an important therapeutic target in the search for anti-Human Immunodeficiency Virus (HIV inhibitors. This enzyme is composed of three domains and is hard to crystallize in its full form. First structural results on IN were obtained on the catalytic core domain (CCD of the avian Rous and Sarcoma Virus strain Schmidt-Ruppin A (RSV-A and on the CCD of HIV-1 IN. A ribonuclease-H like motif was revealed as well as a dimeric interface stabilized by two pairs of α-helices (α1/α5, α5/α1. These structural features have been validated in other structures of IN CCDs. We have determined the crystal structure of the Rous-associated virus type-1 (RAV-1 IN CCD to 1.8 Å resolution. RAV-1 IN shows a standard activity for integration and its CCD differs in sequence from that of RSV-A by a single accessible residue in position 182 (substitution A182T. Surprisingly, the CCD of RAV-1 IN associates itself with an unexpected dimeric interface characterized by three pairs of α-helices (α3/α5, α1/α1, α5/α3. A182 is not involved in this novel interface, which results from a rigid body rearrangement of the protein at its α1, α3, α5 surface. A new basic groove that is suitable for single-stranded nucleic acid binding is observed at the surface of the dimer. We have subsequently determined the structure of the mutant A182T of RAV-1 IN CCD and obtained a RSV-A IN CCD-like structure with two pairs of buried α-helices at the interface. Our results suggest that the CCD of avian INs can dimerize in more than one state. Such flexibility can further explain the multifunctionality of retroviral INs, which beside integration of dsDNA are implicated in different steps of the retroviral cycle in presence of viral ssRNA.

  16. A crystal structure of the catalytic core domain of an avian sarcoma and leukemia virus integrase suggests an alternate dimeric assembly.

    Science.gov (United States)

    Ballandras, Allison; Moreau, Karen; Robert, Xavier; Confort, Marie-Pierre; Merceron, Romain; Haser, Richard; Ronfort, Corinne; Gouet, Patrice

    2011-01-01

    Integrase (IN) is an important therapeutic target in the search for anti-Human Immunodeficiency Virus (HIV) inhibitors. This enzyme is composed of three domains and is hard to crystallize in its full form. First structural results on IN were obtained on the catalytic core domain (CCD) of the avian Rous and Sarcoma Virus strain Schmidt-Ruppin A (RSV-A) and on the CCD of HIV-1 IN. A ribonuclease-H like motif was revealed as well as a dimeric interface stabilized by two pairs of α-helices (α1/α5, α5/α1). These structural features have been validated in other structures of IN CCDs. We have determined the crystal structure of the Rous-associated virus type-1 (RAV-1) IN CCD to 1.8 Å resolution. RAV-1 IN shows a standard activity for integration and its CCD differs in sequence from that of RSV-A by a single accessible residue in position 182 (substitution A182T). Surprisingly, the CCD of RAV-1 IN associates itself with an unexpected dimeric interface characterized by three pairs of α-helices (α3/α5, α1/α1, α5/α3). A182 is not involved in this novel interface, which results from a rigid body rearrangement of the protein at its α1, α3, α5 surface. A new basic groove that is suitable for single-stranded nucleic acid binding is observed at the surface of the dimer. We have subsequently determined the structure of the mutant A182T of RAV-1 IN CCD and obtained a RSV-A IN CCD-like structure with two pairs of buried α-helices at the interface. Our results suggest that the CCD of avian INs can dimerize in more than one state. Such flexibility can further explain the multifunctionality of retroviral INs, which beside integration of dsDNA are implicated in different steps of the retroviral cycle in presence of viral ssRNA.

  17. Synthesis and Characterization of Iron-impregnated Pre-oxidized Activated Carbon Prepared by Microwave Radiation for As(V) Removal from Water

    Science.gov (United States)

    Yurum, Yuda; Yurum, Alp; Ozlem Kocabas, Zuleyha; Semiat, Raphael

    2013-04-01

    One of the most efficient ways to treat water is probably by adsorption and catalytic oxidation. Surely, for such a process to be economical, the catalyst and the adsorber should have a high catalytic activity and adsorption capacity, and be inexpensive. One of these materials is iron oxide, which is studied and used in areas like catalysis and environmental applications. It is known that synthesizing iron oxides in nano size enhances the catalytic activity. Pre-oxidized activated carbons impregnated with iron-based nanoparticles are prepared in a single step under hydrothermal conditions with microwave radiation. The hydrothermal treatment provides an important advantage by forming fine particles that can easily impregnate deep in to the porous support by the help of water. Their efficiency for the removal of As(V) from water was compared with the pure pre-oxidized activated carbon and iron oxide nanoparticles impregnated without microwave radiation. The synthesized nanomaterials with different iron oxide loadings were characterized by x-ray diffraction (XRD), scanning electron microscopy (SEM), and Brunauer-Emmett-Teller (BET) surface area analyzer. Iron loadings were calculated using flame atomic absorbance. Microwave radiation provided much faster iron impregnation on the active carbon surface. At the first stage of microwave radiation iron oxide impregnation is low but after 6 minutes, iron oxide nanoparticles of 100 nm size started to cover the surface homogeneously. Further treatment with microwave increased the size of particles and the amount of surface coverage. Additionally, with microwave hydrothermal treatment, relatively higher iron oxide loadings were achieved within 10 minutes. From the XRD characterization it was seen that at the first stage of radiation, iron deposited in the form of β-FeOOH, but after the first stage the structure became Fe2O3. While radiation increased the surface area of the material during the first stages, at the last stage

  18. Evidence for the horizontal transfer of an integrase gene from a fusellovirus to a pRN-like plasmid within a single strain of Sulfolobus and the implications for plasmid survival

    DEFF Research Database (Denmark)

    Peng, Xu

    2008-01-01

    A fusellovirus SSV4 and a pRN-like plasmid pXZ1 were co-isolated from a single strain of Sulfolobus. In contrast to the previously characterized virus-plasmid hybrids pSSVx and pSSVi, which can coexist intracellulary with a fusellovirus, pXZ1 is not packaged into viral particles and shows no viral...... infectivity. The virus and plasmid carry genomes of 15 135 and 6970 bp, respectively. For SSV4, 33 predicted ORFs are compactly organized with a strong preference for UGA stop codons, three-quarters of which overlap with either the Shine-Dalgarno motif or the start codon of the following gene. pXZ1 carries...... seven ORFs, three of which encode an atypical RepA, a PlrA and a CopG protein. A fourth ORF exhibits a high nucleotide sequence identity to the SSV4 integrase gene, which suggests that it has been transferred to the plasmid from SSV4. A single point mutation within an otherwise identical 500 bp region...

  19. The effect of adaptive servo ventilation (ASV) on objective and subjective outcomes in Cheyne-Stokes respiration (CSR) with central sleep apnea (CSA) in heart failure (HF): A systematic review.

    Science.gov (United States)

    Yang, Hyunju; Sawyer, Amy M

    2016-01-01

    To summarize the current evidence for adaptive servo ventilation (ASV) in Cheyne-Stokes respiration (CSR) with central sleep apnea (CSA) in heart failure (HF) and advance a research agenda and clinical considerations for ASV-treated CSR-CSA in HF. CSR-CSA in HF is associated with higher overall mortality, worse outcomes and lower quality of life (QOL) than HF without CSR-CSA. Five databases were searched using key words (n = 234). Randomized controlled trials assessed objective sleep quality, cardiac, and self-reported outcomes in adults (≥18 years) with HF (n = 10). ASV has a beneficial effect on the reduction of central sleep apnea in adult patients with CSR-CSA in HF, but it is not be superior to CPAP, bilevel PPV, or supplemental oxygen in terms of sleep quality defined by polysomnography, cardiovascular outcomes, subjective daytime sleepiness, and quality of life. ASV is not recommended for CSR-CSA in HF. It is important to continue to refer HF patients for sleep evaluation to clearly discern OSA from CSR-CSA. Symptom management research, inclusive of objective and subjective outcomes, in CSR-CSA in HF adults is needed.

  20. The effect of adaptive servo ventilation (ASV) on objective and subjective outcomes in Cheyne-Stokes respiration (CSR) with central sleep apnea (CSA) in heart failure (HF): A systematic review.

    Science.gov (United States)

    Yang, Hyunju; Sawyer, Amy M

    2016-01-01

    To summarize the current evidence for adaptive servo ventilation (ASV) in Cheyne-Stokes respiration (CSR) with central sleep apnea (CSA) in heart failure (HF) and advance a research agenda and clinical considerations for ASV-treated CSR-CSA in HF. CSR-CSA in HF is associated with higher overall mortality, worse outcomes and lower quality of life (QOL) than HF without CSR-CSA. Five databases were searched using key words (n = 234). Randomized controlled trials assessed objective sleep quality, cardiac, and self-reported outcomes in adults (≥18 years) with HF (n = 10). ASV has a beneficial effect on the reduction of central sleep apnea in adult patients with CSR-CSA in HF, but it is not be superior to CPAP, bilevel PPV, or supplemental oxygen in terms of sleep quality defined by polysomnography, cardiovascular outcomes, subjective daytime sleepiness, and quality of life. ASV is not recommended for CSR-CSA in HF. It is important to continue to refer HF patients for sleep evaluation to clearly discern OSA from CSR-CSA. Symptom management research, inclusive of objective and subjective outcomes, in CSR-CSA in HF adults is needed. PMID:26995256

  1. Production of transgenic cattle highly expressing human serum albumin in milk by phiC31 integrase-mediated gene delivery.

    Science.gov (United States)

    Luo, Yan; Wang, Yongsheng; Liu, Jun; Lan, Hui; Shao, Minghao; Yu, Yuan; Quan, Fusheng; Zhang, Yong

    2015-10-01

    Transgenic cattle expressing high levels of recombinant human serum albumin (HSA) in their milk may as an alternative source for commercial production. Our objective was to produce transgenic cattle highly expressing HSA in milk by using phiC31 integrase system and somatic cell nuclear transfer (SCNT). The mammary-specific expression plasmid pIACH(-), containing the attB recognition site for phiC31 integrase, were co-transfected with integrase expression plasmid pCMVInt into bovine fetal fibroblast cells (BFFs). PhiC31 integrase-mediated integrations in genome of BFFs were screened by nested inverse PCR. After analysis of sequence of the PCR products, 46.0% (23/50) of the both attB-genome junction sites (attL and attR) were confirmed, and four pseudo attP sites were identified. The integration rates in BF3, BF11, BF19 and BF4 sites were 4.0% (2/50), 6.0% (3/50), 16.0% (8/50) and 20.0% (10/50), respectively. BF3 is located in the bovine chromosome 3 collagen alpha-3 (VI) chain isomer 2 gene, while the other three sites are located in the non-coding region. The transgenic cell lines from BF11, BF19 and BF4 sites were used as donors for SCNT. Two calves from transgenic cells BF19 were born, one died within a few hours after birth, and another calf survived healthy. PCR and Southern blot analysis revealed integration of the transgene in the genome of cloned calves. The nested reverse PCR confirmed that the integration site in cloned calves was identical to the donor cells. The western blotting assessment indicated that recombinant HSA was expressed in the milk of transgenic cattle and the expression level was about 4-8 mg/mL. The present study demonstrated that phiC31 integrase system was an efficient and safety gene delivery tool for producing HSA transgenic cattle. The production of recombinant HSA in the milk of cattle may provide a large-scale and cost-effective resource.

  2. Implications of integrase inhibitors for HIV-infected transplantation recipients: raltegravir and dolutegravir (S/GSK 1349572).

    Science.gov (United States)

    Waki, Kayo; Sugawara, Yasuhiko

    2011-01-01

    In the modern era of highly active antiretroviral therapy (HAART), reluctance to perform transplantation (Tx) in HIV-infected individuals is no longer justified. Non-nucleoside reverse transcriptase inhibitors (NNRTIs) or protease inhibitors (PIs), the current first line regimens of HAART, are metabolized by the cytochrome P450 family (CYP3A4). Most NNRTIs induce CYP3A4, whereas PIs inhibit it. Calcinuerin inhibitors (CNIs), which are mandatory for Tx, need the same enzyme complex for their clearance. Therefore, a significant drug-drug interaction (DDI) is encountered between current HAART and CNIs. This results in extreme difficulty in adjusting the optimal dose of CNIs, for which the therapeutic range is narrow. Of interest, integrase inhibitors (INIs) - novel, potent anti-HIV drugs - are mainly metabolized by uridine diphosphate glucuronosyltransferase (UGT) 1A1 and do not induce or inhibit CYP3A4. DDI is presumably absent when NNTRIs or PIs are replaced by INIs. Raltegravir (RAL), a first generation INI, has been introduced into kidney and liver Tx. There is increasing evidence that rejection is well controlled without renal impairment due to CNI over-exposure while persistent, robust suppression of HIV is achieved. Global phase III clinical trials of dolutegravir (DTG), a second generation INI, are currently in progress. In vitro data has suggested that DTG may be less prone to resistance than RAL (referred to as having a higher genetic barrier). The time has come to extensively discuss the implications of INIs in Tx for HIV positive patients.

  3. The Need for Development of New HIV-1 Reverse Transcriptase and Integrase Inhibitors in the Aftermath of Antiviral Drug Resistance

    Directory of Open Access Journals (Sweden)

    Mark A. Wainberg

    2012-01-01

    Full Text Available The use of highly active antiretroviral therapy (HAART involves combinations of drugs to achieve maximal virological response and reduce the potential for the emergence of antiviral resistance. There are two broad classes of reverse transcriptase inhibitors, the nucleoside reverse transcriptase inhibitors (NRTIs and nonnucleoside reverse transcriptase inhibitors (NNRTIs. Since the first classes of such compounds were developed, viral resistance against them has necessitated the continuous development of novel compounds within each class. This paper considers the NRTIs and NNRTIs currently in both preclinical and clinical development or approved for second line therapy and describes the patterns of resistance associated with their use, as well as the underlying mechanisms that have been described. Due to reasons of both affordability and availability, some reverse transcriptase inhibitors with low genetic barrier are more commonly used in resource-limited settings. Their use results to the emergence of specific patterns of antiviral resistance and so may require specific actions to preserve therapeutic options for patients in such settings. More recently, the advent of integrase strand transfer inhibitors represents another major step forward toward control of HIV infection, but these compounds are also susceptible to problems of HIV drug resistance.

  4. Anti-HIV-1 protease- and HIV-1 integrase activities of Thai medicinal plants known as Hua-Khao-Yen.

    Science.gov (United States)

    Tewtrakul, Supinya; Itharat, Arunporn; Rattanasuwan, Pranee

    2006-04-21

    Ethanolic- and water extracts from five species of Thai medicinal plants known as Hua-Khao-Yen were tested for their inhibitory effects against HIV-1 protease (HIV-PR) and HIV-1 integrase (HIV-1 IN). The result revealed that the ethanolic (EtOH) extract of Smilax corbularia exhibited anti-HIV-1 IN activity with an IC50 value of 1.9 microg/ml, followed by the water extract of Dioscorea birmanica (IC50 = 4.5 microg/ml), the EtOH extract of Dioscorea birmanica (IC50 = 4.7 microg/ml), the water extract of Smilax corbularia (IC50 = 5.4 microg/ml), the EtOH extract of Smilax glabra (IC50 = 6.7 microg/ml) and the water extract of Smilax glabra (IC50 = 8.5 microg/ml). The extracts of Pygmaeopremna herbacea and Dioscorea membranacea were apparently inactive (IC50 > 100 microg/ml). Interestingly, only the EtOH extract of Dioscorea membranacea showed appreciable activity (IC50 = 48 microg/ml) against HIV-1 PR, while the other extracts possessed mild activity. This result strongly supported the basis for the use of Smilax corbularia and Dioscorea membranacea for AIDS treatment by Thai traditional doctors. PMID:16406414

  5. Drug interaction profile of the HIV integrase inhibitor cabotegravir: assessment from in vitro studies and a clinical investigation with midazolam.

    Science.gov (United States)

    Reese, Melinda J; Bowers, Gary D; Humphreys, Joan E; Gould, Elizabeth P; Ford, Susan L; Webster, Lindsey O; Polli, Joseph W

    2016-01-01

    1. Cabotegravir (CAB; GSK1265744) is a potent HIV integrase inhibitor in clinical development as an oral lead-in tablet and long-acting injectable for the treatment and prevention of HIV infection. 2. This work investigated if CAB was a substrate for efflux transporters, the potential for CAB to interact with drug-metabolizing enzymes and transporters to cause clinical drug interactions, and the effect of CAB on the pharmacokinetics of midazolam, a CYP3A4 probe substrate, in humans. 3. CAB is a substrate for Pgp and BCRP; however, its high intrinsic membrane permeability limits the impact of these transporters on its intestinal absorption. 4. At clinically relevant concentrations, CAB did not inhibit or induce any of the CYP or UGT enzymes evaluated in vitro and had no effect on the clinical pharmacokinetics of midazolam. 5. CAB is an inhibitor of OAT1 (IC50 0.81 µM) and OAT3 (IC50 0.41 µM) but did not or only weakly inhibited Pgp, BCRP, MRP2, MRP4, MATE1, MATE2-K, OATP1B1, OATP1B3, OCT1, OCT2 or BSEP. 6. Based on regulatory guidelines and quantitative extrapolations, CAB has a low propensity to cause clinically significant drug interactions, except for coadministration with OAT1 or OAT3 substrates. PMID:26340566

  6. Q148N, a Novel Integrase Inhibitor Resistance Mutation Associated with Low-Level Reduction in Elvitegravir Susceptibility.

    Science.gov (United States)

    Varghese, Vici; Pinsky, Benjamin A; Smith, Darvin S; Klein, Daniel; Shafer, Robert W

    2016-07-01

    The integrase strand transfer inhibitor (INSTI)-resistance mutations Q148H/K/R are arguably the most important INSTI-resistance mutations as they represented the first step to high-level dolutegravir cross-resistance. We describe an individual with transmitted four-class drug resistance whose virus sequence had the previously uncharacterized mutation Q148N. Infectious molecular HIV-1 clones containing Q148N alone and in combination with G140S demonstrated ∼2.4-4.5 reduced elvitegravir susceptibility depending on the virus's genetic context but retained susceptibility to raltegravir and dolutegravir. This level of reduced elvitegravir susceptibility is lower than that observed with Q148H/K/R and in fact the infected individual responded to an initial treatment regimen containing tenofovir/emtricitabine/elvitegravir/cobicistat. Q148N was associated with a higher replication capacity than Q148H, suggesting that this mutation may be more fit in the absence of selective INSTI therapy. PMID:27009474

  7. Rational Design, Synthesis and Evaluation of Coumarin Derivatives as Protein-protein Interaction Inhibitors.

    Science.gov (United States)

    De Luca, Laura; Agharbaoui, Fatima E; Gitto, Rosaria; Buemi, Maria Rosa; Christ, Frauke; Debyser, Zeger; Ferro, Stefania

    2016-09-01

    Herein we describe the design and synthesis of a new series of coumarin derivatives searching for novel HIV-1 integrase (IN) allosteric inhibitors. All new obtained compounds were tested in order to evaluate their ability to inhibit the interaction between the HIV-1 IN enzyme and the nuclear protein lens epithelium growth factor LEDGF/p75. A combined approach of docking and molecular dynamic simulations has been applied to clarify the activity of the new compounds. Specifically, the binding free energies by using the method of molecular mechanics-generalized Born surface area (MM-GBSA) was calculated, whereas hydrogen bond occupancies were monitored throughout simulations methods.

  8. Advances of screening methods in vitro for HIV-1 integrase inhibitors%HIV-1整合酶抑制剂体外筛选方法研究进展

    Institute of Scientific and Technical Information of China (English)

    张旋; 杨柳萌; 郑永唐

    2013-01-01

    整合酶是HIV基因表达和复制所必需的酶,而且宿主细胞内不存在该酶的类似物.因此,HIV-1整合酶已成为设计、筛选抗HIV药物的理想靶点.迄今为止,Raltegravir仍是唯一上市的HIV整合酶抑制剂,而且临床上也已经出现耐药问题.研发新一代整合酶抑制剂非常必要.高通量、高灵敏度、简单易行的筛选方法是研究开发新一代HIV-1整合酶抑制剂的关键.目前,HIV-1整合酶抑制剂筛选方法有多种,各有优缺点,该文将对文献报道的整合酶抑制剂体外筛选方法的最新进展做一介绍.%Integrase is an essential enzyme for HIV-1 replication and has no functional analogue in host cells. The integrase is an ideal target for designing and screening anti-HIV drugs. Ralte-gravir is the only marked HIV-1 integrase inhibitor so far, and has caused drug resistance. It is necessary to developing new generation of HIV-1 integrase inhibitors. High-throughput, highly sensitive, easy and feasible screening methods are the key to developing new HIV-1 integrase inhibitors. This review introduces the various HIV-1 integrase inhibitors screening methods that were reported recently.

  9. Synthesis of green nano iron particles (GnIP) and their application in adsorptive removal of As(III) and As(V) from aqueous solution

    Science.gov (United States)

    Prasad, Kumar Suranjit; Gandhi, Pooja; Selvaraj, Kaliaperumal

    2014-10-01

    The present study reports a new approach to synthesise nano iron particles using leaf extract of Mint (Mentha spicata L.) plant. The synthesised GnIPs were subjected to detailed adsorption studies for removal of arsenite and arsenate from aqueous solution of defined concentration. Iron nanoparticles synthesised using leaf extract showed UV-vis absorption peaks at 360 and 430 nm. TEM result showed the formation of polydispersed nanoparticles of size ranging from 20 to 45 nm. Nanoparticles were found to have core-shell structure. The planer reflection of selected area electron diffraction (SAED) and XRD analysis suggested that iron particles were crystalline and belonged to fcc (face centred cubic) type. Energy-dispersive X-ray analysis (EDAX) shows that Fe was an integral component of synthesised nanoparticles. The content of Fe in nanoparticles was found to be 40%, in addition to other elements like C (16%), O (19%) and Cl (23%). FT-IR study suggested that functional groups like sbnd NH, sbnd Cdbnd O, sbnd Cdbnd N and sbnd Cdbnd C were involved in particle formation. The removal efficiency of GnIP-chitosan composite for As(III) and As(V) was found to be 98.79 and 99.65%. Regeneration of adsorbent suggested that synthesised green GnIP may work as an effective tool for removal of arsenic from contaminated water.

  10. Implications of integrase inhibitors for HIV-infected transplantation recipients: raltegravir and dolutegravir (S/GSK 1349572).

    Science.gov (United States)

    Waki, Kayo; Sugawara, Yasuhiko

    2011-01-01

    In the modern era of highly active antiretroviral therapy (HAART), reluctance to perform transplantation (Tx) in HIV-infected individuals is no longer justified. Non-nucleoside reverse transcriptase inhibitors (NNRTIs) or protease inhibitors (PIs), the current first line regimens of HAART, are metabolized by the cytochrome P450 family (CYP3A4). Most NNRTIs induce CYP3A4, whereas PIs inhibit it. Calcinuerin inhibitors (CNIs), which are mandatory for Tx, need the same enzyme complex for their clearance. Therefore, a significant drug-drug interaction (DDI) is encountered between current HAART and CNIs. This results in extreme difficulty in adjusting the optimal dose of CNIs, for which the therapeutic range is narrow. Of interest, integrase inhibitors (INIs) - novel, potent anti-HIV drugs - are mainly metabolized by uridine diphosphate glucuronosyltransferase (UGT) 1A1 and do not induce or inhibit CYP3A4. DDI is presumably absent when NNTRIs or PIs are replaced by INIs. Raltegravir (RAL), a first generation INI, has been introduced into kidney and liver Tx. There is increasing evidence that rejection is well controlled without renal impairment due to CNI over-exposure while persistent, robust suppression of HIV is achieved. Global phase III clinical trials of dolutegravir (DTG), a second generation INI, are currently in progress. In vitro data has suggested that DTG may be less prone to resistance than RAL (referred to as having a higher genetic barrier). The time has come to extensively discuss the implications of INIs in Tx for HIV positive patients. PMID:22101373

  11. Substrate recognition and motion mode analyses of PFV integrase in complex with viral DNA via coarse-grained models.

    Directory of Open Access Journals (Sweden)

    Jianping Hu

    Full Text Available HIV-1 integrase (IN is an important target in the development of drugs against the AIDS virus. Drug design based on the structure of IN was markedly hampered due to the lack of three-dimensional structure information of HIV-1 IN-viral DNA complex. The prototype foamy virus (PFV IN has a highly functional and structural homology with HIV-1 IN. Recently, the X-ray crystal complex structure of PFV IN with its cognate viral DNA has been obtained. In this study, both Gaussian network model (GNM and anisotropy network model (ANM have been applied to comparatively investigate the motion modes of PFV DNA-free and DNA-bound IN. The results show that the motion mode of PFV IN has only a slight change after binding with DNA. The motion of this enzyme is in favor of association with DNA, and the binding ability is determined by its intrinsic structural topology. Molecular docking experiments were performed to gain the binding modes of a series of diketo acid (DKA inhibitors with PFV IN obtained from ANM, from which the dependability of PFV IN-DNA used in the drug screen for strand transfer (ST inhibitors was confirmed. It is also found that the functional groups of keto-enol, bis-diketo, tetrazole and azido play a key role in aiding the recognition of viral DNA, and thus finally increase the inhibition capability for the corresponding DKA inhibitor. Our study provides some theoretical information and helps to design anti-AIDS drug based on the structure of IN.

  12. A novel high-throughput format assay for HIV-1 integrase strand transfer reaction using magnetic beads

    Institute of Scientific and Technical Information of China (English)

    Hong-qiu HE; Xiao-hui MA; Bin LIU; Wei-zu CHEN; Cun-xin WANG; Shao-hui CHENG

    2008-01-01

    Aim:To develop a novel high-throughput format assay to monitor the integrase (IN) strand transfer (ST) reaction in vitro and apply it to a reaction character study and the identification of antiviral drugs.Methods:The donor DNA duplex,with a sequence identical to the U5 end of HIV-1 long terminal repeats,is labeled at its 5' end with biotin (BIO).The target DNA duplex is labeled at its 3' end with digoxin (DIG).IN mediates the integration of donor DNA into target DNA and results in a 5' BIO and 3' DIG-labeled duplex DNA product.Streptavidin-coated magnetic beads were used to capture the product,and the amount of DIG was measured as the ST reaction product.The assay was optimized in 96-well microplate format for high-throughput screening purpose.Moreover,the assay was applied in a ST reaction character study,and the efficiency of the assay in the identification of antiviral compounds was tested.Results:The end-point values,measured as absorbance at 405 nm was approximately 1.5 for the IN-mediated ST reaction as compared with no more than 0.05 of background readings.The ST reaction char-acter and the half maximal inhibitory concentration (IC50) values of 2 known IN inhibitors obtained in our assay were similar to previously reported results using other assays.The evaluation parameter Z' factor for this assay ranged from 0.6 to 0.9.Conclusion:The assay presented here has been proven to be rapid,sensitive,and specific for the detection of IN ST activity,the reaction character study,as well as for the identification of antiviral drugs targeting IN.

  13. Application of granular activated carbon/MnFe2O4 composite immobilized on C. glutamicum MTCC 2745 to remove As(III) and As(V): Kinetic, mechanistic and thermodynamic studies

    Science.gov (United States)

    Podder, M. S.; Majumder, C. B.

    2016-01-01

    The main objective of the present study was to investigate the efficiency of Corynebacterium glutamicum MTCC 2745 immobilized on granular activated carbon/MnFe2O4 (GAC/MnFe2O4) composite to treat high concentration of arsenic bearing wastewater. Non-linear regression analysis was done for determining the best-fit kinetic model on the basis of three correlation coefficients and three error functions and also for predicting the parameters involved in kinetic models. The results showed that Fractal-like mixed 1,2 order model for As(III) and Brouser-Weron-Sototlongo as well as Fractal-like pseudo second order models for As(V) were proficient to provide realistic description of biosorption/bioaccumulation kinetic. Applicability of mechanistic models in the current study exhibited that the rate governing step in biosorption/bioaccumulation of both As(III) and As(V) was film diffusion rather than intraparticle diffusion. The evaluated thermodynamic parameters ΔG0, ΔH0 and ΔS0 revealed that biosorption/bioaccumulation of both As(III) and As(V) was feasible, spontaneous and exothermic under studied conditions.

  14. Determination of As(III) and As(V) species in some natural water and food samples by solid-phase extraction on Streptococcus pyogenes immobilized on Sepabeads SP 70 and hydride generation atomic absorption spectrometry.

    Science.gov (United States)

    Uluozlu, Ozgur Dogan; Tuzen, Mustafa; Mendil, Durali; Soylak, Mustafa

    2010-05-01

    The speciation of arsenic(III) and arsenic(V) by using Streptococcus pyogenes immobilized on Sepabeads SP 70 resin has been investigated with solid-phase extraction method. The arsenic levels were determined hydride generation atomic absorption spectrometry (HGAAS) in sample solutions. The procedure presented based on quantitative recoveries of As(III) as >95%. Also the As(V) recoveries were obtained as method. After reduction of As(V) by using KI and ascorbic acid and waiting 1h later, the system was applied to determination of total arsenic. As(V) was found as the difference between the total As and As(III) content. Various experimental parameters such as pH, amount of microorganism, sample volume, etc. were investigated. The capacity of biosorbent for arsenic(III) was calculated as 7.3 mg/g. The preconcentration factor was found as 36. The relative standard deviation was calculated below 8%. Limit of detection was calculated as 13 ng/L. The validation of the presented procedure was tested by analysis of standard reference materials (NIST SRM 1568a Rice floor and GBW 07605 Tea) and obtained fairly compatible results. The procedure was also successfully applied to arsenic speciation and determination of some natural water and food samples.

  15. Application of granular activated carbon/MnFe₂O₄ composite immobilized on C. glutamicum MTCC 2745 to remove As(III) and As(V): Kinetic, mechanistic and thermodynamic studies.

    Science.gov (United States)

    Podder, M S; Majumder, C B

    2016-01-15

    The main objective of the present study was to investigate the efficiency of Corynebacterium glutamicum MTCC 2745 immobilized on granular activated carbon/MnFe2O4 (GAC/MnFe2O4) composite to treat high concentration of arsenic bearing wastewater. Non-linear regression analysis was done for determining the best-fit kinetic model on the basis of three correlation coefficients and three error functions and also for predicting the parameters involved in kinetic models. The results showed that Fractal-like mixed 1,2 order model for As(III) and Brouser-Weron-Sototlongo as well as Fractal-like pseudo second order models for As(V) were proficient to provide realistic description of biosorption/bioaccumulation kinetic. Applicability of mechanistic models in the current study exhibited that the rate governing step in biosorption/bioaccumulation of both As(III) and As(V) was film diffusion rather than intraparticle diffusion. The evaluated thermodynamic parameters ΔG(0), ΔH(0) and ΔS(0) revealed that biosorption/bioaccumulation of both As(III) and As(V) was feasible, spontaneous and exothermic under studied conditions. PMID:26322840

  16. Development of Lentiviral Vectors for Targeted Integration and Protein Delivery.

    Science.gov (United States)

    Schenkwein, Diana; Ylä-Herttuala, Seppo

    2016-01-01

    The method in this chapter describes the design of human immunodeficiency virus type 1 (HIV-1) integrase (IN)-fusion proteins which we have developed to transport different proteins into the nuclei of lentiviral vector (LV)-transduced cells. The IN-fusion protein cDNA is incorporated into the LV packaging plasmid, which leads to its incorporation into vector particles as part of a large Gag-Pol polyprotein. This specific feature of protein packaging enables also the incorporation of cytotoxic and proapoptotic proteins, such as frequently cutting endonucleases and P53. The vectors can hence be used for various protein transduction needs. An outline of the necessary methods is also given to study the functionality of a chosen IN-fusion protein in a cell culture assay. PMID:27317182

  17. Short communication: analysis of the integrase gene from HIV type 1-positive patients living in a rural area of West Cameroon.

    Science.gov (United States)

    Turriziani, Ombretta; Montagna, Claudia; Falasca, Francesca; Bucci, Mauro; Russo, Gianluca; Lichtner, Miriam; Sobze, Martin Sanou; Vullo, Vincenzo; Pistello, Mauro; Antonelli, Guido

    2012-12-01

    Major mutations associated with HIV-I integrase inhibitors (INI) resistance are rare in INI-naive patients. However, polymorphisms at positions that may influence the genetic barrier and/or drive the selection of specific INI resistance pathways are common in HIV non-B subtypes. The aim was to evaluate the presence of natural polymorphisms and/or INI resistance mutations in HIV-1 non-B subtype samples obtained from INI-naive patients living in rural west Cameroon. Thirty-three HIV-1 non-B samples were obtained from INI-naive African women and, as controls, 15 samples of HIV-1 subtype B were obtained from antiretroviral-naive Italian patients. The integrase gene was amplified and sequenced using Trugene Core Reagents. Several amino acid positions in B and non-B subtypes were found to be polymorphic. Interestingly, two patients infected with the CRF02_AG subtype had the resistance mutations N155H and E157Q/E and 12% of African samples had an amino acid substitution at position 143. Silent mutations leading to a higher increment of genetic barriers were detected at 140 and 151 positions in non B-subtypes. Although most polymorphisms may have little effect on INI susceptibility, the IN gene variations found in the present study should be taken into consideration as they may facilitate or delay the emergence of variants fully resistant to INIs. PMID:22214532

  18. Short communication: analysis of the integrase gene from HIV type 1-positive patients living in a rural area of West Cameroon.

    Science.gov (United States)

    Turriziani, Ombretta; Montagna, Claudia; Falasca, Francesca; Bucci, Mauro; Russo, Gianluca; Lichtner, Miriam; Sobze, Martin Sanou; Vullo, Vincenzo; Pistello, Mauro; Antonelli, Guido

    2012-12-01

    Major mutations associated with HIV-I integrase inhibitors (INI) resistance are rare in INI-naive patients. However, polymorphisms at positions that may influence the genetic barrier and/or drive the selection of specific INI resistance pathways are common in HIV non-B subtypes. The aim was to evaluate the presence of natural polymorphisms and/or INI resistance mutations in HIV-1 non-B subtype samples obtained from INI-naive patients living in rural west Cameroon. Thirty-three HIV-1 non-B samples were obtained from INI-naive African women and, as controls, 15 samples of HIV-1 subtype B were obtained from antiretroviral-naive Italian patients. The integrase gene was amplified and sequenced using Trugene Core Reagents. Several amino acid positions in B and non-B subtypes were found to be polymorphic. Interestingly, two patients infected with the CRF02_AG subtype had the resistance mutations N155H and E157Q/E and 12% of African samples had an amino acid substitution at position 143. Silent mutations leading to a higher increment of genetic barriers were detected at 140 and 151 positions in non B-subtypes. Although most polymorphisms may have little effect on INI susceptibility, the IN gene variations found in the present study should be taken into consideration as they may facilitate or delay the emergence of variants fully resistant to INIs.

  19. Eliminación de As (V y producción simultánea de polvo de cobre de un electrolito de Cu(II - As(V - H2SO4 mediante electrodiálisis reactiva

    Directory of Open Access Journals (Sweden)

    Ibáñez, J. P.

    2012-06-01

    Full Text Available The removal of As(V and the simultaneous generation of powder of copper from an electrolyte made of As(V - Cu(II - H2SO4 was studied by using electro dialysis at several current densities, temperatures and aeration of the electrolyte. The removal of arsenic was proportional to the current density, temperature and aeration used. The removal of arsenic reached a value of 0.14 mmol/h at 500 A/m2, 25 °C and without aeration, this value increased to 0.31 mmol/h by increasing the aeration to 6.6 l/h. The Cu(II was recovered in a 98 % as a fine arsenic free powder of metallic copper with oxides of copper. The arsenic was removed from the electrolyte by adsorption onto the anodic slimes generated from the lead anode oxidation.Se investigó la utilización de la electrodiálisis reactiva para eliminar arsénico y producir en forma simultánea polvo de cobre de una solución de Cu(II - As(V - H2SO4, a distintas densidades de corriente, temperatura y nivel de aireación de la solución. La eliminación de arsénico fue directamente proporcional a la densidad de corriente, aireación y temperatura. La eliminación alcanzada de As(V fue de 0,14 mmol/h a 500 A/m2, 25 °C y sin aireación, al introducir un flujo de 6,6 l/h de aire a la solución de trabajo se incrementa la eliminación a 0,31 mmol/h. El Cu(II se recupera en un 98 % en la forma de polvo de cobre metálico y óxidos de cobre libres de arsénico. El As(V es eliminado de la solución de trabajo mediante su adsorción en la borra anódica generada por la oxidación del ánodo de plomo empleado.

