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Sample records for binding requirements compared

  1. Comparative Ellipsis and Variable Binding

    OpenAIRE

    Lerner, Jan; Pinkal, Manfred

    1995-01-01

    In this paper, we discuss the question whether phrasal comparatives should be given a direct interpretation, or require an analysis as elliptic constructions, and answer it with Yes and No. The most adequate analysis of wide reading attributive (WRA) comparatives seems to be as cases of ellipsis, while a direct (but asymmetric) analysis fits the data for narrow scope attributive comparatives. The question whether it is a syntactic or a semantic process which provides the missing linguistic ma...

  2. Comparative Ellipsis and Variable Binding

    CERN Document Server

    Lerner, J; Lerner, Jan; Pinkal, Manfred

    1995-01-01

    In this paper, we discuss the question whether phrasal comparatives should be given a direct interpretation, or require an analysis as elliptic constructions, and answer it with Yes and No. The most adequate analysis of wide reading attributive (WRA) comparatives seems to be as cases of ellipsis, while a direct (but asymmetric) analysis fits the data for narrow scope attributive comparatives. The question whether it is a syntactic or a semantic process which provides the missing linguistic material in the complement of WRA comparatives is also given a complex answer: Linguistic context is accessed by combining a reconstruction operation and a mechanism of anaphoric reference. The analysis makes only few and straightforward syntactic assumptions. In part, this is made possible because the use of Generalized Functional Application as a semantic operation allows us to model semantic composition in a flexible way.

  3. Solution structure of the Equine Infectious Anemia Virus p9 protein: a rationalization of its different ALIX binding requirements compared to the analogous HIV-p6 protein

    Directory of Open Access Journals (Sweden)

    Henklein Peter

    2009-12-01

    Full Text Available Abstract Background The equine infection anemia virus (EIAV p9 Gag protein contains the late (L- domain required for efficient virus release of nascent virions from the cell membrane of infected cell. Results In the present study the p9 protein and N- and C-terminal fragments (residues 1-21 and 22-51, respectively were chemically synthesized and used for structural analyses. Circular dichroism and 1H-NMR spectroscopy provide the first molecular insight into the secondary structure and folding of this 51-amino acid protein under different solution conditions. Qualitative 1H-chemical shift and NOE data indicate that in a pure aqueous environment p9 favors an unstructured state. In its most structured state under hydrophobic conditions, p9 adopts a stable helical structure within the C-terminus. Quantitative NOE data further revealed that this α-helix extends from Ser-27 to Ser-48, while the N-terminal residues remain unstructured. The structural elements identified for p9 differ substantially from that of the functional homologous HIV-1 p6 protein. Conclusions These structural differences are discussed in the context of the different types of L-domains regulating distinct cellular pathways in virus budding. EIAV p9 mediates virus release by recruiting the ALG2-interacting protein X (ALIX via the YPDL-motif to the site of virus budding, the counterpart of the YPXnL-motif found in p6. However, p6 contains an additional PTAP L-domain that promotes HIV-1 release by binding to the tumor susceptibility gene 101 (Tsg101. The notion that structures found in p9 differ form that of p6 further support the idea that different mechanisms regulate binding of ALIX to primary versus secondary L-domains types.

  4. COMPARING LEGAL REQUIREMENTS AND USER NEEDS

    Directory of Open Access Journals (Sweden)

    S. Gristina

    2016-10-01

    Full Text Available Road transport has always played an important role in a country’s growth and, in order to manage road networks and ensure a high standard of road performance (e.g. durability, efficiency and safety, both public and private road inventories have been implemented using databases and Geographical Information Systems. They enable registering and managing significant amounts of different road information, but to date do not focus on 3D road information, data integration and interoperability. In an increasingly complex 3D urban environment, and in the age of smart cities, however, applications including intelligent transport systems, mobility and traffic management, road maintenance and safety require digital data infrastructures to manage road data: thus new inventories based on integrated 3D road models (queryable, updateable and shareable on line are required. This paper outlines the first step towards the implementation of 3D GIS-based road inventories. Focusing on the case study of the “Road Cadastre” (the Italian road inventory as established by law, it investigates current limitations and required improvements, and also compares the required data structure imposed by cadastral legislation with real road users’ needs. The study aims to: a determine whether 3D GIS would improve road cadastre (for better management of data through the complete life-cycle infrastructure projects; b define a conceptual model for a 3D road cadastre for Italy (whose general principles may be extended also to other countries.

  5. Comparative structural analysis of lipid binding START domains.

    Directory of Open Access Journals (Sweden)

    Ann-Gerd Thorsell

    Full Text Available BACKGROUND: Steroidogenic acute regulatory (StAR protein related lipid transfer (START domains are small globular modules that form a cavity where lipids and lipid hormones bind. These domains can transport ligands to facilitate lipid exchange between biological membranes, and they have been postulated to modulate the activity of other domains of the protein in response to ligand binding. More than a dozen human genes encode START domains, and several of them are implicated in a disease. PRINCIPAL FINDINGS: We report crystal structures of the human STARD1, STARD5, STARD13 and STARD14 lipid transfer domains. These represent four of the six functional classes of START domains. SIGNIFICANCE: Sequence alignments based on these and previously reported crystal structures define the structural determinants of human START domains, both those related to structural framework and those involved in ligand specificity. ENHANCED VERSION: This article can also be viewed as an enhanced version in which the text of the article is integrated with interactive 3D representations and animated transitions. Please note that a web plugin is required to access this enhanced functionality. Instructions for the installation and use of the web plugin are available in Text S1.

  6. Comparative studies on insulin binding human erythrocytes by immunoradiometric techniques

    International Nuclear Information System (INIS)

    Blood cells have been widely used to evaluate the status of the insulin receptor in man. The receptors exhibited competitive inhibition curves and nonlinear Scatchard plots similar to those reported for insulin target tissues, such as the hepatocyte and the adipocytes. This study demonstrated specific insulin binding by the erythrocytes (RBCs) of infants, children and adults. The total insulin bound by the RBCs from both children and adults gave a small difference over the physiologic range of insulin concentrations. blood RBCs of infants showed greater numbers of insulin receptors per cell and significant increase in the total amount of insulin binding than that in either children (ps for of infants, children and adults were similar to each other. It is clear that, the measurement of insulin binding by RBCs may be particularly useful in the study of infants and children with disorders of carbohydrate metabolism to elucidate the role, if any, of abnormal receptor function in their conditions

  7. Exploring the Structural Requirements of Collagen-Binding Peptides

    OpenAIRE

    Abd-Elgaliel, Wael R; Tung, Ching-Hsuan

    2013-01-01

    Collagen synthesis and tissue remodeling are involved in many diseases; therefore collagen specific binding agents have been developed to study collagen changes in various tissues. Based on a recently reported collagen binding peptide, which contains unnatural Biphenylalanine (Bip) amino acid residue, constructs with various structure variations were synthesized to explore the contributions of unnatural Bip residue, conformational restrain, and amino acid sequence in collagen recognition. The...

  8. Characterization of the comparative drug binding to intra- (liver fatty acid binding protein) and extra- (human serum albumin) cellular proteins.

    Science.gov (United States)

    Rowland, Andrew; Hallifax, David; Nussio, Matthew R; Shapter, Joseph G; Mackenzie, Peter I; Brian Houston, J; Knights, Kathleen M; Miners, John O

    2015-01-01

    1. This study compared the extent, affinity, and kinetics of drug binding to human serum albumin (HSA) and liver fatty acid binding protein (LFABP) using ultrafiltration and surface plasmon resonance (SPR). 2. Binding of basic and neutral drugs to both HSA and LFABP was typically negligible. Binding of acidic drugs ranged from minor (fu > 0.8) to extensive (fu LFABP was observed for the acidic drugs torsemide and sulfinpyrazone, and for β-estradiol (a polar, neutral compound). 3. The extent of binding of acidic drugs to HSA was up to 40% greater than binding to LFABP. SPR experiments demonstrated comparable kinetics and affinity for the binding of representative acidic drugs (naproxen, sulfinpyrazone, and torsemide) to HSA and LFABP. 4. Simulations based on in vitro kinetic constants derived from SPR experiments and a rapid equilibrium model were undertaken to examine the impact of binding characteristics on compartmental drug distribution. Simulations provided mechanistic confirmation that equilibration of intracellular unbound drug with the extracellular unbound drug is attained rapidly in the absence of active transport mechanisms for drugs bound moderately or extensively to HSA and LFABP. PMID:25801059

  9. Glucagon and adenylate cyclase: binding studies and requirements for activation.

    Science.gov (United States)

    Levey, G S; Fletcher, M A; Klein, I

    1975-01-01

    Solubilization of myocardial adenylate cyclase abolished responsiveness to glucagon and catecholamines, two of the hormones which activate the membrane-bound enzyme. Adenylate cyclase freed of detergent by DEAE-cellulose chromatography continues to remain unresponsive to hormone stimulation. However, adding purified bovine brain phospholipids--phosphotidylserine and monophosphatidylinositol--restored responsiveness to glucagon and catecholamines, respectively. 125-i-glucagon binding appeared to be independent of phospholipid, since equal binding was observed in the presence or absence of detergent and in the presence or absence of phospholipids. Chromatography of the solubilized preparation on Sephadex G-100 WAS CHARACTERIZED BY 125-I-glucagon binding and fluoride-stimulatable adenylate cyclase activity appearing in the fractions consistent with the void volume, suggesting a molecular weight greater than 100,000 for the receptor-adenylate cyclase complex. Prior incubation of the binding peak with 125-I-glucagon and rechromatography of the bound glucagon on Sephadex G-100 shifted its elution to a later fraction consistent with a smaller-molecular-weight peak. The molecular weight of this material was 24,000 to 28,000, as determined by SDS polyacrylamide gel electrophoresis. The latter findings are consistent with a dissociable receptor site for glucagon on myocardial adenylate cyclase. PMID:165684

  10. Comparative study of methyl-CpG-binding domain proteins

    Directory of Open Access Journals (Sweden)

    Ropers H Hilger

    2003-01-01

    Full Text Available Abstract Background Methylation at CpG dinucleotides in genomic DNA is a fundamental epigenetic mechanism of gene expression control in vertebrates. Proteins with a methyl-CpG-binding domain (MBD can bind to single methylated CpGs and most of them are involved in transcription control. So far, five vertebrate MBD proteins have been described as MBD family members: MBD1, MBD2, MBD3, MBD4 and MECP2. Results We performed database searches for new proteins containing an MBD and identified six amino acid sequences which are different from the previously described ones. Here we present a comparison of their MBD sequences, additional protein motifs and the expression of the encoding genes. A calculated unrooted dendrogram indicates the existence of at least four different groups of MBDs within these proteins. Two of these polypeptides, KIAA1461 and KIAA1887, were only present as predicted amino acid sequences based on a partial human cDNA. We investigated their expression by Northern blot analysis and found transcripts of ~8 kb and ~5 kb respectively, in all eight normal tissues studied. Conclusions Eleven polypeptides with a MBD could be identified in mouse and man. The analysis of protein domains suggests a role in transcriptional regulation for most of them. The knowledge of additional existing MBD proteins and their expression pattern is important in the context of Rett syndrome.