  20. HIV整合酶抑制剂的应用及耐药研究进展%Research progress in appbication of HIV-1 integrase inhibitor and drug resistance

    Institute of Scientific and Technical Information of China (English)

    王欣; 程绍辉

    2012-01-01

    Objective HIV integrase is one of the most important enzymes to virus reproduction. It can catalyzes the course of integration involved in HIV replication cycle, by which the reverse transcribed cDNA of HIV is inserted into the host genome. Raltegravir, which is a new drug for treating AIDS, belongs to integrase inhibitor. With the clinical application of integrase inhibitor, drug-resistance is emerging inevitably. This review describes the recent development in researches on HIV integrase inhibitor resistance and its clinical implications.%目的 艾滋病病毒(HIV)整合酶是病毒复制所必需的基本酶之一.它能够催化病毒复制周期中的整合过程,将病毒的cDNA整合入宿主基因组中.以雷替格韦(Raltegravir,RAL)为代表的整合酶抑制剂,是新一代治疗艾滋病的药物.随着其在临床上的应用,也不可避免地出现了耐药问题.文章综述了目前HIV整合酶抑制剂在临床上的应用以及耐药情况的最新进展.

  1. Synthesis of green nano iron particles (GnIP) and their application in adsorptive removal of As(III) and As(V) from aqueous solution

    Energy Technology Data Exchange (ETDEWEB)

    Prasad, Kumar Suranjit, E-mail: suranjit@gmail.com [Department of Environmental Studies, Faculty of Science, The M. S. University of Baroda, Vadodara, 390002, Gujarat (India); Gandhi, Pooja, E-mail: poojagandhi.3090@gmail.com [Department of Environmental Sciences, Ashok and Rita Patel Institute of Integrated Study and Research in Biotechnology and Allied Sciences (ARIBAS), New Vallabh Vidyanagar, Anand, Gujarat, 388121 (India); Selvaraj, Kaliaperumal, E-mail: k.selvaraj@ncl.res.in [Nano and Computational Materials Lab, Catalysis Division, National Chemical Laboratory, Council of Scientific and Industrial Research, Pune, 411008 (India)

    2014-10-30

    Graphical abstract: - Highlights: • Colloidal GnIP synthesised using extract of Mint leaves were entrapped in chitosan beads. • GnIP loaded beads were employed for removal of As ions, showed excellent removal efficiency. • Iron and chitosan are cost effective materials hence can be a good adsorbent for removal of arsenic. - Abstract: The present study reports a new approach to synthesise nano iron particles using leaf extract of Mint (Mentha spicata L.) plant. The synthesised GnIPs were subjected to detailed adsorption studies for removal of arsenite and arsenate from aqueous solution of defined concentration. Iron nanoparticles synthesised using leaf extract showed UV–vis absorption peaks at 360 and 430 nm. TEM result showed the formation of polydispersed nanoparticles of size ranging from 20 to 45 nm. Nanoparticles were found to have core–shell structure. The planer reflection of selected area electron diffraction (SAED) and XRD analysis suggested that iron particles were crystalline and belonged to fcc (face centred cubic) type. Energy-dispersive X-ray analysis (EDAX) shows that Fe was an integral component of synthesised nanoparticles. The content of Fe in nanoparticles was found to be 40%, in addition to other elements like C (16%), O (19%) and Cl (23%). FT-IR study suggested that functional groups like -NH, -C=O, -C=N and -C=C were involved in particle formation. The removal efficiency of GnIP-chitosan composite for As(III) and As(V) was found to be 98.79 and 99.65%. Regeneration of adsorbent suggested that synthesised green GnIP may work as an effective tool for removal of arsenic from contaminated water.

  2. Natural polymorphisms of HIV-1 CRF01_AE integrase coding region in ARV-naïve individuals in Cambodia, Thailand and Vietnam: an ANRS AC12 working group study.

    Science.gov (United States)

    Nouhin, Janin; Donchai, Tawee; Hoang, Khanh Thu Huynh; Ken, Sreymom; Kamkorn, Jiraporn; Tran, Ton; Ayouba, Ahidjo; Peeters, Martine; Chaix, Marie-Laure; Lien, Truong Xuan; Nerrienet, Eric; Ngo-Giang-Huong, Nicole

    2011-01-01

    The HIV integrase enzyme is essential for the HIV life cycle as it mediates integration of HIV-1 proviral DNA into the infected cell's genome. Recently, the development of drugs capable of inhibiting integrase has provided major new options for HIV-infected, treatment-experienced patients with multidrug resistant virus, as well treatment-naïve patients. More than 40 amino acid substitutions within integrase have been described as associated mostly with resistance of HIV B-subtypes to currently available integrase inhibitors (INIs). We have analyzed the natural polymorphisms of the integrase coding region in 87 antiretroviral-naïve subjects (32 from Cambodia, 37 from Thailand and 18 from Vietnam) infected with CRF01_AE virus, the predominant HIV-1 strain circulating in Southeast Asia. The 864bp integrase coding region was sequenced using the ANRS consensus sequencing technique from plasma samples, and amino acid results were interpreted for drug resistance according to the ANRS (Updated July 2009, version 18) and Stanford algorithms (Version November 6, 2009). Alignment of the 87 amino acid sequences against the 2004 Los Alamos HIV-1 clade B consensus sequence showed that overall, 119 of 288 (41.3%) amino acid positions presented at least one polymorphism each. Substitutions found in >60% of study subjects occurred at: K14, A21, V31, S39, I72, T112, T124, T125, G134, I135, K136, D167, V201, L234 and S283. Also, new amino acid substitutions of as yet unknown significance were identified: E152K/H, S153F/L, N155I and E157G. None of the known integrase resistance mutations were observed, except E157Q found in one Cambodian subject (1.1%, CI 95% 0.02-6.3%). The clinical impact of this substitution on resistance of B and nonB-viruses to the licensed INI raltegravir is unclear. If this substitution is confirmed to compromise the virologic response to raltegravir, further studies will be needed to better assess the prevalence of this substitution among CRF01_AE virus

  3. Natural polymorphisms of HIV-1 CRF01_AE integrase coding region in ARV-naïve individuals in Cambodia, Thailand and Vietnam: an ANRS AC12 working group study.

    Science.gov (United States)

    Nouhin, Janin; Donchai, Tawee; Hoang, Khanh Thu Huynh; Ken, Sreymom; Kamkorn, Jiraporn; Tran, Ton; Ayouba, Ahidjo; Peeters, Martine; Chaix, Marie-Laure; Lien, Truong Xuan; Nerrienet, Eric; Ngo-Giang-Huong, Nicole

    2011-01-01

    The HIV integrase enzyme is essential for the HIV life cycle as it mediates integration of HIV-1 proviral DNA into the infected cell's genome. Recently, the development of drugs capable of inhibiting integrase has provided major new options for HIV-infected, treatment-experienced patients with multidrug resistant virus, as well treatment-naïve patients. More than 40 amino acid substitutions within integrase have been described as associated mostly with resistance of HIV B-subtypes to currently available integrase inhibitors (INIs). We have analyzed the natural polymorphisms of the integrase coding region in 87 antiretroviral-naïve subjects (32 from Cambodia, 37 from Thailand and 18 from Vietnam) infected with CRF01_AE virus, the predominant HIV-1 strain circulating in Southeast Asia. The 864bp integrase coding region was sequenced using the ANRS consensus sequencing technique from plasma samples, and amino acid results were interpreted for drug resistance according to the ANRS (Updated July 2009, version 18) and Stanford algorithms (Version November 6, 2009). Alignment of the 87 amino acid sequences against the 2004 Los Alamos HIV-1 clade B consensus sequence showed that overall, 119 of 288 (41.3%) amino acid positions presented at least one polymorphism each. Substitutions found in >60% of study subjects occurred at: K14, A21, V31, S39, I72, T112, T124, T125, G134, I135, K136, D167, V201, L234 and S283. Also, new amino acid substitutions of as yet unknown significance were identified: E152K/H, S153F/L, N155I and E157G. None of the known integrase resistance mutations were observed, except E157Q found in one Cambodian subject (1.1%, CI 95% 0.02-6.3%). The clinical impact of this substitution on resistance of B and nonB-viruses to the licensed INI raltegravir is unclear. If this substitution is confirmed to compromise the virologic response to raltegravir, further studies will be needed to better assess the prevalence of this substitution among CRF01_AE virus.

  4. Major intrinsic proteins and arsenic transport in plants: new players and their potential role.

    Science.gov (United States)

    Bienert, Gerd P; Jahn, Thomas P

    2010-01-01

    Arsenic (As) is a toxic and highly abundant metalloid that endangers human health through drinking water and the food chain. The most common forms of As in the environment re arsenate [As(V)] and arsenite [As(III)]. As(V) is a nonfunctional phosphate analog that enters the food chain via plant phosphate transporters. Recently, evidence was provided that uptake of As(III)--the second most abundant As species in soils--is mediated by plant nodulin26-like intrinsic proteins (NIPs), a subfamily of plant major intrinsic proteins (MIPs). Specific NIPs are also essential for the uptake of the metalloids boron and silicon and aquaglyceroporins from microbes and mammals were shown to be the major routes of As uptake. Therefore As(III) transport through MIPs is a conserved and ancient feature. In this chapter we summarize the current view on As transport in plants and address the potential physiological significance of As(III) transport through NIPs.

  5. Multiple cellular proteins interact with LEDGF/p75 through a conserved unstructured consensus motif.

    Science.gov (United States)

    Tesina, Petr; Čermáková, Kateřina; Hořejší, Magdalena; Procházková, Kateřina; Fábry, Milan; Sharma, Subhalakshmi; Christ, Frauke; Demeulemeester, Jonas; Debyser, Zeger; De Rijck, Jan; Veverka, Václav; Řezáčová, Pavlína

    2015-01-01

    Lens epithelium-derived growth factor (LEDGF/p75) is an epigenetic reader and attractive therapeutic target involved in HIV integration and the development of mixed lineage leukaemia (MLL1) fusion-driven leukaemia. Besides HIV integrase and the MLL1-menin complex, LEDGF/p75 interacts with various cellular proteins via its integrase binding domain (IBD). Here we present structural characterization of IBD interactions with transcriptional repressor JPO2 and domesticated transposase PogZ, and show that the PogZ interaction is nearly identical to the interaction of LEDGF/p75 with MLL1. The interaction with the IBD is maintained by an intrinsically disordered IBD-binding motif (IBM) common to all known cellular partners of LEDGF/p75. In addition, based on IBM conservation, we identify and validate IWS1 as a novel LEDGF/p75 interaction partner. Our results also reveal how HIV integrase efficiently displaces cellular binding partners from LEDGF/p75. Finally, the similar binding modes of LEDGF/p75 interaction partners represent a new challenge for the development of selective interaction inhibitors.

  6. Speciation of the immediately mobilisable As(III), As(V), MMA and DMA in river sediments by high performance liquid chromatography-hydride generation-atomic fluorescence spectrometry following ultrasonic extraction

    International Nuclear Information System (INIS)

    In this work, a fast method is developed for the speciation of As(III), As(V), MMA and DMA in the immediately mobilisable fraction of river sediments (i.e. water-soluble and phosphate-exchangeable) by high performance liquid chromatography-hydride generation-atomic fluorescence detection (HPLC-HG-AFD) after extraction using focused ultrasound. The influence of relevant parameters influencing an ion-pairing chromatographic separation following isocratic elution (i.e. amount of MeOH in the mobile phase, ion pair reagent concentration, pH, flow rate) was studied. Focused ultrasound transmitted from an ultrasonic probe provided the same extractable contents as conventional extraction with no changes in the species distribution. The effect of the drying step over extraction of As species was investigated. The following drying procedures were compared: freeze-, oven-, microwave- and air-drying. No influence of the drying operation on the water-extractable fraction was observed. However, freeze- and air-drying yielded significantly higher phosphate-extractable amounts of As(III) and As(V) as compared to oven and microwaves. Detection limits for the As species were in the range 1.3-4.1 ng/g for the water-soluble fraction and 1.6-4.8 ng/g for the phosphate buffer exchangeable fraction. The method was applied to the speciation of immediately mobilisable As(III), As(V), DMA and MMA in 11 sediment samples collected along the beds of the Louro River (southern Galicia, Spain)

  7. Speciation of As(III) and As(V) in water samples by graphite furnace atomic absorption spectrometry after solid phase extraction combined with dispersive liquid-liquid microextraction based on the solidification of floating organic drop.

    Science.gov (United States)

    Shamsipur, Mojtaba; Fattahi, Nazir; Assadi, Yaghoub; Sadeghi, Marzieh; Sharafi, Kiomars

    2014-12-01

    A solid phase extraction (SPE) coupled with dispersive liquid-liquid microextraction based on the solidification of floating organic drop (DLLME-SFO) method, using diethyldithiphosphate (DDTP) as a proper chelating agent, has been developed as an ultra preconcentration technique for the determination of inorganic arsenic in water samples prior to graphite furnace atomic absorption spectrometry (GFAAS). Variables affecting the performance of both steps were thoroughly investigated. Under optimized conditions, 100mL of As(ΙΙΙ) solution was first concentrated using a solid phase sorbent. The extract was collected in 2.0 mL of acetone and 60.0 µL of 1-undecanol was added into the collecting solvent. The mixture was then injected rapidly into 5.0 mL of pure water for further DLLME-SFO. Total inorganic As(III, V) was extracted similarly after reduction of As(V) to As(III) with potassium iodide and sodium thiosulfate and As(V) concentration was calculated by difference. A mixture of Pd(NO3)2 and Mg(NO3)2 was used as a chemical modifier in GFAAS. The analytical characteristics of the method were determined. The calibration graph was linear in the rage of 10-100 ng L(-1) with detection limit of 2.5 ng L(-1). Repeatability (intra-day) and reproducibility (inter-day) of method based on seven replicate measurements of 80 ng L(-1) of As(ΙΙΙ) were 6.8% and 7.5%, respectively. The method was successfully applied to speciation of As(III), As(V) and determination of the total amount of As in water samples and in a certified reference material (NIST RSM 1643e). PMID:25159375

  8. Speciation of the immediately mobilisable As(III), As(V), MMA and DMA in river sediments by high performance liquid chromatography-hydride generation-atomic fluorescence spectrometry following ultrasonic extraction

    Energy Technology Data Exchange (ETDEWEB)

    Huerga, A. [Departamento de Quimica Analitica y Alimentaria, Area de Quimica Analitica, Facultad de Ciencias (Quimica), Universidad de Vigo, As Lagoas-Marcosende s/n, 36200 Vigo (Spain); Lavilla, I. [Departamento de Quimica Analitica y Alimentaria, Area de Quimica Analitica, Facultad de Ciencias (Quimica), Universidad de Vigo, As Lagoas-Marcosende s/n, 36200 Vigo (Spain); Bendicho, C. [Departamento de Quimica Analitica y Alimentaria, Area de Quimica Analitica, Facultad de Ciencias (Quimica), Universidad de Vigo, As Lagoas-Marcosende s/n, 36200 Vigo (Spain)]. E-mail: bendicho@uvigo.es

    2005-04-04

    In this work, a fast method is developed for the speciation of As(III), As(V), MMA and DMA in the immediately mobilisable fraction of river sediments (i.e. water-soluble and phosphate-exchangeable) by high performance liquid chromatography-hydride generation-atomic fluorescence detection (HPLC-HG-AFD) after extraction using focused ultrasound. The influence of relevant parameters influencing an ion-pairing chromatographic separation following isocratic elution (i.e. amount of MeOH in the mobile phase, ion pair reagent concentration, pH, flow rate) was studied. Focused ultrasound transmitted from an ultrasonic probe provided the same extractable contents as conventional extraction with no changes in the species distribution. The effect of the drying step over extraction of As species was investigated. The following drying procedures were compared: freeze-, oven-, microwave- and air-drying. No influence of the drying operation on the water-extractable fraction was observed. However, freeze- and air-drying yielded significantly higher phosphate-extractable amounts of As(III) and As(V) as compared to oven and microwaves. Detection limits for the As species were in the range 1.3-4.1 ng/g for the water-soluble fraction and 1.6-4.8 ng/g for the phosphate buffer exchangeable fraction. The method was applied to the speciation of immediately mobilisable As(III), As(V), DMA and MMA in 11 sediment samples collected along the beds of the Louro River (southern Galicia, Spain)

  9. Mutations in the catalytic core or the C-terminus of murine leukemia virus (MLV) integrase disrupt virion infectivity and exert diverse effects on reverse transcription

    International Nuclear Information System (INIS)

    Understanding of the structures and functions of the retroviral integrase (IN), a key enzyme in the viral replication cycle, is essential for developing antiretroviral treatments and facilitating the development of safer gene therapy vehicles. Thus, four MLV IN-mutants were constructed in the context of a retroviral vector system, harbouring either a substitution in the catalytic centre, deletions in the C-terminus, or combinations of both modifications. IN-mutants were tested for their performance in different stages of the viral replication cycle: RNA-packaging; RT-activity; transient and stable infection efficiency; dynamics of reverse transcription and nuclear entry. All mutant vectors packaged viral RNA with wild-type efficiencies and displayed only slight reductions in RT-activity. Deletion of either the IN C-terminus alone, or in addition to part of the catalytic domain exerted contrasting effects on intracellular viral DNA levels, implying that IN influences reverse transcription in more than one direction

  10. Detection of integrase in retroviral particles by a specific DNA sensor with a potential for adaptation to point-of-care diagnosis

    DEFF Research Database (Denmark)

    Wang, Jing

    2016-01-01

    might be controlled by rational drug development and combination therapy. Early detection of HIV-positive individuals has a direct impact on therapeutic efficacy and can therefore reduce the risk of transmission. With regard to the disadvantage of the current routine HIV screening, it is therefore......The acquired immunodeficiency syndrome (AIDS) caused by HIV is one of the greatest threats to human health. Currently, HIV-infected individuals are treated by FDA approved highly active antiretroviral therapy that targets three steps of the HIV life cycle. HAART includes a protease inhibitor...... or a nonnucleoside reverse transcriptase inhibitor in combination with two nucleoside/nucleotide reverse transcriptase inhibitors. More recently, also an integrase (IN) inhibitor was included in the treatment protocol. If the HIV-infected individuals could be identified and treated immediately after infection, AIDS...

  11. 2-hydroxyisoquinoline-1,3(2H,4H)-diones (HIDs) as human immunodeficiency virus type 1 integrase inhibitors: Influence of the alkylcarboxamide substitution of position 4.

    Science.gov (United States)

    Billamboz, Muriel; Suchaud, Virginie; Bailly, Fabrice; Lion, Cedric; Andréola, Marie-Line; Christ, Frauke; Debyser, Zeger; Cotelle, Philippe

    2016-07-19

    Herein, we report further insight into the biological activities displayed by the 2-hydroxyisoquinoline-1,3(2H,4H)-dione (HID) scaffold. Previous studies have evidenced the marked fruitful effect of substitution of this two-metal binding pharmacophore at position 4 by phenyl and benzyl carboxamido chains. Strong human immunodeficiency virus type 1 integrase (HIV-1 IN) inhibitors in the low nanomolar range with micromolar (even down to low nanomolar) anti-HIV activities were obtained. Keeping this essential 4-carboxamido function, we investigated the influence of the replacement of phenyl and benzyl groups by various alkyl chains. This study shows that the recurrent halogenobenzyl pharmacophore found in the INSTIs can be efficiently replaced by an n-alkyl group. With an optimal length of six carbons, we observed a biological profile and a high barrier to resistance equivalent to those of a previously reported hit compound bearing a 4-fluorobenzyl group. PMID:27105029

  12. Synthesis and anti-HIV activity of some [Nucleoside Reverse Transcriptase Inhibitor]-C5'-linker-[Integrase Inhibitor] heterodimers as inhibitors of HIV replication.

    Science.gov (United States)

    Sugeac, Elena; Fossey, Christine; Ladurée, Daniel; Schmidt, Sylvie; Laumond, Geraldine; Aubertin, Anne-Marie

    2004-12-01

    Selected for their expected ability to inhibit HIV replication, a series of eight heterodimers containing a Nucleoside Reverse Transcriptase Inhibitor (NRTI) and an Integrase Inhibitor (INI), bound by a linker, were designed and synthesized. For the NRTIs, d4U, d2U and d4T were chosen. For the INIs, 4-[1-(4-fluorobenzyl)-1H-pyrrol-2-yl]-2,4-dioxobutyric acid (6) and 4-(3,5-dibenzyloxyphenyl)-2,4-dioxobutyric acid (9) (belonging to the beta-diketo acids class) were chosen. The conjugation of the two different inhibitors (NRTI and INI) was performed using an amino acid (glycine or beta-alanine) as a cleavable linker. PMID:15662954

  13. 4-Substituted 2-Hydroxyisoquinoline-1,3(2H,4H)-diones as a Novel Class of HIV-1 Integrase Inhibitors.

    Science.gov (United States)

    Billamboz, Muriel; Suchaud, Virginie; Bailly, Fabrice; Lion, Cedric; Demeulemeester, Jonas; Calmels, Christina; Andréola, Marie-Line; Christ, Frauke; Debyser, Zeger; Cotelle, Philippe

    2013-07-11

    A series of 2-hydroxy-1,3-dioxoisoquinoline-4-carboxamides featuring an N-hydroxyimide chelating functionality was evaluated for their inhibitory properties against human immunodeficiency virus type 1 integrase (HIV-1 IN). Several derivatives displayed low nanomolar IC50 values comparable to that of the clinically used raltegravir. A marked effect of one compound on both primary IN-catalyzed reactions, strand transfer (ST), and 3' processing (3'-P), emphasizes a novel IN inhibition mechanism establishing it as a potential new generation IN inhibitor. Substitution of the 2-hydroxyisoquinoline-1,3-dione scaffold at position 4 by carboxamido chains was beneficial for antiviral activity since reproducible low micromolar anti-HIV activities were obtained for the first time within this scaffold. PMID:24900718

  14. Synthesis and anti-HIV activity of some [Nucleoside Reverse Transcriptase Inhibitor]-C5'-linker-[Integrase Inhibitor] heterodimers as inhibitors of HIV replication.

    Science.gov (United States)

    Sugeac, Elena; Fossey, Christine; Ladurée, Daniel; Schmidt, Sylvie; Laumond, Geraldine; Aubertin, Anne-Marie

    2004-12-01

    Selected for their expected ability to inhibit HIV replication, a series of eight heterodimers containing a Nucleoside Reverse Transcriptase Inhibitor (NRTI) and an Integrase Inhibitor (INI), bound by a linker, were designed and synthesized. For the NRTIs, d4U, d2U and d4T were chosen. For the INIs, 4-[1-(4-fluorobenzyl)-1H-pyrrol-2-yl]-2,4-dioxobutyric acid (6) and 4-(3,5-dibenzyloxyphenyl)-2,4-dioxobutyric acid (9) (belonging to the beta-diketo acids class) were chosen. The conjugation of the two different inhibitors (NRTI and INI) was performed using an amino acid (glycine or beta-alanine) as a cleavable linker.

  15. Construction of transformed, cultured silkworm cells and transgenic silkworm using the site-specific integrase system from phage φC31.

    Science.gov (United States)

    Yin, Yajuan; Cao, Guangli; Xue, Renyu; Gong, Chengliang

    2014-10-01

    The Streptomyces bacteriophage, φC31, uses a site-specific integrase enzyme to perform efficient recombination. The recombination system uses specific sequences to integrate exogenous DNA from the phage into a host. The sequences are known as the attP site in the phage and the attB site in the host. The system can be used as a genetic manipulation tool. In this study it has been applied to the transformation of cultured BmN cells and the construction of transgenic Bombyx mori individuals. A plasmid, pSK-attB/Pie1-EGFP/Zeo-PASV40, containing a cassette designed to express a egfp-zeocin fusion gene, was co-transfected into cultured BmN cells with a helper plasmid, pSK-Pie1/NLS-Int/NSL. Expression of the egfp-zeocin fusion gene was driven by an ie-1 promoter, downstream of a φC31 attB site. The helper plasmid encoded the φC31 integrase enzyme, which was flanked by two nuclear localization signals. Expression of the egfp-zeocin fusion gene could be observed in transformed cells. The two plasmids were also transferred into silkworm eggs to obtain transgenic silkworms. Successful integration of the fusion gene was indicated by the detection of green fluorescence, which was emitted by the silkworms. Nucleotide sequence analysis demonstrated that the attB site had been cut, to allow recombination between the attB and endogenous pseudo attP sites in the cultured silkworm cells and silkworm individuals.

  16. Inorganic and organic fertilizers impact the abundance and proportion of antibiotic resistance and integron-integrase genes in agricultural grassland soil.

    Science.gov (United States)

    Nõlvak, Hiie; Truu, Marika; Kanger, Kärt; Tampere, Mailiis; Espenberg, Mikk; Loit, Evelin; Raave, Henn; Truu, Jaak

    2016-08-15

    Soil fertilization with animal manure or its digestate may facilitate an important antibiotic resistance dissemination route from anthropogenic sources to the environment. This study examines the effect of mineral fertilizer (NH4NO3), cattle slurry and cattle slurry digestate amendment on the abundance and proportion dynamics of five antibiotic resistance genes (ARGs) and two classes of integron-integrase genes (intI1 and intI2) in agricultural grassland soil. Fertilization was performed thrice throughout one vegetation period. The targeted ARGs (sul1, tetA, blaCTX-M, blaOXA2 and qnrS) encode resistance to several major antibiotic classes used in veterinary medicine such as sulfonamides, tetracycline, cephalosporins, penicillin and fluoroquinolones, respectively. The non-fertilized grassland soil contained a stable background of tetA, blaCTX-M and sul1 genes. The type of applied fertilizer significantly affected ARGs and integron-integrase genes abundances and proportions in the bacterial community (porganic fertilizer's application event, but this increase was followed by a stage of decrease, suggesting that microbes possessing these genes were predominantly entrained into soil via cattle slurry or its digestate application and had somewhat limited survival potential in a soil environment. However, the abundance of these three target genes did not decrease to a background level by the end of the study period. TetA was most abundant in mineral fertilizer treated soil and blaCTX-M in cattle slurry digestate amended soil. Despite significantly different abundances, the abundance dynamics of bacteria possessing these genes were similar (p<0.05 in all cases) in different treatments and resembled the dynamics of the whole bacterial community abundance in each soil treatment. PMID:27115621

  17. Genetic variation of the HIV-1 integrase region in newly diagnosed anti-retroviral drug-naïve patients with HIV/AIDS in Korea.

    Science.gov (United States)

    Kim, J-Y; Kim, E-J; Choi, J-Y; Kwon, O-K; Kim, G J; Choi, S Y; Kim, S S

    2011-08-01

    The survival time of HIV/AIDS patients in Korea has increased since HAART (highly active anti-retroviral therapy) was introduced. However, the occurrence of drug-resistant strains requires new anti-retroviral drugs, one of which, an integrase inhibitor (INI), was approved by the US Food and Drug Administration (FDA) in 2007. INIs have been used for therapy in many countries and are about to be employed in Korea. Therefore, it is important to identify basic mutant variants prior to the introduction of INIs in order to estimate their efficacy. To monitor potential drug-resistant INI mutations in Korean HIV/AIDS patients, the polymorphism of the int gene was investigated together with the pol gene using a genotypic assay for 75 randomly selected Korean HIV-1 patients newly diagnosed in 2007. The drug-resistant mutation sequences were analysed using the Stanford HIV DB and the International AIDS Society resistance testing-USA panel (IAS-USA). Seventy strains of Korean subtype B were compared with foreign subtype-B strains, and there were no significantly different variants of the int gene region in the study population. Major mutation sites in the integrase (E92Q, F121Y, G140A/S, Y143C/R, Q148H/R/K and N155H) were not detected, and only a few minor mutation sites (L74M, V151I, E157Q, V165I, I203M, S230N and D232N) were identified in 21 strains (28%). Resistance due to mutations in the pol gene was observed in a single strain (1.3%) resistant to protease inhibitors (PIs) and in four strains (5.3%) resistant to reverse transcriptase inhibitors (RTIs). In summary, this demonstrates that INIs will be susceptible to drug naïve HIV/AIDS patients in Korea. PMID:20946407

  18. Genetic variation of the HIV-1 integrase region in newly diagnosed anti-retroviral drug-naïve patients with HIV/AIDS in Korea.

    Science.gov (United States)

    Kim, J-Y; Kim, E-J; Choi, J-Y; Kwon, O-K; Kim, G J; Choi, S Y; Kim, S S

    2011-08-01

    The survival time of HIV/AIDS patients in Korea has increased since HAART (highly active anti-retroviral therapy) was introduced. However, the occurrence of drug-resistant strains requires new anti-retroviral drugs, one of which, an integrase inhibitor (INI), was approved by the US Food and Drug Administration (FDA) in 2007. INIs have been used for therapy in many countries and are about to be employed in Korea. Therefore, it is important to identify basic mutant variants prior to the introduction of INIs in order to estimate their efficacy. To monitor potential drug-resistant INI mutations in Korean HIV/AIDS patients, the polymorphism of the int gene was investigated together with the pol gene using a genotypic assay for 75 randomly selected Korean HIV-1 patients newly diagnosed in 2007. The drug-resistant mutation sequences were analysed using the Stanford HIV DB and the International AIDS Society resistance testing-USA panel (IAS-USA). Seventy strains of Korean subtype B were compared with foreign subtype-B strains, and there were no significantly different variants of the int gene region in the study population. Major mutation sites in the integrase (E92Q, F121Y, G140A/S, Y143C/R, Q148H/R/K and N155H) were not detected, and only a few minor mutation sites (L74M, V151I, E157Q, V165I, I203M, S230N and D232N) were identified in 21 strains (28%). Resistance due to mutations in the pol gene was observed in a single strain (1.3%) resistant to protease inhibitors (PIs) and in four strains (5.3%) resistant to reverse transcriptase inhibitors (RTIs). In summary, this demonstrates that INIs will be susceptible to drug naïve HIV/AIDS patients in Korea.

  19. Inorganic and organic fertilizers impact the abundance and proportion of antibiotic resistance and integron-integrase genes in agricultural grassland soil.

    Science.gov (United States)

    Nõlvak, Hiie; Truu, Marika; Kanger, Kärt; Tampere, Mailiis; Espenberg, Mikk; Loit, Evelin; Raave, Henn; Truu, Jaak

    2016-08-15

    Soil fertilization with animal manure or its digestate may facilitate an important antibiotic resistance dissemination route from anthropogenic sources to the environment. This study examines the effect of mineral fertilizer (NH4NO3), cattle slurry and cattle slurry digestate amendment on the abundance and proportion dynamics of five antibiotic resistance genes (ARGs) and two classes of integron-integrase genes (intI1 and intI2) in agricultural grassland soil. Fertilization was performed thrice throughout one vegetation period. The targeted ARGs (sul1, tetA, blaCTX-M, blaOXA2 and qnrS) encode resistance to several major antibiotic classes used in veterinary medicine such as sulfonamides, tetracycline, cephalosporins, penicillin and fluoroquinolones, respectively. The non-fertilized grassland soil contained a stable background of tetA, blaCTX-M and sul1 genes. The type of applied fertilizer significantly affected ARGs and integron-integrase genes abundances and proportions in the bacterial community (porganic fertilizer's application event, but this increase was followed by a stage of decrease, suggesting that microbes possessing these genes were predominantly entrained into soil via cattle slurry or its digestate application and had somewhat limited survival potential in a soil environment. However, the abundance of these three target genes did not decrease to a background level by the end of the study period. TetA was most abundant in mineral fertilizer treated soil and blaCTX-M in cattle slurry digestate amended soil. Despite significantly different abundances, the abundance dynamics of bacteria possessing these genes were similar (p<0.05 in all cases) in different treatments and resembled the dynamics of the whole bacterial community abundance in each soil treatment.

  20. Potential disruption of protein-protein interactions by graphene oxide.

    Science.gov (United States)

    Feng, Mei; Kang, Hongsuk; Yang, Zaixing; Luan, Binquan; Zhou, Ruhong

    2016-06-14

    Graphene oxide (GO) is a promising novel nanomaterial with a wide range of potential biomedical applications due to its many intriguing properties. However, very little research has been conducted to study its possible adverse effects on protein-protein interactions (and thus subsequent toxicity to human). Here, the potential cytotoxicity of GO is investigated at molecular level using large-scale, all-atom molecular dynamics simulations to explore the interaction mechanism between a protein dimer and a GO nanosheet oxidized at different levels. Our theoretical results reveal that GO nanosheet could intercalate between the two monomers of HIV-1 integrase dimer, disrupting the protein-protein interactions and eventually lead to dimer disassociation as graphene does [B. Luan et al., ACS Nano 9(1), 663 (2015)], albeit its insertion process is slower when compared with graphene due to the additional steric and attractive interactions. This study helps to better understand the toxicity of GO to cell functions which could shed light on how to improve its biocompatibility and biosafety for its wide potential biomedical applications.

  1. Potential disruption of protein-protein interactions by graphene oxide

    Science.gov (United States)

    Feng, Mei; Kang, Hongsuk; Yang, Zaixing; Luan, Binquan; Zhou, Ruhong

    2016-06-01

    Graphene oxide (GO) is a promising novel nanomaterial with a wide range of potential biomedical applications due to its many intriguing properties. However, very little research has been conducted to study its possible adverse effects on protein-protein interactions (and thus subsequent toxicity to human). Here, the potential cytotoxicity of GO is investigated at molecular level using large-scale, all-atom molecular dynamics simulations to explore the interaction mechanism between a protein dimer and a GO nanosheet oxidized at different levels. Our theoretical results reveal that GO nanosheet could intercalate between the two monomers of HIV-1 integrase dimer, disrupting the protein-protein interactions and eventually lead to dimer disassociation as graphene does [B. Luan et al., ACS Nano 9(1), 663 (2015)], albeit its insertion process is slower when compared with graphene due to the additional steric and attractive interactions. This study helps to better understand the toxicity of GO to cell functions which could shed light on how to improve its biocompatibility and biosafety for its wide potential biomedical applications.

  2. Progress of φC31 integrase system in site-specific integration%φC31整合酶系统介导的位点特异性整合研究进展

    Institute of Scientific and Technical Information of China (English)

    马晴雯

    2011-01-01

    来源于链霉菌(Streptomyces)噬菌体φC31的整合酶可介导链霉菌附着位点(attB)和噬菌体附着位点(attP)之间的同源重组,这种重组亦可在多种动植物细胞内进行;而且,该整合酶还可介导含attB位点的载体以位点特异性方式整合于多种真核生物基因组内的假attP位点,并使转基因持续高效表达.因此,φC31整合酶在基因修饰、基因治疗及转基因动物研制等方面得到了广泛的应用.文章就近年来φC31整合酶整合规律、提高效率方面的改进及安全性等相关领域的研究进展进行了综述.%Integrase of phage φC31 catalyses the homologous recombination between Streptomyces attachment site attB and the phage attachment site attP.Meanwhile, this integrase can mediate integration of attB-containing donor plasmids into the pseudo attP sites in eukaryotic genomes by a site-specific manner and resulting long-term and robust expression of integrated genes.Nowadays, φC31 integrase system is becoming a potential tool for genome modification, gene therapy and transgenic research.Recent progress of φC31 integrase system in integration mode in mammalian genomes, efficiency improvement and researches concemed on transgenic safety were summarized in this review.