  11. The XRCC1 phosphate-binding pocket binds poly (ADP-ribose) and is required for XRCC1 function.

    Science.gov (United States)

    Breslin, Claire; Hornyak, Peter; Ridley, Andrew; Rulten, Stuart L; Hanzlikova, Hana; Oliver, Antony W; Caldecott, Keith W

    2015-08-18

    Poly (ADP-ribose) is synthesized at DNA single-strand breaks and can promote the recruitment of the scaffold protein, XRCC1. However, the mechanism and importance of this process has been challenged. To address this issue, we have characterized the mechanism of poly (ADP-ribose) binding by XRCC1 and examined its importance for XRCC1 function. We show that the phosphate-binding pocket in the central BRCT1 domain of XRCC1 is required for selective binding to poly (ADP-ribose) at low levels of ADP-ribosylation, and promotes interaction with cellular PARP1. We also show that the phosphate-binding pocket is required for EGFP-XRCC1 accumulation at DNA damage induced by UVA laser, H2O2, and at sites of sub-nuclear PCNA foci, suggesting that poly (ADP-ribose) promotes XRCC1 recruitment both at single-strand breaks globally across the genome and at sites of DNA replication stress. Finally, we show that the phosphate-binding pocket is required following DNA damage for XRCC1-dependent acceleration of DNA single-strand break repair, DNA base excision repair, and cell survival. These data support the hypothesis that poly (ADP-ribose) synthesis promotes XRCC1 recruitment at DNA damage sites and is important for XRCC1 function. PMID:26130715

  12. The XRCC1 phosphate-binding pocket binds poly (ADP-ribose) and is required for XRCC1 function

    Science.gov (United States)

    Breslin, Claire; Hornyak, Peter; Ridley, Andrew; Rulten, Stuart L.; Hanzlikova, Hana; Oliver, Antony W.; Caldecott, Keith W.

    2015-01-01

    Poly (ADP-ribose) is synthesized at DNA single-strand breaks and can promote the recruitment of the scaffold protein, XRCC1. However, the mechanism and importance of this process has been challenged. To address this issue, we have characterized the mechanism of poly (ADP-ribose) binding by XRCC1 and examined its importance for XRCC1 function. We show that the phosphate-binding pocket in the central BRCT1 domain of XRCC1 is required for selective binding to poly (ADP-ribose) at low levels of ADP-ribosylation, and promotes interaction with cellular PARP1. We also show that the phosphate-binding pocket is required for EGFP-XRCC1 accumulation at DNA damage induced by UVA laser, H2O2, and at sites of sub-nuclear PCNA foci, suggesting that poly (ADP-ribose) promotes XRCC1 recruitment both at single-strand breaks globally across the genome and at sites of DNA replication stress. Finally, we show that the phosphate-binding pocket is required following DNA damage for XRCC1-dependent acceleration of DNA single-strand break repair, DNA base excision repair, and cell survival. These data support the hypothesis that poly (ADP-ribose) synthesis promotes XRCC1 recruitment at DNA damage sites and is important for XRCC1 function. PMID:26130715

  13. Stage-specific adhesion of Leishmania promastigotes to sand fly midguts assessed using an improved comparative binding assay.

    Directory of Open Access Journals (Sweden)

    Raymond Wilson

    Full Text Available BACKGROUND: The binding of Leishmania promastigotes to the midgut epithelium is regarded as an essential part of the life-cycle in the sand fly vector, enabling the parasites to persist beyond the initial blood meal phase and establish the infection. However, the precise nature of the promastigote stage(s that mediate binding is not fully understood. METHODOLOGY/PRINCIPAL FINDINGS: To address this issue we have developed an in vitro gut binding assay in which two promastigote populations are labelled with different fluorescent dyes and compete for binding to dissected sand fly midguts. Binding of procyclic, nectomonad, leptomonad and metacyclic promastigotes of Leishmania infantum and L. mexicana to the midguts of blood-fed, female Lutzomyia longipalpis was investigated. The results show that procyclic and metacyclic promastigotes do not bind to the midgut epithelium in significant numbers, whereas nectomonad and leptomonad promastigotes both bind strongly and in similar numbers. The assay was then used to compare the binding of a range of different parasite species (L. infantum, L. mexicana, L. braziliensis, L. major, L. tropica to guts dissected from various sand flies (Lu. longipalpis, Phlebotomus papatasi, P. sergenti. The results of these comparisons were in many cases in line with expectations, the natural parasite binding most effectively to its natural vector, and no examples were found where a parasite was unable to bind to its natural vector. However, there were interesting exceptions: L. major and L. tropica being able to bind to Lu. longipalpis better than L. infantum; L. braziliensis was able to bind to P. papatasi as well as L. major; and significant binding of L. major to P. sergenti and L. tropica to P. papatasi was observed. CONCLUSIONS/SIGNIFICANCE: The results demonstrate that Leishmania gut binding is strictly stage-dependent, is a property of those forms found in the middle phase of development (nectomonad and leptomonad

  14. Monomeric rhodopsin is the minimal functional unit required for arrestin binding*

    OpenAIRE

    Tsukamoto, Hisao; Sinha, Abhinav; DeWitt, Mark; Farrens, David L.

    2010-01-01

    We have tested if arrestin binding requires the G protein-coupled receptor (GPCR) be a dimer or multimer. To do this, we encapsulated single rhodopsin molecules into nanoscale phospholipids particles (so called nanodiscs) and measured their ability to bind arrestin. Our data clearly show that both visual arrestin and β-arrestin 1 can bind to monomeric rhodopsin and stabilize the active metarhodopsin II form. Interestingly, we find the monomeric rhodopsin in nanodiscs has a higher affinity for...

  15. Comparing AHP and ELECTRE I for prioritizing software requirements

    OpenAIRE

    Fernandes, João M.; Rodrigues, Susana Prozil; Costa, L.

    2015-01-01

    Requirement prioritization is a process that allows selection of the “key” candidate requirements, the ones that are the most important for the construction of quality and cost-controlled software. Requirement prioritization brings certain issues and challenges related with the different stakeholders involved in the project, as well as with the prioritization techniques used, which differ in procedures, criteria and metrics. This manuscript compares two multi-criteria decision methods (MCD...

  16. Defining the stoichiometry of inositol 1,4,5-trisphosphate binding required to initiate Ca2+ release.

    Science.gov (United States)

    Alzayady, Kamil J; Wang, Liwei; Chandrasekhar, Rahul; Wagner, Larry E; Van Petegem, Filip; Yule, David I

    2016-04-05

    Inositol 1,4,5-trisphosphate (IP3) receptors (IP3Rs) are tetrameric intracellular Ca(2+)-release channels with each subunit containing a binding site for IP3in the amino terminus. We provide evidence that four IP3molecules are required to activate the channel under diverse conditions. Comparing the concentration-response relationship for binding and Ca(2+)release suggested that IP3Rs are maximally occupied by IP3before substantial Ca(2+)release occurs. We showed that ligand binding-deficient subunits acted in a dominant-negative manner when coexpressed with wild-type monomers in the chicken immune cell line DT40-3KO, which lacks all three genes encoding IP3R subunits, and confirmed the same effect in an IP3R-null human cell line (HEK-3KO) generated by CRISPR/Cas9 technology. Using dimeric and tetrameric concatenated IP3Rs with increasing numbers of binding-deficient subunits, we addressed the obligate ligand stoichiometry. The concatenated IP3Rs with four ligand-binding sites exhibited Ca(2+)release and electrophysiological properties of native IP3Rs. However, IP3failed to activate IP3Rs assembled from concatenated dimers consisting of one binding-competent and one binding-deficient mutant subunit. Similarly, IP3Rs containing two monomers of IP3R2short, an IP3binding-deficient splice variant, were nonfunctional. Concatenated tetramers containing only three binding-competent ligand-binding sites were nonfunctional under a wide range of activating conditions. These data provide definitive evidence that IP3-induced Ca(2+)release only occurs when each IP3R monomer within the tetramer is occupied by IP3, thereby ensuring fidelity of Ca(2+)release.

  17. Dynamic binding of replication protein a is required for DNA repair

    Science.gov (United States)

    Chen, Ran; Subramanyam, Shyamal; Elcock, Adrian H.; Spies, Maria; Wold, Marc S.

    2016-01-01

    Replication protein A (RPA), the major eukaryotic single-stranded DNA (ssDNA) binding protein, is essential for replication, repair and recombination. High-affinity ssDNA-binding by RPA depends on two DNA binding domains in the large subunit of RPA. Mutation of the evolutionarily conserved aromatic residues in these two domains results in a separation-of-function phenotype: aromatic residue mutants support DNA replication but are defective in DNA repair. We used biochemical and single-molecule analyses, and Brownian Dynamics simulations to determine the molecular basis of this phenotype. Our studies demonstrated that RPA binds to ssDNA in at least two modes characterized by different dissociation kinetics. We also showed that the aromatic residues contribute to the formation of the longer-lived state, are required for stable binding to short ssDNA regions and are needed for RPA melting of partially duplex DNA structures. We conclude that stable binding and/or the melting of secondary DNA structures by RPA is required for DNA repair, including RAD51 mediated DNA strand exchange, but is dispensable for DNA replication. It is likely that the binding modes are in equilibrium and reflect dynamics in the RPA–DNA complex. This suggests that dynamic binding of RPA to DNA is necessary for different cellular functions. PMID:27131385

  18. A Novel Alignment-Free Method for Comparing Transcription Factor Binding Site Motifs

    OpenAIRE

    Minli Xu; Zhengchang Su

    2010-01-01

    BACKGROUND: Transcription factor binding site (TFBS) motifs can be accurately represented by position frequency matrices (PFM) or other equivalent forms. We often need to compare TFBS motifs using their PFMs in order to search for similar motifs in a motif database, or cluster motifs according to their binding preference. The majority of current methods for motif comparison involve a similarity metric for column-to-column comparison and a method to find the optimal position alignment between ...

  19. Intracellular binding site kinetics of 201 Tl binding compared to β-adrenergic analog receptors in dog myocardium

    International Nuclear Information System (INIS)

    It has been demonstrated with the multiple indicator dilution technique (MID) in an isolated dog heart preparation, that the permeation of thallium ions across the sarcolemma is about ten times larger compared to potassium ions (cellular permeability surface area product PSM 8.90 +- 4.60 vs. 0.65 +- 0.46 ml/min gsup(-1)). Similarly, the intracellular (IC) distribution space of Tlsup(+) is larger compared to that of Ksup(+). These properties may explain in part the rapid and large extraction of Tl in the myocardium. To explain the slow washout rate of Tl from the myocardium (T 1/2>600 sec determined with an on-line residue detection) we proposed a temporary binding of Tl to an IC protein. In experiments the permeation properties of 201 Tl were compared to 125 I-cyanopinodolol (I-CP) and 131 I metabenzylquanidin (I-MBG) by means of MID. The latter two substances act at the β-adrenergic receptor site. Both substances have a lower capillary permeability surface area product PSC of 0.43 +- 0.37 ml/min gsup(-1) compared to that of 201 Tl (1.37 +- 0.49 ml/min gsup(-1)). I-CP and I-MBG are sequestered extracellularly in contrast to Tl, which permeates intracellularly. However, the relation between time and instantaneous extraction during a single bolus passage of 201 Tl is very comparable to that of those receptor substances suggesting also a receptor-type kinetics for Tl with intracellular binding which may elucidate its prolonged washout. (Author)

  20. Computational predictions suggest that structural similarity in viral polymerases may lead to comparable allosteric binding sites.

    Science.gov (United States)

    Brown, Jodian A; Espiritu, Marie V; Abraham, Joel; Thorpe, Ian F

    2016-08-15

    The identification of ligand-binding sites is often the first step in drug targeting and design. To date there are numerous computational tools available to predict ligand binding sites. These tools can guide or mitigate the need for experimental methods to identify binding sites, which often require significant resources and time. Here, we evaluate four ligand-binding site predictor (LBSP) tools for their ability to predict allosteric sites within the Hepatitis C Virus (HCV) polymerase. Our results show that the LISE LBSP is able to identify all three target allosteric sites within the HCV polymerase as well as a known allosteric site in the Coxsackievirus polymerase. LISE was then employed to identify novel binding sites within the polymerases of the Dengue, West Nile, and Foot-and-mouth Disease viruses. Our results suggest that all three viral polymerases have putative sites that share structural or chemical similarities with allosteric pockets of the HCV polymerase. Thus, these binding locations may represent an evolutionarily conserved structural feature of several viral polymerases that could be exploited for the development of small molecule therapeutics. PMID:27262620

  1. Transition of arrestin into the active receptor-binding state requires an extended interdomain hinge.

    Science.gov (United States)

    Vishnivetskiy, Sergey A; Hirsch, Joel A; Velez, Maria-Gabriela; Gurevich, Yulia V; Gurevich, Vsevolod V

    2002-11-15

    Arrestins selectively bind to the phosphorylated activated form of G protein-coupled receptors, thereby blocking further G protein activation. Structurally, arrestins consist of two domains topologically connected by a 12-residue long loop, which we term the "hinge" region. Both domains contain receptor-binding elements. The relative size and shape of arrestin and rhodopsin suggest that dramatic changes in arrestin conformation are required to bring all of its receptor-binding elements in contact with the cytoplasmic surface of the receptor. Here we use the visual arrestin/rhodopsin system to test the hypothesis that the transition of arrestin into its active receptor-binding state involves a movement of the two domains relative to each other that might be limited by the length of the hinge. We have introduced three insertions and 24 deletions in the hinge region and measured the binding of all of these mutants to light-activated phosphorylated (P-Rh*), dark phosphorylated (P-Rh), dark unphosphorylated (Rh), and light-activated unphosphorylated rhodopsin (Rh*). The addition of 1-3 extra residues to the hinge has no effect on arrestin function. In contrast, sequential elimination of 1-8 residues results in a progressive decrease in P-Rh* binding without changing arrestin selectivity for P-Rh*. These results suggest that there is a minimum length of the hinge region necessary for high affinity binding, consistent with the idea that the two domains move relative to each other in the process of arrestin transition into its active receptor-binding state. The same length of the hinge is also necessary for the binding of "constitutively active" arrestin mutants to P-Rh*, dark P-Rh, and Rh*, suggesting that the active (receptor-bound) arrestin conformation is essentially the same in both wild type and mutant forms.

  2. IsdB-dependent hemoglobin binding is required for acquisition of heme by Staphylococcus aureus.

    Science.gov (United States)

    Pishchany, Gleb; Sheldon, Jessica R; Dickson, Claire F; Alam, Md Tauqeer; Read, Timothy D; Gell, David A; Heinrichs, David E; Skaar, Eric P

    2014-06-01

    Staphylococcus aureus is a Gram-positive pathogen responsible for tremendous morbidity and mortality. As with most bacteria, S. aureus requires iron to cause disease, and it can acquire iron from host hemoglobin. The current model for staphylococcal hemoglobin-iron acquisition proposes that S. aureus binds hemoglobin through the surface-exposed hemoglobin receptor IsdB. IsdB removes heme from bound hemoglobin and transfers this cofactor to other proteins of the Isd system, which import and degrade heme to release iron in the cytoplasm. Here we demonstrate that the individual components of the Isd system are required for growth on low nanomolar concentrations of hemoglobin as a sole source of iron. An in-depth study of hemoglobin binding by IsdB revealed key residues that are required for hemoglobin binding. Further, we show that these residues are necessary for heme extraction from hemoglobin and growth on hemoglobin as a sole iron source. These processes are found to contribute to the pathogenicity of S. aureus in a murine model of infection. Together these results build on the model for Isd-mediated hemoglobin binding and heme-iron acquisition during the pathogenesis of S. aureus infection.

  3. The acyl-CoA binding protein is required for normal epidermal barrier function in mice.

    Science.gov (United States)

    Bloksgaard, Maria; Bek, Signe; Marcher, Ann-Britt; Neess, Ditte; Brewer, Jonathan; Hannibal-Bach, Hans Kristian; Helledie, Torben; Fenger, Christina; Due, Marianne; Berzina, Zane; Neubert, Reinhard; Chemnitz, John; Finsen, Bente; Clemmensen, Anders; Wilbertz, Johannes; Saxtorph, Henrik; Knudsen, Jens; Bagatolli, Luis; Mandrup, Susanne

    2012-10-01

    The acyl-CoA binding protein (ACBP) is a 10 kDa intracellular protein expressed in all eukaryotic species. Mice with targeted disruption of Acbp (ACBP(-/-) mice) are viable and fertile but present a visible skin and fur phenotype characterized by greasy fur and development of alopecia and scaling with age. Morphology and development of skin and appendages are normal in ACBP(-/-) mice; however, the stratum corneum display altered biophysical properties with reduced proton activity and decreased water content. Mass spectrometry analyses of lipids from epidermis and stratum corneum of ACBP(+/+) and ACBP(-/-) mice showed very similar composition, except for a significant and specific decrease in the very long chain free fatty acids (VLC-FFA) in stratum corneum of ACBP(-/-) mice. This finding indicates that ACBP is critically involved in the processes that lead to production of stratum corneum VLC-FFAs via complex phospholipids in the lamellar bodies. Importantly, we show that ACBP(-/-) mice display a ∼50% increased transepidermal water loss compared with ACBP(+/+) mice. Furthermore, skin and fur sebum monoalkyl diacylglycerol (MADAG) levels are significantly increased, suggesting that ACBP limits MADAG synthesis in sebaceous glands. In summary, our study shows that ACBP is required for production of VLC-FFA for stratum corneum and for maintaining normal epidermal barrier function. PMID:22829653

  4. Normocyte-binding protein required for human erythrocyte invasion by the zoonotic malaria parasitePlasmodium knowlesi

    KAUST Repository

    Moon, Robert W.

    2016-06-15

    The dominant cause of malaria in Malaysia is now Plasmodium knowlesi, a zoonotic parasite of cynomolgus macaque monkeys found throughout South East Asia. Comparative genomic analysis of parasites adapted to in vitro growth in either cynomolgus or human RBCs identified a genomic deletion that includes the gene encoding normocyte-binding protein Xa (NBPXa) in parasites growing in cynomolgus RBCs but not in human RBCs. Experimental deletion of the NBPXa gene in parasites adapted to growth in human RBCs (which retain the ability to grow in cynomolgus RBCs) restricted them to cynomolgus RBCs, demonstrating that this gene is selectively required for parasite multiplication and growth in human RBCs. NBPXa-null parasites could bind to human RBCs, but invasion of these cells was severely impaired. Therefore, NBPXa is identified as a key mediator of P. knowlesi human infection and may be a target for vaccine development against this emerging pathogen.

  5. Comparative study on ChIP-seq data: normalization and binding pattern characterization

    OpenAIRE

    Taslim, Cenny; Wu, Jiejun; Yan, Pearlly; Singer, Greg; Parvin, Jeffrey; Huang, Tim; Lin, Shili; Huang, Kun

    2009-01-01

    Motivation: Antibody-based Chromatin Immunoprecipitation assay followed by high-throughput sequencing technology (ChIP-seq) is a relatively new method to study the binding patterns of specific protein molecules over the entire genome. ChIP-seq technology allows scientist to get more comprehensive results in shorter time. Here, we present a non-linear normalization algorithm and a mixture modeling method for comparing ChIP-seq data from multiple samples and characterizing genes based on their ...

  6. A novel alignment-free method for comparing transcription factor binding site motifs.

    Directory of Open Access Journals (Sweden)

    Minli Xu

    Full Text Available BACKGROUND: Transcription factor binding site (TFBS motifs can be accurately represented by position frequency matrices (PFM or other equivalent forms. We often need to compare TFBS motifs using their PFMs in order to search for similar motifs in a motif database, or cluster motifs according to their binding preference. The majority of current methods for motif comparison involve a similarity metric for column-to-column comparison and a method to find the optimal position alignment between the two compared motifs. In some applications, alignment-free methods might be preferred; however, few such methods with high accuracy have been described. METHODOLOGY/PRINCIPAL FINDINGS: Here we describe a novel alignment-free method for quantifying the similarity of motifs using their PFMs by converting PFMs into k-mer vectors. The motifs could then be compared by measuring the similarity among their corresponding k-mer vectors. CONCLUSIONS/SIGNIFICANCE: We demonstrate that our method in general achieves similar performance or outperforms the existing methods for clustering motifs according to their binding preference and identifying similar motifs of transcription factors of the same family.

  7. A Novel Alignment-Free Method for Comparing Transcription Factor Binding Site Motifs

    Science.gov (United States)

    Xu, Minli; Su, Zhengchang

    2010-01-01

    Background Transcription factor binding site (TFBS) motifs can be accurately represented by position frequency matrices (PFM) or other equivalent forms. We often need to compare TFBS motifs using their PFMs in order to search for similar motifs in a motif database, or cluster motifs according to their binding preference. The majority of current methods for motif comparison involve a similarity metric for column-to-column comparison and a method to find the optimal position alignment between the two compared motifs. In some applications, alignment-free methods might be preferred; however, few such methods with high accuracy have been described. Methodology/Principal Findings Here we describe a novel alignment-free method for quantifying the similarity of motifs using their PFMs by converting PFMs into k-mer vectors. The motifs could then be compared by measuring the similarity among their corresponding k-mer vectors. Conclusions/Significance We demonstrate that our method in general achieves similar performance or outperforms the existing methods for clustering motifs according to their binding preference and identifying similar motifs of transcription factors of the same family. PMID:20098703

  8. Comparing the Affinity of GTPase-binding Proteins using Competition Assays.

    Science.gov (United States)

    Williamson, Rosalind C; Bass, Mark D

    2015-01-01

    In this protocol we demonstrate a method for comparing the competition between GTPase-binding proteins. Such an approach is important for determining the binding capabilities of GTPases for two reasons: The fact that all interactions involve the same face of the GTPases means that binding events must be considered in the context of competitors, and the fact that the bound nucleotide must also be controlled means that conventional approaches such as immunoprecipitation are unsuitable for GTPase biochemistry. The assay relies on the use of purified proteins. Purified Rac1 immobilized on beads is used as the bait protein, and can be loaded with GDP, a non-hydrolyzable version of GTP or left nucleotide free, so that the signaling stage to be investigated can be controlled. The binding proteins to be investigated are purified from mammalian cells, to allow correct folding, by means of a GFP tag. Use of the same tag on both proteins is important because not only does it allow rapid purification and elution, but also allows detection of both competitors with the same antibody during elution. This means that the relative amounts of the two bound proteins can be determined accurately.