  3. Mapping the λ Integrase bridges in the nucleoprotein Holliday junction intermediates of viral integrative and excisive recombination

    OpenAIRE

    Tong, Wenjun; Warren, David; Seah, Nicole E.; Laxmikanthan, Gurunathan; Van Duyne, Gregory D.; Landy, Arthur

    2014-01-01

    The bacteriophage encoded λ Int protein is distinguished from other well-studied and widely exploited tyrosine recombinase family members as a heterobivalent DNA binding protein. With the help of accessory DNA bending proteins, Int bridges two different classes of DNA sites within the unique 400-kDa recombinogenic complexes of integrative and excisive recombination. The absence of any overarching investigations or structural models for these key complexes stems from the inability to determine...

  4. Expression, Purification, Crystallization and Molecular Replacement Studies of TorI, an Inhibition Protein of Tor System

    Institute of Scientific and Technical Information of China (English)

    HUANG Wei; YUAN Cai; Ansaldi Mireille; Morelli Xavier; Edward J. Meehan; CHEN Li-Qing; HUANG Ming-Dong

    2007-01-01

    TorI, a Tor system inhibitor acting through protein-protein interaction with the TorR response regulator, is an excisionase that interacts with the integrase and DNA during prophage excision. It has been crystallized by the vapor-diffusion method using polyethylene glycol 3350 as a precipitant at pH 8.5. The X-ray diffraction data sets from the TorI crystal was collected at a resolution of 2.1 (A), using a synchrotron source. The crystal belongs to primitive monoclinic lattice with cell parameters of 46.210(A) × 53.992(A) × 73.561(A)

  5. Characterization of the binding sites of two proteins involved in the bacteriophage P2 site-specific recombination system.

    OpenAIRE

    Yu, A.; Haggård-Ljungquist, E

    1993-01-01

    Integration of the bacteriophage P2 genome into the Escherichia coli host chromosome occurs by site-specific recombination between the phage attP and E. coli attB sites. The phage-encoded 38-kDa protein, integrase, is known to be necessary for both phage integration as well as excision. In order to begin the molecular characterization of this recombination event, we have cloned the int gene and overproduced and partially purified the Int protein and an N-terminal truncated form of Int. Both t...

  6. Human immunodeficiency virus integrase inhibitors efficiently suppress feline immunodeficiency virus replication in vitro and provide a rationale to redesign antiretroviral treatment for feline AIDS

    Directory of Open Access Journals (Sweden)

    Ciervo Alessandra

    2007-10-01

    Full Text Available Abstract Background Treatment of feline immunodeficiency virus (FIV infection has been hampered by the absence of a specific combination antiretroviral treatment (ART. Integrase strand transfer inhibitors (INSTIs are emerging as a promising new drug class for HIV-1 treatment, and we evaluated the possibility of inhibiting FIV replication using INSTIs. Methods Phylogenetic analysis of lentiviral integrase (IN sequences was carried out using the PAUP* software. A theoretical three-dimensional structure of the FIV IN catalytic core domain (CCD was obtained by homology modeling based on a crystal structure of HIV-1 IN CCD. The interaction of the transferred strand of viral DNA with the catalytic cavity of FIV IN was deduced from a crystal structure of a structurally similar transposase complexed with transposable DNA. Molecular docking simulations were conducted using a genetic algorithm (GOLD. Antiviral activity was tested in feline lymphoblastoid MBM cells acutely infected with the FIV Petaluma strain. Circular and total proviral DNA was quantified by real-time PCR. Results The calculated INSTI-binding sites were found to be nearly identical in FIV and HIV-1 IN CCDs. The close similarity of primate and feline lentivirus IN CCDs was also supported by phylogenetic analysis. In line with these bioinformatic analyses, FIV replication was efficiently inhibited in acutely infected cell cultures by three investigational INSTIs, designed for HIV-1 and belonging to different classes. Of note, the naphthyridine carboxamide INSTI, L-870,810 displayed an EC50 in the low nanomolar range. Inhibition of FIV integration in situ was shown by real-time PCR experiments that revealed accumulation of circular forms of FIV DNA within cells treated with L-870,810. Conclusion We report a drug class (other than nucleosidic reverse transcriptase inhibitors that is capable of inhibiting FIV replication in vitro. The present study helped establish L-870,810, a compound

  7. The BET Family of Proteins Targets Moloney Murine Leukemia Virus Integration near Transcription Start Sites

    Directory of Open Access Journals (Sweden)

    Jan De Rijck

    2013-11-01

    Full Text Available A hallmark of retroviral replication is integration of the viral genome into host cell DNA. This characteristic makes retrovirus-based vectors attractive delivery vehicles for gene therapy. However, adverse events in gene therapeutic trials, caused by activation of proto-oncogenes due to murine leukemia virus (MLV-derived vector integration, hamper their application. Here, we show that bromodomain and extraterminal (BET proteins (BRD2, BRD3, and BRD4 and MLV integrase specifically interact and colocalize within the nucleus of the cell. Inhibition of the BET proteins’ chromatin interaction via specific bromodomain inhibitors blocks MLV virus replication at the integration step. MLV integration site distribution parallels the chromatin binding profile of BET proteins, and expression of an artificial fusion protein of the BET integrase binding domain with the chromatin interaction domain of the lentiviral targeting factor LEDGF/p75 retargets MLV integration away from transcription start sites and into the body of actively transcribed genes, conforming to the HIV integration pattern. Together, these data validate BET proteins as MLV integration targeting factors.

  8. Studies on voltammetric determination of cadmium in samples containing native and digested proteins

    Energy Technology Data Exchange (ETDEWEB)

    Drozd, Marcin; Pietrzak, Mariusz, E-mail: mariusz@ch.pw.edu.pl; Malinowska, Elżbieta

    2014-03-01

    Highlights: • Proteins exhibit diverse impact on the DPASV cadmium signals. • Proteins subjected to HNO{sub 3} introduce less interference, than the native ones. • Optimal amount of SDS depends on the kind of protein. • Presence of thiolated coating agents of QDs do not influence the analysis. - Abstract: This work focuses on determination of cadmium ions using anodic stripping voltammetry (ASV) on thin film mercury electrode in conditions corresponding to those obtained after digestion of cadmium-based quantum dots and their conjugates. It presents the impact of selected proteins, including potential receptors and surface blocking agents on the voltammetric determination of cadmium. Experiments regarding elimination of interferences related to proteins presence using sodium dodecyl sulfate (SDS) are also shown. Effect of SDS on selected analytical parameters and simplicity of analyses carried out was investigated in the framework of current studies. The significant differences of influence among tested proteins on ASV cadmium determination, as well as the variability in SDS effectiveness as the antifouling agent were observed and explained. This work is especially important for those, who design new bioassays and biosensors with a use of quantum dots as electrochemical labels, as it shows what problems may arise from presence of native and digested proteins in tested samples.

  9. Study on multielemental speciation analysis of Cr(VI), As(III) and As(V) in water by advanced hyphenated technique HPLC/ICP-DRC-MS. Fast and reliable procedures.

    Science.gov (United States)

    Marcinkowska, Monika; Komorowicz, Izabela; Barałkiewicz, Danuta

    2015-11-01

    Analytical procedures for multielemental speciation analysis of arsenite - As(III), arsenate - As(V) and hexavalent chromium - Cr(VI) in water using high performance liquid chromatography inductively coupled plasma mass spectrometry (HPLC/ICP-DRC-MS) hyphenated technique have been developed. A dynamic reaction cell (DRC) for spectral interferences elimination has been involved in the experiment. Application of oxygen and ammonia as reaction gases in the DRC under various conditions has been verified. Optimization of the following chromatographic conditions: mobile phase composition, concentration of particular components, mobile phase pH and flow rate, injection volume and column temperature, has been conducted. Species separation has been carried out on anion-exchange chromatographic column using isocratic elution with: 22 mM (NH4)2HPO4, 25 mM NH4NO3 (for procedure A) and 22 mM (NH4)2HPO4, 65 mM NH4NO3 (for procedure B) as mobile phases. Detection limits (LD) for procedure A were 0.16 µg L(-1), 0.090 µg L(-1), 0.073 µg L(-1), and for procedure B were 0.14 µg L(-1), 0.062 µg L(-1), 0.15 µg L(-1) for As(III), As(V) and Cr(VI), respectively. Linearity was tested at two concentration ranges of: (0.5-10.0) µg L(-1) for procedure A and (5-50) µg L(-1) for procedure B, and showed correlation coefficients for each of the analytes to be above 0.999. Repeatability values obtained for spiked real water samples were in the range of (4.2-7.6)% and (2.0-2.4)% for procedure A and B respectively. Accuracy was calculated based on the analysis of spiked real water samples at three concentration levels. Obtained recoveries were from 94% to 102%. PMID:26452815

  10. The Microbiota and Abundance of the Class 1 Integron-Integrase Gene in Tropical Sewage Treatment Plant Influent and Activated Sludge.

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    Magna C Paiva

    Full Text Available Bacteria are assumed to efficiently remove organic pollutants from sewage in sewage treatment plants, where antibiotic-resistance genes can move between species via mobile genetic elements known as integrons. Nevertheless, few studies have addressed bacterial diversity and class 1 integron abundance in tropical sewage. Here, we describe the extant microbiota, using V6 tag sequencing, and quantify the class 1 integron-integrase gene (intI1 in raw sewage (RS and activated sludge (AS. The analysis of 1,174,486 quality-filtered reads obtained from RS and AS samples revealed complex and distinct bacterial diversity in these samples. The RS sample, with 3,074 operational taxonomic units, exhibited the highest alpha-diversity indices. Among the 25 phyla, Proteobacteria, Bacteroidetes and Firmicutes represented 85% (AS and 92% (RS of all reads. Increased relative abundance of Micrococcales, Myxococcales, and Sphingobacteriales and reduced pathogen abundance were noted in AS. At the genus level, differences were observed for the dominant genera Simplicispira and Diaphorobacter (AS as well as for Enhydrobacter (RS. The activated sludge process decreased (55% the amount of bacteria harboring the intI1 gene in the RS sample. Altogether, our results emphasize the importance of biological treatment for diminishing pathogenic bacteria and those bearing the intI1 gene that arrive at a sewage treatment plant.

  11. From ligand to complexes. Part 2. Remarks on human immunodeficiency virus type 1 integrase inhibition by beta-diketo acid metal complexes.

    Science.gov (United States)

    Bacchi, Alessia; Biemmi, Mariano; Carcelli, Mauro; Carta, Fabrizio; Compari, Carlotta; Fisicaro, Emilia; Rogolino, Dominga; Sechi, Mario; Sippel, Martin; Sotriffer, Christoph A; Sanchez, Tino W; Neamati, Nouri

    2008-11-27

    Previously, we synthesized a series of beta-diketo acid metal complexes as novel HIV-1 integrase (IN) inhibitors (J. Med. Chem. 2006, 46, 4248-4260). Herein, a further extension of this study is reported. First, detailed docking studies were performed in order to investigate the mode of binding in the active site of the free ligands and of their metal complexes. Second, a series of potentiometric measurements were conducted for two diketo acids chosen as model ligands, with Mn(2+) and Ca(2+), in order to outline a speciation model. Third, we designed and synthesized a new set of complexes with different stoichiometries and tested them in an in vitro assay specific for IN. Finally, we obtained the first X-ray structure of a metal complex with HIV-1 IN inhibition activity. Analysis of these results supports the hypothesis that the diketo acids could act as complexes and form complexes with the metal ions on the active site of the enzyme.

  12. Study on integrase gene polymorphism and drug resistance among antiretroviral treatment-naive patients in Tianjin City%天津市HIV/AIDS未治疗人群整合酶基因多态性及耐药性分析

    Institute of Scientific and Technical Information of China (English)

    王欣; 董笑月; 于茂河; 柳忠泉; 周宁; 程绍辉

    2013-01-01

    目的 了解天津市HIV/AIDS未治疗人群整合酶基因的变异情况,比较不同基因亚型之间整合酶基因多态性及耐药突变的分布差异.方法 收集2010年天津市HIV/AIDS未治疗人群的血液样本50例,并提取DNA,用巢式聚合酶链式反应扩增病毒整合酶基因,并进行亚型及耐药基因型分析.结果 在50例样本中,扩增并得到有效整合酶基因序列的共有38例.共发现2种基因亚型,其中CRF01 _AE为主要亚型,占73.68%(28/38),其次是B亚型,占26.32% (10/38).检出1例针对埃替格韦存在低度耐药,占2.63% (1/38),其耐药相关突变位点为R263KR.不同基因亚型中,整合酶基因变异存在差异.结论 天津市不同HIV-1基因亚型中,整合酶基因多态性及耐药突变情况均存在一定差异,应深入研究整合酶基因序列特征,掌握基线数据,从而为更好地开展抗病毒治疗提供科学依据.%Objective To investigate background knowledge of drug-resistant mutation among antiretroviral treatment-naive patients in Tianjin City, and compare the integrase gene polymorphism among different genetic subtype of HIV-1. Methods 50 blood samples of antiretroviral treatment-naive patients were collected randomly in 2010. Nested polymer-ase chain reaction (PCR) method was used to amplify integrase genes sequences of HIV-1 after DNA extracted. The nucle-otide sequences of amplification products were determined, and the genetic subtype was identified by comparative analysis. Results Integrase gene fragments of HIV-1 were successfully amplified for 38 samples and 2 kinds of HIV-1 subtypes and recombinant gene were found. CRF01_AE was major subtype, accounted for 73.68% (28/38) of all positives. B was the second, accounted for 26. 32% (10/38). Only one individual was drug resistant, and the rate of drug resistance was 2.63% (1/38). It was low-level resistance for elvitegravir. The genotype was R263KR. The integrase gene polymorphism was different in different

  13. Design, Synthesis and Anti-HIV Integrase Evaluation of 1,2,3-Triazol-4-yl-substituted 1,4-Dihydro-4-oxo-1,5-napthyridine-3-carboxylic Acids

    Institute of Scientific and Technical Information of China (English)

    ZENG,Jia; L(U),Xiuhua; ZENG,Chengchu; HU,Liming; ZHONG,Rugang

    2009-01-01

    Copper(I)-eatalyzed terminal alkyne-azide 1,3-cycloaddition reaction has emerged as one of the main strategies for the rapid creation and screening of a small molecular library. The present work describes the design and synthe-sis of a series of 1,2,3-triazol-4-yl-substituted 1,4-dihydro-4-oxo-1,5-napthyridine-3-carboxylic acids in which the hydrophobic and hydrophilic domains were efficiently incorporated by a click reaction. The structures of the desired products 8 and 12 were characterized by spectroscopic methods and their HIV integrase inhibitory activities were also screened.

  14. Hologram quantitative structure-activity relationship and comparative molecular field analysis studies within a series of tricyclic phthalimide HIV-1 integrase inhibitors.

    Science.gov (United States)

    Magalhães, Uiaran de Oliveira; Souza, Alessandra Mendonça Teles de; Albuquerque, Magaly Girão; Brito, Monique Araújo de; Bello, Murilo Lamim; Cabral, Lucio Mendes; Rodrigues, Carlos Rangel

    2013-01-01

    Acquired immunodeficiency syndrome is a public health problem worldwide caused by the Human immunodeficiency virus (HIV). Treatment with antiretroviral drugs is the best option for viral suppression, reducing morbidity and mortality. However, viral resistance in HIV-1 therapy has been reported. HIV-1 integrase (IN) is an essential enzyme for effective viral replication and an attractive target for the development of new inhibitors. In the study reported here, two- and three-dimensional quantitative structure-activity relationship (2D/3D-QSAR) studies, applying hologram quantitative structure-activity relationship (HQSAR) and comparative molecular field analysis (CoMFA) methods, respectively, were performed on a series of tricyclic phthalimide HIV-1 IN inhibitors. The best HQSAR model (q (2) = 0.802, r (2) = 0.972) was obtained using atoms, bonds, and connectivity as the fragment distinction, a fragment size of 2-5 atoms, hologram length of 61 bins, and six components. The best CoMFA model (q (2) = 0.748, r (2) = 0.974) was obtained with alignment of all atoms of the tricyclic phthalimide moiety (alignment II). The HQSAR contribution map identified that the carbonyl-hydroxy-aromatic nitrogen motif made a positive contribution to the activity of the compounds. Furthermore, CoMFA contour maps suggested that bulky groups in meta and para positions in the phenyl ring would increase the biological activity of this class. The conclusions of this work may lead to a better understanding of HIV-1 IN inhibition and contribute to the design of new and more potent derivatives.

  15. Antiviral activity of dolutegravir in subjects with failure on an integrase inhibitor-based regimen: week 24 phase 3 results from VIKING-3

    Science.gov (United States)

    Nichols, G; Mills, A; Grossberg, R; Lazzarin, A; Maggiolo, F; Molina, J; Pialoux, G; Wright, D; Ait-Khaled, M; Huang, J; Vavro, C; Wynne, B; Yeo, J

    2012-01-01

    Background VIKING-3 aimed to examine efficacy and safety of dolutegravir (DTG) 50 mg twice daily in patients with resistance to multiple ARV classes, including integrase inhibitors (INI). Methods RAL and/or EVG-resistant (current or historical) adult subjects with screening plasma HIV-1 RNA ≥500 c/mL and resistance to ≥2 other ART classes received open-label DTG 50 mg BID while continuing their failing regimen (without RAL/EVG). At Day 8 the background regimen was optimised and DTG continued. Activity of the optimized background regimen (OBR) was determined by Monogram Net Assessment. Primary endpoints were antiviral efficacy at Day 8 and Week 24. Results 183 subjects enrolled, 124 with INI-resistance at screening and 59 with historical (but no screening) resistance. Population was advanced: at BL, median CD4 140, prior ART 13 yrs, 56% CDC Class C; 79% had >2 NRTI, 75% >1 NNRTI, and 70% >2 PI resistance-associated mutations, and 61% had non-R5 HIV detected. Of the 114 subjects who had the opportunity to complete 24 weeks on study before data cutoff, 72 (63%) had 1 log HIV RNA decline of 2, respectively. Discontinuations due to adverse events were uncommon (6/183, 3%); the most common drug-related AEs were diarrhoea, nausea and headache, each reported in only 5% of subjects. Conclusion A majority of the highly treatment-experienced subjects in VIKING-3 achieved suppression with DTG-based therapy. Responses were associated with Baseline IN genotype but not OSS, highlighting the importance and independence of DTG antiviral activity. DTG had a low rate of discontinuation due to adverse events at 50 mg BID in this advanced patient population.

  16. Urinary Eicosanoid Metabolites in HIV-Infected Women with Central Obesity Switching to Raltegravir: An Analysis from the Women, Integrase, and Fat Accumulation Trial

    Directory of Open Access Journals (Sweden)

    Todd Hulgan

    2014-01-01

    Full Text Available Chronic inflammation is a hallmark of HIV infection. Eicosanoids reflect inflammation, oxidant stress, and vascular health and vary by sex and metabolic parameters. Raltegravir (RAL is an HIV-1 integrase inhibitor that may have limited metabolic effects. We assessed urinary F2-isoprostanes (F2-IsoPs, prostaglandin E2 (PGE-M, prostacyclin (PGI-M, and thromboxane B2 (TxB2 in HIV-infected women switching to RAL-containing antiretroviral therapy (ART. Thirty-seven women (RAL = 17; PI/NNRTI = 20 with a median age of 43 years and BMI 32 kg/m2 completed week 24. TxB2 increased in the RAL versus PI/NNRTI arm (+0.09 versus −0.02; P=0.06. Baseline PGI-M was lower in the RAL arm (P=0.005; no other between-arm cross-sectional differences were observed. In the PI/NNRTI arm, 24-week visceral adipose tissue change correlated with PGI-M (rho=0.45; P=0.04 and TxB2 (rho=0.44; P=0.005 changes, with a trend seen for PGE-M (rho=0.41; P=0.07. In an adjusted model, age ≥ 50 years (N=8 was associated with increased PGE-M (P=0.04. In this randomized trial, a switch to RAL did not significantly affect urinary eicosanoids over 24 weeks. In women continuing PI/NNRTI, increased visceral adipose tissue correlated with increased PGI-M and PGE-M. Older age (≥50 was associated with increased PGE-M. Relationships between aging, adiposity, ART, and eicosanoids during HIV-infection require further study.

  17. Establishment and optimization of a method for screening HIV-1 integrase 3′-processing inhibitor%HIV-1整合酶3′端加工抑制剂筛选方法的建立与优化

    Institute of Scientific and Technical Information of China (English)

    陆翠林; 张旋; 詹金彪; 杨柳萌; 郑永唐

    2014-01-01

    Aim To establish and optimize a method for screening HIV-1 integrase 3′-processing inhibitor. Methods Fluorescence resonance energy transfer ( FRET) was used to create an assay for screening in-tegrase 3′-processing inhibitors; wavelength was de-fined by DNaseⅠ; factors affecting IN activity were optimized, including buffer composition, substrate con-centration, enzyme concentration, metal ion concentra-tion. Results Integrase 3′-processing optimizing reac-tion conditions were buffer 1 , 500 nmol · L-1 sub-strate, 1 μmol·L-1 integrase, 20mmol·L-1 magne-sium ion. Positive drug raltegravir and myricetin could effectively inhibit integrase 3′-processing activity using this assay. Two integrase 3′-processing inhibitors were screened by this method. Conclusion The method for screening HIV-1 integrase 3′-processing inhibitor is successfully established and optimized.%目的:建立与优化HIV-1整合酶3′端加工抑制剂筛选方法。方法利用荧光共振能量转移原理建立筛选方法,用DNaseⅠ切割底物 DNA确定底物检测波长,在该检测波长下,对缓冲液成分、底物浓度、酶浓度、金属离子浓度等影响整合酶活性的条件进行优化,并用阳性药物雷特格韦和杨梅黄素进行方法验证。结果检测波长为495 nm/525 nm,使用缓冲液1、底物浓度500 nmol·L-1,整合酶浓度1μmol· L-1,镁离子浓度20 mmol · L-1时整合酶3′端加工活性最强。在此反应条件下雷特格韦和杨梅黄素能有效地抑制整合酶3′端加工活性。用所建立的方法筛选到2个有较强抑制整合酶3′端加工活性的抑制剂。结论该文成功建立并优化了HIV-1整合酶3′端加工抑制剂筛选方法。

  18. Hologram quantitative structure–activity relationship and comparative molecular field analysis studies within a series of tricyclic phthalimide HIV-1 integrase inhibitors

    Directory of Open Access Journals (Sweden)

    Magalhães UO

    2013-09-01

    Full Text Available Uiaran de Oliveira Magalhães,1 Alessandra Mendonça Teles de Souza,1 Magaly Girão Albuquerque,2 Monique Araújo de Brito,3 Murilo Lamim Bello,1 Lucio Mendes Cabral,4 Carlos Rangel Rodrigues1 1Universidade Federal do Rio de Janeiro (UFRJ, Faculdade de Farmácia, Laboratório de Modelagem Molecular and QSAR (ModMolQSAR, Rio de Janeiro, RJ, Brazil; 2Universidade Federal do Rio de Janeiro (UFRJ, Instituto de Química, Laboratório de Modelagem Molecular (LabMMol, Rio de Janeiro, RJ, Brazil; 3Universidade Federal Fluminense (UFF, Faculdade de Farmácia, Laboratório de Química Medicinal Computacional (LabQMC, Niterói, RJ, Brazil; 4Universidade Federal do Rio de Janeiro (UFRJ, Faculdade de Farmácia, Laboratório de Tecnologia Farmacêutica Industrial (LabTIF, Rio de Janeiro, RJ, Brazil Abstract: Acquired immunodeficiency syndrome is a public health problem worldwide caused by the Human immunodeficiency virus (HIV. Treatment with antiretroviral drugs is the best option for viral suppression, reducing morbidity and mortality. However, viral resistance in HIV-1 therapy has been reported. HIV-1 integrase (IN is an essential enzyme for effective viral replication and an attractive target for the development of new inhibitors. In the study reported here, two- and three-dimensional quantitative structure–activity relationship (2D/3D-QSAR studies, applying hologram quantitative structure–activity relationship (HQSAR and comparative molecular field analysis (CoMFA methods, respectively, were performed on a series of tricyclic phthalimide HIV-1 IN inhibitors. The best HQSAR model (q2 = 0.802, r2 = 0.972 was obtained using atoms, bonds, and connectivity as the fragment distinction, a fragment size of 2–5 atoms, hologram length of 61 bins, and six components. The best CoMFA model (q2 = 0.748, r2 = 0.974 was obtained with alignment of all atoms of the tricyclic phthalimide moiety (alignment II. The HQSAR contribution map identified that the carbonyl

  19. Antiviral activity of dolutegravir in subjects with failure on an integrase inhibitor-based regimen: week 24 phase 3 results from VIKING-3

    Directory of Open Access Journals (Sweden)

    Mills A

    2012-11-01

    Full Text Available Background VIKING-3 aimed to examine efficacy and safety of dolutegravir (DTG 50 mg twice daily in patients with resistance to multiple ARV classes, including integrase inhibitors (INI. Methods RAL and/or EVG-resistant (current or historical adult subjects with screening plasma HIV-1 RNA ≥500 c/mL and resistance to ≥2 other ART classes received open-label DTG 50 mg BID while continuing their failing regimen (without RAL/EVG. At Day 8 the background regimen was optimised and DTG continued. Activity of the optimized background regimen (OBR was determined by Monogram Net Assessment. Primary endpoints were antiviral efficacy at Day 8 and Week 24. Results 183 subjects enrolled, 124 with INI-resistance at screening and 59 with historical (but no screening resistance. Population was advanced: at BL, median CD4 140, prior ART 13 yrs, 56% CDC Class C; 79% had >2 NRTI, 75% >1 NNRTI, and 70% >2 PI resistance-associated mutations, and 61% had non-R5 HIV detected. Of the 114 subjects who had the opportunity to complete 24 weeks on study before data cutoff, 72 (63% had <50 c/mL RNA at Week 24 (SNAPSHOT algorithm. Mean HIV RNA declined by 1.4 log10 c/mL (95% CI: 1.3, 1.5; p < 0.001 at Day 8; response differed by genotype pathway (Table. In subjects with Q148 pathway mutations, virologic response decreased with increasing number of secondary mutations. Background overall susceptibility score (OSS was not associated with Wk 24 response: % <50 c/mL were 83%, 63%, 59% and 69% for OSS 0, 1, 2 and >2, respectively. Discontinuations due to adverse events were uncommon (6/183, 3%; the most common drug-related AEs were diarrhoea, nausea and headache, each reported in only 5% of subjects. Conclusion A majority of the highly treatment-experienced subjects in VIKING-3 achieved suppression with DTG-based therapy. Responses were associated with Baseline IN genotype but not OSS, highlighting the importance and independence of DTG antiviral activity. DTG had a low rate

  20. A novel integrase-containing element may interact with Laem-Singh virus (LSNV to cause slow growth in giant tiger shrimp

    Directory of Open Access Journals (Sweden)

    Sriurairatana Siriporn

    2011-05-01

    Full Text Available Abstract Background From 2001-2003 monodon slow growth syndrome (MSGS caused severe economic losses for Thai shrimp farmers who cultivated the native, giant tiger shrimp, and this led them to adopt exotic stocks of the domesticated whiteleg shrimp as the species of cultivation choice, despite the higher value of giant tiger shrimp. In 2008, newly discovered Laem-Singh virus (LSNV was proposed as a necessary but insufficient cause of MSGS, and this stimulated the search for the additional component cause(s of MSGS in the hope that discovery would lead to preventative measures that could revive cultivation of the higher value native shrimp species. Results Using a universal shotgun cloning protocol, a novel RNA, integrase-containing element (ICE was found in giant tiger shrimp from MSGS ponds (GenBank accession number FJ498866. In situ hybridization probes and RT-PCR tests revealed that ICE and Laem-Singh virus (LSNV occurred together in lymphoid organs (LO of shrimp from MSGS ponds but not in shrimp from normal ponds. Tissue homogenates of shrimp from MSGS ponds yielded a fraction that gave positive RT-PCR reactions for both ICE and LSNV and showed viral-like particles by transmission electron microscopy (TEM. Bioassays of this fraction with juvenile giant tiger shrimp resulted in retarded growth with gross signs of MSGS, and in situ hybridization assays revealed ICE and LSNV together in LO, eyes and gills. Viral-like particles similar to those seen in tissue extracts from natural infections were also seen by TEM. Conclusions ICE and LSNV were found together only in shrimp from MSGS ponds and only in shrimp showing gross signs of MSGS after injection with a preparation containing ICE and LSNV. ICE was never found in the absence of LSNV although LSNV was sometimes found in normal shrimp in the absence of ICE. The results suggest that ICE and LSNV may act together as component causes of MSGS, but this cannot be proven conclusively without single

  1. Potent inhibitors of HIV-1 integrase display a two-step, slow-binding inhibition mechanism which is absent in a drug-resistant T66I/M154I mutant.

    Science.gov (United States)

    Garvey, Edward P; Schwartz, Benjamin; Gartland, Margaret J; Lang, Scott; Halsey, Wendy; Sathe, Ganesh; Carter, H Luke; Weaver, Kurt L

    2009-02-24

    Two-metal binding HIV-1 integrase inhibitors (INIs) are potent inhibitors of HIV-1 in vitro and in patients. We report here for the first time the kinetics of inhibition of integrase-catalyzed strand transfer. First, the IC(50) values for each of six structurally distinct INIs decreased when a preincubation was included: S-1360 (1.3 microM vs 0.12 microM), L-731,988 (130 nM vs 9 nM), L-870,810 (130 nM vs 4 nM), raltegravir (300 nM vs 9 nM), elvitegravir (90 nM vs 6 nM), and GSK364735 (90 nM vs 6 nM). When reactions with these INIs were initiated with integrase, progress curve analyses indicated time-dependent inhibition, which could be fitted to a two-step mechanism of binding. Overall fitted K(i) values matched the IC(50) values measured with a preincubation: S-1360 (0.17 microM), L-731,988 (34 nM), L-870,810 (2.4 nM), raltegravir (10 nM), elvitegravir (4.0 nM), and GSK364735 (2.5 nM). To begin to understand the mechanism for this slow onset of inhibition and its possible impact on drug resistance, studies of resistance mutations were initiated. T66I/M154I exhibited little if any time-dependent inhibition by any of the six INIs, as measured by differences in potency upon preincubation or by progress curve analysis. These data demonstrate that slow binding is a signature of two-metal binding INIs, and that the second slow step is required for full potency. We discuss a possible structural explanation of the second slow step of inhibition and also the relationship between loss of time-dependent inhibition and drug resistance of this important new class of HIV-1 antiretroviral drugs. PMID:19178153

  2. Potent inhibitors of HIV-1 integrase display a two-step, slow-binding inhibition mechanism which is absent in a drug-resistant T66I/M154I mutant.

    Science.gov (United States)

    Garvey, Edward P; Schwartz, Benjamin; Gartland, Margaret J; Lang, Scott; Halsey, Wendy; Sathe, Ganesh; Carter, H Luke; Weaver, Kurt L

    2009-02-24

    Two-metal binding HIV-1 integrase inhibitors (INIs) are potent inhibitors of HIV-1 in vitro and in patients. We report here for the first time the kinetics of inhibition of integrase-catalyzed strand transfer. First, the IC(50) values for each of six structurally distinct INIs decreased when a preincubation was included: S-1360 (1.3 microM vs 0.12 microM), L-731,988 (130 nM vs 9 nM), L-870,810 (130 nM vs 4 nM), raltegravir (300 nM vs 9 nM), elvitegravir (90 nM vs 6 nM), and GSK364735 (90 nM vs 6 nM). When reactions with these INIs were initiated with integrase, progress curve analyses indicated time-dependent inhibition, which could be fitted to a two-step mechanism of binding. Overall fitted K(i) values matched the IC(50) values measured with a preincubation: S-1360 (0.17 microM), L-731,988 (34 nM), L-870,810 (2.4 nM), raltegravir (10 nM), elvitegravir (4.0 nM), and GSK364735 (2.5 nM). To begin to understand the mechanism for this slow onset of inhibition and its possible impact on drug resistance, studies of resistance mutations were initiated. T66I/M154I exhibited little if any time-dependent inhibition by any of the six INIs, as measured by differences in potency upon preincubation or by progress curve analysis. These data demonstrate that slow binding is a signature of two-metal binding INIs, and that the second slow step is required for full potency. We discuss a possible structural explanation of the second slow step of inhibition and also the relationship between loss of time-dependent inhibition and drug resistance of this important new class of HIV-1 antiretroviral drugs.

  3. Ce-Fe-modified zeolite-rich tuff to remove Ba(2+)-like (226)Ra(2+) in presence of As(V) and F(-) from aqueous media as pollutants of drinking water.

    Science.gov (United States)

    Olguín, María Teresa; Deng, Shuguang

    2016-01-25

    The sorption behavior of the Ba(2+)-like (226)Ra(2+) in the presence of H2AsO4(-)/HAsO4(2-) and F(-) from aqueous media using Ce-Fe-modified zeolite-rich tuff was investigated in this work. The Na-modified zeolite-rich tuff was also considered for comparison purposes. The zeolite-rich tuff collected from Wyoming (US) was in contact with NaCl and CeCl3-FeCl3 solutions to obtain the Na- and Ce-Fe-modified zeolite-rich tuffs (ZUSNa and ZUSCeFe). These zeolites were characterized by scanning electron microscopy and X-ray diffraction. The BET-specific surface and the points of zero charge were determined as well as the content of Na, Ce and Fe by neutron activation analysis. The textural characteristics and the point of zero charge were changed by the presence of Ce and Fe species in the zeolitic network. A linear model described the Ba(2+)-like (226)Ra(2+) sorption isotherms and the distribution coefficients (Kd) varied with respect to the metallic species present in the zeolitic material. The As(V) oxianionic chemical species and F(-) affected this parameter when the Ba(2+)-like (226)Ra(2+)-As(V)-F(-) solutions were in contact with ZUSCeFe. The H2AsO4(-)/HAsO4(2-) and F(-) were adsorbed by ZUSCeFe in the same amount, independent of the concentration of Ba(2+)-like (226)Ra(2+) in the initial solution.

  4. Synthesis and anti-HIV evaluation of hybrid-type prodrugs conjugating HIV integrase inhibitors with d4t by self-cleavable spacers containing an amino acid residue.