  9. COMPARATIVE ANALYSIS FOR METAL BINDING CAPACITY OF CYSTEINE BY USING UV-VIS SPECTROPHOTOMETER

    Directory of Open Access Journals (Sweden)

    Shivendu Ranjan

    2012-05-01

    Full Text Available The metal binding capacity of cysteine with three different metals Nickel, Copper and Lead was studied using UV-Vis spectrophotometer for which absorbance values were taken after interaction of cysteine with metal salt solutions (10ppm and 100ppm. Before taking above absorbance dilution factor was set using cysteine stock. The increase in peak intensity was observed when metal salt solution and metal saltcysteine solution were compared. Based on peak shift and peak intensity finally it can be concluded that the binding capacity of cysteine with Nickel is more, followed by lead and copper. The normal chromophore activity in cysteine is due to the sulphur in which the transition takes place from non bonding orbital’s to the excited antibonding orbital in the range of 210-215nm range. The binding of the metals with cysteine may affect the chromophore activity and may also lead to structural damage of the chromophore. This can give the decrease in the peak intensity or the complete shift in the peak. These results suggest that cysteine metal binding ability can be used for the removal of the metals in water purification. Also this property can be used in removal of metals from our body considering the fact that cysteine may not show adverse effect in the system. So we can go for designing a new type of drug containing cysteine which helps to prevent the accumulation of such metals and thus prevent us from adverse effect.

  10. Wiz binds active promoters and CTCF-binding sites and is required for normal behaviour in the mouse

    Science.gov (United States)

    Isbel, Luke; Prokopuk, Lexie; Wu, Haoyu; Daxinger, Lucia; Oey, Harald; Spurling, Alex; Lawther, Adam J; Hale, Matthew W; Whitelaw, Emma

    2016-01-01

    We previously identified Wiz in a mouse screen for epigenetic modifiers. Due to its known association with G9a/GLP, Wiz is generally considered a transcriptional repressor. Here, we provide evidence that it may also function as a transcriptional activator. Wiz levels are high in the brain, but its function and direct targets are unknown. ChIP-seq was performed in adult cerebellum and Wiz peaks were found at promoters and transcription factor CTCF binding sites. RNA-seq in Wiz mutant mice identified genes differentially regulated in adult cerebellum and embryonic brain. In embryonic brain most decreased in expression and included clustered protocadherin genes. These also decreased in adult cerebellum and showed strong Wiz ChIP-seq enrichment. Because a precise pattern of protocadherin gene expression is required for neuronal development, behavioural tests were carried out on mutant mice, revealing an anxiety-like phenotype. This is the first evidence of a role for Wiz in neural function. DOI: http://dx.doi.org/10.7554/eLife.15082.001 PMID:27410475

  11. Fibronectin Binding Is Required for Acquisition of Mesenchymal/Endothelial Differentiation Potential in Human Circulating Monocytes

    Directory of Open Access Journals (Sweden)

    Noriyuki Seta

    2012-01-01

    Full Text Available We previously reported monocyte-derived multipotential cells (MOMCs, which include progenitors capable of differentiating into a variety of mesenchymal cells and endothelial cells. In vitro generation of MOMCs from circulating CD14+ monocytes requires their binding to extracellular matrix (ECM protein and exposure to soluble factor(s derived from circulating CD14- cells. Here, we investigated the molecular factors involved in MOMC generation by examining the binding of monocytes to ECM proteins. We found that MOMCs were obtained on the fibronectin, but not on type I collagen, laminin, or poly-L-lysine. MOMC generation was followed by changes in the expression profiles of transcription factors and was completely inhibited by either anti-α5 integrin antibody or a synthetic peptide that competed with the RGD domain for the β1-integrin binding site. These results indicate that acquisition of the multidifferentiation potential by circulating monocytes depends on their binding to the RGD domain of fibronectin via cell-surface α5β1 integrin.

  12. 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.

  13. Activation of phenylalanine hydroxylase by phenylalanine does not require binding in the active site.

    Science.gov (United States)

    Roberts, Kenneth M; Khan, Crystal A; Hinck, Cynthia S; Fitzpatrick, Paul F

    2014-12-16

    Phenylalanine hydroxylase (PheH), a liver enzyme that catalyzes the hydroxylation of excess phenylalanine in the diet to tyrosine, is activated by phenylalanine. The lack of activity at low levels of phenylalanine has been attributed to the N-terminus of the protein's regulatory domain acting as an inhibitory peptide by blocking substrate access to the active site. The location of the site at which phenylalanine binds to activate the enzyme is unknown, and both the active site in the catalytic domain and a separate site in the N-terminal regulatory domain have been proposed. Binding of catecholamines to the active-site iron was used to probe the accessibility of the active site. Removal of the regulatory domain increases the rate constants for association of several catecholamines with the wild-type enzyme by ∼2-fold. Binding of phenylalanine in the active site is effectively abolished by mutating the active-site residue Arg270 to lysine. The k(cat)/K(phe) value is down 10⁴ for the mutant enzyme, and the K(m) value for phenylalanine for the mutant enzyme is >0.5 M. Incubation of the R270K enzyme with phenylalanine also results in a 2-fold increase in the rate constants for catecholamine binding. The change in the tryptophan fluorescence emission spectrum seen in the wild-type enzyme upon activation by phenylalanine is also seen with the R270K mutant enzyme in the presence of phenylalanine. Both results establish that activation of PheH by phenylalanine does not require binding of the amino acid in the active site. This is consistent with a separate allosteric site, likely in the regulatory domain.

  14. Comparative vitamin E requirements and metabolism in livestock.

    Science.gov (United States)

    Hidiroglou, N; Cave, N; Atwall, A S; Farnworth, E R; McDowell, L R

    1992-01-01

    It has been over 50 years since vitamin E was originally described as a lipid-soluble dietary constituent required for normal reproduction in rats. Vitamin E is recognized as an essential vitamin required for all classes of animals functioning predominantly as an intracellular antioxidant in maintaining the integrity of biological cell membranes. Although a wealth of information has been gathered on clinical signs of vitamin E deficiency, establishing its requirements for animals has been exceedingly difficult because of interrelationships with other dietary constituents. Vitamin E requirements for animals cannot be defined in isolation. Requirements are influenced by the amount and type of fat (particularly with monogastrics) and degree of fat oxidation in the diet; the presence of antioxidants; dietary selenium (closely interrelated with vitamin E), iron, copper, and sulphur amino acids, as well as the physiological status of the animal. Other factors to be considered in assessing vitamin E needs of animals under commercial production conditions include: a) variability of vitamin E content in feedstuffs; b) poor stability of vitamin E during processing and storage of feeds; and c) management practices resulting in overstressed animals. Information on the function of or requirements for vitamin E in animals is very incomplete. Estimated dietary vitamin E requirements for most animal species are in the range of 10-40 IU/kg of diet. Of particular concern is the lack of vitamin E requirement information regarding young dairy and beef calves. Although good experimental evidence indicates a beneficial role of supplemental vitamin E above physiological levels on overall performance, enhanced immunocompetence and preservation of meat and milk products, levels of vitamin E required to produce these desired effects needs to be firmly established. Present estimated dietary requirements for vitamin E across species may need to be redefined as new information becomes

  15. Randomized crossover study comparing the phosphate-binding efficacy of calcium ketoglutarate versus calcium carbonate in patients on chronic hemodialysis

    DEFF Research Database (Denmark)

    Bro, S; Rasmussen, R A; Handberg, J;

    1998-01-01

    into the study. Calculations based on median doses after 12 weeks showed that the cost of the therapy in Denmark was 10 times higher for calcium ketoglutarate compared with calcium carbonate (US$6.00/d v US$0.65/d). Calcium ketoglutarate may be an effective and safe alternative to treatment with aluminum......The objective of the study was to evaluate the phosphate-binding efficacy, side effects, and cost of therapy of calcium ketoglutarate granulate as compared with calcium carbonate tablets in patients on chronic hemodialysis. The study design used was a randomized, crossover open trial, and the main...... outcome measurements were plasma ionized calcium levels, plasma phosphate levels, plasma intact parathyroid hormone (PTH) levels, requirements for supplemental aluminum-aminoacetate therapy, patient tolerance, and cost of therapy. Nineteen patients on chronic hemodialysis were treated with a dialysate...

  16. Binding of transcription factor GabR to DNA requires recognition of DNA shape at a location distinct from its cognate binding site

    Science.gov (United States)

    Al-Zyoud, Walid A.; Hynson, Robert MG.; Ganuelas, Lorraine A.; Coster, Adelle CF.; Duff, Anthony P.; Baker, Matthew AB.; Stewart, Alastair G.; Giannoulatou, Eleni; Ho, Joshua WK.; Gaus, Katharina; Liu, Dali; Lee, Lawrence K.; Böcking, Till

    2016-01-01

    Mechanisms for transcription factor recognition of specific DNA base sequences are well characterized and recent studies demonstrate that the shape of these cognate binding sites is also important. Here, we uncover a new mechanism where the transcription factor GabR simultaneously recognizes two cognate binding sites and the shape of a 29 bp DNA sequence that bridges these sites. Small-angle X-ray scattering and multi-angle laser light scattering are consistent with a model where the DNA undergoes a conformational change to bend around GabR during binding. In silico predictions suggest that the bridging DNA sequence is likely to be bendable in one direction and kinetic analysis of mutant DNA sequences with biolayer interferometry, allowed the independent quantification of the relative contribution of DNA base and shape recognition in the GabR–DNA interaction. These indicate that the two cognate binding sites as well as the bendability of the DNA sequence in between these sites are required to form a stable complex. The mechanism of GabR–DNA interaction provides an example where the correct shape of DNA, at a clearly distinct location from the cognate binding site, is required for transcription factor binding and has implications for bioinformatics searches for novel binding sites. PMID:26681693

  17. A comparative study of recombinant and native frutalin binding to human prostate tissues

    Directory of Open Access Journals (Sweden)