    Science.gov (United States)

    Fossey, Christine; Huynh, Ngoc-Trinh; Vu, Anh-Hoang; Vidu, Anamaria; Zarafu, Irina; Laduree, Daniel; Schmidt, Sylvie; Laumond, Geraldine; Aubertin, Anne-Marie

    2007-10-01

    In an attempt to combine the anti-HIV inhibitory capacity of reverse transcriptase (RT) inhibitors (NRTIs) and integrase (IN) inhibitors (INIs), several heterodimer analogues of the previously reported [d4T]-PABC-[INI] and [d4T]-OABC-[INI] prototypes have been prepared. In these novel series, we wished to extend our results to conjugates which incorporated an enzymatically labile aminoacid unit (L-alanine) connected to d4T through a self-immolative para- or ortho-aminobenzyl carbonate (PABC or OABC) spacer. Among the novel heterodimers, several derivatives show a potent anti-HIV-1 activity, which proved comparable to that of the [L-708,906]-PABC-[d4T] Heterodimer A prototype. However, although the compounds proved inhibitory to HIV-1, they were less potent than the parent compounds from which they were derived. PMID:18035829

  5. Synthesis and molecular docking towards HIV-1 integrase of the new dihydrobenzofuran%苯并二氢呋喃新化合物的合成与 HIV-1整合酶分子对接研究

    Institute of Scientific and Technical Information of China (English)

    罗杨; 范晔; 杨旭超; 马成

    2016-01-01

    Objective With the HIV-1 integrase enzyme molecular docking analysis synthetic dihydrobenzo-furan compounds (3)and the integrase binding mode and inhibition.Methods From vanillin and malonic acid as raw material,the Knoevenagel condensation,esterification,oxidation coupling reaction,synthesis of dihydro-benzofuran new compounds (3)2-(4-hydroxy-3-methoxy)phenyl-7-methoxy-5-[3-(2-methoxy-2-oxoethoxy)-3-oxopropenyl ]-2,3-dihydrobenzofuran-3-carboxylic acid methoxycarbonyl methyl ester. Using the computer software of Autodock compound (3)by molecular docking,prediction and analysis of the compounds have inhibition of HIV-1 integrase.Results The yield of the synthetic target compound (3)was 21.4%,which was determined by the structural identification data (3),Molecular docking showed that the compounds by docking analysis to HIV integrase (PDB:1QS4)indicates that the amino acids resi-due of asparagine,glutamine and histidine interact with the enzyme through hydrogen bond,π-π coinci-dence and hydrophobic force.Conclusion The synthesis route of the oxidative coupling reaction is short, the reaction condition is mild,the operation is simple and easy to control,the target compound is obtained by column chromatography,docking displayed that compounds inhibited the HIV-1 integration enzyme.%目的:通过与 HIV-1整合酶的分子对接,分析合成的苯并二氢呋喃类化合物(3)与整合酶的结合方式和抑制作用。方法以香草醛和丙二酸为原料,经 Knoevenagel 缩合、酯化、氧化偶联反应,合成苯并二氢呋喃新化合物(3)2-(4-羟基-3-甲氧基)苯基-7-甲氧基-5-[3-(2-甲氧基-2-氧代乙氧基)-3-氧代丙烯基]-2,3-二氢苯并呋喃-3-羧酸甲氧羰基甲酯,采用计算机 Autodock 软件对化合物(3)进行分子对接,预测分析该化合物是否有抑制 HIV-1整合酶的作用。结果合成目标化合物(3)的产率为21.4%,经结构鉴定数据确定为化合物(3),

  6. Synthesis and anti-HIV evaluation of hybrid-type prodrugs conjugating HIV integrase inhibitors with d4t by self-cleavable spacers containing an amino acid residue.

    Science.gov (United States)

    Fossey, Christine; Huynh, Ngoc-Trinh; Vu, Anh-Hoang; Vidu, Anamaria; Zarafu, Irina; Laduree, Daniel; Schmidt, Sylvie; Laumond, Geraldine; Aubertin, Anne-Marie

    2007-10-01

    In an attempt to combine the anti-HIV inhibitory capacity of reverse transcriptase (RT) inhibitors (NRTIs) and integrase (IN) inhibitors (INIs), several heterodimer analogues of the previously reported [d4T]-PABC-[INI] and [d4T]-OABC-[INI] prototypes have been prepared. In these novel series, we wished to extend our results to conjugates which incorporated an enzymatically labile aminoacid unit (L-alanine) connected to d4T through a self-immolative para- or ortho-aminobenzyl carbonate (PABC or OABC) spacer. Among the novel heterodimers, several derivatives show a potent anti-HIV-1 activity, which proved comparable to that of the [L-708,906]-PABC-[d4T] Heterodimer A prototype. However, although the compounds proved inhibitory to HIV-1, they were less potent than the parent compounds from which they were derived.

  7. Identification of proteolytic cleavage sites within the gag-analogue protein of Ty1 virus-like particles.

    Science.gov (United States)

    Martin-Rendon, E; Hurd, D W; Marfany, G; Kingsman, S M; Kingsman, A J

    1996-12-01

    Like retroviruses, the yeast retrotransposon Ty1 produces its proteins as precursors that are subsequently cleaved by a protease encoded by the element. These cleavage events are essential for transposition as they release the active reverse transcriptase and integrase and they modify the structure of the virus-like particles in a way that is analogous to the morphological changes that occur during retrovirus core maturation. Using a combination of epitope tagging, amino acid analysis and mutagenesis, we have identified the major cleavage sites for the Ty1 protease within the particle-forming protein, p1, at 407S/408N. In addition, we present evidence indicating that the Ty1 protease may be a 17 kDa protein. PMID:8971723

  8. Basic Principles of Anodic Stripping Voltammetry (ASV)

    OpenAIRE

    2012-01-01

    In this interactive exercise, the basic principles of Anodic Stripping Voltammetry are shown. Each step of the voltammetric process is described using simulated animations. This activity illustrates what takes place in the voltammetric cell when this technique is applied to the determination of cadmium as well as to the simultaneous determination of copper and cadmium.

  9. Marine ASV Range Surveillance System Project

    Data.gov (United States)

    National Aeronautics and Space Administration — United States spaceports carry out the impressive task of launching and recovering spacecrafts and payloads which represent extremely unique and expensive assets....

  10. Marine ASV Range Surveillance System Project

    Data.gov (United States)

    National Aeronautics and Space Administration — United States spaceports carry out the critical task of launching and recovering vehicles and payloads. These are extremely unique and expensive assets, and their...

  11. Stabilization of noncondensed (As(III)S3)(3-) anions by coordinating to [Mn(II)(phen)](2+) complex cations: a mixed-valent thioarsenate (III, V) {[Mn(phen)]3(As(V)S4)(As(III)S3)}(n)·nH2O showing the coexistence of antiferromagnetic order, photoluminescence, and nonlinear optical properties.

    Science.gov (United States)

    Liu, Guang-Ning; Jiang, Xiao-Ming; Wu, Mei-Feng; Wang, Guan-E; Guo, Guo-Cong; Huang, Jin-Shun

    2011-06-20

    A novel one-dimensional (1-D) mixed-valent thioarsenate (III, V), {[Mn(phen)](3)(As(V)S(4))(As(III)S(3))}(n)·nH(2)O (1), with a noncentrosymmetric (NCS) polar packing arrangement has been obtained under solvothermal conditions. The noncondensed (As(III)S(3))(3-) anion in 1 is stabilized by coordinating to [Mn(II)(phen)](2+) complex cations and exhibits an unprecedented μ(3)-1,2κS:2,3κS':3κS'' linkage mode. Compound 1 represents the first example of the stabilization of noncondensed (MQ(3))(3-) (M = As, Sb; Q = S, Se) species only in the coordination of TM(II) complex cations (TM = transition-metal) and the first observation of the coexistence of the (As(V)S(4))(3-) tetrahedron and the noncondensed (As(III)S(3))(3-) pyramid in a single compound. Of particular interest, compound 1 is also an antiferromagnet with T(N) = 31 K, and exhibits photoluminescence (PL) with a maximum emission at about 438 nm and a second harmonic generation (SHG) response.

  12. HIV-1逆转录酶和整合酶双靶点抑制剂研究进展%Research progress of dual inhibitors targeting HIV-1 reverse transcriptase and integrase

    Institute of Scientific and Technical Information of China (English)

    刘鸿; 展鹏; 刘新泳

    2013-01-01

    在HIV-1 (human immunodeficiency virus type l)的复制周期中,逆转录酶(reverse transcriptase,RT)和整合酶(integrase,IN)均发挥着非常重要的作用,它们一直是抗HIV药物研究的热门靶点.逆转录酶抑制剂(RT inhibitors)是目前临床上治疗艾滋病(AIDS)患者和HIV感染者的一线药物,而整合酶抑制剂(IN inhibitors)新近也有药物上市,两方面的单独研究均取得了可喜的成果.近年来,多靶点药物研究策略渐渐引起科研工作者的浓厚兴趣,经过合理药物设计及随机筛选发现了多类具有HIV逆转录酶和整合酶双重抑制活性的化合物.本文主要就HIV逆转录酶和整合酶双靶点抑制剂的发现与结构修饰的最新研究进展进行介绍,以期对抗HIV药物研究有所启示.

  13. Protein Foods

    Science.gov (United States)

    ... Text Size: A A A Listen En Español Protein Foods Foods high in protein such as fish, ... the vegetarian proteins, whether they have carbohydrate. Best Protein Choices The best choices are: Plant-based proteins ...

  14. Labeling of multiple HIV-1 proteins with the biarsenical-tetracysteine system.

    Directory of Open Access Journals (Sweden)

    Cândida F Pereira

    Full Text Available Due to its small size and versatility, the biarsenical-tetracysteine system is an attractive way to label viral proteins for live cell imaging. This study describes the genetic labeling of the human immunodeficiency virus type 1 (HIV-1 structural proteins (matrix, capsid and nucleocapsid, enzymes (protease, reverse transcriptase, RNAse H and integrase and envelope glycoprotein 120 with a tetracysteine tag in the context of a full-length virus. We measure the impact of these modifications on the natural virus infection and, most importantly, present the first infectious HIV-1 construct containing a fluorescently-labeled nucleocapsid protein. Furthermore, due to the high background levels normally associated with the labeling of tetracysteine-tagged proteins we have also optimized a metabolic labeling system that produces infectious virus containing the natural envelope glycoproteins and specifically labeled tetracysteine-tagged proteins that can easily be detected after virus infection of T-lymphocytes. This approach can be adapted to other viral systems for the visualization of the interplay between virus and host cell during infection.

  15. Identification of multiple integration sites for Stx-phage Phi24B in the Escherichia coli genome, description of a novel integrase and evidence for a functional anti-repressor.

    Science.gov (United States)

    Fogg, Paul C M; Gossage, Sharon M; Smith, Darren L; Saunders, Jon R; McCarthy, Alan J; Allison, Heather E

    2007-12-01

    The key virulence factor in Shiga-toxigenic Escherichia coli is the expression of Shiga toxin (Stx), which is conferred by Stx-encoding temperate lambdoid phages (Stx-phages). It had been assumed that Stx-phages would behave similarly to lambda phage. However, contrary to the lambda superinfection immunity model, it has been demonstrated that double lysogens can be produced with the Stx-phage Phi24(B). Here, the Phi24(B) integrase gene is identified, and the preferred site of integration defined. Although an E. coli int gene was identified close to the Phi24(B) integration site, it was shown not to be involved in the phage integration event. An additional six potential integration sites were identified in the E. coli genome, and three of these were confirmed experimentally. Two of the other potential sites lie within genes predicted to be essential to E. coli and are therefore unlikely to support phage integration. A Phi24(B) gene, possessing similarity to the well-characterized P22 ant gene, was identified. RT-PCR was used to demonstrate that ant is transcribed in a Phi24(B) E. coli lysogen, and expression of an anti-repressor is the likely explanation for the absence of immunity to superinfection. Demonstration of the ability of Phi24(B) to form multiple lysogens has two potentially serious impacts. First, multiple integrated prophages will drive the evolution of bacterial pathogens as novel Stx-phages emerge following intracellular mutation/recombination events. Second, multiple copies of the stx gene may lead to an increase in toxin production and consequently increased virulence. PMID:18048923

  16. Protein-protein interactions

    DEFF Research Database (Denmark)

    Byron, Olwyn; Vestergaard, Bente

    2015-01-01

    . The biophysical and structural investigations of PPIs consequently demand hybrid approaches, implementing orthogonal methods and strategies for global data analysis. Currently, impressive developments in hardware and software within several methodologies define a new era for the biostructural community. Data can......Responsive formation of protein:protein interaction (PPI) upon diverse stimuli is a fundament of cellular function. As a consequence, PPIs are complex, adaptive entities, and exist in structurally heterogeneous interplays defined by the energetic states of the free and complexed protomers...

  17. Detection of Integrase Gone and Analysis of Gone Cassette Structure of Variable Region in Acinetobactor Baumannii%鲍曼不动杆菌整合酶基因测定及可变区基因盒结构分析

    Institute of Scientific and Technical Information of China (English)

    马全玲; 武大伟; 魏殿军; 张坚磊; 胡静仪

    2011-01-01

    Objective: To investigate the distribution and prevalence of integrase gene of multidrug-resistance Acinetobacter baumannii in Tianjin, and to analyze the relationship between integron and multidrug-resisitance of Acinetobacter baumannii. Methods: Fifty-five multidrug- resistance Acinetobacter baumannn were collected from three hospitals in Tianjin.The drug sensitivation was detected by K-B test. The integrase genes were detected by PCR. The relationship of clinical isolates was analysed by multigene cluster typing. Results: There were 47 strains of Ⅰ class integrase genes, 23 contained variable regions, but no Ⅱ and Ⅲ class integrase gene in 55 multidrug- resistance Acinetobacter baumannii. The mam reason caused aminoglycosides resistance was the aacA4 and aadAI cassette in the variable region. Three clone strains were found in 55 multidrug-resistance Acinetohacter haumannii by multigene cluster typing. Conclusion: It was found that Ⅰ class integrase gene was existed extensively in multidrug- resistance Acinetobacter haumannii in Tianjin. All strains could be typed by multigene cluster typing.%目的:了解整合酶基因在天津地区多重耐药鲍曼不动杆菌株中的分布和流行情况,分析整合子与鲍曼不动杆菌多重耐药性的关系.方法:收集天津地区3家医院55株多重耐药鲍曼不动杆菌,以K-B法进行抗生素敏感试验,用PCR方法检测整合酶基因,结合以往检测的耐药基因,采用聚类法对55株多重耐药鲍曼不动杆菌进行菌株亲缘性分析.结果:55株多重耐药鲍曼不动杆菌共检出47株含有Ⅰ类整合酶基因,其中有23株检出可变区结构,未检出Ⅱ、Ⅲ类整合酶基因,可变区所携带的aacA4和aadA1基因盒是引起鲍曼不动杆菌对氨基糖苷类抗生素耐药的主要原因.55株多重耐药鲍曼不动杆菌共含有3个克隆株.结论:天津地区多重耐药鲍曼不动杆菌中主要存在Ⅰ类整合子,聚类分析方法可对所有菌株分型.

  18. Effect of Ethanol on the Metabolic Characteristics of HIV-1 Integrase Inhibitor Elvitegravir and Elvitegravir/Cobicistat with CYP3A: An Analysis Using a Newly Developed LC-MS/MS Method.

    Science.gov (United States)

    Midde, Narasimha M; Rahman, Mohammad A; Rathi, Chetan; Li, Junhao; Meibohm, Bernd; Li, Weihua; Kumar, Santosh

    2016-01-01

    Elvitegravir (EVG), an integrase inhibitor for the treatment HIV infection, is increasingly becoming the part of first-line antiretroviral therapy (ART) regimen. EVG is mainly metabolized through cytochrome P450 (CYP) 3A4. Previously, we have shown that ethanol alters ART-CYP3A4 interactions with protease inhibitors thereby altering their metabolisms. However, as EVG is a fairly new class of drug, its kinetic characteristics and the effect of ethanol on EVG-CYPP3A4 interaction is poorly understood. In this study, we characterized EVG and cobicistat (COBI)-boosted EVG metabolism in human microsomes followed by ethanol-EVG, ethanol-COBI-EVG interaction with CYP3A. First, we developed and validated a simple, sensitive, and robust liquid chromatography-tandem mass spectrometry (LC-MS/MS) method for the quantification of EVG in the human liver microsomes. The lower limit of quantification for the drug was at 0.003 μM (1.34 ng/ml). Extraction yield, matrix effects, drug stability, and calibration curves for the proposed method were validated according to the FDA guidelines. Time dependent kinetics data showed that 20mM ethanol decreases the apparent half-life of EVG degradation by ~50% compared to EVG alone. Our substrate kinetic results revealed that ethanol mildly decreases the catalytic efficiency for EVG metabolism. Inhibition studies demonstrated that EVG inhibits CYP3A4, and 20 mM ethanol causes a decrease in the IC50 of EVG. However, in the presence of COBI we were unable to determine these parameters effectively because COBI, being a strong inhibitor of CYP3A4, blocked the EVG/ethanol-CYP3A4 interactions. Docking studies predicted a shift of EVG or COBI binding to the active site of CYP3A4 in the presence of ethanol. Taken together, these results suggest that ethanol interacts with microsomal CYP3A and alters EVG-CYP3A4 interaction thereby altering EVG metabolism and inhibition of CYP3A4 by EVG. This finding has clinical significance because alcohol use is

  19. 用分子模拟方法研究HIV-1整合酶突变体的耐药性机理%A Study on Drug Resistance Mechanism of HIV-1 Integrase Mutants by Molecular Modeling

    Institute of Scientific and Technical Information of China (English)

    张小轶; 何红秋; 刘斌; 王存新

    2009-01-01

    二酮酸类化合物(DKAs)是目前最有前景的HIV-1整合酶(integrase,IN)抑制剂.为了解DKAs引起的多种耐药株共有的耐药性机理,选择3种S-1360引起的IN耐药突变体,用分子对接和分子动力学模拟,研究了野生型和突变型IN与S-1360的结合模式,基于该结合模式探讨了3种耐药突变体所共有的耐药性机理.结果表明:在突变体中,S-1360结合到耐药突变IN核心区中的位置靠近功能loop 3区却远离与DNA结合的关键残基,结合位置不同导致S-1360的抑制作用部分丧失;残基138到166区域的柔性对IN发挥生物学功能很重要,S-1360能与DNA结合的关键残基N155及K159形成氢键,这2个氢键作用降低了该区域的柔性,突变体中无类似氢键,因而该区域柔性增高;在突变体中,S-1360的苯环远离病毒DNA结合区,不能阻止病毒DNA末端暴露给宿主DNA;T66I突变导敛残基Ⅰ的长侧链占据IN的活性口袋,阻止抑制剂以与野生型中相同的方式结合到活性中心,这均是产生抗药性的重要原因.这些模拟结果与实验结果吻合,可为抗IN的抑制剂设计和改造提供帮助.%The drug resistant mutations in human immunodefieiency virus type 1 (HIV-1) are a major impediment to successful highly active antiretrovirai therapy (HAART) and new drug design. In order to understand the drug resistance mechanism of HIV-1 integrase (IN) mutually existed for multiple drug-resistant strains to the most potent IN inhibitors diketo acids (DKAs), three S-1360-resistant HIV-1 strains were selected and molecular docking and molecular dynamics (MD) simulations were performed to obtain the inhibitor binding modes. Based on the binding modes, compelling differences between the wild-type and the 3 mutants for IN have been observed. The results showed that: 1) In the mutants, the inhibitor is close to the funetional loop 3 region but far away from the DNA binding site. Different binding sites lead to the decrease in

  20. Adsorption of As(Ⅲ) and As(V) in Water by Nano-MnO2 with Three Kinds of Crystal Structures%三种晶型纳米二氧化锰对水中As(Ⅲ)和As(Ⅴ)的吸附性能研究

    Institute of Scientific and Technical Information of China (English)

    卞战强; 谢莉; 田向红; 樊伟; 张娟

    2012-01-01

    Objective To investigate the efficiency of nano-MnO2 with three kinds of crystal structures for removing As(IH) and As(V) from aqueous solutions. Methods By means of static adsorption experiments, a-MnO2, β-MnO2 and 8-MnO2 was prepared successfully by using manganese acetate tetrahydrate and manganous sulfate, respectively. The adsorption capability of As(Ⅲ) and As(V) from aqueous solutions on α-MnO2, β-MnO2 and 8-MnO2 with the concentration of 100 mg/L was investigated. A batch equilibrium data were tested using the isotherm equations. Determination of As (Ⅲ) and As(V)in water by high performance liquid chromatography coupled with hydride generation-atomic fluorescence spectrometry. The optimal reaction conditions were studied. Results Under the optimum condition [pH =2, reacting 10 min, As (Ⅲ) and As (V) initial concentration 0.5 mg/L], the saturated adsorption capacity of nano-Mn02 for As (Ⅲ) and As (V) were calculated from the Langmuir-type isotherm equations, which were 103.1 and 106.4 mg/g for a-MnO2, 50 and 49.8 mg/g for β-MnO2, 31.3 and 34.9 mg/g for 8-MnO2. Equilibrium data for the adsorption of As (HI) and As (V) by a-MnO2 were obtained and correlated with Langmuir-type and Freundlich-type isotherm equations, equilibrium data for the adsorption of As(Ⅲ) and As(V) by β-MnO2 and 8—MnO2 were obtained and correlated with Langmuir—type isotherm equations. Conclusion Nano—MnO2 can be used as a sorbent for removal of As(Ⅲ) and As(V) from aqueous solutions.%目的 考察三种晶型纳米二氧化锰对水中As(Ⅲ)和As(V)的去除效果.方法 采用静态吸附实验,分别采用Mn(Ac)2·4H2O和硫酸锰制备α-MnO2、β-MnO2和δ-MnO2,观察100 mg/L的α-MnO2、β -MnO2和δ-MnO2三种晶型纳米二氧化锰吸附剂对水中As(Ⅲ)和As(V)的吸附效果,并进行吸附等温线拟合.采用高效液相氢化物发生原子荧光法测定水中As(Ⅲ)和As(Ⅴ)的含量,并研究最佳反应条件.结果 最佳反应条件为pH=2,

  1. Total protein

    Science.gov (United States)

    ... page: //medlineplus.gov/ency/article/003483.htm Total protein To use the sharing features on this page, please enable JavaScript. The total protein test measures the total amount of two classes ...

  2. Protein Structure

    Science.gov (United States)

    Asmus, Elaine Garbarino

    2007-01-01

    Individual students model specific amino acids and then, through dehydration synthesis, a class of students models a protein. The students clearly learn amino acid structure, primary, secondary, tertiary, and quaternary structure in proteins and the nature of the bonds maintaining a protein's shape. This activity is fun, concrete, inexpensive and…

  3. in-silico analysis suggests alterations in the function of XisA protein as a possible mechanism of butachlor toxicity in the nitrogen fixing cyanobacterium Anabaena sp. PCC 7120.

    Science.gov (United States)

    Singh, Shilpi; Singh, Prem Pal

    2013-01-01

    Butachlor, a commonly used herbicide adversely affects the nitrogen fixing capability of Anabaena, an acclaimed nitrogen fixer in the Indian paddy fields. The nitrogen fixation in Anabaena is triggered by the excision of nifD element by xisA gene leading to rearrangement of nifD forming nifHDK operon in the heterocyst of Anabaena sp. PCC7120. Functional elucidation adjudged through in-silico analysis revealed that xisA belongs to integrase family of tyrosine recombinase. The predicted functional partners with XisA protein that have shown cooccurence with this protein in a network are mainly hypothetical proteins with unknown functions except psaK1 whose exact function in photosystem I is not yet known. The focus of this study was to find out the relation between XisA and butachlor using in-silico approaches. The XisA protein was modeled and its active sites were identified. Docking studies revealed that butachlor binds at the active site of XisA protein hampering its excision ability vis-à-vis nif genes in Anabaena sp. PCC7120. This study reveals that butachlor is not directly involved in hampering the nitrogen fixing ability of Anabaena sp. PCC7120 but by arresting the excision ability of XisA protein necessary for the functioning of nif gene and nitrogen fixation.

  4. Flotation removal of As(V) onto goethite.

    Science.gov (United States)

    Matis, K A; Zouboulis, A I; Malamas, F B; Ramos Afonso, M D; Hudson, M J

    1997-01-01

    Arsenic oxyanions, considered as priority pollutants, were removed from dilute aqueous solutions by sorption onto synthetic goethite, a typical inorganic adsorbent. Flotation was subsequently applied as an effective solid/liquid separation method. The combined process produced a foam concentrate, containing the arsenic-loaded goethite particles. The dispersed-air flotation technique was used for the generation of fine gas bubbles. The main parameters affecting the process were studied and promising results, in terms of arsenic removal and of goethite separation, were obtained. PMID:15093361

  5. Principles of protein-protein interactions.

    OpenAIRE

    Jones, S; Thornton, J. M.

    1996-01-01

    This review examines protein complexes in the Brookhaven Protein Databank to gain a better understanding of the principles governing the interactions involved in protein-protein recognition. The factors that influence the formation of protein-protein complexes are explored in four different types of protein-protein complexes--homodimeric proteins, heterodimeric proteins, enzyme-inhibitor complexes, and antibody-protein complexes. The comparison between the complexes highlights differences tha...

  6. Tau protein

    DEFF Research Database (Denmark)

    Frederiksen, Jette Lautrup Battistini; Kristensen, Kim; Bahl, Jmc;

    2011-01-01

    Background: Tau protein has been proposed as biomarker of axonal damage leading to irreversible neurological impairment in MS. CSF concentrations may be useful when determining risk of progression from ON to MS. Objective: To investigate the association between tau protein concentration and 14......-3-3 protein in the cerebrospinal fluid (CSF) of patients with monosymptomatic optic neuritis (ON) versus patients with monosymptomatic onset who progressed to multiple sclerosis (MS). To evaluate results against data found in a complete literature review. Methods: A total of 66 patients with MS and/or ON from...... the Department of Neurology of Glostrup Hospital, University of Copenhagen, Denmark, were included. CSF samples were analysed for tau protein and 14-3-3 protein, and clinical and paraclinical information was obtained from medical records. Results: The study shows a significantly increased concentration of tau...

  7. Protein-Protein Interaction Databases

    DEFF Research Database (Denmark)

    Szklarczyk, Damian; Jensen, Lars Juhl

    2015-01-01

    of research are explored. Here we present an overview of the most widely used protein-protein interaction databases and the methods they employ to gather, combine, and predict interactions. We also point out the trade-off between comprehensiveness and accuracy and the main pitfall scientists have to be aware...

  8. Human Polycomb group EED protein negatively affects HIV-1 assembly and release

    Directory of Open Access Journals (Sweden)

    Darlix Jean-Luc

    2007-06-01

    Full Text Available Abstract Background The human EED protein, a member of the superfamily of Polycomb group (PcG proteins with WD-40 repeats, has been found to interact with three HIV-1 components, namely the structural Gag matrix protein (MA, the integrase enzyme (IN and the Nef protein. The aim of the present study was to analyze the possible biological role of EED in HIV-1 replication, using the HIV-1-based vector HIV-Luc and EED protein expressed by DNA transfection of 293T cells. Results During the early phase of HIV-1 infection, a slight negative effect on virus infectivity occurred in EED-expressing cells, which appeared to be dependent on EED-MA interaction. At late times post infection, EED caused an important reduction of virus production, from 20- to 25-fold as determined by CAp24 immunoassay, to 10- to 80-fold based on genomic RNA levels, and this decrease was not due to a reduction of Gag protein synthesis. Coexpression of WTNef, or the non-N-myristoylated mutant NefG2A, restored virus yields to levels obtained in the absence of exogenous EED protein. This effect was not observed with mutant NefΔ57 mimicking the Nef core, or with the lipid raft-retargeted fusion protein LAT-Nef. LATAA-Nef, a mutant defective in the lipid raft addressing function, had the same anti-EED effect as WTNef. Cell fractionation and confocal imaging showed that, in the absence of Nef, EED mainly localized in membrane domains different from the lipid rafts. Upon co-expression with WTNef, NefG2A or LATAA-Nef, but not with NefΔ57 or LAT-Nef, EED was found to relocate into an insoluble fraction along with Nef protein. Electron microscopy of HIV-Luc producer cells overexpressing EED showed significant less virus budding at the cell surface compared to control cells, and ectopic assembly and clustering of nuclear pore complexes within the cytoplasm. Conclusion Our data suggested that EED exerted an antiviral activity at the late stage of HIV-1 replication, which included genomic

  9. Protein Crystallization

    Science.gov (United States)

    Chernov, Alexander A.

    2005-01-01

    Nucleation, growth and perfection of protein crystals will be overviewed along with crystal mechanical properties. The knowledge is based on experiments using optical and force crystals behave similar to inorganic crystals, though with a difference in orders of magnitude in growing parameters. For example, the low incorporation rate of large biomolecules requires up to 100 times larger supersaturation to grow protein, rather than inorganic crystals. Nucleation is often poorly reproducible, partly because of turbulence accompanying the mixing of precipitant with protein solution. Light scattering reveals fluctuations of molecular cluster size, its growth, surface energies and increased clustering as protein ages. Growth most often occurs layer-by-layer resulting in faceted crystals. New molecular layer on crystal face is terminated by a step where molecular incorporation occurs. Quantitative data on the incorporation rate will be discussed. Rounded crystals with molecularly disordered interfaces will be explained. Defects in crystals compromise the x-ray diffraction resolution crucially needed to find the 3D atomic structure of biomolecules. The defects are immobile so that birth defects stay forever. All lattice defects known for inorganics are revealed in protein crystals. Contribution of molecular conformations to lattice disorder is important, but not studied. This contribution may be enhanced by stress field from other defects. Homologous impurities (e.g., dimers, acetylated molecules) are trapped more willingly by a growing crystal than foreign protein impurities. The trapped impurities induce internal stress eliminated in crystals exceeding a critical size (part of mni for ferritin, lysozyme). Lesser impurities are trapped from stagnant, as compared to the flowing, solution. Freezing may induce much more defects unless quickly amorphysizing intracrystalline water.

  10. A microarray study of gene and protein regulation in human and rat brain following middle cerebral artery occlusion

    Directory of Open Access Journals (Sweden)

    Kumar Pat

    2007-11-01

    Full Text Available Abstract Background Altered gene expression is an important feature of ischemic cerebral injury and affects proteins of many functional classes. We have used microarrays to investigate the changes in gene expression at various times after middle cerebral artery occlusion in human and rat brain. Results Our results demonstrated a significant difference in the number of genes affected and the time-course of expression between the two cases. The total number of deregulated genes in the rat was 335 versus 126 in the human, while, of 393 overlapping genes between the two array sets, 184 were changed only in the rat and 36 in the human with a total of 41 genes deregulated in both cases. Interestingly, the mean fold changes were much higher in the human. The expression of novel genes, including p21-activated kinase 1 (PAK1, matrix metalloproteinase 11 (MMP11 and integrase interactor 1, was further analyzed by RT-PCR, Western blotting and immunohistochemistry. Strong neuronal staining was seen for PAK1 and MMP11. Conclusion Our findings confirmed previous studies reporting that gene expression screening can detect known and unknown transcriptional features of stroke and highlight the importance of research using human brain tissue in the search for novel therapeutic agents.

  11. Optimizing HIV-1 protease production in Escherichia coli as fusion protein

    Directory of Open Access Journals (Sweden)

    Piubelli Luciano

    2011-06-01

    Full Text Available Abstract Background Human immunodeficiency virus (HIV is the etiological agent in AIDS and related diseases. The aspartyl protease encoded by the 5' portion of the pol gene is responsible for proteolytic processing of the gag-pol polyprotein precursor to yield the mature capsid protein and the reverse transcriptase and integrase enzymes. The HIV protease (HIV-1Pr is considered an attractive target for designing inhibitors which could be used to tackle AIDS and therefore it is still the object of a number of investigations. Results A recombinant human immunodeficiency virus type 1 protease (HIV-1Pr was overexpressed in Escherichia coli cells as a fusion protein with bacterial periplasmic protein dithiol oxidase (DsbA or glutathione S-transferase (GST, also containing a six-histidine tag sequence. Protein expression was optimized by designing a suitable HIV-1Pr cDNA (for E. coli expression and to avoid autoproteolysis and by screening six different E. coli strains and five growth media. The best expression yields were achieved in E. coli BL21-Codon Plus(DE3-RIL host and in TB or M9 medium to which 1% (w/v glucose was added to minimize basal expression. Among the different parameters assayed, the presence of a buffer system (based on phosphate salts and a growth temperature of 37°C after adding IPTG played the main role in enhancing protease expression (up to 10 mg of chimeric DsbA:HIV-1Pr/L fermentation broth. GST:HIVPr was in part (50% produced as soluble protein while the overexpressed DsbA:HIV-1Pr chimeric protein largely accumulated in inclusion bodies as unprocessed fusion protein. A simple refolding procedure was developed on HiTrap Chelating column that yielded a refolded DsbA:HIV-1Pr with a > 80% recovery. Finally, enterokinase digestion of resolubilized DsbA:HIV-1Pr gave more than 2 mg of HIV-1Pr per liter of fermentation broth with a purity ≤ 80%, while PreScission protease cleavage of soluble GST:HIVPr yielded ~ 0.15 mg of pure HIV-1

  12. Function and regulation of plant major intrinsic proteins

    DEFF Research Database (Denmark)

    Popovic, Milan

    inorganic forms of arsenic in the environment, can be taken up by plants and thus enter the food chain. Once inside the root cells, As(V) is reduced to As(III) which is then extruded to the soil solution or bound to phytochelatins (PCs) and transported to the vacuole in an effort to accomplish...

  13. Prediction of Protein-Protein Interactions Related to Protein Complexes Based on Protein Interaction Networks

    OpenAIRE

    Peng Liu; Lei Yang; Daming Shi; Xianglong Tang

    2015-01-01

    A method for predicting protein-protein interactions based on detected protein complexes is proposed to repair deficient interactions derived from high-throughput biological experiments. Protein complexes are pruned and decomposed into small parts based on the adaptive k-cores method to predict protein-protein interactions associated with the complexes. The proposed method is adaptive to protein complexes with different structure, number, and size of nodes in a protein-protein interaction net...

  14. NCBI nr-aa BLAST: CBRC-SARA-01-1986 [SEVENS

    Lifescience Database Archive (English)

    Full Text Available CBRC-SARA-01-1986 ref|NP_223335.1| INTEGRASE-RECOMBINASE PROTEIN (XERCD FAMILY) [Helicobacter... pylori J99] gb|AAD06197.1| INTEGRASE-RECOMBINASE PROTEIN (XERCD FAMILY) [Helicobacter pylori J99] NP_223335.1 5.2 32% ...

  15. Protein electrophoresis - serum

    Science.gov (United States)

    ... of protein and fat, called lipoproteins (such as LDL cholesterol). ... globulin proteins may indicate: Abnormally low level of LDL cholesterol Malnutrition Increased gamma globulin proteins may indicate: Bone ...

  16. Grafting of protein-protein binding sites

    Institute of Scientific and Technical Information of China (English)

    2000-01-01

    A strategy for grafting protein-protein binding sites is described. Firstly, key interaction residues at the interface of ligand protein to be grafted are identified and suitable positions in scaffold protein for grafting these key residues are sought. Secondly, the scaffold proteins are superposed onto the ligand protein based on the corresponding Ca and Cb atoms. The complementarity between the scaffold protein and the receptor protein is evaluated and only matches with high score are accepted. The relative position between scaffold and receptor proteins is adjusted so that the interface has a reasonable packing density. Then the scaffold protein is mutated to corresponding residues in ligand protein at each candidate position. And the residues having bad steric contacts with the receptor proteins, or buried charged residues not involved in the formation of any salt bridge are mutated. Finally, the mutated scaffold protein in complex with receptor protein is co-minimized by Charmm. In addition, we deduce a scoring function to evaluate the affinity between mutated scaffold protein and receptor protein by statistical analysis of rigid binding data sets.