    Domingues Lucília

    2009-09-01

    Full Text Available Abstract Background Numerous studies indicate that cancer cells present an aberrant glycosylation pattern that can be detected by lectin histochemistry. Lectins have shown the ability to recognise these modifications in several carcinomas, namely in the prostate carcinoma, one of the most lethal diseases in man. Thus, the aim of this work was to investigate if the α-D-galactose-binding plant lectin frutalin is able to detect such changes in the referred carcinoma. Frutalin was obtained from different sources namely, its natural source (plant origin and a recombinant source (Pichia expression system. Finally, the results obtained with the two lectins were compared and their potential use as prostate tumour biomarkers was discussed. Results The binding of recombinant and native frutalin to specific glycoconjugates expressed in human prostate tissues was assessed by using an immuhistochemical technique. A total of 20 cases of prostate carcinoma and 25 cases of benign prostate hyperplasia were studied. Lectins bound directly to the tissues and anti-frutalin polyclonal antibody was used as the bridge to react with the complex biotinilated anti-rabbit IgG plus streptavidin-conjugated peroxidase. DAB was used as visual indicator to specifically localise the binding of the lectins to the tissues. Both lectins bound to the cells cytoplasm of the prostate carcinoma glands. The binding intensity of native frutalin was stronger in the neoplasic cells than in hyperplasic cells; however no significant statistical correlation could be found (P = 0.051. On the other hand, recombinant frutalin bound exclusively to the neoplasic cells and a significant positive statistical correlation was obtained (P Conclusion Native and recombinant frutalin yielded different binding responses in the prostate tissues due to their differences in carbohydrate-binding affinities. Also, this study shows that both lectins may be used as histochemical biomarkers for the prostate

  18. Mutational definition of binding requirements of an hnRNP-like protein in Arabidopsis using fluorescence correlation spectroscopy

    Energy Technology Data Exchange (ETDEWEB)

    Leder, Verena [Molecular Cell Physiology, Faculty of Biology, Bielefeld University (Germany); Biomolecular Photonics, Faculty of Physics, Bielefeld University (Germany); Lummer, Martina [Molecular Cell Physiology, Faculty of Biology, Bielefeld University (Germany); Tegeler, Kathrin [Molecular Cell Physiology, Faculty of Biology, Bielefeld University (Germany); Biomolecular Photonics, Faculty of Physics, Bielefeld University (Germany); Humpert, Fabian [Biomolecular Photonics, Faculty of Physics, Bielefeld University (Germany); Lewinski, Martin [Molecular Cell Physiology, Faculty of Biology, Bielefeld University (Germany); Schüttpelz, Mark [Biomolecular Photonics, Faculty of Physics, Bielefeld University (Germany); Staiger, Dorothee, E-mail: dorothee.staiger@uni-bielefeld.de [Molecular Cell Physiology, Faculty of Biology, Bielefeld University (Germany)

    2014-10-10

    Highlights: • We use FCS to investigate binding site requirements for the hnRNP-like protein AtGRP7. • We identify three nucleotides critical for AtGRP7 binding to its own intron. • Mutation of the conserved R{sup 49} abolishes binding altogether. • The paralogue AtGRP8 binds to an overlapping motif with different sequence requirement. • The glycine-rich stretch of a plant hnRNP-like protein contributes to binding. - Abstract: Arabidopsis thaliana glycine-rich RNA binding protein 7 (AtGRP7) is part of a negative feedback loop through which it regulates alternative splicing and steady-state abundance of its pre-mRNA. Here we use fluorescence correlation spectroscopy to investigate the requirements for AtGRP7 binding to its intron using fluorescently-labelled synthetic oligonucleotides. By systematically introducing point mutations we identify three nucleotides that lead to an increased K{sub d} value when mutated and thus are critical for AtGRP7 binding. Simultaneous mutation of all three residues abrogates binding. The paralogue AtGRP8 binds to an overlapping motif but with a different sequence preference, in line with overlapping but not identical functions of this protein pair. Truncation of the glycine-rich domain reduces the binding affinity of AtGRP7, showing for the first time that the glycine-rich stretch of a plant hnRNP-like protein contributes to binding. Mutation of the conserved R{sup 49} that is crucial for AtGRP7 function in pathogen defence and splicing abolishes binding.

  19. Mutational definition of binding requirements of an hnRNP-like protein in Arabidopsis using fluorescence correlation spectroscopy

    International Nuclear Information System (INIS)

    Highlights: • We use FCS to investigate binding site requirements for the hnRNP-like protein AtGRP7. • We identify three nucleotides critical for AtGRP7 binding to its own intron. • Mutation of the conserved R49 abolishes binding altogether. • The paralogue AtGRP8 binds to an overlapping motif with different sequence requirement. • The glycine-rich stretch of a plant hnRNP-like protein contributes to binding. - Abstract: Arabidopsis thaliana glycine-rich RNA binding protein 7 (AtGRP7) is part of a negative feedback loop through which it regulates alternative splicing and steady-state abundance of its pre-mRNA. Here we use fluorescence correlation spectroscopy to investigate the requirements for AtGRP7 binding to its intron using fluorescently-labelled synthetic oligonucleotides. By systematically introducing point mutations we identify three nucleotides that lead to an increased Kd value when mutated and thus are critical for AtGRP7 binding. Simultaneous mutation of all three residues abrogates binding. The paralogue AtGRP8 binds to an overlapping motif but with a different sequence preference, in line with overlapping but not identical functions of this protein pair. Truncation of the glycine-rich domain reduces the binding affinity of AtGRP7, showing for the first time that the glycine-rich stretch of a plant hnRNP-like protein contributes to binding. Mutation of the conserved R49 that is crucial for AtGRP7 function in pathogen defence and splicing abolishes binding

  20. Fatty Acid-binding Proteins Interact with Comparative Gene Identification-58 Linking Lipolysis with Lipid Ligand Shuttling.

    Science.gov (United States)

    Hofer, Peter; Boeszoermenyi, Andras; Jaeger, Doris; Feiler, Ursula; Arthanari, Haribabu; Mayer, Nicole; Zehender, Fabian; Rechberger, Gerald; Oberer, Monika; Zimmermann, Robert; Lass, Achim; Haemmerle, Guenter; Breinbauer, Rolf; Zechner, Rudolf; Preiss-Landl, Karina

    2015-07-24

    The coordinated breakdown of intracellular triglyceride (TG) stores requires the exquisitely regulated interaction of lipolytic enzymes with regulatory, accessory, and scaffolding proteins. Together they form a dynamic multiprotein network designated as the "lipolysome." Adipose triglyceride lipase (Atgl) catalyzes the initiating step of TG hydrolysis and requires comparative gene identification-58 (Cgi-58) as a potent activator of enzyme activity. Here, we identify adipocyte-type fatty acid-binding protein (A-Fabp) and other members of the fatty acid-binding protein (Fabp) family as interaction partners of Cgi-58. Co-immunoprecipitation, microscale thermophoresis, and solid phase assays proved direct protein/protein interaction between A-Fabp and Cgi-58. Using nuclear magnetic resonance titration experiments and site-directed mutagenesis, we located a potential contact region on A-Fabp. In functional terms, A-Fabp stimulates Atgl-catalyzed TG hydrolysis in a Cgi-58-dependent manner. Additionally, transcriptional transactivation assays with a luciferase reporter system revealed that Fabps enhance the ability of Atgl/Cgi-58-mediated lipolysis to induce the activity of peroxisome proliferator-activated receptors. Our studies identify Fabps as crucial structural and functional components of the lipolysome.

  1. Fatty Acid-binding Proteins Interact with Comparative Gene Identification-58 Linking Lipolysis with Lipid Ligand Shuttling.

    Science.gov (United States)

    Hofer, Peter; Boeszoermenyi, Andras; Jaeger, Doris; Feiler, Ursula; Arthanari, Haribabu; Mayer, Nicole; Zehender, Fabian; Rechberger, Gerald; Oberer, Monika; Zimmermann, Robert; Lass, Achim; Haemmerle, Guenter; Breinbauer, Rolf; Zechner, Rudolf; Preiss-Landl, Karina

    2015-07-24

    The coordinated breakdown of intracellular triglyceride (TG) stores requires the exquisitely regulated interaction of lipolytic enzymes with regulatory, accessory, and scaffolding proteins. Together they form a dynamic multiprotein network designated as the "lipolysome." Adipose triglyceride lipase (Atgl) catalyzes the initiating step of TG hydrolysis and requires comparative gene identification-58 (Cgi-58) as a potent activator of enzyme activity. Here, we identify adipocyte-type fatty acid-binding protein (A-Fabp) and other members of the fatty acid-binding protein (Fabp) family as interaction partners of Cgi-58. Co-immunoprecipitation, microscale thermophoresis, and solid phase assays proved direct protein/protein interaction between A-Fabp and Cgi-58. Using nuclear magnetic resonance titration experiments and site-directed mutagenesis, we located a potential contact region on A-Fabp. In functional terms, A-Fabp stimulates Atgl-catalyzed TG hydrolysis in a Cgi-58-dependent manner. Additionally, transcriptional transactivation assays with a luciferase reporter system revealed that Fabps enhance the ability of Atgl/Cgi-58-mediated lipolysis to induce the activity of peroxisome proliferator-activated receptors. Our studies identify Fabps as crucial structural and functional components of the lipolysome. PMID:25953897

  2. Rat embryo fibroblasts require both the cell-binding and the heparin-binding domains of fibronectin for survival

    DEFF Research Database (Denmark)

    Jeong, J; Han, I; Lim, Y;

    2001-01-01

    -terminal heparin-binding domain, HepII. REFs on FN maintained a well-spread fibroblastic shape and even proliferated in serum-free medium at 20 h after plating. In contrast, previously well-spread REFs on FN120 started losing fibroblastic shape with time and detached from FN120-coated plates after approx. 8 h...

  3. Probing the human estrogen receptor-α binding requirements for phenolic mono- and di-hydroxyl compounds: a combined synthesis, binding and docking study.

    Science.gov (United States)

    McCullough, Christopher; Neumann, Terrence S; Gone, Jayapal Reddy; He, Zhengjie; Herrild, Christian; Wondergem Nee Lukesh, Julie; Pandey, Rajesh K; Donaldson, William A; Sem, Daniel S

    2014-01-01

    Various estrogen analogs were synthesized and tested for binding to human ERα using a fluorescence polarization displacement assay. Binding affinity and orientation were also predicted using docking calculations. Docking was able to accurately predict relative binding affinity and orientation for estradiol, but only if a tightly bound water molecule bridging Arg394/Glu353 is present. Di-hydroxyl compounds sometimes bind in two orientations, which are flipped in terms of relative positioning of their hydroxyl groups. Di-hydroxyl compounds were predicted to bind with their aliphatic hydroxyl group interacting with His524 in ERα. One nonsteroid-based dihdroxyl compound was 1000-fold specific for ERβ over ERα, and was also 25-fold specific for agonist ERβ versus antagonist activity. Docking predictions suggest this specificity may be due to interaction of the aliphatic hydroxyl with His475 in the agonist form of ERβ, versus with Thr299 in the antagonist form. But, the presence of this aliphatic hydroxyl is not required in all compounds, since mono-hydroxyl (phenolic) compounds bind ERα with high affinity, via hydroxyl hydrogen bonding interactions with the ERα Arg394/Glu353/water triad, and van der Waals interactions with the rest of the molecule.

  4. Functional requirements of AID's higher order structures and their interaction with RNA-binding proteins.

    Science.gov (United States)

    Mondal, Samiran; Begum, Nasim A; Hu, Wenjun; Honjo, Tasuku

    2016-03-15

    Activation-induced cytidine deaminase (AID) is essential for the somatic hypermutation (SHM) and class-switch recombination (CSR) of Ig genes. Although both the N and C termini of AID have unique functions in DNA cleavage and recombination, respectively, during SHM and CSR, their molecular mechanisms are poorly understood. Using a bimolecular fluorescence complementation (BiFC) assay combined with glycerol gradient fractionation, we revealed that the AID C terminus is required for a stable dimer formation. Furthermore, AID monomers and dimers form complexes with distinct heterogeneous nuclear ribonucleoproteins (hnRNPs). AID monomers associate with DNA cleavage cofactor hnRNP K whereas AID dimers associate with recombination cofactors hnRNP L, hnRNP U, and Serpine mRNA-binding protein 1. All of these AID/ribonucleoprotein associations are RNA-dependent. We propose that AID's structure-specific cofactor complex formations differentially contribute to its DNA-cleavage and recombination functions. PMID:26929374

  5. The AUXIN BINDING PROTEIN 1 is required for differential auxin responses mediating root growth.

    Directory of Open Access Journals (Sweden)

    Alexandre Tromas

    Full Text Available BACKGROUND: In plants, the phytohormone auxin is a crucial regulator sustaining growth and development. At the cellular level, auxin is interpreted differentially in a tissue- and dose-dependent manner. Mechanisms of auxin signalling are partially unknown and the contribution of the AUXIN BINDING PROTEIN 1 (ABP1 as an auxin receptor is still a matter of debate. METHODOLOGY/PRINCIPAL FINDINGS: Here we took advantage of the present knowledge of the root biological system to demonstrate that ABP1 is required for auxin response. The use of conditional ABP1 defective plants reveals that the protein is essential for maintenance of the root meristem and acts at least on the D-type CYCLIN/RETINOBLASTOMA pathway to control entry into the cell cycle. ABP1 affects PLETHORA gradients and confers auxin sensitivity to root cells thus defining the competence of the cells to be maintained within the meristem or to elongate. ABP1 is also implicated in the regulation of gene expression in response to auxin. CONCLUSIONS/SIGNIFICANCE: Our data support that ABP1 is a key regulator for root growth and is required for auxin-mediated responses. Differential effects of ABP1 on various auxin responses support a model in which ABP1 is the major regulator for auxin action on the cell cycle and regulates auxin-mediated gene expression and cell elongation in addition to the already well known TIR1-mediated ubiquitination pathway.

  6. Comparative in silico analysis identifies bona fide MyoD binding sites within the Myocyte Stress 1 gene promoter

    Directory of Open Access Journals (Sweden)

    Imbalzano Anthony N

    2008-05-01

    Full Text Available Abstract Background Myocyte stress 1 (MS1 is a striated muscle actin binding protein required for the muscle specific activity of the evolutionary ancient myocardin related transcription factor (MRTF/serum response factor (SRF transcriptional pathway. To date, little is known about the molecular mechanisms that govern skeletal muscle specific expression of MS1. Such mechanisms are likely to play a major role in modulating SRF activity and therefore muscle determination, differentiation and regeneration. In this study we employed a comparative in silico analysis coupled with an experimental promoter characterisation to delineate these mechanisms. Results Analysis of MS1 expression in differentiating C2C12 muscle cells demonstrated a temporal differentiation dependent up-regulation in ms1 mRNA. An in silico comparative sequence analysis identified two conserved putative myogenic regulatory domains within the proximal 1.5 kbp of 5' upstream sequence. Co-transfecting C2C12 myoblasts with ms1 promoter/luciferase reporters and myogenic regulatory factor (MRF over-expression plasmids revealed specific sensitivity of the ms1 promoter to MyoD. Subsequent mutagenesis and EMSA analysis demonstrated specific targeting of MyoD at two distinct E-Boxes (E1 and E2 within identified evolutionary conserved regions (ECRs, α and β. Chromatin immunoprecipitation (ChIP analysis indicates that co-ordinated binding of MyoD at E-Boxes located within ECRs α and β correlates with the temporal induction in ms1 mRNA. Conclusion These findings suggest that the tissue specific and differentiation dependent up-regulation in ms1 mRNA is mediated by temporal binding of MyoD at distinct evolutionary conserved E-Boxes within the ms1 5' upstream sequence. We believe, through its activation of ms1, this is the first study to demonstrate a direct link between MyoD activity and SRF transcriptional signalling, with clear implications for the understanding of muscle determination

  7. Comparative thermodynamic studies on substrate and product binding of O-Acetylserine Sulfhydrylase reveals two different ligand recognition modes†

    Directory of Open Access Journals (Sweden)

    Kumaran Sangaralingam

    2011-06-01

    Full Text Available Abstract Background The importance of understanding the detailed mechanism of cysteine biosynthesis in bacteria is underscored by the fact that cysteine is the only sulfur donor for all cellular components containing reduced sulfur. O-acetylserine sulfhydrylase (OASS catalyzes this crucial last step in the cysteine biosynthesis and has been recognized as an important gene for the survival and virulence of pathogenic bacteria. Structural and kinetic studies have contributed to the understanding of mechanistic aspects of OASS, but details of ligand recognition features of OASS are not available. In the absence of any detailed study on the energetics of ligand binding, we have studied the thermodynamics of OASS from Salmonella typhimurium (StOASS, Haemophilus influenzae (HiOASS, and Mycobacterium tuberculosis (MtOASS binding to their substrate O-acetylserine (OAS, substrate analogue (methionine, and product (cysteine. Results Ligand binding properties of three OASS enzymes are studied under defined solution conditions. Both substrate and product binding is an exothermic reaction, but their thermodynamic signatures are very different. Cysteine binding to OASS shows that both enthalpy and entropy contribute significantly to the binding free energy at all temperatures (10-30°C examined. The analyses of interaction between OASS with OAS (substrate or methionine (substrate analogue revealed a completely different mode of binding. Binding of both OAS and methionine to OASS is dominated by a favorable entropy change, with minor contribution from enthalpy change (ΔHSt-Met = -1.5 ± 0.1 kJ/mol; TΔSSt-Met = 8.2 kJ/mol at 20°C. Our salt dependent ligand binding studies indicate that methionine binding affinity is more sensitive to [NaCl] as compared to cysteine affinity. Conclusions We show that OASS from three different pathogenic bacteria bind substrate and product through two different mechanisms. Results indicate that predominantly entropy driven

  8. rVISTA for Comparative Sequence-Based Discovery of Functional Transcription Factor Binding Sites

    Energy Technology Data Exchange (ETDEWEB)

    Loots, Gabriela G.; Ovcharenko, Ivan; Pachter, Lior; Dubchak, Inna; Rubin, Edward M.

    2002-03-08

    Identifying transcriptional regulatory elements represents a significant challenge in annotating the genomes of higher vertebrates. We have developed a computational tool, rVISTA, for high-throughput discovery of cis-regulatory elements that combines transcription factor binding site prediction and the analysis of inter-species sequence conservation. Here, we illustrate the ability of rVISTA to identify true transcription factor binding sites through the analysis of AP-1 and NFAT binding sites in the 1 Mb well-annotated cytokine gene cluster1 (Hs5q31; Mm11). The exploitation of orthologous human-mouse data set resulted in the elimination of 95 percent of the 38,000 binding sites predicted upon analysis of the human sequence alone, while it identified 87 percent of the experimentally verified binding sites in this region.

  9. A comparative study of density functional and density functional tight binding calculations of defects in graphene

    Energy Technology Data Exchange (ETDEWEB)

    Zobelli, Alberto [Laboratoire de Physique des Solides, Univ. Paris Sud, CNRS UMR, Orsay (France); Ivanovskaya, Viktoria; Wagner, Philipp; Yaya, Abu; Ewels, Chris P. [Institut des Materiaux Jean Rouxel (IMN), CNRS UMR, University of Nantes (France); Suarez-Martinez, Irene [Nanochemistry Research Institute, Curtin University of Technology, Perth, Western Australia (Australia)

    2012-02-15

    The density functional tight binding approach (DFTB) is well adapted for the study of point and line defects in graphene based systems. After briefly reviewing the use of DFTB in this area, we present a comparative study of defect structures, energies, and dynamics between DFTB results obtained using the dftb+ code, and density functional results using the localized Gaussian orbital code, AIMPRO. DFTB accurately reproduces structures and energies for a range of point defect structures such as vacancies and Stone-Wales defects in graphene, as well as various unfunctionalized and hydroxylated graphene sheet edges. Migration barriers for the vacancy and Stone-Wales defect formation barriers are accurately reproduced using a nudged elastic band approach. Finally we explore the potential for dynamic defect simulations using DFTB, taking as an example electron irradiation damage in graphene. DFTB-MD derived sputtering energy threshold map for a carbon atom in a graphene plane. (Copyright copyright 2012 WILEY-VCH Verlag GmbH and Co. KGaA, Weinheim)

  10. Model-based Comparative Prediction of Transcription-Factor Binding Motifs in Anabolic Responses in Bone

    Institute of Scientific and Technical Information of China (English)

    Andy; B.; Chen; Kazunori; Hamamura; Guohua; Wang; Weirong; Xing; Subburaman; Mohan; Hiroki; Yokota; Yunlong; Liu

    2007-01-01

    Understanding the regulatory mechanism that controls the alteration of global gene expression patterns continues to be a challenging task in computational biology. We previously developed an ant algorithm, a biologically-inspired computational technique for microarray data, and predicted putative transcription-factor binding motifs (TFBMs) through mimicking interactive behaviors of natural ants. Here we extended the algorithm into a set of web-based software, Ant Modeler, and applied it to investigate the transcriptional mechanism underlying bone formation. Mechanical loading and administration of bone morphogenic proteins (BMPs) are two known treatments to strengthen bone. We addressed a question: Is there any TFBM that stimulates both "anabolic responses of mechanical loading" and "BMP-mediated osteogenic signaling"? Although there is no significant overlap among genes in the two responses, a comparative model-based analysis suggests that the two independent osteogenic processes employ common TFBMs, such as a stress responsive element and a motif for peroxisome proliferator-activated recep- tor (PPAR). The post-modeling in vitro analysis using mouse osteoblast cells sup- ported involvements of the predicted TFBMs such as PPAR, Ikaros 3, and LMO2 in response to mechanical loading. Taken together, the results would be useful to derive a set of testable hypotheses and examine the role of specific regulators in complex transcriptional control of bone formation.

  11. Identification and comparative expression analysis of odorant binding protein genes in the tobacco cutworm Spodoptera litura.