  17. A protein ballet around the viral genome orchestrated by HIV-1 reverse transcriptase leads to an architectural switch: from nucleocapsid-condensed RNA to Vpr-bridged DNA

    Science.gov (United States)

    Lyonnais, Sébastien; Gorelick, Robert J.; Heniche-Boukhalfa, Fatima; Bouaziz, Serge; Parissi, Vincent; Mouscadet, Jean-François; Restle, Tobias; Gatell, Jose Maria; Le Cam, Eric; Mirambeau, Gilles

    2012-01-01

    Summary HIV-1 reverse transcription is achieved in the newly infected cell before viral DNA (vDNA) nuclear import. Reverse transcriptase (RT) has previously been shown to function as a molecular motor, dismantling the nucleocapsid complex that binds the viral genome as soon as plus-strand DNA synthesis initiates. We first propose a detailed model of this dismantling in close relationship with the sequential conversion from RNA to double-stranded (ds) DNA, focusing on the nucleocapsid protein (NCp7). The HIV-1 DNA-containing preintegration complex (PIC) resulting from completion of reverse transcription is translocated through the nuclear pore. The PIC nucleoprotein architecture is poorly understood but contains at least two HIV-1 proteins initially from the virion core, namely Integrase (IN) and the viral protein r (Vpr). We next present a set of electron micrographs supporting that Vpr behaves as a DNA architectural protein, initiating multiple DNA bridges over more than 500 base pairs (bp). These complexes are shown to interact with NCp7 bound to single-stranded nucleic acid regions that are thought to maintain IN binding during dsDNA synthesis, concurrently with nucleocapsid complex dismantling. This unexpected binding of Vpr conveniently leads to a compacted but filamentous folding of the vDNA that should favor its nuclear import. Finally, nucleocapsid-like aggregates engaged in dsDNA synthesis appear to efficiently bind to F-actin filaments, a property that may be involved in targeting complexes to the nuclear envelope. More generally, this article highlights unique possibilities offered by in vitro reconstitution approaches combined with macromolecular imaging to gain insights into the mechanisms that alter the nucleoprotein architecture of the HIV-1 genome, ultimately enabling its insertion into the nuclear chromatin. PMID:23017337

  18. Mapping of immunogenic and protein-interacting regions at the surface of the seven-bladed β-propeller domain of the HIV-1 cellular interactor EED

    Directory of Open Access Journals (Sweden)

    Gouet Patrice

    2008-02-01

    Full Text Available Abstract Background The human EED protein, a member of the superfamily of Polycomb group proteins, is involved in multiple cellular protein complexes. Its C-terminal domain, which is common to the four EED isoforms, contains seven repeats of a canonical WD-40 motif. EED is an interactor of three HIV-1 proteins, matrix (MA, integrase (IN and Nef. An antiviral activity has been found to be associated with isoforms EED3 and EED4 at the late stage of HIV-1 replication, due to a negative effect on virus assembly and genomic RNA packaging. The aim of the present study was to determine the regions of the EED C-terminal core domain which were accessible and available to protein interactions, using three-dimensional (3D protein homology modelling with a WD-40 protein of known structure, and epitope mapping of anti-EED antibodies. Results Our data suggested that the C-terminal domain of EED was folded as a seven-bladed β-propeller protein. During the completion of our work, crystallographic data of EED became available from co-crystals of the EED C-terminal core with the N-terminal domain of its cellular partner EZH2. Our 3D-model was in good congruence with the refined structural model determined from crystallographic data, except for a unique α-helix in the fourth β-blade. More importantly, the position of flexible loops and accessible β-strands on the β-propeller was consistent with our mapping of immunogenic epitopes and sites of interaction with HIV-1 MA and IN. Certain immunoreactive regions were found to overlap with the EZH2, MA and IN binding sites, confirming their accessibility and reactivity at the surface of EED. Crystal structure of EED showed that the two discrete regions of interaction with MA and IN did not overlap with each other, nor with the EZH2 binding pocket, but were contiguous, and formed a continuous binding groove running along the lateral face of the β-propeller. Conclusion Identification of antibody-, MA-, IN- and EZH2

  19. Exploring the Genome and Proteome of Desulfitobacterium hafniense DCB2 for its Protein Complexes Involved in Metal Reduction and Dechlorination

    Energy Technology Data Exchange (ETDEWEB)

    Sang-Hoon, Kim; Hardzman, Christina; Davis, John k.; Hutcheson, Rachel; Broderick, Joan B.; Marsh, Terence L.; Tiedje, James M.

    2012-09-27

    Desulfitobacteria are of interest to DOE mission because of their ability to reduce many electron acceptors including Fe(III), U(VI), Cr(VI), As(V), Mn(IV), Se(VI), NO3- and well as CO2, sulfite, fumarate and humates, their ability to colonize more stressful environments because they form spores, fix nitrogen and they have the more protective Gram positive cell walls. Furthermore at least some of them reductively dechlorinate aromatic and aliphatic pollutants. Importantly, most of the metals and the organochlorine reductions are coupled to ATP production and support growth providing for the organism's natural selection at DOE's contaminant sites. This work was undertaken to gain insight into the genetic and metabolic pathways involved in dissimilatory metal reduction and reductive dechlorination, (ii) to discern the commonalities among these electron-accepting processes, (iii) to identify multi-protein complexes catalyzing these functions and (iv) to elucidate the coordination in expression of these pathways and processes.

  20. Multiple sites of the cleavage of 21- and 25-mer encephalytogenic oligopeptides corresponding to human myelin basic protein (MBP by specific anti-MBP antibodies from patients with systemic lupus erythematosus.

    Directory of Open Access Journals (Sweden)

    Anna M Timofeeva

    Full Text Available IgGs from patients with multiple sclerosis and systemic lupus erythematosus (SLE purified on MBP-Sepharose in contrast to canonical proteases hydrolyze effectively only myelin basic protein (MBP, but not many other tested proteins. Here we have shown for the first time that anti-MBP SLE IgGs hydrolyze nonspecific tri- and tetrapeptides with an extreme low efficiency and cannot effectively hydrolyze longer 20-mer nonspecific oligopeptides corresponding to antigenic determinants (AGDs of HIV-1 integrase. At the same time, anti-MBP SLE IgGs efficiently hydrolyze oligopeptides corresponding to AGDs of MBP. All sites of IgG-mediated proteolysis of 21-and 25-mer encephalytogenic oligopeptides corresponding to two known AGDs of MBP were found by a combination of reverse-phase chromatography, TLC, and MALDI spectrometry. Several clustered major, moderate, and minor sites of cleavage were revealed in the case of 21- and 25-mer oligopeptides. The active sites of anti-MBP abzymes are localised on their light chains, while heavy chains are responsible for the affinity of protein substrates. Interactions of intact globular proteins with both light and heavy chains of abzymes provide high affinity to MBP and specificity of this protein hydrolysis. The affinity of anti-MBP abzymes for intact MBP is approximately 1000-fold higher than for the oligopeptides. The data suggest that all oligopeptides interact mainly with the light chains of different monoclonal abzymes of total pool of IgGs, which possesses a lower affinity for substrates, and therefore, depending on the oligopeptide sequences, their hydrolysis may be less specific than globular protein and can occur in several sites.

  1. Detecting overlapping protein complexes in protein-protein interaction networks

    OpenAIRE

    Nepusz, Tamás; Yu, Haiyuan; Paccanaro, Alberto

    2012-01-01

    We introduce clustering with overlapping neighborhood expansion (ClusterONE), a method for detecting potentially overlapping protein complexes from protein-protein interaction data. ClusterONE-derived complexes for several yeast data sets showed better correspondence with reference complexes in the Munich Information Center for Protein Sequence (MIPS) catalog and complexes derived from the Saccharomyces Genome Database (SGD) than the results of seven popular methods. The results also showed a...

  2. Fusion-protein-assisted protein crystallization.

    Science.gov (United States)

    Kobe, Bostjan; Ve, Thomas; Williams, Simon J

    2015-07-01

    Fusion proteins can be used directly in protein crystallization to assist crystallization in at least two different ways. In one approach, the `heterologous fusion-protein approach', the fusion partner can provide additional surface area to promote crystal contact formation. In another approach, the `fusion of interacting proteins approach', protein assemblies can be stabilized by covalently linking the interacting partners. The linker connecting the proteins plays different roles in the two applications: in the first approach a rigid linker is required to reduce conformational heterogeneity; in the second, conversely, a flexible linker is required that allows the native interaction between the fused proteins. The two approaches can also be combined. The recent applications of fusion-protein technology in protein crystallization from the work of our own and other laboratories are briefly reviewed.

  3. EDITORIAL: Precision proteins Precision proteins

    Science.gov (United States)

    Demming, Anna

    2010-06-01

    Since the birth of modern day medicine, during the times of Hippocrates in ancient Greece, the profession has developed from the rudimentary classification of disease into a rigorous science with an inspiring capability to treat and cure. Scientific methodology has distilled clinical diagnostic tools from the early arts of prognosis, which used to rely as much on revelation and prophecy, as intuition and judgement [1]. Over the past decade, research into the interactions between proteins and nanosystems has provided some ingenious and apt techniques for delving into the intricacies of anatomical systems. In vivo biosensing has emerged as a vibrant field of research, as much of medical diagnosis relies on the detection of substances or an imbalance in the chemicals in the body. The inherent properties of nanoscale structures, such as cantilevers, make them well suited to biosensing applications that demand the detection of molecules at very low concentrations. Measurable deflections in cantilevers functionalised with antibodies provide quantitative indicators of the presence of specific antigens when the two react. Such developments have roused mounting interest in the interactions of proteins with nanostructures, such as carbon nanotubes [3], which have demonstrated great potential as generic biomarkers. Plasmonic properties are also being exploited in sensing applications, such as the molecular sentinel recently devised by researchers in the US. The device uses the plasmonic properties of a silver nanoparticle linked to a Raman labelled hairpin DNA probe to signal changes in the probe geometry resulting from interactions with substances in the environment. Success stories so far include the detection of two specific genes associated with breast cancer [4]. A greater understanding of how RNA interference regulates gene expression has highlighted the potential of using this natural process as another agent for combating disease in personalized medicine. However, the

  4. Protein Crystal Based Nanomaterials

    Science.gov (United States)

    Bell, Jeffrey A.; VanRoey, Patrick

    2001-01-01

    This is the final report on a NASA Grant. It concerns a description of work done, which includes: (1) Protein crystals cross-linked to form fibers; (2) Engineering of protein to favor crystallization; (3) Better knowledge-based potentials for protein-protein contacts; (4) Simulation of protein crystallization.

  5. Shotgun protein sequencing.

    Energy Technology Data Exchange (ETDEWEB)

    Faulon, Jean-Loup Michel; Heffelfinger, Grant S.

    2009-06-01

    A novel experimental and computational technique based on multiple enzymatic digestion of a protein or protein mixture that reconstructs protein sequences from sequences of overlapping peptides is described in this SAND report. This approach, analogous to shotgun sequencing of DNA, is to be used to sequence alternative spliced proteins, to identify post-translational modifications, and to sequence genetically engineered proteins.

  6. Protein-Protein Interaction Analysis by Docking

    OpenAIRE

    Stephan Ederer; Florian Fink; Wolfram Gronwald

    2009-01-01

    Based on a protein-protein docking approach we have developed a procedure to verify or falsify protein-protein interactions that were proposed by other methods such as yeast-2-hybrid assays. Our method currently utilizes intermolecular energies but can be expanded to incorporate additional terms such as amino acid based pair-potentials. We show some early results that demonstrate the general applicability of our approach.

  7. Protein folding, protein homeostasis, and cancer

    Institute of Scientific and Technical Information of China (English)

    John H. Van Drie

    2011-01-01

    Proteins fold into their functional 3-dimensional structures from a linear amino acid sequence. In vitro this process is spontaneous; while in vivo it is orchestrated by a specialized set of proteins, called chaperones. Protein folding is an ongoing cellular process, as cellular proteins constantly undergo synthesis and degradation. Here emerging links between this process and cancer are reviewed. This perspective both yields insights into the current struggle to develop novel cancer chemotherapeutics and has implications for future chemotherapy discovery.

  8. Protein-protein complexation in bioluminescence

    OpenAIRE

    Titushin, Maxim S.; Feng, Yingang; Lee, John; Vysotski, Eugene S.; Liu, Zhi-jie

    2011-01-01

    In this review we summarize the progress made towards understanding the role of protein-protein interactions in the function of various bioluminescence systems of marine organisms, including bacteria, jellyfish and soft corals, with particular focus on methodology used to detect and characterize these interactions. In some bioluminescence systems, protein-protein interactions involve an “accessory protein” whereby a stored substrate is efficiently delivered to the bioluminescent enzyme lucife...

  9. Protein-losing enteropathy

    Science.gov (United States)

    ... this page: //medlineplus.gov/ency/article/007338.htm Protein-losing enteropathy To use the sharing features on this page, please enable JavaScript. Protein-losing enteropathy is an abnormal loss of protein ...

  10. Protein and Heart Health

    Science.gov (United States)

    ... Recognition & Awards Healthy Workplace Food and Beverage Toolkit Protein and Heart Health Updated:May 5,2015 Protein ... said. What’s the harm in getting too much protein? The main problem is that often the extra ...

  11. Oligomeric protein structure networks: insights into protein-protein interactions

    Directory of Open Access Journals (Sweden)

    Brinda KV

    2005-12-01

    Full Text Available Abstract Background Protein-protein association is essential for a variety of cellular processes and hence a large number of investigations are being carried out to understand the principles of protein-protein interactions. In this study, oligomeric protein structures are viewed from a network perspective to obtain new insights into protein association. Structure graphs of proteins have been constructed from a non-redundant set of protein oligomer crystal structures by considering amino acid residues as nodes and the edges are based on the strength of the non-covalent interactions between the residues. The analysis of such networks has been carried out in terms of amino acid clusters and hubs (highly connected residues with special emphasis to protein interfaces. Results A variety of interactions such as hydrogen bond, salt bridges, aromatic and hydrophobic interactions, which occur at the interfaces are identified in a consolidated manner as amino acid clusters at the interface, from this study. Moreover, the characterization of the highly connected hub-forming residues at the interfaces and their comparison with the hubs from the non-interface regions and the non-hubs in the interface regions show that there is a predominance of charged interactions at the interfaces. Further, strong and weak interfaces are identified on the basis of the interaction strength between amino acid residues and the sizes of the interface clusters, which also show that many protein interfaces are stronger than their monomeric protein cores. The interface strengths evaluated based on the interface clusters and hubs also correlate well with experimentally determined dissociation constants for known complexes. Finally, the interface hubs identified using the present method correlate very well with experimentally determined hotspots in the interfaces of protein complexes obtained from the Alanine Scanning Energetics database (ASEdb. A few predictions of interface hot

  12. Drugging Membrane Protein Interactions.

    Science.gov (United States)

    Yin, Hang; Flynn, Aaron D

    2016-07-11

    The majority of therapeutics target membrane proteins, accessible on the surface of cells, to alter cellular signaling. Cells use membrane proteins to transduce signals into cells, transport ions and molecules, bind cells to a surface or substrate, and catalyze reactions. Newly devised technologies allow us to drug conventionally "undruggable" regions of membrane proteins, enabling modulation of protein-protein, protein-lipid, and protein-nucleic acid interactions. In this review, we survey the state of the art of high-throughput screening and rational design in drug discovery, and we evaluate the advances in biological understanding and technological capacity that will drive pharmacotherapy forward against unorthodox membrane protein targets. PMID:26863923

  13. Protein sequence comparison and protein evolution

    Energy Technology Data Exchange (ETDEWEB)

    Pearson, W.R. [Univ. of Virginia, Charlottesville, VA (United States). Dept. of Biochemistry

    1995-12-31

    This tutorial was one of eight tutorials selected to be presented at the Third International Conference on Intelligent Systems for Molecular Biology which was held in the United Kingdom from July 16 to 19, 1995. This tutorial examines how the information conserved during the evolution of a protein molecule can be used to infer reliably homology, and thus a shared proteinfold and possibly a shared active site or function. The authors start by reviewing a geological/evolutionary time scale. Next they look at the evolution of several protein families. During the tutorial, these families will be used to demonstrate that homologous protein ancestry can be inferred with confidence. They also examine different modes of protein evolution and consider some hypotheses that have been presented to explain the very earliest events in protein evolution. The next part of the tutorial will examine the technical aspects of protein sequence comparison. Both optimal and heuristic algorithms and their associated parameters that are used to characterize protein sequence similarities are discussed. Perhaps more importantly, they survey the statistics of local similarity scores, and how these statistics can both be used to improve the selectivity of a search and to evaluate the significance of a match. They them examine distantly related members of three protein families, the serine proteases, the glutathione transferases, and the G-protein-coupled receptors (GCRs). Finally, the discuss how sequence similarity can be used to examine internal repeated or mosaic structures in proteins.

  14. Inferring Protein Associations Using Protein Pulldown Assays

    Energy Technology Data Exchange (ETDEWEB)

    Sharp, Julia L.; Anderson, Kevin K.; Daly, Don S.; Auberry, Deanna L.; Borkowski, John J.; Cannon, William R.

    2007-02-01

    Background: One method to infer protein-protein associations is through a “bait-prey pulldown” assay using a protein affinity agent and an LC-MS (liquid chromatography-mass spectrometry)-based protein identification method. False positive and negative protein identifications are not uncommon, however, leading to incorrect inferences. Methods: A pulldown experiment generates a protein association matrix wherein each column represents a sample from one bait protein, each row represents one prey protein and each cell contains a presence/absence association indicator. Our method evaluates the presence/absence pattern across a prey protein (row) with a Likelihood Ratio Test (LRT), computing its p-value with simulated LRT test statistic distributions after a check with simulated binomial random variates disqualified the large sample 2 test. A pulldown experiment often involves hundreds of tests so we apply the false discovery rate method to control the false positive rate. Based on the p-value, each prey protein is assigned a category (specific association, non-specific association, or not associated) and appraised with respect to the pulldown experiment’s goal and design. The method is illustrated using a pulldown experiment investigating the protein complexes of Shewanella oneidensis MR-1. Results: The Monte Carlo simulated LRT p-values objectively reveal specific and ubiquitous prey, as well as potential systematic errors. The example analysis shows the results to be biologically sensible and more realistic than the ad hoc screening methods previously utilized. Conclusions: The method presented appears to be informative for screening for protein-protein associations.

  15. Prediction of Protein-Protein Interactions Using Protein Signature Profiling

    Institute of Scientific and Technical Information of China (English)

    Mahmood; A.; Mahdavi; Yen-Han; Lin

    2007-01-01

    Protein domains are conserved and functionally independent structures that play an important role in interactions among related proteins. Domain-domain inter- actions have been recently used to predict protein-protein interactions (PPI). In general, the interaction probability of a pair of domains is scored using a trained scoring function. Satisfying a threshold, the protein pairs carrying those domains are regarded as "interacting". In this study, the signature contents of proteins were utilized to predict PPI pairs in Saccharomyces cerevisiae, Caenorhabditis ele- gans, and Homo sapiens. Similarity between protein signature patterns was scored and PPI predictions were drawn based on the binary similarity scoring function. Results show that the true positive rate of prediction by the proposed approach is approximately 32% higher than that using the maximum likelihood estimation method when compared with a test set, resulting in 22% increase in the area un- der the receiver operating characteristic (ROC) curve. When proteins containing one or two signatures were removed, the sensitivity of the predicted PPI pairs in- creased significantly. The predicted PPI pairs are on average 11 times more likely to interact than the random selection at a confidence level of 0.95, and on aver- age 4 times better than those predicted by either phylogenetic profiling or gene expression profiling.

  16. Polymer Directed Protein Assemblies

    NARCIS (Netherlands)

    van Rijn, Patrick

    2013-01-01

    Protein aggregation and protein self-assembly is an important occurrence in natural systems, and is in some form or other dictated by biopolymers. Very obvious influences of biopolymers on protein assemblies are, e. g., virus particles. Viruses are a multi-protein assembly of which the morphology is

  17. Mirror image proteins.

    Science.gov (United States)

    Zhao, Le; Lu, Wuyuan

    2014-10-01

    Proteins composed entirely of unnatural d-amino acids and the achiral amino acid glycine are mirror image forms of their native l-protein counterparts. Recent advances in chemical protein synthesis afford unique and facile synthetic access to domain-sized mirror image d-proteins, enabling protein research to be conducted through 'the looking glass' and in a way previously unattainable. d-Proteins can facilitate structure determination of their native l-forms that are difficult to crystallize (racemic X-ray crystallography); d-proteins can serve as the bait for library screening to ultimately yield pharmacologically superior d-peptide/d-protein therapeutics (mirror-image phage display); d-proteins can also be used as a powerful mechanistic tool for probing molecular events in biology. This review examines recent progress in the application of mirror image proteins to structural biology, drug discovery, and immunology.

  18. Protein- protein interaction detection system using fluorescent protein microdomains

    Science.gov (United States)

    Waldo, Geoffrey S.; Cabantous, Stephanie

    2010-02-23

    The invention provides a protein labeling and interaction detection system based on engineered fragments of fluorescent and chromophoric proteins that require fused interacting polypeptides to drive the association of the fragments, and further are soluble and stable, and do not change the solubility of polypeptides to which they are fused. In one embodiment, a test protein X is fused to a sixteen amino acid fragment of GFP (.beta.-strand 10, amino acids 198-214), engineered to not perturb fusion protein solubility. A second test protein Y is fused to a sixteen amino acid fragment of GFP (.beta.-strand 11, amino acids 215-230), engineered to not perturb fusion protein solubility. When X and Y interact, they bring the GFP strands into proximity, and are detected by complementation with a third GFP fragment consisting of GFP amino acids 1-198 (strands 1-9). When GFP strands 10 and 11 are held together by interaction of protein X and Y, they spontaneous association with GFP strands 1-9, resulting in structural complementation, folding, and concomitant GFP fluorescence.

  19. [Protein expression and purification].

    Science.gov (United States)

    Růčková, E; Müller, P; Vojtěšek, B

    2014-01-01

    Production of recombinant proteins is essential for many applications in both basic research and also in medicine, where recombinant proteins are used as pharmaceuticals. This review summarizes procedures involved in recombinant protein expression and purification, including molecular cloning of target genes into expression vectors, selection of the appropriate expression system, and protein purification techniques. Recombinant DNA technology allows protein engineering to modify protein stability, activity and function or to facilitate protein purification by affinity tag fusions. A wide range of cloning systems enabling fast and effective design of expression vectors is currently available. A first choice of protein expression system is usually the bacteria Escherichia coli. The main advantages of this prokaryotic expression system are low cost and simplicity; on the other hand this system is often unsuitable for production of complex mammalian proteins. Protein expression mediated by eukaryotic cells (yeast, insect and mammalian cells) usually produces properly folded and posttranslationally modified proteins. How-ever, cultivation of insect and, especially, mammalian cells is time consuming and expensive. Affinity tagged recombinant proteins are purified efficiently using affinity chromatography. An affinity tag is a protein or peptide that mediates specific binding to a chromatography column, unbound proteins are removed during a washing step and pure protein is subsequently eluted. PMID:24945544

  20. Urine Protein and Urine Protein to Creatinine Ratio

    Science.gov (United States)

    ... limited. Home Visit Global Sites Search Help? Urine Protein and Urine Protein to Creatinine Ratio Share this page: Was this page helpful? Also known as: 24-Hour Urine Protein; Urine Total Protein; Urine Protein to Creatinine Ratio; ...

  1. IGSF9 Family Proteins

    DEFF Research Database (Denmark)

    Hansen, Maria; Walmod, Peter Schledermann

    2013-01-01

    The Drosophila protein Turtle and the vertebrate proteins immunoglobulin superfamily (IgSF), member 9 (IGSF9/Dasm1) and IGSF9B are members of an evolutionarily ancient protein family. A bioinformatics analysis of the protein family revealed that invertebrates contain only a single IGSF9 family gene......, the longest isoforms of the proteins have the same general organization as the neural cell adhesion molecule family of cell adhesion molecule proteins, and like this family of proteins, IGSF9 family members are expressed in the nervous system. A review of the literature revealed that Drosophila Turtle...... facilitates homophilic cell adhesion. Moreover, IGSF9 family proteins have been implicated in the outgrowth and branching of neurites, axon guidance, synapse maturation, self-avoidance, and tiling. However, despite the few published studies on IGSF9 family proteins, reports on the functions of both Turtle...

  2. Discover protein sequence signatures from protein-protein interaction data

    Directory of Open Access Journals (Sweden)

    Haasl Ryan J

    2005-11-01

    Full Text Available Abstract Background The development of high-throughput technologies such as yeast two-hybrid systems and mass spectrometry technologies has made it possible to generate large protein-protein interaction (PPI datasets. Mining these datasets for underlying biological knowledge has, however, remained a challenge. Results A total of 3108 sequence signatures were found, each of which was shared by a set of guest proteins interacting with one of 944 host proteins in Saccharomyces cerevisiae genome. Approximately 94% of these sequence signatures matched entries in InterPro member databases. We identified 84 distinct sequence signatures from the remaining 172 unknown signatures. The signature sharing information was then applied in predicting sub-cellular localization of yeast proteins and the novel signatures were used in identifying possible interacting sites. Conclusion We reported a method of PPI data mining that facilitated the discovery of novel sequence signatures using a large PPI dataset from S. cerevisiae genome as input. The fact that 94% of discovered signatures were known validated the ability of the approach to identify large numbers of signatures from PPI data. The significance of these discovered signatures was demonstrated by their application in predicting sub-cellular localizations and identifying potential interaction binding sites of yeast proteins.

  3. Protein and protein hydrolysates in sports nutrition.

    Science.gov (United States)

    van Loon, Luc J C; Kies, Arie K; Saris, Wim H M

    2007-08-01

    With the increasing knowledge about the role of nutrition in increasing exercise performance, it has become clear over the last 2 decades that amino acids, protein, and protein hydrolysates can play an important role. Most of the attention has been focused on their effects at a muscular level. As these nutrients are ingested, however, it also means that gastrointestinal digestibility and absorption can modulate their efficacy significantly. Therefore, discussing the role of amino acids, protein, and protein hydrolysates in sports nutrition entails holding a discussion on all levels of the metabolic route. On May 28-29, 2007, a small group of researchers active in the field of exercise science and protein metabolism presented an overview of the different aspects of the application of protein and protein hydrolysates in sports nutrition. In addition, they were asked to share their opinions on the future progress in their fields of research. In this overview, an introduction to the workshop and a short summary of its outcome is provided.

  4. Protein Data Bank (PDB)

    Data.gov (United States)

    U.S. Department of Health & Human Services — The Protein Data Bank (PDB) archive is the single worldwide repository of information about the 3D structures of large biological molecules, including proteins and...

  5. Learning about Proteins

    Science.gov (United States)

    ... need from peanuts alone, but if you have peanut butter on whole-grain bread, you're set. Likewise, ... protein in a day: 2 tablespoons (15 milliliters) peanut butter (7 grams protein) 1 cup (240 milliliters) low- ...

  6. Abnormal protein aggregationand neurodegenerativediseases

    Institute of Scientific and Technical Information of China (English)

    2001-01-01

    Abnormal protein aggregation or amyloid is the major cause ofmany neurodegenerative disorders. The present review focuses on the correlation between sequence and structure features of proteins related to the diseases and abnormal protein aggregation. Recent progress has improved our knowledge on understand-ing the mechanism of amyloid formation. We suggest a nucleation model for ordered protein aggregation, which can also explain pathogenesis mechanisms of these neurodegenerative diseases in vivo.

  7. Destabilized bioluminescent proteins

    Science.gov (United States)

    Allen, Michael S.; Rakesh, Gupta; Gary, Sayler S.

    2007-07-31

    Purified nucleic acids, vectors and cells containing a gene cassette encoding at least one modified bioluminescent protein, wherein the modification includes the addition of a peptide sequence. The duration of bioluminescence emitted by the modified bioluminescent protein is shorter than the duration of bioluminescence emitted by an unmodified form of the bioluminescent protein.

  8. CSF total protein

    Science.gov (United States)

    CSF total protein is a test to determine the amount of protein in your spinal fluid, also called cerebrospinal fluid (CSF). ... The normal protein range varies from lab to lab, but is typically about 15 to 60 mg/dL. Note: mg/dL = ...

  9. Modeling Protein Domain Function

    Science.gov (United States)

    Baker, William P.; Jones, Carleton "Buck"; Hull, Elizabeth

    2007-01-01

    This simple but effective laboratory exercise helps students understand the concept of protein domain function. They use foam beads, Styrofoam craft balls, and pipe cleaners to explore how domains within protein active sites interact to form a functional protein. The activity allows students to gain content mastery and an understanding of the…

  10. Protein domain prediction

    NARCIS (Netherlands)

    Ingolfsson, Helgi; Yona, Golan

    2008-01-01

    Domains are considered to be the building blocks of protein structures. A protein can contain a single domain or multiple domains, each one typically associated with a specific function. The combination of domains determines the function of the protein, its subcellular localization and the interacti

  11. ASV_SEISMICSHOT500 - Shot Points at 500 Shot Intervals for Seismic Data Collected with Autonomous Surface Vehicle (ASV) IRIS in Apalachicola Bay and St. George Sound, FL.

    Data.gov (United States)

    National Oceanic and Atmospheric Administration, Department of Commerce — These data were collected under a cooperative mapping program between the U.S. Geological Survey (USGS), the National Oceanic and Atmospheric Administration Office...

  12. Protein - Which is Best?

    Science.gov (United States)

    Hoffman, Jay R; Falvo, Michael J

    2004-09-01

    Protein intake that exceeds the recommended daily allowance is widely accepted for both endurance and power athletes. However, considering the variety of proteins that are available much less is known concerning the benefits of consuming one protein versus another. The purpose of this paper is to identify and analyze key factors in order to make responsible recommendations to both the general and athletic populations. Evaluation of a protein is fundamental in determining its appropriateness in the human diet. Proteins that are of inferior content and digestibility are important to recognize and restrict or limit in the diet. Similarly, such knowledge will provide an ability to identify proteins that provide the greatest benefit and should be consumed. The various techniques utilized to rate protein will be discussed. Traditionally, sources of dietary protein are seen as either being of animal or vegetable origin. Animal sources provide a complete source of protein (i.e. containing all essential amino acids), whereas vegetable sources generally lack one or more of the essential amino acids. Animal sources of dietary protein, despite providing a complete protein and numerous vitamins and minerals, have some health professionals concerned about the amount of saturated fat common in these foods compared to vegetable sources. The advent of processing techniques has shifted some of this attention and ignited the sports supplement marketplace with derivative products such as whey, casein and soy. Individually, these products vary in quality and applicability to certain populations. The benefits that these particular proteins possess are discussed. In addition, the impact that elevated protein consumption has on health and safety issues (i.e. bone health, renal function) are also reviewed. Key PointsHigher protein needs are seen in athletic populations.Animal proteins is an important source of protein, however potential health concerns do exist from a diet of protein

  13. Protopia: a protein-protein interaction tool

    Science.gov (United States)

    Real-Chicharro, Alejandro; Ruiz-Mostazo, Iván; Navas-Delgado, Ismael; Kerzazi, Amine; Chniber, Othmane; Sánchez-Jiménez, Francisca; Medina, Miguel Ángel; Aldana-Montes, José F

    2009-01-01

    Background Protein-protein interactions can be considered the basic skeleton for living organism self-organization and homeostasis. Impressive quantities of experimental data are being obtained and computational tools are essential to integrate and to organize this information. This paper presents Protopia, a biological tool that offers a way of searching for proteins and their interactions in different Protein Interaction Web Databases, as a part of a multidisciplinary initiative of our institution for the integration of biological data . Results The tool accesses the different Databases (at present, the free version of Transfac, DIP, Hprd, Int-Act and iHop), and results are expressed with biological protein names or databases codes and can be depicted as a vector or a matrix. They can be represented and handled interactively as an organic graph. Comparison among databases is carried out using the Uniprot codes annotated for each protein. Conclusion The tool locates and integrates the current information stored in the aforementioned databases, and redundancies among them are detected. Results are compatible with the most important network analysers, so that they can be compared and analysed by other world-wide known tools and platforms. The visualization possibilities help to attain this goal and they are especially interesting for handling multiple-step or complex networks. PMID:19828077

  14. Protein hydration and dynamics

    International Nuclear Information System (INIS)

    Inelastic neutron scattering can measure the protein thermal fluctuations under the physiological aqueous environment, especially it is powerful to observe the low-energy protein dynamics in THz region, which are revealed theoretically to be coupled with solvations. Neutron enables the selective observation of protein and hydration water by deuteration. The complementary analysis with molecular dynamics simulation is also effective for the study of protein hydration. Some examples of the application toward the understanding of molecular basis of protein functions will be introduced. (author)

  15. Highly thermostable fluorescent proteins

    Science.gov (United States)

    Bradbury, Andrew M.; Waldo, Geoffrey S.; Kiss, Csaba

    2011-03-22

    Thermostable fluorescent proteins (TSFPs), methods for generating these and other stability-enhanced proteins, polynucleotides encoding such proteins, and assays and method for using the TSFPs and TSFP-encoding nucleic acid molecules are provided. The TSFPs of the invention show extremely enhanced levels of stability and thermotolerance. In one case, for example, a TSFP of the invention is so stable it can be heated to 99.degree. C. for short periods of time without denaturing, and retains 85% of its fluorescence when heated to 80.degree. C. for several minutes. The invention also provides a method for generating stability-enhanced variants of a protein, including but not limited to fluorescent proteins.

  16. Racemic protein crystallography.

    Science.gov (United States)

    Yeates, Todd O; Kent, Stephen B H

    2012-01-01

    Although natural proteins are chiral and are all of one "handedness," their mirror image forms can be prepared by chemical synthesis. This opens up new opportunities for protein crystallography. A racemic mixture of the enantiomeric forms of a protein molecule can crystallize in ways that natural proteins cannot. Recent experimental data support a theoretical prediction that this should make racemic protein mixtures highly amenable to crystallization. Crystals obtained from racemic mixtures also offer advantages in structure determination strategies. The relevance of these potential advantages is heightened by advances in synthetic methods, which are extending the size limit for proteins that can be prepared by chemical synthesis. Recent ideas and results in the area of racemic protein crystallography are reviewed.

  17. Protein kinesis: The dynamics of protein trafficking and stability

    Energy Technology Data Exchange (ETDEWEB)

    NONE

    1995-12-31

    The purpose of this conference is to provide a multidisciplinary forum for exchange of state-of-the-art information on protein kinesis. This volume contains abstracts of papers in the following areas: protein folding and modification in the endoplasmic reticulum; protein trafficking; protein translocation and folding; protein degradation; polarity; nuclear trafficking; membrane dynamics; and protein import into organelles.

  18. Protein: FBA3 [TP Atlas

    Lifescience Database Archive (English)

    Full Text Available FBA3 Atg1 kinase complex ATG1 APG1, AUT3, CVT10 Serine/threonine-protein kinase ATG1 Autophagy protein... 3, Autophagy-related protein 1, Cytoplasm to vacuole targeting protein 10 559292 Sacchar

  19. Protein: FEA4 [TP Atlas

    Lifescience Database Archive (English)

    Full Text Available FEA4 Proteins in gibberellin signaling GID2 F-box protein GID2 Gibberellin-insensitive dwarf protein... 2, Protein GIBBERELLIN INSENSITIVE DWARF2 39947 Oryza sativa subsp. japonica Q7XAK4 ...

  20. Protein Electrophoresis/Immunofixation Electrophoresis

    Science.gov (United States)

    ... be limited. Home Visit Global Sites Search Help? Protein Electrophoresis Immunofixation Electrophoresis Share this page: Was this page helpful? Also known as: Serum Protein Electrophoresis; Protein ELP; SPE; SPEP; Urine Protein Electrophoresis; ...

  1. NMR of unfolded proteins

    Indian Academy of Sciences (India)

    Amarnath Chtterjee; Ashutosh Kumar; Jeetender Chugh; Sudha Srivastava; Neel S Bhavesh; Ramakrishna V Hosur

    2005-01-01

    In the post-genomic era, as more and more genome sequences are becoming known and hectic efforts are underway to decode the information content in them, it is becoming increasingly evident that flexibility in proteins plays a crucial role in many of the biological functions. Many proteins have intrinsic disorder either wholly or in specific regions. It appears that this disorder may be important for regulatory functions of the proteins, on the one hand, and may help in directing the folding process to reach the compact native state, on the other. Nuclear magnetic resonance (NMR) has over the last two decades emerged as the sole, most powerful technique to help characterize these disordered protein systems. In this review, we first discuss the significance of disorder in proteins and then describe the recent developments in NMR methods for their characterization. A brief description of the results obtained on several disordered proteins is presented at the end.

  2. Computational Protein Design

    DEFF Research Database (Denmark)

    Johansson, Kristoffer Enøe

    Proteins are the major functional group of molecules in biology. The impact of protein science on medicine and chemical productions is rapidly increasing. However, the greatest potential remains to be realized. The fi eld of protein design has advanced computational modeling from a tool of support...... to a central method that enables new developments. For example, novel enzymes with functions not found in natural proteins have been de novo designed to give enough activity for experimental optimization. This thesis presents the current state-of-the-art within computational design methods together...... with a novel method based on probability theory. With the aim of assembling a complete pipeline for protein design, this work touches upon several aspects of protein design. The presented work is the computational half of a design project where the other half is dedicated to the experimental part...