    Science.gov (United States)

    Gu, Shao-Hua; Zhou, Jing-Jiang; Gao, Shang; Wang, Da-Hai; Li, Xian-Chun; Guo, Yu-Yuan; Zhang, Yong-Jun

    2015-01-01

    Insect odorant binding proteins (OBPs) are thought to involve in insects' olfaction perception. In the present study, we identified 38 OBP genes from the antennal transcriptomes of Spodoptera litura. Tissue expression profiles analysis revealed that 17 of the 38 SlitOBP transcripts were uniquely or primarily expressed in the antennae of both sexes, suggesting their putative role in chemoreception. The RPKM value analysis revealed that seven OBPs (SlitPBP1-3, SlitGOBP1-2, SlitOBP3 and SlitOBP5) are highly abundant in male and female antennae. Most S. litura antennal unigenes had high homology with Lepidoptera insects, especially genes of the genus Spodoptera. Phylogenetic analysis of the Lepidoptera OBPs demonstrated that the OBP genes from the genus Spodoptera (S. litura, Spodoptera littoralis and Spodoptera exigua) had a relatively close evolutionary relationship. Some regular patterns and key conserved motifs of OBPs in genus Spodoptera are identified by MEME, and their putative roles in detecting odorants are discussed here. The motif-patterns between Lepidoptera OBPs and CSPs are also compared. The SlitOBPs identified here provide a starting point to facilitate functional studies of insect OBPs at the molecular level both in vivo and in vitro. PMID:26346731

  12. Comparative Analysis of Regulatory Motif Discovery Tools for Transcription Factor Binding Sites

    Institute of Scientific and Technical Information of China (English)

    2007-01-01

    In the post-genomic era, identification of specific regulatory motifs or transcription factor binding sites (TFBSs) in non-coding DNA sequences, which is essential to elucidate transcriptional regulatory networks, has emerged as an obstacle that frustrates many researchers. Consequently, numerous motif discovery tools and correlated databases have been applied to solving this problem. However, these existing methods, based on different computational algorithms, show diverse motif prediction efficiency in non-coding DNA sequences. Therefore, understanding the similarities and differences of computational algorithms and enriching the motif discovery literatures are important for users to choose the most appropriate one among the online available tools. Moreover, there still lacks credible criterion to assess motif discovery tools and instructions for researchers to choose the best according to their own projects. Thus integration of the related resources might be a good approach to improve accuracy of the application. Recent studies integrate regulatory motif discovery tools with experimental methods to offer a complementary approach for researchers, and also provide a much-needed model for current researches on transcriptional regulatory networks. Here we present a comparative analysis of regulatory motif discovery tools for TFBSs.

  13. β-lactoglobulin's conformational requirements for ligand binding at the calyx and the dimer interphase: a flexible docking study.

    Directory of Open Access Journals (Sweden)

    Lenin Domínguez-Ramírez

    Full Text Available β-lactoglobulin (BLG is an abundant milk protein relevant for industry and biotechnology, due significantly to its ability to bind a wide range of polar and apolar ligands. While hydrophobic ligand sites are known, sites for hydrophilic ligands such as the prevalent milk sugar, lactose, remain undetermined. Through the use of molecular docking we first, analyzed the known fatty acid binding sites in order to dissect their atomistic determinants and second, predicted the interaction sites for lactose with monomeric and dimeric BLG. We validated our approach against BLG structures co-crystallized with ligands and report a computational setup with a reduced number of flexible residues that is able to reproduce experimental results with high precision. Blind dockings with and without flexible side chains on BLG showed that: i 13 experimentally-determined ligands fit the calyx requiring minimal movement of up to 7 residues out of the 23 that constitute this binding site. ii Lactose does not bind the calyx despite conformational flexibility, but binds the dimer interface and an alternate Site C. iii Results point to a probable lactolation site in the BLG dimer interface, at K141, consistent with previous biochemical findings. In contrast, no accessible lysines are found near Site C. iv lactose forms hydrogen bonds with residues from both monomers stabilizing the dimer through a claw-like structure. Overall, these results improve our understanding of BLG's binding sites, importantly narrowing down the calyx residues that control ligand binding. Moreover, our results emphasize the importance of the dimer interface as an insufficiently explored, biologically relevant binding site of particular importance for hydrophilic ligands. Furthermore our analyses suggest that BLG is a robust scaffold for multiple ligand-binding, suitable for protein design, and advance our molecular understanding of its ligand sites to a point that allows manipulation to control

  14. A comparative analysis on the binding characteristics of various mammalian albumins towards a multitherapeutic agent, pinostrobin.

    Science.gov (United States)

    Feroz, Shevin R; Sumi, Rumana A; Malek, Sri N A; Tayyab, Saad

    2015-01-01

    The interaction of pinostrobin (PS), a multitherapeutic agent with serum albumins of various mammalian species namely, goat, bovine, human, porcine, rabbit, sheep and dog was investigated using fluorescence quench titration and competitive drug displacement experiments. Analysis of the intrinsic fluorescence quenching data revealed values of the association constant, K(a) in the range of 1.49 - 6.12 × 10(4) M(-1), with 1:1 binding stoichiometry. Based on the PS-albumin binding characteristics, these albumins were grouped into two classes. Ligand displacement studies using warfarin as the site I marker ligand correlated well with the binding data. Albumins from goat and bovine were found to be closely similar to human albumin on the basis of PS binding characteristics. PMID:25519455

  15. Stable MCC binding to the APC/C is required for a functional spindle assembly checkpoint

    DEFF Research Database (Denmark)

    Hein, Jamin B; Nilsson, Jakob

    2014-01-01

    The spindle assembly checkpoint (SAC) delays progression into anaphase until all chromosomes have aligned on the metaphase plate by inhibiting Cdc20, the mitotic co-activator of the APC/C. Mad2 and BubR1 bind and inhibit Cdc20, thereby forming the mitotic checkpoint complex (MCC), which can bind...

  16. A comparative analysis on the binding characteristics of various mammalian albumins towards a multitherapeutic agent, pinostrobin

    OpenAIRE

    Feroz, Shevin R.; SUMI, Rumana A.; Sri N A Malek; Tayyab, Saad

    2014-01-01

    The interaction of pinostrobin (PS), a multitherapeutic agent with serum albumins of various mammalian species namely, goat, bovine, human, porcine, rabbit, sheep and dog was investigated using fluorescence quench titration and competitive drug displacement experiments. Analysis of the intrinsic fluorescence quenching data revealed values of the association constant, Ka in the range of 1.49 – 6.12 × 104 M−1, with 1:1 binding stoichiometry. Based on the PS–albumin binding characteristics, thes...

  17. Sequence and structural requirements for high-affinity DNA binding by the WT1 gene product.

    OpenAIRE

    Nakagama, H; Heinrich, G.; Pelletier, J; Housman, D E

    1995-01-01

    The Wilms' tumor suppressor gene, WT1, encodes a zinc finger polypeptide which plays a key role regulating cell growth and differentiation in the urogenital system. Using the whole-genome PCR approach, we searched murine genomic DNA for high-affinity WT1 binding sites and identified a 10-bp motif 5'GCGTGGGAGT3' which we term WTE). The WTE motif is similar to the consensus binding sequence 5'GCG(G/T)GGGCG3' recognized by EGR-1 and is also suggested to function as a binding site for WT1, settin...

  18. Alteration of methotrexate binding to human serum albumin induced by oxidative stress. Spectroscopic comparative study

    Science.gov (United States)

    Maciążek-Jurczyk, M.; Sułkowska, A.; Równicka-Zubik, J.

    2016-01-01

    Changes of oxidative modified albumin conformation by comparison of non-modified (HSA) and modified (oHSA) human serum albumin absorption spectra, Red Edge Excitation Shift (REES) effect and fluorescence synchronous spectra were investigated. Studies of absorption spectra indicated that changes in the value of absorbance associated with spectral changes in the region from 200 to 250 nm involve structural alterations related to variations in peptide backbone conformation. Analysis of the REES effect allowed for the observation of changes caused by oxidation in the region of the hydrophobic pocket containing the tryptophanyl residue. Synchronous fluorescence spectroscopy confirmed changes of the position of the tryptophanyl and tyrosil residues fluorescent band. Effect of oxidative stress on binding of methotrexate (MTX) was investigated by spectrofluorescence, UV-VIS and 1HNMR spectroscopy. MTX caused the fluorescence quenching of non-modified (HSA) and modified (oHSA) human serum albumin molecule. The values of binding constants, Hill's coefficients and a number of binding sites in the protein molecule in the high affinity binding site were calculated for the binary MTX-HSA and MTX-oHSA systems. For these systems, qualitative analysis in the low affinity binding sites was performed with the use of the 1HNMR technique.

  19. Azaflavones compared to flavones as ligands to the benzodiazepine binding site of brain GABAA receptors

    DEFF Research Database (Denmark)

    Nilsson, Jakob; Nielsen, Elsebet Østergaard; Liljefors, Tommy;

    2008-01-01

    A series of azaflavone derivatives and analogues were prepared and evaluated for their affinity to the benzodiazepine binding site of the GABA(A) receptor, and compared to their flavone counterparts. Three of the compounds, the azaflavones 9 and 12 as well as the new flavone 13, were also assayed...

  20. Calcium-binding capacity of centrin2 is required for linear POC5 assembly but not for nucleotide excision repair.

    Directory of Open Access Journals (Sweden)

    Tiago J Dantas

    Full Text Available Centrosomes, the principal microtubule-organising centres in animal cells, contain centrins, small, conserved calcium-binding proteins unique to eukaryotes. Centrin2 binds to xeroderma pigmentosum group C protein (XPC, stabilising it, and its presence slightly increases nucleotide excision repair (NER activity in vitro. In previous work, we deleted all three centrin isoforms present in chicken DT40 cells and observed delayed repair of UV-induced DNA lesions, but no centrosome abnormalities. Here, we explore how centrin2 controls NER. In the centrin null cells, we expressed centrin2 mutants that cannot bind calcium or that lack sites for phosphorylation by regulatory kinases. Expression of any of these mutants restored the UV sensitivity of centrin null cells to normal as effectively as expression of wild-type centrin. However, calcium-binding-deficient and T118A mutants showed greatly compromised localisation to centrosomes. XPC recruitment to laser-induced UV-like lesions was only slightly slower in centrin-deficient cells than in controls, and levels of XPC and its partner HRAD23B were unaffected by centrin deficiency. Interestingly, we found that overexpression of the centrin interactor POC5 leads to the assembly of linear, centrin-dependent structures that recruit other centrosomal proteins such as PCM-1 and NEDD1. Together, these observations suggest that assembly of centrins into complex structures requires calcium binding capacity, but that such assembly is not required for centrin activity in NER.