  3. Staining Proteins in Gels

    OpenAIRE

    Gallagher, Sean; Chakavarti, Deb

    2008-01-01

    Following separation by electrophoretic methods, proteins in a gel can be detected by several staining methods. This unit describes protocols for detecting proteins by four popular methods. Coomassie blue staining is an easy and rapid method. Silver staining, while more time consuming, is considerably more sensitive and can thus be used to detect smaller amounts of protein. Fluorescent staining is a popular alternative to traditional staining procedures, mainly because it is more sensitive th...

  4. Pressure cryocooling protein crystals

    Science.gov (United States)

    Kim, Chae Un; Gruner, Sol M.

    2011-10-04

    Preparation of cryocooled protein crystal is provided by use of helium pressurizing and cryocooling to obtain cryocooled protein crystal allowing collection of high resolution data and by heavier noble gas (krypton or xenon) binding followed by helium pressurizing and cryocooling to obtain cryocooled protein crystal for collection of high resolution data and SAD phasing simultaneously. The helium pressurizing is carried out on crystal coated to prevent dehydration or on crystal grown in aqueous solution in a capillary.

  5. Proteins at interfaces

    OpenAIRE

    Evers, Florian

    2011-01-01

    Protein adsorption is a fundamental and ubiquitous phenomenon, which has severe implications in the fields of biomaterials as well as bio- and nanotechnology, e.g., in drug delivery, biofouling, the biocompatibility of implants, food chemistry, and biosensors. Therefore, the mechanisms of protein adsorption and controlling the interfacial affinity of proteins have become intriguing and interdisciplinary research topics. In this work, X-ray and neutron reflectometry are the main...

  6. Acanthamoeba castellanii STAT protein.

    Directory of Open Access Journals (Sweden)

    Anna Kicinska

    Full Text Available STAT (signal transducers and activators of transcription proteins are one of the important mediators of phosphotyrosine-regulated signaling in metazoan cells. We described the presence of STAT protein in a unicellular, free-living amoebae with a simple life cycle, Acanthamoeba castellanii. A. castellanii is the only, studied to date, Amoebozoan that does not belong to Mycetozoa but possesses STATs. A sequence of the A. castellanii STAT protein includes domains similar to those of the Dictyostelium STAT proteins: a coiled coil (characteristic for Dictyostelium STAT coiled coil, a STAT DNA-binding domain and a Src-homology domain. The search for protein sequences homologous to A. castellanii STAT revealed 17 additional sequences from lower eukaryotes. Interestingly, all of these sequences come from Amoebozoa organisms that belong to either Mycetozoa (slime molds or Centramoebida. We showed that there are four separated clades within the slime mold STAT proteins. The A. castellanii STAT protein branches next to a group of STATc proteins from Mycetozoa. We also demonstrate that Amoebozoa form a distinct monophyletic lineage within the STAT protein world that is well separated from the other groups.

  7. Acanthamoeba castellanii STAT protein.

    Science.gov (United States)

    Kicinska, Anna; Leluk, Jacek; Jarmuszkiewicz, Wieslawa

    2014-01-01

    STAT (signal transducers and activators of transcription) proteins are one of the important mediators of phosphotyrosine-regulated signaling in metazoan cells. We described the presence of STAT protein in a unicellular, free-living amoebae with a simple life cycle, Acanthamoeba castellanii. A. castellanii is the only, studied to date, Amoebozoan that does not belong to Mycetozoa but possesses STATs. A sequence of the A. castellanii STAT protein includes domains similar to those of the Dictyostelium STAT proteins: a coiled coil (characteristic for Dictyostelium STAT coiled coil), a STAT DNA-binding domain and a Src-homology domain. The search for protein sequences homologous to A. castellanii STAT revealed 17 additional sequences from lower eukaryotes. Interestingly, all of these sequences come from Amoebozoa organisms that belong to either Mycetozoa (slime molds) or Centramoebida. We showed that there are four separated clades within the slime mold STAT proteins. The A. castellanii STAT protein branches next to a group of STATc proteins from Mycetozoa. We also demonstrate that Amoebozoa form a distinct monophyletic lineage within the STAT protein world that is well separated from the other groups. PMID:25338074

  8. Moonlighting proteins in cancer.

    Science.gov (United States)

    Min, Kyung-Won; Lee, Seong-Ho; Baek, Seung Joon

    2016-01-01

    Since the 1980s, growing evidence suggested that the cellular localization of proteins determined their activity and biological functions. In a classical view, a protein is characterized by the single cellular compartment where it primarily resides and functions. It is now believed that when proteins appear in different subcellular locations, the cells surpass the expected activity of proteins given the same genomic information to fulfill complex biological behavior. Many proteins are recognized for having the potential to exist in multiple locations in cells. Dysregulation of translocation may cause cancer or contribute to poorer cancer prognosis. Thus, quantitative and comprehensive assessment of dynamic proteins and associated protein movements could be a promising indicator in determining cancer prognosis and efficiency of cancer treatment and therapy. This review will summarize these so-called moonlighting proteins, in terms of a coupled intracellular cancer signaling pathway. Determination of the detailed biological intracellular and extracellular transit and regulatory activity of moonlighting proteins permits a better understanding of cancer and identification of potential means of molecular intervention.

  9. Engineering therapeutic protein disaggregases

    Science.gov (United States)

    Shorter, James

    2016-01-01

    Therapeutic agents are urgently required to cure several common and fatal neurodegenerative disorders caused by protein misfolding and aggregation, including amyotrophic lateral sclerosis (ALS), Parkinson’s disease (PD), and Alzheimer’s disease (AD). Protein disaggregases that reverse protein misfolding and restore proteins to native structure, function, and localization could mitigate neurodegeneration by simultaneously reversing 1) any toxic gain of function of the misfolded form and 2) any loss of function due to misfolding. Potentiated variants of Hsp104, a hexameric AAA+ ATPase and protein disaggregase from yeast, have been engineered to robustly disaggregate misfolded proteins connected with ALS (e.g., TDP-43 and FUS) and PD (e.g., α-synuclein). However, Hsp104 has no metazoan homologue. Metazoa possess protein disaggregase systems distinct from Hsp104, including Hsp110, Hsp70, and Hsp40, as well as HtrA1, which might be harnessed to reverse deleterious protein misfolding. Nevertheless, vicissitudes of aging, environment, or genetics conspire to negate these disaggregase systems in neurodegenerative disease. Thus, engineering potentiated human protein disaggregases or isolating small-molecule enhancers of their activity could yield transformative therapeutics for ALS, PD, and AD. PMID:27255695

  10. MicroProteins

    DEFF Research Database (Denmark)

    Eguen, Teinai Ebimienere; Straub, Daniel; Graeff, Moritz;

    2015-01-01

    MicroProteins (miPs) are short, usually single-domain proteins that, in analogy to miRNAs, heterodimerize with their targets and exert a dominant-negative effect. Recent bioinformatic attempts to identify miPs have resulted in a list of potential miPs, many of which lack the defining characterist......MicroProteins (miPs) are short, usually single-domain proteins that, in analogy to miRNAs, heterodimerize with their targets and exert a dominant-negative effect. Recent bioinformatic attempts to identify miPs have resulted in a list of potential miPs, many of which lack the defining...

  11. How Many Protein-Protein Interactions Types Exist in Nature?

    OpenAIRE

    Leonardo Garma; Srayanta Mukherjee; Pralay Mitra; Yang Zhang

    2012-01-01

    "Protein quaternary structure universe" refers to the ensemble of all protein-protein complexes across all organisms in nature. The number of quaternary folds thus corresponds to the number of ways proteins physically interact with other proteins. This study focuses on answering two basic questions: Whether the number of protein-protein interactions is limited and, if yes, how many different quaternary folds exist in nature. By all-to-all sequence and structure comparisons, we grouped the pro...

  12. Poxviral Ankyrin Proteins

    Directory of Open Access Journals (Sweden)

    Michael H. Herbert

    2015-02-01

    Full Text Available Multiple repeats of the ankyrin motif (ANK are ubiquitous throughout the kingdoms of life but are absent from most viruses. The main exception to this is the poxvirus family, and specifically the chordopoxviruses, with ANK repeat proteins present in all but three species from separate genera. The poxviral ANK repeat proteins belong to distinct orthologue groups spread over different species, and align well with the phylogeny of their genera. This distribution throughout the chordopoxviruses indicates these proteins were present in an ancestral vertebrate poxvirus, and have since undergone numerous duplication events. Most poxviral ANK repeat proteins contain an unusual topology of multiple ANK motifs starting at the N-terminus with a C-terminal poxviral homologue of the cellular F-box enabling interaction with the cellular SCF ubiquitin ligase complex. The subtle variations between ANK repeat proteins of individual poxviruses suggest an array of different substrates may be bound by these protein-protein interaction domains and, via the F-box, potentially directed to cellular ubiquitination pathways and possible degradation. Known interaction partners of several of these proteins indicate that the NF-κB coordinated anti-viral response is a key target, whilst some poxviral ANK repeat domains also have an F-box independent affect on viral host-range.

  13. Brushes and proteins

    NARCIS (Netherlands)

    Bosker, W.T.E.

    2011-01-01

      Brushes and Proteins   Wouter T. E. Bosker         Protein adsorption at solid surfaces can be prevented by applying a polymer brush at the surface. A polymer brush consists of polymer chains end-grafted to the surface at such a grafting density that th

  14. Proteins in biomass streams

    NARCIS (Netherlands)

    Mulder, W.J.

    2010-01-01

    The focus of this study is to give an overview of traditional and new biomasses and biomass streams that contain proteins. When information was available, the differences in molecular structure and physical and chemical properties for the different proteins is given. For optimal biomass use, isolati

  15. Protein Attachment on Nanodiamonds.

    Science.gov (United States)

    Lin, Chung-Lun; Lin, Cheng-Huang; Chang, Huan-Cheng; Su, Meng-Chih

    2015-07-16

    A recent advance in nanotechnology is the scale-up production of small and nonaggregated diamond nanoparticles suitable for biological applications. Using detonation nanodiamonds (NDs) with an average diameter of ∼4 nm as the adsorbents, we have studied the static attachment of three proteins (myoglobin, bovine serum albumin, and insulin) onto the nanoparticles by optical spectroscopy, mass spectrometry, and dynamic light scattering, and electrophoretic zeta potential measurements. Results show that the protein surface coverage is predominantly determined by the competition between protein-protein and protein-ND interactions, giving each protein a unique and characteristic structural configuration in its own complex. Specifically, both myoglobin and bovine serum albumin show a Langmuir-type adsorption behavior, forming 1:1 complexes at saturation, whereas insulin folds into a tightly bound multimer before adsorption. The markedly different adsorption patterns appear to be independent of the protein concentration and are closely related to the affinity of the individual proteins for the NDs. The present study provides a fundamental understanding for the use of NDs as a platform for nanomedical drug delivery. PMID:25815400

  16. Protein Unfolding and Alzheimer's

    Science.gov (United States)

    Cheng, Kelvin

    2012-10-01

    Early interaction events of beta-amyloid (Aβ) proteins with neurons have been associated with the pathogenesis of Alzheimer's disease. Knowledge pertaining to the role of lipid molecules, particularly cholesterol, in modulating the single Aβ interactions with neurons at the atomic length and picosecond time resolutions, remains unclear. In our research, we have used atomistic molecular dynamics simulations to explore early molecular events including protein insertion kinetics, protein unfolding, and protein-induced membrane disruption of Aβ in lipid domains that mimic the nanoscopic raft and non-raft regions of the neural membrane. In this talk, I will summarize our current work on investigating the role of cholesterol in regulating the Aβ interaction events with membranes at the molecular level. I will also explain how our results will provide new insights into understanding the pathogenesis of Alzheimer's disease associated with the Aβ proteins.

  17. Sensitizing properties of proteins

    DEFF Research Database (Denmark)

    Poulsen, Lars K.; Ladics, Gregory S; McClain, Scott;

    2014-01-01

    The scope of allergy risk is diverse considering the myriad ways in which protein allergenicity is affected by physiochemical characteristics of proteins. The complexity created by the matrices of foods and the variability of the human immune system add additional challenges to understanding...... the relationship between sensitization potential and allergy disease. To address these and other issues, an April 2012 international symposium was held in Prague, Czech Republic, to review and discuss the state-of-the-science of sensitizing properties of protein allergens. The symposium, organized by the Protein...... Allergenicity Technical Committee of the International Life Sciences Institute's Health and Environmental Sciences Institute, featured presentations on current methods, test systems, research trends, and unanswered questions in the field of protein sensitization. A diverse group of over 70 interdisciplinary...

  18. Bacterial Ice Crystal Controlling Proteins

    OpenAIRE

    Lorv, Janet S. H.; Rose, David R; Glick, Bernard R.

    2014-01-01

    Across the world, many ice active bacteria utilize ice crystal controlling proteins for aid in freezing tolerance at subzero temperatures. Ice crystal controlling proteins include both antifreeze and ice nucleation proteins. Antifreeze proteins minimize freezing damage by inhibiting growth of large ice crystals, while ice nucleation proteins induce formation of embryonic ice crystals. Although both protein classes have differing functions, these proteins use the same ice binding mechanisms. R...

  19. The centrality of cancer proteins in human protein-protein interaction network: a revisit.

    Science.gov (United States)

    Xiong, Wei; Xie, Luyu; Zhou, Shuigeng; Liu, Hui; Guan, Jihong

    2014-01-01

    Topological analysis of protein-protein interaction (PPI) networks has been widely applied to the investigation on cancer mechanisms. However, there is still a debate on whether cancer proteins exhibit more topological centrality compared to the other proteins in the human PPI network. To resolve this debate, we first identified four sets of human proteins, and then mapped these proteins into the yeast PPI network by homologous genes. Finally, we compared these proteins' properties in human and yeast PPI networks. Experiments over two real datasets demonstrated that cancer proteins tend to have higher degree and smaller clustering coefficient than non-cancer proteins. Experimental results also validated that cancer proteins have larger betweenness centrality compared to the other proteins on the STRING dataset. However, on the BioGRID dataset, the average betweenness centrality of cancer proteins is larger than that of disease and control proteins, but smaller than that of essential proteins. PMID:24878726

  20. PSC: protein surface classification.

    Science.gov (United States)

    Tseng, Yan Yuan; Li, Wen-Hsiung

    2012-07-01

    We recently proposed to classify proteins by their functional surfaces. Using the structural attributes of functional surfaces, we inferred the pairwise relationships of proteins and constructed an expandable database of protein surface classification (PSC). As the functional surface(s) of a protein is the local region where the protein performs its function, our classification may reflect the functional relationships among proteins. Currently, PSC contains a library of 1974 surface types that include 25,857 functional surfaces identified from 24,170 bound structures. The search tool in PSC empowers users to explore related surfaces that share similar local structures and core functions. Each functional surface is characterized by structural attributes, which are geometric, physicochemical or evolutionary features. The attributes have been normalized as descriptors and integrated to produce a profile for each functional surface in PSC. In addition, binding ligands are recorded for comparisons among homologs. PSC allows users to exploit related binding surfaces to reveal the changes in functionally important residues on homologs that have led to functional divergence during evolution. The substitutions at the key residues of a spatial pattern may determine the functional evolution of a protein. In PSC (http://pocket.uchicago.edu/psc/), a pool of changes in residues on similar functional surfaces is provided.

  1. Protein oxidation in aquatic foods

    DEFF Research Database (Denmark)

    Baron, Caroline P.

    2014-01-01

    The chapter discusses general considerations about protein oxidation and reviews the mechanisms involved in protein oxidation and consequences of protein oxidation on fish proteins. It presents two case studies, the first deals with protein and lipid oxidation in frozen rainbow trout......, and the second with oxidation in salted herring. The mechanisms responsible for initiation of protein oxidation are unclear, but it is generally accepted that free radical species initiating lipid oxidation can also initiate protein oxidation. The chapter focuses on interaction between protein and lipid...... oxidation. The protein carbonyl group measurement is the widely used method for estimating protein oxidation in foods and has been used in fish muscle. The chapter also talks about the impact of protein oxidation on protein functionality, fish muscle texture, and food nutritional value. Protein oxidation...

  2. Bacterial ice crystal controlling proteins.

    Science.gov (United States)

    Lorv, Janet S H; Rose, David R; Glick, Bernard R

    2014-01-01

    Across the world, many ice active bacteria utilize ice crystal controlling proteins for aid in freezing tolerance at subzero temperatures. Ice crystal controlling proteins include both antifreeze and ice nucleation proteins. Antifreeze proteins minimize freezing damage by inhibiting growth of large ice crystals, while ice nucleation proteins induce formation of embryonic ice crystals. Although both protein classes have differing functions, these proteins use the same ice binding mechanisms. Rather than direct binding, it is probable that these protein classes create an ice surface prior to ice crystal surface adsorption. Function is differentiated by molecular size of the protein. This paper reviews the similar and different aspects of bacterial antifreeze and ice nucleation proteins, the role of these proteins in freezing tolerance, prevalence of these proteins in psychrophiles, and current mechanisms of protein-ice interactions. PMID:24579057

  3. Anchored design of protein-protein interfaces.

    Directory of Open Access Journals (Sweden)

    Steven M Lewis

    Full Text Available BACKGROUND: Few existing protein-protein interface design methods allow for extensive backbone rearrangements during the design process. There is also a dichotomy between redesign methods, which take advantage of the native interface, and de novo methods, which produce novel binders. METHODOLOGY: Here, we propose a new method for designing novel protein reagents that combines advantages of redesign and de novo methods and allows for extensive backbone motion. This method requires a bound structure of a target and one of its natural binding partners. A key interaction in this interface, the anchor, is computationally grafted out of the partner and into a surface loop on the design scaffold. The design scaffold's surface is then redesigned with backbone flexibility to create a new binding partner for the target. Careful choice of a scaffold will bring experimentally desirable characteristics into the new complex. The use of an anchor both expedites the design process and ensures that binding proceeds against a known location on the target. The use of surface loops on the scaffold allows for flexible-backbone redesign to properly search conformational space. CONCLUSIONS AND SIGNIFICANCE: This protocol was implemented within the Rosetta3 software suite. To demonstrate and evaluate this protocol, we have developed a benchmarking set of structures from the PDB with loop-mediated interfaces. This protocol can recover the correct loop-mediated interface in 15 out of 16 tested structures, using only a single residue as an anchor.

  4. An Algorithm for Finding Functional Modules and Protein Complexes in Protein-Protein Interaction Networks

    OpenAIRE

    Guangyu Cui; Yu Chen; De-Shuang Huang; Kyungsook Han

    2008-01-01

    Biological processes are often performed by a group of proteins rather than by individual proteins, and proteins in a same biological group form a densely connected subgraph in a protein-protein interaction network. Therefore, finding a densely connected subgraph provides useful information to predict the function or protein complex of uncharacterized proteins in the highly connected subgraph. We have developed an efficient algorithm and program for finding cliques and near-cliques in a prote...

  5. Quantification of the Influence of Protein-Protein Interactions on Adsorbed Protein Structure and Bioactivity

    OpenAIRE

    Wei, Yang; Thyparambil, Aby A.; Latour, Robert A.

    2013-01-01

    While protein-surface interactions have been widely studied, relatively little is understood at this time regarding how protein-surface interaction effects are influenced by protein-protein interactions and how these effects combine with the internal stability of a protein to influence its adsorbed-state structure and bioactivity. The objectives of this study were to develop a method to study these combined effects under widely varying protein-protein interaction conditions using hen egg-whit...

  6. Protein: FEB6 [TP Atlas

    Lifescience Database Archive (English)

    Full Text Available FEB6 Photoresponse regulatory proteins HD1 SE1 Zinc finger protein HD1 Protein CONSTANS-like, Protein... HEADING DATE 1, Protein PHOTOPERIOD SENSITIVITY 1 39947 Oryza sativa subsp. japonica 4340746 Q9FDX8 21952207, 19246394 #shimamoto ...

  7. Protein: MPA6 [TP Atlas

    Lifescience Database Archive (English)

    Full Text Available in 30 kDa adipocyte complement-related protein, Adipocyte complement-related 30 kDa protein, Adipocyte, C1q ...and collagen domain-containing protein, Adipose most abundant gene transcript 1 protein, Gelatin-binding protein 9606 Homo sapiens Q15848 9370 9370 Q15848 18054335, 19646806 ...

  8. Protein crystallography prescreen kit

    Science.gov (United States)

    Segelke, Brent W.; Krupka, Heike I.; Rupp, Bernhard

    2005-07-12

    A kit for prescreening protein concentration for crystallization includes a multiplicity of vials, a multiplicity of pre-selected reagents, and a multiplicity of sample plates. The reagents and a corresponding multiplicity of samples of the protein in solutions of varying concentrations are placed on sample plates. The sample plates containing the reagents and samples are incubated. After incubation the sample plates are examined to determine which of the sample concentrations are too low and which the sample concentrations are too high. The sample concentrations that are optimal for protein crystallization are selected and used.

  9. Human Protein Z.

    OpenAIRE

    Broze, G J; Miletich, J P

    1984-01-01

    Protein Z was purified from human plasma by a four-step procedure which included barium citrate adsorption, ammonium sulfate fractionation, DEAE-Sepharose chromatography, and blue agarose chromatography with a yield of 20%. It is a 62,000 mol wt protein with an extinction coefficient of 12.0. The concentration of Protein Z in pooled, citrated plasma is 2.2 micrograms/ml and its half-life in patients starting warfarin anticoagulation therapy is estimated to be less than 2.5 d. The NH2-terminal...

  10. Piezoelectric allostery of protein.

    Science.gov (United States)

    Ohnuki, Jun; Sato, Takato; Takano, Mitsunori

    2016-07-01

    Allostery is indispensable for a protein to work, where a locally applied stimulus is transmitted to a distant part of the molecule. While the allostery due to chemical stimuli such as ligand binding has long been studied, the growing interest in mechanobiology prompts the study of the mechanically stimulated allostery, the physical mechanism of which has not been established. By molecular dynamics simulation of a motor protein myosin, we found that a locally applied mechanical stimulus induces electrostatic potential change at distant regions, just like the piezoelectricity. This novel allosteric mechanism, "piezoelectric allostery", should be of particularly high value for mechanosensor/transducer proteins. PMID:27575163

  11. Evolution of proteins.

    Science.gov (United States)

    Dayhoff, M. O.

    1971-01-01

    The amino acid sequences of proteins from living organisms are dealt with. The structure of proteins is first discussed; the variation in this structure from one biological group to another is illustrated by the first halves of the sequences of cytochrome c, and a phylogenetic tree is derived from the cytochrome c data. The relative geological times associated with the events of this tree are discussed. Errors which occur in the duplication of cells during the evolutionary process are examined. Particular attention is given to evolution of mutant proteins, globins, ferredoxin, and transfer ribonucleic acids (tRNA's). Finally, a general outline of biological evolution is presented.

  12. Protein oxidation and ageing

    DEFF Research Database (Denmark)

    Linton, S; Davies, Michael Jonathan; Dean, R T

    2001-01-01

    of redox-active metal ions that could catalyse oxidant formation. As a result of this decrease in antioxidant defences, and increased rate of ROS formation, it is possible that the impact of ROS increases with age. ROS are known to oxidise biological macromolecules, with proteins an important target....... If the argument that the impact of ROS increases with age is true, then proteins would be expected to accumulate oxidised materials with age, and the rate of such accumulation should increase with time, reflecting impaired inefficiency of homeostasis. Here we review the evidence for the accumulation of oxidised......, or modified, extra- and intra-cellular proteins in vivo....

  13. Sound of proteins

    DEFF Research Database (Denmark)

    2007-01-01

    In my group we work with Molecular Dynamics to model several different proteins and protein systems. We submit our modelled molecules to changes in temperature, changes in solvent composition and even external pulling forces. To analyze our simulation results we have so far used visual inspection...... and statistical analysis of the resulting molecular trajectories (as everybody else!). However, recently I started assigning a particular sound frequency to each amino acid in the protein, and by setting the amplitude of each frequency according to the movement amplitude we can "hear" whenever two aminoacids...

  14. Alpha Shapes and Proteins

    DEFF Research Database (Denmark)

    Winter, Pawel; Sterner, Henrik; Sterner, Peter

    2009-01-01

    We provide a unified description of (weighted) alpha shapes, beta shapes and the corresponding simplicialcomplexes. We discuss their applicability to various protein-related problems. We also discuss filtrations of alpha shapes and touch upon related persistence issues.We claim that the full...... potential of alpha-shapes and related geometrical constructs in protein-related problems yet remains to be realized and verified. We suggest parallel algorithms for (weighted) alpha shapes, and we argue that future use of filtrations and kinetic variants for larger proteins will need such implementation....

  15. New approach for predicting protein-protein interactions

    Institute of Scientific and Technical Information of China (English)

    2007-01-01

    @@ Protein-protein interactions (PPIs) are of vital importance for virtually all processes of a living cell. The study of these associations of protein molecules could improve people's understanding of diseases and provide basis for therapeutic approaches.

  16. Analysis of correlations between protein complex and protein-protein interaction and mRNA expression

    Institute of Scientific and Technical Information of China (English)

    CAI Lun; XUE Hong; LU Hongchao; ZHAO Yi; ZHU Xiaopeng; BU Dongbo; LING Lunjiang; CHEN Runsheng

    2003-01-01

    Protein-protein interaction is a physical interaction of two proteins in living cells. In budding yeast Saccharomyces cerevisiae, large-scale protein-protein interaction data have been obtained through high-throughput yeast two-hybrid systems (Y2H) and protein complex purification techniques based on mass-spectrometry. Here, we collect 11855 interactions between total 2617 proteins. Through seriate genome-wide mRNA expression data, similarity between two genes could be measured. Protein complex data can also be obtained publicly and can be translated to pair relationship that any two proteins can only exist in the same complex or not. Analysis of protein complex data, protein-protein interaction data and mRNA expression data can elucidate correlations between them. The results show that proteins that have interactions or similar expression patterns have a higher possibility to be in the same protein complex than randomized selected proteins, and proteins which have interactions and similar expression patterns are even more possible to exist in the same protein complex. The work indicates that comprehensive integration and analysis of public large-scale bioinformatical data, such as protein complex data, protein-protein interaction data and mRNA expression data, may help to uncover their relationships and common biological information underlying these data. The strategies described here may help to integrate and analyze other functional genomic and proteomic data, such as gene expression profiling, protein-localization mapping and large-scale phenotypic data, both in yeast and in other organisms.

  17. A Bayesian Estimator of Protein-Protein Association Probabilities

    Energy Technology Data Exchange (ETDEWEB)

    Gilmore, Jason M.; Auberry, Deanna L.; Sharp, Julia L.; White, Amanda M.; Anderson, Kevin K.; Daly, Don S.

    2008-07-01

    The Bayesian Estimator of Protein-Protein Association Probabilities (BEPro3) is a software tool for estimating probabilities of protein-protein association between bait and prey protein pairs using data from multiple-bait, multiple-replicate, protein pull-down LC-MS assay experiments. BEPro3 is open source software that runs on both Windows XP and Mac OS 10.4 or newer versions, and is freely available from http://www.pnl.gov/statistics/BEPro3.

  18. Whey Protein- The Role of Protein Supplementation in Resistance Training

    OpenAIRE

    Zimmer, Raymond

    2005-01-01

    Adequate protein intake is an important concern for many athletes who are undergoing strength-training programs. Many athletes choose to take a protein supplement, such as whey protein, in order to help them build lean muscle mass more efficiently. But the benefit of very high levels of dietary protein in resistance training remains questionable. This paper examines the effectiveness of whey protein, and other forms of protein supplements, in helping athletes augment their muscle mass. A comp...

  19. Protein-protein interaction databases: keeping up with growing interactomes

    OpenAIRE

    Lehne Benjamin; Schlitt Thomas

    2009-01-01

    Abstract Over the past few years, the number of known protein-protein interactions has increased substantially. To make this information more readily available, a number of publicly available databases have set out to collect and store protein-protein interaction data. Protein-protein interactions have been retrieved from six major databases, integrated and the results compared. The six databases (the Biological General Repository for Interaction Datasets [BioGRID], the Molecular INTeraction ...

  20. Interactive protein manipulation

    Energy Technology Data Exchange (ETDEWEB)

    SNCrivelli@lbl.gov

    2003-07-01

    We describe an interactive visualization and modeling program for the creation of protein structures ''from scratch''. The input to our program is an amino acid sequence -decoded from a gene- and a sequence of predicted secondary structure types for each amino acid-provided by external structure prediction programs. Our program can be used in the set-up phase of a protein structure prediction process; the structures created with it serve as input for a subsequent global internal energy minimization, or another method of protein structure prediction. Our program supports basic visualization methods for protein structures, interactive manipulation based on inverse kinematics, and visualization guides to aid a user in creating ''good'' initial structures.

  1. C-reactive protein

    Science.gov (United States)

    ... body. It is one of a group of proteins called "acute phase reactants" that go up in response to inflammation. This article discusses the blood test done to measure the amount of CRP in your blood.

  2. Polymers for Protein Conjugation

    Directory of Open Access Journals (Sweden)

    Gianfranco Pasut

    2014-01-01

    Full Text Available Polyethylene glycol (PEG at the moment is considered the leading polymer for protein conjugation in view of its unique properties, as well as to its low toxicity in humans, qualities which have been confirmed by its extensive use in clinical practice. Other polymers that are safe, biodegradable and custom-designed have, nevertheless, also been investigated as potential candidates for protein conjugation. This review will focus on natural polymers and synthetic linear polymers that have been used for protein delivery and the results associated with their use. Genetic fusion approaches for the preparation of protein-polypeptide conjugates will be also reviewed and compared with the best known chemical conjugation ones.

  3. The Pentapeptide Repeat Proteins

    Energy Technology Data Exchange (ETDEWEB)

    Vetting,M.; Hegde, S.; Fajardo, J.; Fiser, A.; Roderick, S.; Takiff, H.; Blanchard, J.

    2006-01-01

    The Pentapeptide Repeat Protein (PRP) family has over 500 members in the prokaryotic and eukaryotic kingdoms. These proteins are composed of, or contain domains composed of, tandemly repeated amino acid sequences with a consensus sequence of [S, T,A, V][D, N][L, F]-[S, T,R][G]. The biochemical function of the vast majority of PRP family members is unknown. The three-dimensional structure of the first member of the PRP family was determined for the fluoroquinolone resistance protein (MfpA) from Mycobacterium tuberculosis. The structure revealed that the pentapeptide repeats encode the folding of a novel right-handed quadrilateral {beta}-helix. MfpA binds to DNA gyrase and inhibits its activity. The rod-shaped, dimeric protein exhibits remarkable size, shape and electrostatic similarity to DNA.

  4. Protein Colloidal Aggregation Project

    Science.gov (United States)

    Oliva-Buisson, Yvette J. (Compiler)

    2014-01-01

    To investigate the pathways and kinetics of protein aggregation to allow accurate predictive modeling of the process and evaluation of potential inhibitors to prevalent diseases including cataract formation, chronic traumatic encephalopathy, Alzheimer's Disease, Parkinson's Disease and others.

  5. Egg protein hydrolysates

    NARCIS (Netherlands)

    Amerongen, van A.; Beelen, M.J.C.; Wolbers, L.A.M.; Gilst, van W.H.; Buikema, J.H.; Nelissen, J.W.P.M.

    2009-01-01

    The present invention provides egg-protein hydrolysates with DPP-IV inhibitory activity which are particularly suited for the treatment of diabetes. Particularly advantageous is to use hydrolysate of lysozyme for the treatment of diabetes.

  6. Plant protein glycosylation

    Science.gov (United States)

    Strasser, Richard

    2016-01-01

    Protein glycosylation is an essential co- and post-translational modification of secretory and membrane proteins in all eukaryotes. The initial steps of N-glycosylation and N-glycan processing are highly conserved between plants, mammals and yeast. In contrast, late N-glycan maturation steps in the Golgi differ significantly in plants giving rise to complex N-glycans with β1,2-linked xylose, core α1,3-linked fucose and Lewis A-type structures. While the essential role of N-glycan modifications on distinct mammalian glycoproteins is already well documented, we have only begun to decipher the biological function of this ubiquitous protein modification in different plant species. In this review, I focus on the biosynthesis and function of different protein N-linked glycans in plants. Special emphasis is given on glycan-mediated quality control processes in the ER and on the biological role of characteristic complex N-glycan structures. PMID:26911286

  7. Markers of protein oxidation

    DEFF Research Database (Denmark)

    Headlam, Henrietta A; Davies, Michael Jonathan

    2004-01-01

    Exposure of proteins to radicals in the presence of O2 gives both side-chain oxidation and backbone fragmentation. These processes can be interrelated, with initial side-chain oxidation giving rise to backbone damage via transfer reactions. We have shown previously that alkoxyl radicals formed...... of this process depends on the extent of oxidation at C-3 compared with other sites. HO*, generated by gamma radiolysis, gave the highest total carbonyl yield, with protein-bound carbonyls predominating over released. In contrast, metal ion/H2O2 systems, gave more released than bound carbonyls, with this ratio...... modulated by EDTA. This is ascribed to metal ion-protein interactions affecting the sites of initial oxidation. Hypochlorous acid gave low concentrations of released carbonyls, but high yields of protein-bound material. The peroxyl radical generator 2,2'-azobis(2-amidinopropane) hydrochloride...

  8. Protein tyrosine nitration

    Science.gov (United States)

    Chaki, Mounira; Leterrier, Marina; Barroso, Juan B

    2009-01-01

    Nitric oxide metabolism in plant cells has a relative short history. Nitration is a chemical process which consists of introducing a nitro group (-NO2) into a chemical compound. in biological systems, this process has been found in different molecules such as proteins, lipids and nucleic acids that can affect its function. This mini-review offers an overview of this process with special emphasis on protein tyrosine nitration in plants and its involvement in the process of nitrosative stress. PMID:19826215

  9. Digestibility of sorghum proteins.

    OpenAIRE

    Axtell, J D; Kirleis, A. W.; Hassen, M M; D'Croz Mason, N; Mertz, E T; Munck, L.

    1981-01-01

    Published information indicates that rice, maize, and wheat proteins are much more digestible in children than sorghum proteins are (66-81% compared with 46%). However, this digestibility difference cannot be demonstrated with the weanling rat, which gave digestibility values of 80% for cooked and 85% for uncooked sorghum gruels. Therefore, a search was made for a laboratory system sensitive to the digestibility differences between sorghum and other cereals. We found that porcine pepsin in vi...

  10. Identifying Unknown Proteins

    OpenAIRE

    Barker, Winona C.; Dayhoff, Margaret O.

    1983-01-01

    In this paper we discuss ways to identify a protein, both when its amino acid sequence is known and, particularly, prior to the determination of the complete sequence. If a similar sequence is in the Protein Sequence Database, an unknown may be identified on the basis of partial or ambiguous sequence data, or on the basis of amino acid composition. Identification in the early stages of structural determination can save time and scarce resources by preventing duplicate effort or by suggesting ...

  11. Fish protein hydrolysates

    Energy Technology Data Exchange (ETDEWEB)

    Mackie, I.M.

    1982-01-01

    Proteolytic enzymes now available in commercial quantities can be used to liquefy the fish and fish waste presently considered suitable for conversion to fish meal. The products obtained are readily dispersed or dissolved in water and have a high nutritional value. They have been satisfactorily used as substitutes for milk proteins in milk replacers for young animals. Further research is necessary on means of controlling the degree of hydrolysis to give protein preparations with acceptable functional properties as human food supplements. (Refs. 21).

  12. The Malignant Protein Puzzle.

    Science.gov (United States)

    Walker, Lary C; Jucker, Mathias

    2016-01-01

    When most people hear the words malignant and brain, cancer immediately comes to mind. But our authors argue that proteins can be malignant too, and can spread harmfully through the brain in neurodegenerative diseases that include Alzheimer's, Parkinson's, CTE, and ALS. Studying how proteins such as PrP, amyloid beta, tau, and others aggregate and spread, and kill brain cells, represents a crucial new frontier in neuroscience. PMID:27408676

  13. Recombinant Collagenlike Proteins

    Science.gov (United States)

    Fertala, Andzej

    2007-01-01

    A group of collagenlike recombinant proteins containing high densities of biologically active sites has been invented. The method used to express these proteins is similar to a method of expressing recombinant procollagens and collagens described in U. S. Patent 5,593,859, "Synthesis of human procollagens and collagens in recombinant DNA systems." Customized collagenous proteins are needed for biomedical applications. In particular, fibrillar collagens are attractive for production of matrices needed for tissue engineering and drug delivery. Prior to this invention, there was no way of producing customized collagenous proteins for these and other applications. Heretofore, collagenous proteins have been produced by use of such biological systems as yeasts, bacteria, and transgenic animals and plants. These products are normal collagens that can also be extracted from such sources as tendons, bones, and hides. These products cannot be made to consist only of biologically active, specific amino acid sequences that may be needed for specific applications. Prior to this invention, it had been established that fibrillar collagens consist of domains that are responsible for such processes as interaction with cells, binding of growth factors, and interaction with a number of structural proteins present in the extracellular matrix. A normal collagen consists of a sequence of domains that can be represented by a corresponding sequence of labels, e.g., D1D2D3D4. A collagenlike protein of the present invention contains regions of collagen II that contain multiples of a single domain (e.g., D1D1D1D1 or D4D4D4D4) chosen for its specific biological activity. By virtue of the multiplicity of the chosen domain, the density of sites having that specific biological activity is greater than it is in a normal collagen. A collagenlike protein according to this invention can thus be made to have properties that are necessary for tissue engineering.

  14. The effect of protein-protein and protein-membrane interactions on membrane fouling in ultrafiltration

    NARCIS (Netherlands)

    Huisman, I.H.; Prádanos, P.; Hernández, A.

    2000-01-01

    It was studied how protein-protein and protein-membrane interactions influence the filtration performance during the ultrafiltration of protein solutions over polymeric membranes. This was done by measuring flux, streaming potential, and protein transmission during filtration of bovine serum albumin

  15. Bence-Jones protein - quantitative

    Science.gov (United States)

    Immunoglobulin light chains - urine; Urine Bence-Jones protein ... Bence-Jones proteins are a part of regular antibodies called light chains. These proteins are not normally in urine. Sometimes, when ...

  16. More protein in cereals?

    International Nuclear Information System (INIS)

    Ways in which the protein content of plant crops may be raised by the use of nuclear radiation are to be discussed at a symposium in Vienna in June next year, organized by the joint Food and Agriculture Organization/Agency Division of Atomic Energy in Food and Agriculture. Plant crops - especially cereal grains - are the basic food and protein source of most of the world's population, particularly in less-developed countries. But their natural protein content is low; increasing the quantity and nutritional quality of plant protein is potentially the most feasible way to combat widespread protein malnutrition. This improvement in seed stock can be achieved by plant breeding methods in which nuclear irradiation techniques are used to induce mutations in grain, and other isotopic techniques can be used to select only those mutants which have the desired properties. The scientists who attend the symposium will have an opportunity to review what mutation plant breeders have achieved, the application of nuclear techniques to screening for protein and amino-acid content and nutritional value, and isotopic methods which contribute to research in plant nutrition and physiology. (author)

  17. Hepatitis C virus proteins

    Institute of Scientific and Technical Information of China (English)

    Jean Dubuisson

    2007-01-01

    Hepatitis C virus (HCV) encodes a single polyprotein,which is processed by cellular and viral proteases to generate 10 polypeptides. The HCV genome also contains an overlapping +1 reading frame that may lead to the synthesis of an additional protein. Until recently,studies of HCV have been hampered by the lack of a productive cell culture system. Since the identification of HCV genome approximately 17 years ago, structural,biochemical and biological information on HCV proteins has mainly been obtained with proteins produced by heterologous expression systems. In addition, some functional studies have also been confirmed with replicon systems or with retroviral particles pseudotyped with HCV envelope glycoproteins. The data that have accumulated on HCV proteins begin to provide a framework for understanding the molecular mechanisms involved in the major steps of HCV life cycle. Moreover,the knowledge accumulated on HCV proteins is also leading to the development of antiviral drugs among which some are showing promising results in early-phase clinical trials. This review summarizes the current knowledge on the functions and biochemical features of HCV proteins.

  18. Cardiolipin Interactions with Proteins.

    Science.gov (United States)

    Planas-Iglesias, Joan; Dwarakanath, Himal; Mohammadyani, Dariush; Yanamala, Naveena; Kagan, Valerian E; Klein-Seetharaman, Judith

    2015-09-15

    Cardiolipins (CL) represent unique phospholipids of bacteria and eukaryotic mitochondria with four acyl chains and two phosphate groups that have been implicated in numerous functions from energy metabolism to apoptosis. Many proteins are known to interact with CL, and several cocrystal structures of protein-CL complexes exist. In this work, we describe the collection of the first systematic and, to the best of our knowledge, the comprehensive gold standard data set of all known CL-binding proteins. There are 62 proteins in this data set, 21 of which have nonredundant crystal structures with bound CL molecules available. Using binding patch analysis of amino acid frequencies, secondary structures and loop supersecondary structures considering phosphate and acyl chain binding regions together and separately, we gained a detailed understanding of the general structural and dynamic features involved in CL binding to proteins. Exhaustive docking of CL to all known structures of proteins experimentally shown to interact with CL demonstrated the validity of the docking approach, and provides a rich source of information for experimentalists who may wish to validate predictions.

  19. Predictive and interpretive simulation of green fluorescent protein expression in reporter bacteria.

    Science.gov (United States)

    Leveau, J H; Lindow, S E

    2001-12-01

    We have formulated a numerical model that simulates the accumulation of green fluorescent protein (GFP) in bacterial cells from a generic promoter-gfp fusion. The model takes into account the activity of the promoter, the time it takes GFP to mature into its fluorescent form, the susceptibility of GFP to proteolytic degradation, and the growth rate of the bacteria. From the model, we derived a simple formula with which promoter activity can be inferred easily and quantitatively from actual measurements of GFP fluorescence in growing bacterial cultures. To test the usefulness of the formula, we determined the activity of the LacI-repressible promoter P(A1/O4/O3) in response to increasing concentrations of the inducer IPTG (isopropyl-beta-D-thiogalactopyranoside) and were able to predict cooperativity between the LacI repressors on each of the two operator sites within P(A1/O4/O3). Aided by the model, we also quantified the proteolytic degradation of GFP[AAV], GFP[ASV], and GFP[LVA], which are popular variants of GFP with reduced stability in bacteria. Best described by Michaelis-Menten kinetics, the rate at which these variants were degraded was a function of the activity of the promoter that drives their synthesis: a weak promoter yielded proportionally less GFP fluorescence than a strong one. The degree of disproportionality is species dependent: the effect was more pronounced in Erwinia herbicola than in Escherichia coli. This phenomenon has important implications for the interpretation of fluorescence from bacterial reporters based on these GFP variants. The model furthermore predicted a significant effect of growth rate on the GFP content of individual bacteria, which if not accounted for might lead to misinterpretation of GFP data. In practice, our model will be helpful for prior testing of different combinations of promoter-gfp fusions that best fit the application of a particular bacterial reporter strain, and also for the interpretation of actual GFP

  20. Protein hydrolysates in sports nutrition

    Directory of Open Access Journals (Sweden)

    Manninen Anssi H

    2009-09-01

    Full Text Available Abstract It has been suggested that protein hydrolysates providing mainly di- and tripeptides are superior to intact (whole proteins and free amino acids in terms of skeletal muscle protein anabolism. This review provides a critical examination of protein hydrolysate studies conducted in healthy humans with special reference to sports nutrition. The effects of protein hydrolysate ingestion on blood amino acid levels, muscle protein anabolism, body composition, exercise performance and muscle glycogen resynthesis are discussed.

  1. Protein Functionality in Food Systems

    Institute of Scientific and Technical Information of China (English)

    WANG Panpan

    2010-01-01

    The structure,shape,color,smell and taste of food were decided by protein functionality.The utilization of protein will improve by changing the protein functionality.Protein functionality is also advantage to maintain and utilize the nutrition of food.This paper summarized the nature,classification,factors and prospect of protein functionality.It ccn provide a theoretical basis for application of protein in food industry.

  2. Joint ASV/AUV Range-Based Formation Control: Theory and Experimental Results

    OpenAIRE

    Soares, Jorge Miguel; Aguiar, António Pedro; Pascoal, António; Martinoli, Alcherio

    2013-01-01

    The use of groups of autonomous marine vehicles has enormous potential in numerous marine applications, perhaps the most relevant of which is the surveying and exploration of the oceans, still widely unknown and misunderstood. In many mission scenarios requiring the concerted operation of multiple marine vehicles carrying distinct, yet complementary sensor suites, relative positioning and formation control becomes mandatory. However, the constraints placed by the medium make it hard to both ...

  3. Autonomous Surface Vehicle Sidescan-sonar mosaic from Hotel Bar, Apalachicola Bay, Florida (ASV162.TIF)

    Data.gov (United States)

    U.S. Geological Survey, Department of the Interior — These data were collected under a cooperative mapping program between the U.S. Geological Survey (USGS), the National Oceanic and Atmospheric Administration Coastal...

  4. Autonomous Surface Vehicle Sidescan-sonar mosaic from North Spur, Apalachicola Bay, Florida (ASV178.TIF)

    Data.gov (United States)

    U.S. Geological Survey, Department of the Interior — These data were collected under a cooperative mapping program between the U.S. Geological Survey (USGS), the National Oceanic and Atmospheric Administration Coastal...

  5. Autonomous Surface Vehicle Sidescan-sonar mosaic from Norman's Bar, Apalachicola Bay, Florida (ASV174.TIF)

    Data.gov (United States)

    U.S. Geological Survey, Department of the Interior — These data were collected under a cooperative mapping program between the U.S. Geological Survey (USGS), the National Oceanic and Atmospheric Administration Coastal...

  6. Modeling Mercury in Proteins

    Energy Technology Data Exchange (ETDEWEB)

    Smith, Jeremy C [ORNL; Parks, Jerry M [ORNL

    2016-01-01

    Mercury (Hg) is a naturally occurring element that is released into the biosphere both by natural processes and anthropogenic activities. Although its reduced, elemental form Hg(0) is relatively non-toxic, other forms such as Hg2+ and, in particular, its methylated form, methylmercury, are toxic, with deleterious effects on both ecosystems and humans. Microorganisms play important roles in the transformation of mercury in the environment. Inorganic Hg2+ can be methylated by certain bacteria and archaea to form methylmercury. Conversely, bacteria also demethylate methylmercury and reduce Hg2+ to relatively inert Hg(0). Transformations and toxicity occur as a result of mercury interacting with various proteins. Clearly, then, understanding the toxic effects of mercury and its cycling in the environment requires characterization of these interactions. Computational approaches are ideally suited to studies of mercury in proteins because they can provide a detailed picture and circumvent issues associated with toxicity. Here we describe computational methods for investigating and characterizing how mercury binds to proteins, how inter- and intra-protein transfer of mercury is orchestrated in biological systems, and how chemical reactions in proteins transform the metal. We describe quantum chemical analyses of aqueous Hg(II), which reveal critical factors that determine ligand binding propensities. We then provide a perspective on how we used chemical reasoning to discover how microorganisms methylate mercury. We also highlight our combined computational and experimental studies of the proteins and enzymes of the mer operon, a suite of genes that confers mercury resistance in many bacteria. Lastly, we place work on mercury in proteins in the context of what is needed for a comprehensive multi-scale model of environmental mercury cycling.

  7. PROTEIN SYNTHESIS GAME

    Directory of Open Access Journals (Sweden)

    J.C.Q. Carvalho

    2004-05-01

    Full Text Available The theoretical explanation of biological concepts, associated with the use of teaching games andmodels, intensify the comprehension and increase students interest, stimulating them to participateactively on the teaching-learning process. The sta of dissemination from Centro de BiotecnologiaMolecular Estrutural (CBME, in partnership with the Centro de Divulgac~ao Cientca e Cultural(CDCC, presents, in this work, a new educational resource denoted: Protein Synthesis Game. Theapproach of the game involves the cytological aspects of protein synthesis, directed to high schoolstudents. Students are presented to day-by-day facts related to the function of a given protein in thehuman body. Such task leads players to the goal of solving out a problem through synthesizing aspecied protein. The game comprises: (1 a board illustrated with the transversal section of animalcell, with its main structures and organelles and sequences of hypothetical genes; (2 cards with thedescription of steps and other structures required for protein synthesis in eukaryotic cells; (3 piecesrepresenting nucleotides, polynucleotides, ribosome, amino acids, and polypeptide chains. In order toplay the game, students take cards that sequentially permit them to acquire the necessary pieces forproduction of the protein described in each objective. Players must move the pieces on the board andsimulate the steps of protein synthesis. The dynamic of the game allows students to easily comprehendprocesses of transcription and translation. This game was presented to dierent groups of high schoolteachers and students. Their judgments have been heard and indicated points to be improved, whichhelped us with the game development. Furthermore, the opinions colleted were always favorable forthe application of this game as a teaching resource in classrooms.

  8. Inferring protein function by domain context similarities in protein-protein interaction networks

    OpenAIRE

    Sun Zhirong; Liu Ke; Chen Hu; Zhang Song

    2009-01-01

    Abstract Background Genome sequencing projects generate massive amounts of sequence data but there are still many proteins whose functions remain unknown. The availability of large scale protein-protein interaction data sets makes it possible to develop new function prediction methods based on protein-protein interaction (PPI) networks. Although several existing methods combine multiple information resources, there is no study that integrates protein domain information and PPI networks to pre...

  9. ADSORPTION OF PROTEIN ON NANOPARTICLES

    Institute of Scientific and Technical Information of China (English)

    WU Qi

    1994-01-01

    The adsorption of protein on nanoparticles was studied by using dynamic light scattering to measure the hydrodynamic size of both pure protein and nanoparticles adsorbed with different amounts of protein. The thickness of the adsorbed protein layer increases as protein concentration, but decreases as the initial size of nanoparticles. After properly scaling the thickness with the initial diameter, we are able to fit all experimental data with a single master curve. Our experimental results suggest that the adsorbed proteins form a monolayeron the nanoparticle surface and the adsorbed protein molecules are attached to the particle surface at many points through a possible hydrogen-bonding. Our results also indicate that as protein concentration increases, the overall shape of the adsorbed protein molecule continuously changes from a flat layer on the particle surface to a stretched coil extended into water. During the change, the hydrodynamic volume of the adsorbed protein increases linearly with protein concentration.

  10. Measuring protein breakdown rate in individual proteins in vivo

    DEFF Research Database (Denmark)

    Holm, Lars; Kjaer, Michael

    2010-01-01

    To outline different approaches of how protein breakdown can be quantified and to present a new approach to determine the fractional breakdown rate of individual slow turnover proteins in vivo.......To outline different approaches of how protein breakdown can be quantified and to present a new approach to determine the fractional breakdown rate of individual slow turnover proteins in vivo....

  11. Benchtop Detection of Proteins

    Science.gov (United States)

    Scardelletti, Maximilian C.; Varaljay, Vanessa

    2007-01-01

    A process, and a benchtop-scale apparatus for implementing the process, have been developed to detect proteins associated with specific microbes in water. The process and apparatus may also be useful for detection of proteins in other, more complex liquids. There may be numerous potential applications, including monitoring lakes and streams for contamination, testing of blood and other bodily fluids in medical laboratories, and testing for microbial contamination of liquids in restaurants and industrial food-processing facilities. A sample can be prepared and analyzed by use of this process and apparatus within minutes, whereas an equivalent analysis performed by use of other processes and equipment can often take hours to days. The process begins with the conjugation of near-infrared-fluorescent dyes to antibodies that are specific to a particular protein. Initially, the research has focused on using near-infrared dyes to detect antigens or associated proteins in solution, which has proven successful vs. microbial cells, and streamlining the technique in use for surface protein detection on microbes would theoretically render similar results. However, it is noted that additional work is needed to transition protein-based techniques to microbial cell detection. Consequently, multiple such dye/antibody pairs could be prepared to enable detection of multiple selected microbial species, using a different dye for each species. When excited by near-infrared light of a suitable wavelength, each dye fluoresces at a unique longer wavelength that differs from those of the other dyes, enabling discrimination among the various species. In initial tests, the dye/antibody pairs are mixed into a solution suspected of containing the selected proteins, causing the binding of the dye/antibody pairs to such suspect proteins that may be present. The solution is then run through a microcentrifuge that includes a membrane that acts as a filter in that it retains the dye/antibody/protein

  12. Measuring protein breakdown in individual proteins in vivo

    DEFF Research Database (Denmark)

    Holm, Lars; Kjær, Michael

    2010-01-01

    is that the proteins of interest are the site of measurement. Hence, the application initially demands the proteins to be labeled with stable isotopically labeled amino acids. Subsequently, the loss of label from the proteins will be dependent on the protein breakdown rate when no labeled amino acids......PURPOSE OF REVIEW: To outline different approaches of how protein breakdown can be quantified and to present a new approach to determine the fractional breakdown rate of individual slow turnover proteins in vivo. RECENT FINDINGS: None of the available methods for determining protein breakdown can...

  13. Clustered epitopes within the Gag-Pol fusion protein DNA vaccine enhance immune responses and protection against challenge with recombinant vaccinia viruses expressing HIV-1 Gag and Pol antigens

    International Nuclear Information System (INIS)

    We have generated a codon-optimized hGagp17p24-Polp51 plasmid DNA expressing the human immunodeficiency virus type 1 (HIV-1) Gag-Pol fusion protein that consists of clusters of highly conserved cytotoxic T lymphocyte (CTL) epitopes presented by multiple MHC class I alleles. In the hGagp17p24-Polp51 construct, the ribosomal frameshift site had been deleted together with the potentially immunosuppressive Gag nucleocapsid (p15) as well as Pol protease (p10) and integrase (p31). Analyses of the magnitude and breadth of cellular responses demonstrated that immunization of HLA-A2/Kb transgenic mice with the hGagp17p24-Polp51 construct induced 2- to 5-fold higher CD8+ T-cell responses to Gag p17-, p24-, and Pol reverse transcriptase (RT)-specific CTL epitopes than the full-length hGag-PolΔFsΔPr counterpart. The increases were correlated with higher protection against challenge with recombinant vaccinia viruses (rVVs) expressing gag and pol gene products. Consistent with the profile of Gag- and Pol-specific CD8+ T cell responses, an elevated level of type 1 cytokine production was noted in p24- and RT-stimulated splenocyte cultures established from hGagp17p24-Polp51-immunized mice compared to responses induced with the hGag-PolΔFsΔPr vaccine. Sera of mice immunized with the hGagp17p24-Polp51 vaccine also exhibited an increased titer of p24- and RT-specific IgG2 antibody responses. The results from our studies provide insights into approaches for boosting the breadth of Gag- and Pol-specific immune responses

  14. Ontology integration to identify protein complex in protein interaction networks

    OpenAIRE

    Yang Zhihao; Lin Hongfei; Xu Bo

    2011-01-01

    Abstract Background Protein complexes can be identified from the protein interaction networks derived from experimental data sets. However, these analyses are challenging because of the presence of unreliable interactions and the complex connectivity of the network. The integration of protein-protein interactions with the data from other sources can be leveraged for improving the effectiveness of protein complexes detection algorithms. Methods We have developed novel semantic similarity metho...

  15. Identifying Protein-Protein Interaction Sites Using Covering Algorithm

    OpenAIRE

    Jie Song; Jiaxing Cheng; Xiuquan Du

    2009-01-01

    Identification of protein-protein interface residues is crucial for structural biology. This paper proposes a covering algorithm for predicting protein-protein interface residues with features including protein sequence profile and residue accessible area. This method adequately utilizes the characters of a covering algorithm which have simple, lower complexity and high accuracy for high dimension data. The covering algorithm can achieve a comparable performance (69.62%, Complete dataset; 60....

  16. Protein-Protein Interaction Detection: Methods and Analysis

    OpenAIRE

    V. Srinivasa Rao; Srinivas, K.; Sujini, G. N.; G. N. Sunand Kumar

    2014-01-01

    Protein-protein interaction plays key role in predicting the protein function of target protein and drug ability of molecules. The majority of genes and proteins realize resulting phenotype functions as a set of interactions. The in vitro and in vivo methods like affinity purification, Y2H (yeast 2 hybrid), TAP (tandem affinity purification), and so forth have their own limitations like cost, time, and so forth, and the resultant data sets are noisy and have more false positives to annotate t...

  17. Polarizable protein packing

    KAUST Repository

    Ng, Albert H.

    2011-01-24

    To incorporate protein polarization effects within a protein combinatorial optimization framework, we decompose the polarizable force field AMOEBA into low order terms. Including terms up to the third-order provides a fair approximation to the full energy while maintaining tractability. We represent the polarizable packing problem for protein G as a hypergraph and solve for optimal rotamers with the FASTER combinatorial optimization algorithm. These approximate energy models can be improved to high accuracy [root mean square deviation (rmsd) < 1 kJ mol -1] via ridge regression. The resulting trained approximations are used to efficiently identify new, low-energy solutions. The approach is general and should allow combinatorial optimization of other many-body problems. © 2011 Wiley Periodicals, Inc. J Comput Chem, 2011 Copyright © 2011 Wiley Periodicals, Inc.

  18. Trisulfides in Proteins

    DEFF Research Database (Denmark)

    Nielsen, Rasmus W.; Tachibana, Christine; Hansen, Niels Erik;

    2011-01-01

    Trisulfides and other oligosulfides are widely distributed in the biological world. In plants, e.g., garlic, trisulfides are associated with potentially beneficial properties. However, an extra neutral sulfur atom covalently bound between the two sulfur atoms of a pair of cysteines is not a commo...... post-translational modification, and the number of proteins in which a trisulfide has been unambiguously identified is small. Nevertheless, we believe that its prevalence may be underestimated, particularly with the increasing evidence for significant pools of sulfides in living tissues...... and their possible roles in cellular metabolism. This review focuses on examples of proteins that are known to contain a trisulfide bridge, and gives an overview of the chemistry of trisulfide formation, and the methods by which it is detected in proteins....

  19. Accessory Proteins at ERES

    DEFF Research Database (Denmark)

    Klinkenberg, Rafael David

    proteins. Together these components co‐operate in cargo‐selection as well as forming, loading and releasing budding vesicles from specific regions on the membrane surface of the ER. Coat components furthermore convey vesicle targeting towards the Golgi. However, not much is known about the mechanisms...... that regulate the COPII assembly at the vesicle bud site. This thesis provides the first regulatory mechanism of COPII assembly in relation to ER‐membrane lipid‐signal recognition by the accessory protein p125A (Sec23IP). The aim of the project was to characterize p125A function by dissecting two main domains...... in the protein; a putative lipid‐associating domain termed the DDHD domain that is defined by the four amino acid motif that gives the domain its name; and a ubiquitously found domain termed Sterile α‐motif (SAM), which is mostly associated with oligomerization and polymerization. We first show, that the DDHD...

  20. Electron transfer in proteins.

    Science.gov (United States)

    Gray, H B; Winkler, J R

    1996-01-01

    Electron-transfer (ET) reactions are key steps in a diverse array of biological transformations ranging from photosynthesis to aerobic respiration. A powerful theoretical formalism has been developed that describes ET rates in terms of two parameters: the nuclear reorganization energy (lambda) and the electronic-coupling strength (HAB). Studies of ET reactions in ruthenium-modified proteins have probed lambda and HAB in several metalloproteins (cytochrome c, myoglobin, azurin). This work has shown that protein reorganization energies are sensitive to the medium surrounding the redox sites and that an aqueous environment, in particular, leads to large reorganization energies. Analyses of electronic-coupling strengths suggest that the efficiency of long-range ET depends on the protein secondary structure: beta sheets appear to mediate coupling more efficiently than alpha-helical structures, and hydrogen bonds play a critical role in both. PMID:8811189

  1. Discovery of binding proteins for a protein target using protein-protein docking-based virtual screening.

    Science.gov (United States)

    Zhang, Changsheng; Tang, Bo; Wang, Qian; Lai, Luhua

    2014-10-01

    Target structure-based virtual screening, which employs protein-small molecule docking to identify potential ligands, has been widely used in small-molecule drug discovery. In the present study, we used a protein-protein docking program to identify proteins that bind to a specific target protein. In the testing phase, an all-to-all protein-protein docking run on a large dataset was performed. The three-dimensional rigid docking program SDOCK was used to examine protein-protein docking on all protein pairs in the dataset. Both the binding affinity and features of the binding energy landscape were considered in the scoring function in order to distinguish positive binding pairs from negative binding pairs. Thus, the lowest docking score, the average Z-score, and convergency of the low-score solutions were incorporated in the analysis. The hybrid scoring function was optimized in the all-to-all docking test. The docking method and the hybrid scoring function were then used to screen for proteins that bind to tumor necrosis factor-α (TNFα), which is a well-known therapeutic target for rheumatoid arthritis and other autoimmune diseases. A protein library containing 677 proteins was used for the screen. Proteins with scores among the top 20% were further examined. Sixteen proteins from the top-ranking 67 proteins were selected for experimental study. Two of these proteins showed significant binding to TNFα in an in vitro binding study. The results of the present study demonstrate the power and potential application of protein-protein docking for the discovery of novel binding proteins for specific protein targets.

  2. Modeling Mercury in Proteins.

    Science.gov (United States)

    Parks, J M; Smith, J C

    2016-01-01

    Mercury (Hg) is a naturally occurring element that is released into the biosphere both by natural processes and anthropogenic activities. Although its reduced, elemental form Hg(0) is relatively nontoxic, other forms such as Hg(2+) and, in particular, its methylated form, methylmercury, are toxic, with deleterious effects on both ecosystems and humans. Microorganisms play important roles in the transformation of mercury in the environment. Inorganic Hg(2+) can be methylated by certain bacteria and archaea to form methylmercury. Conversely, bacteria also demethylate methylmercury and reduce Hg(2+) to relatively inert Hg(0). Transformations and toxicity occur as a result of mercury interacting with various proteins. Clearly, then, understanding the toxic effects of mercury and its cycling in the environment requires characterization of these interactions. Computational approaches are ideally suited to studies of mercury in proteins because they can provide a detailed molecular picture and circumvent issues associated with toxicity. Here, we describe computational methods for investigating and characterizing how mercury binds to proteins, how inter- and intraprotein transfer of mercury is orchestrated in biological systems, and how chemical reactions in proteins transform the metal. We describe quantum chemical analyses of aqueous Hg(II), which reveal critical factors that determine ligand-binding propensities. We then provide a perspective on how we used chemical reasoning to discover how microorganisms methylate mercury. We also highlight our combined computational and experimental studies of the proteins and enzymes of the mer operon, a suite of genes that confer mercury resistance in many bacteria. Lastly, we place work on mercury in proteins in the context of what is needed for a comprehensive multiscale model of environmental mercury cycling. PMID:27497164

  3. Protein: FEA3 [TP Atlas

    Lifescience Database Archive (English)

    Full Text Available FEA3 AREB pathway: Signaling proteins AZF1 OZAKGYO, ZF1 At5g67450, Cys2/His2-type zinc finger protein... 1, Zinc finger protein OZAKGYO, Zinc-finger protein 1 3702 Arabidopsis thaliana 836881 Q9SSW1 21852415 ...

  4. Protein: FEA3 [TP Atlas

    Lifescience Database Archive (English)

    Full Text Available FEA3 AREB pathway: Signaling proteins SRK2E OST1, SNRK2.6 Serine/threonine-protein kinase SRK2E Protein... OPEN STOMATA 1, SNF1-related kinase 2.6, Serine/threonine-protein kinase OST1 3702 Arabidopsis thaliana 829541 Q940H6 3UC4, 3ZUT, 3ZUU, 3UDB 19805022 ...

  5. Protein: FBB5 [TP Atlas

    Lifescience Database Archive (English)

    Full Text Available FBB5 RNA silencing TNRC6A CAGH26, KIAA1460, TNRC6 TNRC6A Trinucleotide repeat-containing gene 6A protein... CAG repeat protein 26, EMSY interactor protein, GW182 autoantigen, Glycine-tryptophan protein of 182 kDa 9606 Homo sapiens Q8NDV7 27327 27327 19398495 ...

  6. Ubiquitin domain proteins in disease

    DEFF Research Database (Denmark)

    Klausen, Louise Kjær; Schulze, Andrea; Seeger, Michael;

    2007-01-01

    The human genome encodes several ubiquitin-like (UBL) domain proteins (UDPs). Members of this protein family are involved in a variety of cellular functions and many are connected to the ubiquitin proteasome system, an essential pathway for protein degradation in eukaryotic cells. Despite...... and cancer. Publication history: Republished from Current BioData's Targeted Proteins database (TPdb; http://www.targetedproteinsdb.com)....

  7. Mining protein structure data

    OpenAIRE

    Santos, José Carlos Almeida

    2006-01-01

    The principal topic of this work is the application of data mining techniques, in particular of machine learning, to the discovery of knowledge in a protein database. In the first chapter a general background is presented. Namely, in section 1.1 we overview the methodology of a Data Mining project and its main algorithms. In section 1.2 an introduction to the proteins and its supporting file formats is outlined. This chapter is concluded with section 1.3 which defines that main problem we...

  8. Lipid-transfer proteins.

    Science.gov (United States)

    Ng, Tzi Bun; Cheung, Randy Chi Fai; Wong, Jack Ho; Ye, Xiujuan

    2012-01-01

    Lipid-transfer proteins (LTPs) are basic proteins found in abundance in higher plants. LTPs play lots of roles in plants such as participation in cutin formation, embryogenesis, defense reactions against phytopathogens, symbiosis, and the adaptation of plants to various environmental conditions. In addition, LTPs from field mustard and Chinese daffodil exhibit antiproliferative activity against human cancer cells. LTPs from chili pepper and coffee manifest inhibitory activity against fungi pathogenic to humans such as Candida species. The intent of this article is to review LTPs in the plant kingdom. PMID:23193591

  9. Water-transporting proteins

    DEFF Research Database (Denmark)

    Zeuthen, Thomas

    2010-01-01

    Transport through lipids and aquaporins is osmotic and entirely driven by the difference in osmotic pressure. Water transport in cotransporters and uniporters is different: Water can be cotransported, energized by coupling to the substrate flux by a mechanism closely associated with protein...... is not clear. It is associated with the substrate movements in aqueous pathways within the protein; a conventional unstirred layer mechanism can be ruled out, due to high rates of diffusion in the cytoplasm. The physiological roles of the various modes of water transport are reviewed in relation to epithelial...

  10. Modelling of proteins in membranes

    DEFF Research Database (Denmark)

    Sperotto, Maria Maddalena; May, S.; Baumgaertner, A.

    2006-01-01

    This review describes some recent theories and simulations of mesoscopic and microscopic models of lipid membranes with embedded or attached proteins. We summarize results supporting our understanding of phenomena for which the activities of proteins in membranes are expected to be significantly...... affected by the lipid environment. Theoretical predictions are pointed out, and compared to experimental findings, if available. Among others, the following phenomena are discussed: interactions of interfacially adsorbed peptides, pore-forming amphipathic peptides, adsorption of charged proteins onto...... oppositely charged lipid membranes, lipid-induced tilting of proteins embedded in lipid bilayers, protein-induced bilayer deformations, protein insertion and assembly, and lipid-controlled functioning of membrane proteins....

  11. Conformation Distributions in Adsorbed Proteins.

    Science.gov (United States)

    Meuse, Curtis W.; Hubbard, Joseph B.; Vrettos, John S.; Smith, Jackson R.; Cicerone, Marcus T.

    2007-03-01

    While the structural basis of protein function is well understood in the biopharmaceutical and biotechnology industries, few methods for the characterization and comparison of protein conformation distributions are available. New methods capable of measuring the stability of protein conformations and the integrity of protein-protein, protein-ligand and protein-surface interactions both in solution and on surfaces are needed to help the development of protein-based products. We are developing infrared spectroscopy methods for the characterization and comparison of molecular conformation distributions in monolayers and in solutions. We have extracted an order parameter describing the orientational and conformational variations of protein functional groups around the average molecular values from a single polarized spectrum. We will discuss the development of these methods and compare them to amide hydrogen/deuterium exchange methods for albumin in solution and on different polymer surfaces to show that our order parameter is related to protein stability.

  12. Integral UBL domain proteins: a family of proteasome interacting proteins

    DEFF Research Database (Denmark)

    Hartmann-Petersen, Rasmus; Gordon, Colin

    2004-01-01

    The family of ubiquitin-like (UBL) domain proteins (UDPs) comprises a conserved group of proteins involved in a multitude of different cellular activities. However, recent studies on UBL-domain proteins indicate that these proteins appear to share a common property in their ability to interact with......-domain proteins catalyse the formation of ubiquitin-protein conjugates, whereas others appear to target ubiquitinated proteins for degradation and interact with chaperones. Hence, by binding to the 26S proteasome the UBL-domain proteins seem to tailor and direct the basic proteolytic functions of the particle to...... 26S proteasomes. The 26S proteasome is a multisubunit protease which is responsible for the majority of intracellular proteolysis in eukaryotic cells. Before degradation commences most proteins are first marked for destruction by being coupled to a chain of ubiquitin molecules. Some UBL...

  13. Interaction between plate make and protein in protein crystallisation screening.

    Directory of Open Access Journals (Sweden)

    Gordon J King

    Full Text Available BACKGROUND: Protein crystallisation screening involves the parallel testing of large numbers of candidate conditions with the aim of identifying conditions suitable as a starting point for the production of diffraction quality crystals. Generally, condition screening is performed in 96-well plates. While previous studies have examined the effects of protein construct, protein purity, or crystallisation condition ingredients on protein crystallisation, few have examined the effect of the crystallisation plate. METHODOLOGY/PRINCIPAL FINDINGS: We performed a statistically rigorous examination of protein crystallisation, and evaluated interactions between crystallisation success and plate row/column, different plates of same make, different plate makes and different proteins. From our analysis of protein crystallisation, we found a significant interaction between plate make and the specific protein being crystallised. CONCLUSIONS/SIGNIFICANCE: Protein crystal structure determination is the principal method for determining protein structure but is limited by the need to produce crystals of the protein under study. Many important proteins are difficult to crystallize, so that identification of factors that assist crystallisation could open up the structure determination of these more challenging targets. Our findings suggest that protein crystallisation success may be improved by matching a protein with its optimal plate make.

  14. HIV protein sequence hotspots for crosstalk with host hub proteins.

    Directory of Open Access Journals (Sweden)

    Mahdi Sarmady

    Full Text Available HIV proteins target host hub proteins for transient binding interactions. The presence of viral proteins in the infected cell results in out-competition of host proteins in their interaction with hub proteins, drastically affecting cell physiology. Functional genomics and interactome datasets can be used to quantify the sequence hotspots on the HIV proteome mediating interactions with host hub proteins. In this study, we used the HIV and human interactome databases to identify HIV targeted host hub proteins and their host binding partners (H2. We developed a high throughput computational procedure utilizing motif discovery algorithms on sets of protein sequences, including sequences of HIV and H2 proteins. We identified as HIV sequence hotspots those linear motifs that are highly conserved on HIV sequences and at the same time have a statistically enriched presence on the sequences of H2 proteins. The HIV protein motifs discovered in this study are expressed by subsets of H2 host proteins potentially outcompeted by HIV proteins. A large subset of these motifs is involved in cleavage, nuclear localization, phosphorylation, and transcription factor binding events. Many such motifs are clustered on an HIV sequence in the form of hotspots. The sequential positions of these hotspots are consistent with the curated literature on phenotype altering residue mutations, as well as with existing binding site data. The hotspot map produced in this study is the first global portrayal of HIV motifs involved in altering the host protein network at highly connected hub nodes.

  15. Protein Molecular Structures, Protein SubFractions, and Protein Availability Affected by Heat Processing: A Review

    Directory of Open Access Journals (Sweden)

    Peiqiang Yu

    2007-01-01

    Full Text Available The utilization and availability of protein depended on the types of protein and their specific susceptibility to enzymatic hydrolysis (inhibitory activities in the gastrointestine and was highly associated with protein molecular structures. Studying internal protein structure and protein subfraction profiles leaded to an understanding of the components that make up a whole protein. An understanding of the molecular structure of the whole protein was often vital to understanding its digestive behavior and nutritive value in animals. In this review, recently obtained information on protein molecular structural effects of heat processing was reviewed, in relation to protein characteristics affecting digestive behavior and nutrient utilization and availability. The emphasis of this review was on (1 using the newly advanced synchrotron technology (S-FTIR as a novel approach to reveal protein molecular chemistry affected by heat processing within intact plant tissues; (2 revealing the effects of heat processing on the profile changes of protein subfractions associated with digestive behaviors and kinetics manipulated by heat processing; (3 prediction of the changes of protein availability and supply after heat processing, using the advanced DVE/OEB and NRC-2001 models, and (4 obtaining information on optimal processing conditions of protein as intestinal protein source to achieve target values for potential high net absorbable protein in the small intestine. The information described in this article may give better insight in the mechanisms involved and the intrinsic protein molecular structural changes occurring upon processing.

  16. Characterization of Protein Complexes and Subcomplexes in Protein-Protein Interaction Databases

    OpenAIRE

    Nazar Zaki; Elfadil A. Mohamed; Antonio Mora

    2015-01-01

    The identification and characterization of protein complexes implicated in protein-protein interaction data are crucial to the understanding of the molecular events under normal and abnormal physiological conditions. This paper provides a novel characterization of subcomplexes in protein interaction databases, stressing definition and representation issues, quantification, biological validation, network metrics, motifs, modularity, and gene ontology (GO) terms. The paper introduces the concep...

  17. Protein Thin Film Machines

    OpenAIRE

    Federici, Stefania; Oliviero, Giulio; Hamad-Schifferli, Kimberly; Bergese, Paolo

    2010-01-01

    We report the first example of microcantilever beams that are reversibly driven by protein thin film machines fuelled by cycling the salt concentration of the surrounding solution. We also show that upon the same salinity stimulus the drive can be completely reversed in its direction by introducing a surface coating ligand. Experimental results are throughout discussed within a general yet simple thermodynamic model.

  18. Protein: CAD [Trypanosomes Database

    Lifescience Database Archive (English)

    Full Text Available CAD carbamoyl-phosphate synthetase 2, aspartate transcarbamylase, and dihydroorotaseCA...D trifunctional proteincarbamoylphosphate synthetase 2/aspartate transcarbamylase/dihydroorotasemultifunctional protein CA...D H.sapiens 47458828 18105007 790 P27708 CAD_(gene) 2.1.3.2|3.5.2.3|6.3.5.5 114010 2p22-p21 hsa00250|hsa00240 ...

  19. Cellulose binding domain proteins

    Energy Technology Data Exchange (ETDEWEB)

    Shoseyov, Oded (Karmey Yosef, IL); Shpiegl, Itai (Rehovot, IL); Goldstein, Marc (Davis, CA); Doi, Roy (Davis, CA)

    1998-01-01

    A cellulose binding domain (CBD) having a high affinity for crystalline cellulose and chitin is disclosed, along with methods for the molecular cloning and recombinant production thereof. Fusion products comprising the CBD and a second protein are likewise described. A wide range of applications are contemplated for both the CBD and the fusion products, including drug delivery, affinity separations, and diagnostic techniques.

  20. Electron transfer in proteins

    DEFF Research Database (Denmark)

    Farver, O; Pecht, I

    1991-01-01

    Electron migration between and within proteins is one of the most prevalent forms of biological energy conversion processes. Electron transfer reactions take place between active centers such as transition metal ions or organic cofactors over considerable distances at fast rates and with remarkab...

  1. Protein Requirements during Aging

    Directory of Open Access Journals (Sweden)

    Glenda Courtney-Martin

    2016-08-01

    Full Text Available Protein recommendations for elderly, both men and women, are based on nitrogen balance studies. They are set at 0.66 and 0.8 g/kg/day as the estimated average requirement (EAR and recommended dietary allowance (RDA, respectively, similar to young adults. This recommendation is based on single linear regression of available nitrogen balance data obtained at test protein intakes close to or below zero balance. Using the indicator amino acid oxidation (IAAO method, we estimated the protein requirement in young adults and in both elderly men and women to be 0.9 and 1.2 g/kg/day as the EAR and RDA, respectively. This suggests that there is no difference in requirement on a gender basis or on a per kg body weight basis between younger and older adults. The requirement estimates however are ~40% higher than the current protein recommendations on a body weight basis. They are also 40% higher than our estimates in young men when calculated on the basis of fat free mass. Thus, current recommendations may need to be re-assessed. Potential rationale for this difference includes a decreased sensitivity to dietary amino acids and increased insulin resistance in the elderly compared with younger individuals.

  2. Tuber storage proteins.

    Science.gov (United States)

    Shewry, Peter R

    2003-06-01

    A wide range of plants are grown for their edible tubers, but five species together account for almost 90 % of the total world production. These are potato (Solanum tuberosum), cassava (Manihot esculenta), sweet potato (Ipomoea batatus), yams (Dioscorea spp.) and taro (Colocasia, Cyrtosperma and Xanthosoma spp.). All of these, except cassava, contain groups of storage proteins, but these differ in the biological properties and evolutionary relationships. Thus, patatin from potato exhibits activity as an acylhydrolase and esterase, sporamin from sweet potato is an inhibitor of trypsin, and dioscorin from yam is a carbonic anhydrase. Both sporamin and dioscorin also exhibit antioxidant and radical scavenging activity. Taro differs from the other three crops in that it contains two major types of storage protein: a trypsin inhibitor related to sporamin and a mannose-binding lectin. These characteristics indicate that tuber storage proteins have evolved independently in different species, which contrasts with the highly conserved families of storage proteins present in seeds. Furthermore, all exhibit biological activities which could contribute to resistance to pests, pathogens or abiotic stresses, indicating that they may have dual roles in the tubers.

  3. Protein Requirements during Aging.

    Science.gov (United States)

    Courtney-Martin, Glenda; Ball, Ronald O; Pencharz, Paul B; Elango, Rajavel

    2016-01-01

    Protein recommendations for elderly, both men and women, are based on nitrogen balance studies. They are set at 0.66 and 0.8 g/kg/day as the estimated average requirement (EAR) and recommended dietary allowance (RDA), respectively, similar to young adults. This recommendation is based on single linear regression of available nitrogen balance data obtained at test protein intakes close to or below zero balance. Using the indicator amino acid oxidation (IAAO) method, we estimated the protein requirement in young adults and in both elderly men and women to be 0.9 and 1.2 g/kg/day as the EAR and RDA, respectively. This suggests that there is no difference in requirement on a gender basis or on a per kg body weight basis between younger and older adults. The requirement estimates however are ~40% higher than the current protein recommendations on a body weight basis. They are also 40% higher than our estimates in young men when calculated on the basis of fat free mass. Thus, current recommendations may need to be re-assessed. Potential rationale for this difference includes a decreased sensitivity to dietary amino acids and increased insulin resistance in the elderly compared with younger individuals. PMID:27529275

  4. Protein–protein interactions

    NARCIS (Netherlands)

    Janin, J.; Bonvin, A.M.J.J.

    2013-01-01

    We are proud to present the first edition of the Protein–protein interactions Section of Current Opinion in Structural Biology. The Section is new, but the topic has been present in the journal from the very start. Volume 1, Issue 1, dated February 1991, had a review by Janin entitled Protein–protei

  5. Thermodynamics of meat proteins

    NARCIS (Netherlands)

    Sman, van der R.G.M.

    2012-01-01

    We describe the water activity of meat, being a mixture of proteins, salts and water, by the Free-Volume-Flory–Huggins (FVFH) theory augmented with the equation. Earlier, the FVFH theory is successfully applied to describe the thermodynamics to glucose homopolymers like starch, dextrans and maltodex

  6. AcEST: BP919456 [AcEST

    Lifescience Database Archive (English)

    Full Text Available finition tr|Q9XZR7|Q9XZR7_DROBU Protease, reverse transcriptase, ribonuclease H, integrase (Fragment) OS=Drosophila buzz...e, reverse transcriptase, ribonuclease H, integrase (Fragment) OS=Drosophila buzz...AIIWAVQYFRP 563 >tr|Q7M3L4|Q7M3L4_DROBU Putative uncharacterized protein 2 (Clone Db314) OS=Drosophila buzza

  7. Transient protein-protein interactions visualized by solution NMR.

    Science.gov (United States)

    Liu, Zhu; Gong, Zhou; Dong, Xu; Tang, Chun

    2016-01-01

    Proteins interact with each other to establish their identities in cell. The affinities for the interactions span more than ten orders of magnitude, and KD values in μM-mM regimen are considered transient and are important in cell signaling. Solution NMR including diamagnetic and paramagnetic techniques has enabled atomic-resolution depictions of transient protein-protein interactions. Diamagnetic NMR allows characterization of protein complexes with KD values up to several mM, whereas ultraweak and fleeting complexes can be modeled with the use of paramagnetic NMR especially paramagnetic relaxation enhancement (PRE). When tackling ever-larger protein complexes, PRE can be particularly useful in providing long-range intermolecular distance restraints. As NMR measurements are averaged over the ensemble of complex structures, structural information for dynamic protein-protein interactions besides the stereospecific one can often be extracted. Herein the protein interaction dynamics are exemplified by encounter complexes, alternative binding modes, and coupled binding/folding of intrinsically disordered proteins. Further integration of NMR with other biophysical techniques should allow better visualization of transient protein-protein interactions. In particular, single-molecule data may facilitate the interpretation of ensemble-averaged NMR data. Though same structures of proteins and protein complexes were found in cell as in diluted solution, we anticipate that the dynamics of transient protein protein-protein interactions be different, which awaits awaits exploration by NMR. This article is part of a Special Issue entitled: Physiological Enzymology and Protein Functions. This article is part of a Special Issue entitled: Physiological Enzymology and Protein Functions. PMID:25896389

  8. Bayesian Estimator of Protein-Protein Association Probabilities

    Energy Technology Data Exchange (ETDEWEB)

    2008-05-28

    The Bayesian Estimator of Protein-Protein Association Probabilities (BEPro3) is a software tool for estimating probabilities of protein-protein association between bait and prey protein pairs using data from multiple-bait, multiple-replicate, protein LC-MS/MS affinity isolation experiments. BEPro3 is public domain software, has been tested on Windows XP and version 10.4 or newer of the Mac OS 10.4, and is freely available. A user guide, example dataset with analysis and additional documentation are included with the BEPro3 download.

  9. High quality protein microarray using in situ protein purification

    Directory of Open Access Journals (Sweden)

    Fleischmann Robert D

    2009-08-01

    Full Text Available Abstract Background In the postgenomic era, high throughput protein expression and protein microarray technologies have progressed markedly permitting screening of therapeutic reagents and discovery of novel protein functions. Hexa-histidine is one of the most commonly used fusion tags for protein expression due to its small size and convenient purification via immobilized metal ion affinity chromatography (IMAC. This purification process has been adapted to the protein microarray format, but the quality of in situ His-tagged protein purification on slides has not been systematically evaluated. We established methods to determine the level of purification of such proteins on metal chelate-modified slide surfaces. Optimized in situ purification of His-tagged recombinant proteins has the potential to become the new gold standard for cost-effective generation of high-quality and high-density protein microarrays. Results Two slide surfaces were examined, chelated Cu2+ slides suspended on a polyethylene glycol (PEG coating and chelated Ni2+ slides immobilized on a support without PEG coating. Using PEG-coated chelated Cu2+ slides, consistently higher purities of recombinant proteins were measured. An optimized wash buffer (PBST composed of 10 mM phosphate buffer, 2.7 mM KCl, 140 mM NaCl and 0.05% Tween 20, pH 7.4, further improved protein purity levels. Using Escherichia coli cell lysates expressing 90 recombinant Streptococcus pneumoniae proteins, 73 proteins were successfully immobilized, and 66 proteins were in situ purified with greater than 90% purity. We identified several antigens among the in situ-purified proteins via assays with anti-S. pneumoniae rabbit antibodies and a human patient antiserum, as a demonstration project of large scale microarray-based immunoproteomics profiling. The methodology is compatible with higher throughput formats of in vivo protein expression, eliminates the need for resin-based purification and circumvents

  10. Metabolism of minor isoforms of prion proteins: Cytosolic prion protein and transmembrane prion protein

    OpenAIRE

    Song, Zhiqi; Zhao, Deming; Yang, Lifeng

    2013-01-01

    Transmissible spongiform encephalopathy or prion disease is triggered by the conversion from cellular prion protein to pathogenic prion protein. Growing evidence has concentrated on prion protein configuration changes and their correlation with prion disease transmissibility and pathogenicity. In vivo and in vitro studies have shown that several cytosolic forms of prion protein with specific topological structure can destroy intracellular stability and contribute to prion protein pathogenicit...

  11. Predicting multiplex subcellular localization of proteins using protein-protein interaction network: a comparative study

    OpenAIRE

    Jiang Jonathan Q; Wu Maoying

    2012-01-01

    Abstract Background Proteins that interact in vivo tend to reside within the same or "adjacent" subcellular compartments. This observation provides opportunities to reveal protein subcellular localization in the context of the protein-protein interaction (PPI) network. However, so far, only a few efforts based on heuristic rules have been made in this regard. Results We systematically and quantitatively validate the hypothesis that proteins physically interacting with each other probably shar...

  12. Dairy Proteins and Energy Balance

    DEFF Research Database (Denmark)

    Bendtsen, Line Quist

    High protein diets affect energy balance beneficially through decreased hunger, enhanced satiety and increased energy expenditure. Dairy products are a major source of protein. Dairy proteins are comprised of two classes, casein (80%) and whey proteins (20%), which are both of high quality......, but casein is absorbed slowly and whey is absorbed rapidly. The present PhD study investigated the effects of total dairy proteins, whey, and casein, on energy balance and the mechanisms behind any differences in the effects of the specific proteins. The results do not support the hypothesis that dairy...... proteins, whey or casein are more beneficial than other protein sources in the regulation of energy balance, and suggest that dairy proteins, whey or casein seem to play only a minor role, if any, in the prevention and treatment of obesity....

  13. Coevolution study of mitochondria respiratory chain proteins:Toward the understanding of protein-protein interaction

    Institute of Scientific and Technical Information of China (English)

    Ming Yang; Yan Ge; Jiayan Wu; Jingfa Xiao; Jun Yu

    2011-01-01

    Coevolution can be seen as the interdependency between evolutionary histories. In the context of protein evolution, functional correlation proteins are ever-present coordinated evolutionary characters without disruption of organismal integrity. As to complex system, there are two forms of protein-protein interactions in vivo, which refer to inter-complex interaction and intra-complex interaction. In this paper, we studied the difference of coevolution characters between inter-complex interaction and intra-complex interaction using "Mirror tree" method on the respiratory chain (RC) proteins. We divided the correlation coefficients of every pairwise RC proteins into two groups corresponding to the binary protein-protein interaction in intra-complex and the binary protein-protein interaction in inter-complex, respectively. A dramatical discrepancy is detected between the coevolution characters of the two sets of protein interactions (Wilcoxon test, p-value = 4.4 x 10-6). Our finding reveals some critical information on coevolutionary study and assists the mechanical investigation of protein-protein interaction.Furthermore, the results also provide some unique clue for supramolecular organization of protein complexes in the mitochondrial inner membrane. More detailed binding sites map and genome information of nuclear encoded RC proteins will be extraordinary valuable for the further mitochondria dynamics study.

  14. Protein-protein interaction databases: keeping up with growing interactomes

    Directory of Open Access Journals (Sweden)

    Lehne Benjamin

    2009-04-01

    Full Text Available Abstract Over the past few years, the number of known protein-protein interactions has increased substantially. To make this information more readily available, a number of publicly available databases have set out to collect and store protein-protein interaction data. Protein-protein interactions have been retrieved from six major databases, integrated and the results compared. The six databases (the Biological General Repository for Interaction Datasets [BioGRID], the Molecular INTeraction database [MINT], the Biomolecular Interaction Network Database [BIND], the Database of Interacting Proteins [DIP], the IntAct molecular interaction database [IntAct] and the Human Protein Reference Database [HPRD] differ in scope and content; integration of all datasets is non-trivial owing to differences in data annotation. With respect to human protein-protein interaction data, HPRD seems to be the most comprehensive. To obtain a complete dataset, however, interactions from all six databases have to be combined. To overcome this limitation, meta-databases such as the Agile Protein Interaction Database (APID offer access to integrated protein-protein interaction datasets, although these also currently have certain restrictions.

  15. Mapping the human protein interactome

    Institute of Scientific and Technical Information of China (English)

    Daniel Figeys

    2008-01-01

    Interactions are the essence of all biomolecules because they cannot fulfill their roles without interacting with other molecules. Hence, mapping the interactions of biomolecules can be useful for understanding their roles and functions. Furthermore, the development of molecular based systems biology requires an understanding of the biomolecular interactions. In recent years, the mapping of protein-protein interactions in different species has been reported, but few reports have focused on the large-scale mapping of protein-protein interactions in human. Here, we review the developments in protein interaction mapping and we discuss issues and strategies for the mapping of the human protein interactome.

  16. Hydrogels Constructed from Engineered Proteins.

    Science.gov (United States)

    Li, Hongbin; Kong, Na; Laver, Bryce; Liu, Junqiu

    2016-02-24

    Due to their various potential biomedical applications, hydrogels based on engineered proteins have attracted considerable interest. Benefitting from significant progress in recombinant DNA technology and protein engineering/design techniques, the field of protein hydrogels has made amazing progress. The latest progress of hydrogels constructed from engineered recombinant proteins are presented, mainly focused on biorecognition-driven physical hydrogels as well as chemically crosslinked hydrogels. The various bio-recognition based physical crosslinking strategies are discussed, as well as chemical crosslinking chemistries used to engineer protein hydrogels, and protein hydrogels' various biomedical applications. The future perspectives of this fast evolving field of biomaterials are also discussed.

  17. Hydrogels Constructed from Engineered Proteins.

    Science.gov (United States)

    Li, Hongbin; Kong, Na; Laver, Bryce; Liu, Junqiu

    2016-02-24

    Due to their various potential biomedical applications, hydrogels based on engineered proteins have attracted considerable interest. Benefitting from significant progress in recombinant DNA technology and protein engineering/design techniques, the field of protein hydrogels has made amazing progress. The latest progress of hydrogels constructed from engineered recombinant proteins are presented, mainly focused on biorecognition-driven physical hydrogels as well as chemically crosslinked hydrogels. The various bio-recognition based physical crosslinking strategies are discussed, as well as chemical crosslinking chemistries used to engineer protein hydrogels, and protein hydrogels' various biomedical applications. The future perspectives of this fast evolving field of biomaterials are also discussed. PMID:26707834

  18. Motif-Driven Design of Protein-Protein Interfaces.

    Science.gov (United States)

    Silva, Daniel-Adriano; Correia, Bruno E; Procko, Erik

    2016-01-01

    Protein-protein interfaces regulate many critical processes for cellular function. The ability to accurately control and regulate these molecular interactions is of major interest for biomedical and synthetic biology applications, as well as to address fundamental biological questions. In recent years, computational protein design has emerged as a tool for designing novel protein-protein interactions with functional relevance. Although attractive, these computational tools carry a steep learning curve. In order to make some of these methods more accessible, we present detailed descriptions and examples of ROSETTA computational protocols for the design of functional protein binders using seeded protein interface design. In these protocols, a motif of known structure that interacts with the target site is grafted into a scaffold protein, followed by design of the surrounding interaction surface. PMID:27094298

  19. Competitive Protein Adsorption - Multilayer Adsorption and Surface Induced Protein Aggregation

    DEFF Research Database (Denmark)

    Holmberg, Maria; Hou, Xiaolin

    2009-01-01

    In this study, competitive adsorption of albumin and IgG (immunoglobulin G) from human serum solutions and protein mixtures onto polymer surfaces is studied by means of radioactive labeling. By using two different radiolabels (125I and 131I), albumin and IgG adsorption to polymer surfaces...... is monitored simultaneously and the influence from the presence of other human serum proteins on albumin and IgG adsorption, as well as their mutual influence during adsorption processes, is investigated. Exploring protein adsorption by combining analysis of competitive adsorption from complex solutions...... of high concentration with investigation of single protein adsorption and interdependent adsorption between two specific proteins enables us to map protein adsorption sequences during competitive protein adsorption. Our study shows that proteins can adsorb in a multilayer fashion onto the polymer surfaces...

  20. How do oncoprotein mutations rewire protein-protein interaction networks?

    Science.gov (United States)

    Bowler, Emily H; Wang, Zhenghe; Ewing, Rob M

    2015-01-01

    The acquisition of mutations that activate oncogenes or inactivate tumor suppressors is a primary feature of most cancers. Mutations that directly alter protein sequence and structure drive the development of tumors through aberrant expression and modification of proteins, in many cases directly impacting components of signal transduction pathways and cellular architecture. Cancer-associated mutations may have direct or indirect effects on proteins and their interactions and while the effects of mutations on signaling pathways have been widely studied, how mutations alter underlying protein-protein interaction networks is much less well understood. Systematic mapping of oncoprotein protein interactions using proteomics techniques as well as computational network analyses is revealing how oncoprotein mutations perturb protein-protein interaction networks and drive the cancer phenotype. PMID:26325016

  1. Protein-protein interactions in DNA mismatch repair.

    Science.gov (United States)

    Friedhoff, Peter; Li, Pingping; Gotthardt, Julia

    2016-02-01

    The principal DNA mismatch repair proteins MutS and MutL are versatile enzymes that couple DNA mismatch or damage recognition to other cellular processes. Besides interaction with their DNA substrates this involves transient interactions with other proteins which is triggered by the DNA mismatch or damage and controlled by conformational changes. Both MutS and MutL proteins have ATPase activity, which adds another level to control their activity and interactions with DNA substrates and other proteins. Here we focus on the protein-protein interactions, protein interaction sites and the different levels of structural knowledge about the protein complexes formed with MutS and MutL during the mismatch repair reaction. PMID:26725162

  2. A magnetic protein biocompass

    Science.gov (United States)

    Qin, Siying; Yin, Hang; Yang, Celi; Dou, Yunfeng; Liu, Zhongmin; Zhang, Peng; Yu, He; Huang, Yulong; Feng, Jing; Hao, Junfeng; Hao, Jia; Deng, Lizong; Yan, Xiyun; Dong, Xiaoli; Zhao, Zhongxian; Jiang, Taijiao; Wang, Hong-Wei; Luo, Shu-Jin; Xie, Can

    2016-02-01

    The notion that animals can detect the Earth’s magnetic field was once ridiculed, but is now well established. Yet the biological nature of such magnetosensing phenomenon remains unknown. Here, we report a putative magnetic receptor (Drosophila CG8198, here named MagR) and a multimeric magnetosensing rod-like protein complex, identified by theoretical postulation and genome-wide screening, and validated with cellular, biochemical, structural and biophysical methods. The magnetosensing complex consists of the identified putative magnetoreceptor and known magnetoreception-related photoreceptor cryptochromes (Cry), has the attributes of both Cry- and iron-based systems, and exhibits spontaneous alignment in magnetic fields, including that of the Earth. Such a protein complex may form the basis of magnetoreception in animals, and may lead to applications across multiple fields.

  3. Cow's Milk Protein Allergy.

    Science.gov (United States)

    Mousan, Grace; Kamat, Deepak

    2016-10-01

    Cow's milk protein allergy (CMPA) is a common condition encountered in children with incidence estimated as 2% to 7.5% in the first year of life. Formula and breast-fed babies can present with symptoms of CMPA. It is important to accurately diagnose CMPA to avoid the consequences of either under- or overdiagnosis. CMPA is classically categorized into immunoglobulin E (IgE)- or non-IgE-mediated reaction that vary in clinical manifestations, diagnostic evaluation, and prognosis. The most commonly involved systems in patients with CMPA are gastrointestinal, skin, and respiratory. Evaluation of CMPA starts with good data gathering followed by testing if indicated. Treatment is simply by avoidance of cow's milk protein (CMP) in the child's or mother's diet, if exclusively breast-feeding. This article reviews the definition, epidemiology, risk factors, pathogenesis, clinical presentation, evaluation, management, and prognosis of CMPA and provides an overview of different options for formulas and their indication in the treatment of CMPA.

  4. Protein Functionalized Nanodiamond Arrays

    Directory of Open Access Journals (Sweden)

    Liu YL

    2010-01-01

    Full Text Available Abstract Various nanoscale elements are currently being explored for bio-applications, such as in bio-images, bio-detection, and bio-sensors. Among them, nanodiamonds possess remarkable features such as low bio-cytotoxicity, good optical property in fluorescent and Raman spectra, and good photostability for bio-applications. In this work, we devise techniques to position functionalized nanodiamonds on self-assembled monolayer (SAMs arrays adsorbed on silicon and ITO substrates surface using electron beam lithography techniques. The nanodiamond arrays were functionalized with lysozyme to target a certain biomolecule or protein specifically. The optical properties of the nanodiamond-protein complex arrays were characterized by a high throughput confocal microscope. The synthesized nanodiamond-lysozyme complex arrays were found to still retain their functionality in interacting with E. coli.

  5. Neurocognitive derivation of protein surface property from protein aggregate parameters

    OpenAIRE

    Mishra, Hrishikesh; Lahiri, Tapobrata

    2011-01-01

    Current work targeted to predicate parametric relationship between aggregate and individual property of a protein. In this approach, we considered individual property of a protein as its Surface Roughness Index (SRI) which was shown to have potential to classify SCOP protein families. The bulk property was however considered as Intensity Level based Multi-fractal Dimension (ILMFD) of ordinary microscopic images of heat denatured protein aggregates which was known to have potential to serve as...

  6. Biophysics of protein evolution and evolutionary protein biophysics

    OpenAIRE

    Sikosek, Tobias; Chan, Hue Sun

    2014-01-01

    The study of molecular evolution at the level of protein-coding genes often entails comparing large datasets of sequences to infer their evolutionary relationships. Despite the importance of a protein's structure and conformational dynamics to its function and thus its fitness, common phylogenetic methods embody minimal biophysical knowledge of proteins. To underscore the biophysical constraints on natural selection, we survey effects of protein mutations, highlighting the physical basis for ...

  7. Hub Promiscuity in Protein-Protein Interaction Networks

    OpenAIRE

    Haruki Nakamura; Kengo Kinoshita; Ashwini Patil

    2010-01-01

    Hubs are proteins with a large number of interactions in a protein-protein interaction network. They are the principal agents in the interaction network and affect its function and stability. Their specific recognition of many different protein partners is of great interest from the structural viewpoint. Over the last few years, the structural properties of hubs have been extensively studied. We review the currently known features that are particular to hubs, possibly affecting their binding ...

  8. Geometric De-noising of Protein-Protein Interaction Networks

    OpenAIRE

    Kuchaiev, Oleksii; Rasajski, Marija; Higham, Desmond J.; Przul, Natasa; Przytycka, Teresa Maria

    2009-01-01

    Understanding complex networks of protein-protein interactions (PPIs) is one of the foremost challenges of the post-genomic era. Due to the recent advances in experimental bio-technology, including yeast-2-hybrid (Y2H), tandem affinity purification (TAP) and other high-throughput methods for protein-protein interaction (PPI) detection, huge amounts of PPI network data are becoming available. Of major concern, however, are the levels of noise and incompleteness. For example, for Y2H screens, i...

  9. Statistical thermodynamics of membrane bending mediated protein-protein attraction

    OpenAIRE

    Chou, Tom; Kim, Ken S.; Oster, George

    1999-01-01

    Integral membrane proteins deform the surrounding bilayer creating long-ranged forces that influence distant proteins. These forces can be attractive or repulsive, depending on the proteins' shape, height, contact angle with the bilayer, as well as the local membrane curvature. Although interaction energies are not pairwise additive, for sufficiently low protein density, thermodynamic properties depend only upon pair interactions. Here, we compute pair interaction potentials and entropic cont...

  10. Protein-protein fusion catalyzed by sortase A.

    Science.gov (United States)

    Levary, David A; Parthasarathy, Ranganath; Boder, Eric T; Ackerman, Margaret E

    2011-04-06

    Chimeric proteins boast widespread use in areas ranging from cell biology to drug delivery. Post-translational protein fusion using the bacterial transpeptidase sortase A provides an attractive alternative when traditional gene fusion fails. We describe use of this enzyme for in vitro protein ligation and report the successful fusion of 10 pairs of protein domains with preserved functionality--demonstrating the robust and facile nature of this reaction.

  11. Protein-protein fusion catalyzed by sortase A.

    Directory of Open Access Journals (Sweden)

    David A Levary

    Full Text Available Chimeric proteins boast widespread use in areas ranging from cell biology to drug delivery. Post-translational protein fusion using the bacterial transpeptidase sortase A provides an attractive alternative when traditional gene fusion fails. We describe use of this enzyme for in vitro protein ligation and report the successful fusion of 10 pairs of protein domains with preserved functionality--demonstrating the robust and facile nature of this reaction.

  12. Metabolism of minor isoforms of prion proteins Cytosolic prion protein and transmembrane prion protein*

    Institute of Scientific and Technical Information of China (English)

    Zhiqi Song; Deming Zhao; Lifeng Yang

    2013-01-01

    Transmissible spongiform encephalopathy or prion disease is triggered by the conversion from cellular prion protein to pathogenic prion protein. Growing evidence has concentrated on prion protein configuration changes and their correlation with prion disease transmissibility and pathoge-nicity. In vivo and in vitro studies have shown that several cytosolic forms of prion protein with spe-cific topological structure can destroy intracellular stability and contribute to prion protein pathoge-nicity. In this study, the latest molecular chaperone system associated with endoplasmic reticu-lum-associated protein degradation, the endoplasmic reticulum resident protein quality-control system and the ubiquitination proteasome system, is outlined. The molecular chaperone system directly correlates with the prion protein degradation pathway. Understanding the molecular me-chanisms wil help provide a fascinating avenue for further investigations on prion disease treatment and prion protein-induced neurodegenerative diseases.

  13. HRTEM in protein crystallography

    International Nuclear Information System (INIS)

    Electron microscopy/diffraction (ED/D) using spot-scan and low-dose imaging has been successfully applied to investigate microcrystals of an alpha-helical coiled-coil protein extracted from ootheca of the praying mantis. Fourier transforms of the images show resolution out to 4 Angstroems and can be used to phase the corresponding ED data which shows reflections out to 2 Aangstroems. 5 refs., 3 figs

  14. Tuber Storage Proteins

    OpenAIRE

    Shewry, Peter R.

    2003-01-01

    A wide range of plants are grown for their edible tubers, but five species together account for almost 90 % of the total world production. These are potato (Solanum tuberosum), cassava (Manihot esculenta), sweet potato (Ipomoea batatus), yams (Dioscorea spp.) and taro (Colocasia, Cyrtosperma and Xanthosoma spp.). All of these, except cassava, contain groups of storage proteins, but these differ in the biological properties and evolutionary relationships. Thus, patatin from potato exhibits act...

  15. Neutron protein crystallography

    Energy Technology Data Exchange (ETDEWEB)

    Niimura, Nobuo [Japan Atomic Energy Research Inst., Tokai, Ibaraki (Japan). Tokai Research Establishment

    1998-10-01

    X-ray diffraction of single crystal has enriched the knowledge of various biological molecules such as proteins, DNA, t-RNA, viruses, etc. It is difficult to make structural analysis of hydrogen atoms in a protein using X-ray crystallography, whereas neutron diffraction seems usable to directly determine the location of those hydrogen atoms. Here, neutron diffraction method was applied to structural analysis of hen egg-white lysozyme. Since the crystal size of a protein to analyze is generally small (5 mm{sup 3} at most), the neutron beam at the sample position in monochromator system was set to less than 5 x 5 mm{sup 2} and beam divergence to 0.4 degree or less. Neutron imaging plate with {sup 6}Li or Gd mixed with photostimulated luminescence material was used and about 2500 Bragg reflections were recorded in one crystal setting. A total of 38278 reflections for 2.0 A resolution were collected in less than 10 days. Thus, stereo views of Trp-111 omit map around the indol ring of Trp-111 was presented and the three-dimensional arrangement of 696H and 264D atoms in the lysozyme molecules was determined using the omit map. (M.N.)

  16. Hydrolyzed Proteins in Allergy.

    Science.gov (United States)

    Salvatore, Silvia; Vandenplas, Yvan

    2016-01-01

    Hydrolyzed proteins are used worldwide in the therapeutic management of infants with allergic manifestations and have long been proposed as a dietetic measure to prevent allergy in at risk infants. The degree and method of hydrolysis, protein source and non-nitrogen components characterize different hydrolyzed formulas (HFs) and may determine clinical efficacy, tolerance and nutritional effects. Cow's milk (CM)-based HFs are classified as extensively (eHF) or partially HF (pHF) based on the percentage of small peptides. One whey pHF has been shown to reduce atopic dermatitis in high-risk infants who are not exclusively breastfed. More studies are needed to determine the benefit of these formulas in the prevention of CM allergy (CMA) and in the general population. eHFs represent up to now the treatment of choice for most infants with CMA. However, new developments, such as an extensively hydrolyzed rice protein-based formula, could become alternative options if safety and nutritional and therapeutic efficacy are confirmed as this type of formula is less expensive. In some countries, an extensive soy hydrolysate is available. PMID:27336625

  17. Ontology integration to identify protein complex in protein interaction networks

    Directory of Open Access Journals (Sweden)

    Yang Zhihao

    2011-10-01

    Full Text Available Abstract Background Protein complexes can be identified from the protein interaction networks derived from experimental data sets. However, these analyses are challenging because of the presence of unreliable interactions and the complex connectivity of the network. The integration of protein-protein interactions with the data from other sources can be leveraged for improving the effectiveness of protein complexes detection algorithms. Methods We have developed novel semantic similarity method, which use Gene Ontology (GO annotations to measure the reliability of protein-protein interactions. The protein interaction networks can be converted into a weighted graph representation by assigning the reliability values to each interaction as a weight. Following the approach of that of the previously proposed clustering algorithm IPCA which expands clusters starting from seeded vertices, we present a clustering algorithm OIIP based on the new weighted Protein-Protein interaction networks for identifying protein complexes. Results The algorithm OIIP is applied to the protein interaction network of Sacchromyces cerevisiae and identifies many well known complexes. Experimental results show that the algorithm OIIP has higher F-measure and accuracy compared to other competing approaches.

  18. Yeast Interacting Proteins Database: YJL199C, YJL199C [Yeast Interacting Proteins Database

    Lifescience Database Archive (English)

    Full Text Available d in closely related Saccharomyces species; protein detected in large-scale protein-protein interaction studies...cies; protein detected in large-scale protein-protein interaction studies Rows with this prey as prey (4) Ro...n; not conserved in closely related Saccharomyces species; protein detected in large-scale protein-protein interaction studies... species; protein detected in large-scale protein-protein interaction studies Rows with this prey as prey Ro

  19. Protein: FEA3 [TP Atlas

    Lifescience Database Archive (English)

    Full Text Available FEA3 AREB pathway: AREB transcription factors ABF2 AREB1, BZIP36 ABSCISIC ACID-INSE...NSITIVE 5-like protein 5 ABA-responsive element-binding protein 1, Abscisic acid responsive elements-binding

  20. Protein: FEA3 [TP Atlas

    Lifescience Database Archive (English)

    Full Text Available FEA3 AREB pathway: AREB transcription factors ABF4 AREB2, BZIP38 ABSCISIC ACID-INSE...NSITIVE 5-like protein 7 ABA-responsive element-binding protein 2, Abscisic acid responsive elements-binding