  1. The RNA recognition motif domains of RBM5 are required for RNA binding and cancer cell proliferation inhibition

    International Nuclear Information System (INIS)

    Highlights: • RNA recognition motif domains of RBM5 are essential for cell proliferation inhibition. • RNA recognition motif domains of RBM5 are essential for apoptosis induction. • RNA recognition motif domains of RBM5 are essential for RNA binding. • RNA recognition motif domains of RBM5 are essential for caspase-2 alternative splicing. - Abstract: RBM5 is a known putative tumor suppressor gene that has been shown to function in cell growth inhibition by modulating apoptosis. RBM5 also plays a critical role in alternative splicing as an RNA binding protein. However, it is still unclear which domains of RBM5 are required for RNA binding and related functional activities. We hypothesized the two putative RNA recognition motif (RRM) domains of RBM5 spanning from amino acids 98–178 and 231–315 are essential for RBM5-mediated cell growth inhibition, apoptosis regulation, and RNA binding. To investigate this hypothesis, we evaluated the activities of the wide-type and mutant RBM5 gene transfer in low-RBM5 expressing A549 cells. We found that, unlike wild-type RBM5 (RBM5-wt), a RBM5 mutant lacking the two RRM domains (RBM5-ΔRRM), is unable to bind RNA, has compromised caspase-2 alternative splicing activity, lacks cell proliferation inhibition and apoptosis induction function in A549 cells. These data provide direct evidence that the two RRM domains of RBM5 are required for RNA binding and the RNA binding activity of RBM5 contributes to its function on apoptosis induction and cell growth inhibition

  2. The RNA recognition motif domains of RBM5 are required for RNA binding and cancer cell proliferation inhibition

    Energy Technology Data Exchange (ETDEWEB)

    Zhang, Lei [Key Laboratory of Bioresources and Ecoenvironment (Ministry of Education), College of Life Sciences, Sichuan University, Chengdu (China); Zhang, Qing [Jiangsu Key Laboratory of Biological Cancer Therapy, Xuzhou Medical College, Xuzhou 221002 (China); Yang, Yu [Key Laboratory of Bioresources and Ecoenvironment (Ministry of Education), College of Life Sciences, Sichuan University, Chengdu (China); Wu, Chuanfang, E-mail: wuchuanfangsichuan@gmail.com [Key Laboratory of Bioresources and Ecoenvironment (Ministry of Education), College of Life Sciences, Sichuan University, Chengdu (China)

    2014-02-14

    Highlights: • RNA recognition motif domains of RBM5 are essential for cell proliferation inhibition. • RNA recognition motif domains of RBM5 are essential for apoptosis induction. • RNA recognition motif domains of RBM5 are essential for RNA binding. • RNA recognition motif domains of RBM5 are essential for caspase-2 alternative splicing. - Abstract: RBM5 is a known putative tumor suppressor gene that has been shown to function in cell growth inhibition by modulating apoptosis. RBM5 also plays a critical role in alternative splicing as an RNA binding protein. However, it is still unclear which domains of RBM5 are required for RNA binding and related functional activities. We hypothesized the two putative RNA recognition motif (RRM) domains of RBM5 spanning from amino acids 98–178 and 231–315 are essential for RBM5-mediated cell growth inhibition, apoptosis regulation, and RNA binding. To investigate this hypothesis, we evaluated the activities of the wide-type and mutant RBM5 gene transfer in low-RBM5 expressing A549 cells. We found that, unlike wild-type RBM5 (RBM5-wt), a RBM5 mutant lacking the two RRM domains (RBM5-ΔRRM), is unable to bind RNA, has compromised caspase-2 alternative splicing activity, lacks cell proliferation inhibition and apoptosis induction function in A549 cells. These data provide direct evidence that the two RRM domains of RBM5 are required for RNA binding and the RNA binding activity of RBM5 contributes to its function on apoptosis induction and cell growth inhibition.

  3. Comparative modelling of human β tubulin isotypes and implications for drug binding

    Science.gov (United States)

    Torin Huzil, J.; Ludueña, Richard F.; Tuszynski, Jack

    2006-02-01

    The protein tubulin is a target for several anti-mitotic drugs, which affect microtubule dynamics, ultimately leading to cell cycle arrest and apoptosis. Many of these drugs, including the taxanes and Vinca alkaloids, are currently used clinically in the treatment of several types of cancer. Another tubulin binding drug, colchicine, although too toxic to be used as a chemotherapeutic agent, is commonly used for the treatment of gout. The main disadvantage that all of these drugs share is that they bind tubulin indiscriminately, leading to the death of both cancerous and healthy cells. However, the broad cellular distribution of several tubulin isotypes provides a platform upon which to construct novel chemotherapeutic drugs that could differentiate between different cell types, reducing the undesirable side effects associated with current chemotherapeutic treatments. Here, we report an analysis of ten human β tubulin isotypes and discuss differences within each of the previously characterized paclitaxel, colchicine and vinblastine binding sites.

  4. Comparative study of the binding of trypsin to caffeine and theophylline by spectrofluorimetry

    Energy Technology Data Exchange (ETDEWEB)

    Wang, Ruiyong, E-mail: wangry@zzu.edu.cn [Department of Chemistry, Zhengzhou University, Zhengzhou 450001 (China); Kang, Xiaohui [Department of Chemistry, Zhengzhou University, Zhengzhou 450001 (China); Wang, Ruiqiang [The First Affiliated Hospital of Zhengzhou University, Zhengzhou 450052 (China); Wang, Rui; Dou, Huanjing; Wu, Jing; Song, Chuanjun [Department of Chemistry, Zhengzhou University, Zhengzhou 450001 (China); Chang, Junbiao, E-mail: changjunbiao@zzu.edu.cn [Department of Chemistry, Zhengzhou University, Zhengzhou 450001 (China)

    2013-06-15

    The interactions between trypsin and caffeine/theophylline were investigated by fluorescence spectroscopy, UV–visible absorption spectroscopy, resonance light scattering and synchronous fluorescence spectroscopy under mimic physiological conditions. The results revealed that the fluorescence quenching of trypsin by caffeine and theophylline was the result of the formed complex of caffeine–trypsin and theophylline–trypsin. The binding constants and thermodynamic parameters at three different temperatures were obtained. The hydrophobic interaction was the predominant intermolecular forces to stabilize the complex. Results showed that caffeine was the stronger quencher and bound to trypsin with higher affinity than theophylline. -- Highlights: ► The fluorescence of trypsin can be quenched by caffeine or theophylline via hydrophobic contacts. ► Caffeine binds to trypsin with higher affinity than theophylline. ► The influence of molecular structure on the binding aspects is reported.

  5. Genetic evidence that outer membrane binding of starch is required for starch utilization by Bacteroides thetaiotaomicron.

    OpenAIRE

    Anderson, K.L.; Salyers, A A

    1989-01-01

    Mutagenesis of Bacteroides thetaiotaomicron with the transposon Tn4351 produced five classes of mutants that were not able to grow on amylose or amylopectin. These classes of mutants differed in their ability to grow on maltoheptaose (G7) and in the level of starch-degrading enzymes produced when bacteria were grown on maltose. All of the mutants were deficient in starch binding. Since one class of mutants retained normal levels of starch-degrading enzymes, this indicates that binding of the ...

  6. Efficient translation of alfamovirus RNAs requires the binding of coat protein dimers to the 3' termini of the viral RNAs.

    Science.gov (United States)

    Neeleman, Lyda; Linthorst, Huub J M; Bol, John F

    2004-01-01

    The coat protein (CP) of Alfalfa mosaic virus (AMV) is required to initiate infection by the viral tripartite RNA genome whereas infection by the tripartite Brome mosaic virus (BMV) genome is independent of CP. AMV CP stimulates translation of AMV RNA in vivo 50- to 100-fold. The 3' untranslated region (UTR) of the AMV subgenomic CP messenger RNA 4 contains at least two CP binding sites. A CP binding site in the 3'-terminal 112 nucleotides of RNA 4 was found to be required for efficient translation of the RNA whereas an upstream binding site was not. Binding of CP to the AMV 3' UTR induces a conformational change of the RNA but this change alone was not sufficient to stimulate translation. CP mutant R17A is unable to bind to the 3' UTR and translation in vivo of RNA 4 encoding this mutant occurs at undetectable levels. Replacement of the 3' UTR of this mutant RNA 4 by the 3' UTR of BMV RNA 4 restored translation of R17A-CP to wild-type levels. Apparently, the BMV 3' UTR stimulates translation independently of CP. AMV CP mutant N199 is defective in the formation of CP dimers and did not stimulate translation of RNA 4 in vivo although the mutant CP did bind to the 3' UTR. The finding that N199-CP does not promote AMV infection corroborates the notion that the requirement of CP in the inoculum reflects its role in translation of the viral RNAs. PMID:14718638

  7. Comparative study of heparin-binding proteins profile of Murrah buffalo (Bubalus bubalis semen

    Directory of Open Access Journals (Sweden)

    S. S. Ramteke

    2014-09-01

    Full Text Available Aim: The experiment was conducted to study the total seminal plasma protein (TSPP and heparin-binding proteins (HBPs in relation to initial semen quality of buffalo bull. Materials and Methods: Semen from two Murrah buffalo bulls (bull no. 605 and 790 with mass motility of ≥3+ were used for the study and categorized into three groups (Group I- Mass motility 3+, Group II- Mass motility 4+ and Group III- Mass motility 5+. Seminal plasma from semen was separated by centrifugation. HBPs was isolated and purified from heparin-agarose affinity column by modified elution buffer. TSPP and isolated HBPs concentration was estimated by Lowry’s method. The purified HBPs were resolved on Sodium dodecyl sulfate polyacrylamide gel electrophoresis to check the protein profile of two bulls. Results: The mean values of TSPP concentrations in bull no. 605 and 790 in Group I, II and III were 30.64±0.12, 31.66±0.09, 32.53±0.19 and 28.51±0.09, 29.49±0.15, 30.45±0.17 mg/mL, respectively. The mean values of HBPs concentrations in bull no. 605 and 790 in Group I, II and III were 3.11±0.07, 3.32±0.06, 3.46±0.08 and 2.51±0.08, 2.91±0.05, 3.10±0.03 mg/mL, respectively. Both the values of TSPP and HBPs were significantly higher (p<0.01 in bull no. 605 when compared to 790 in all the three groups. 31 kDa HBP was more intensely present in bull no. 605, thus may indicate its superiority over bull no. 790 in relation to fertility potential. Conclusion: TSPP and HBPs shows variation in concentration with respect to initial semen quality. Furthermore, presence of fertility related 31 kDa HBPs in one of the bull may be an indication of high fertility of a bull. In future, in-vivo and in-vitro correlative study on larger basis is needed for the establishment of fertility-related HBPs in semen which might establish criteria for selection of buffalo bull with high fertility potential.

  8. Randomized crossover study comparing the phosphate-binding efficacy of calcium ketoglutarate versus calcium carbonate in patients on chronic hemodialysis.

    Science.gov (United States)

    Bro, S; Rasmussen, R A; Handberg, J; Olgaard, K; Feldt-Rasmussen, B

    1998-02-01

    The objective of the study was to evaluate the phosphate-binding efficacy, side effects, and cost of therapy of calcium ketoglutarate granulate as compared with calcium carbonate tablets in patients on chronic hemodialysis. The study design used was a randomized, crossover open trial, and the main outcome measurements were plasma ionized calcium levels, plasma phosphate levels, plasma intact parathyroid hormone (PTH) levels, requirements for supplemental aluminum-aminoacetate therapy, patient tolerance, and cost of therapy. Nineteen patients on chronic hemodialysis were treated with a dialysate calcium concentration of 1.25 mmol/L and a fixed alfacalcidol dose for at least 2 months. All had previously tolerated therapy with calcium carbonate. Of the 19 patients included, 10 completed both treatment arms. After 12 weeks of therapy, the mean (+/-SEM) plasma ionized calcium level was significantly lower in the ketoglutarate arm compared with the calcium carbonate arm (4.8+/-0.1 mg/dL v 5.2+/-0.1 mg/dL; P = 0.004), whereas the mean plasma phosphate (4.5+/-0.3 mg/dL v 5.1+/-0.1 mg/dL) and PTH levels (266+/-125 pg/mL v 301+/-148 pg/mL) did not differ significantly between the two treatment arms. Supplemental aluminum-aminoacetate was not required during calcium ketoglutarate treatment, while two patients needed this supplement when treated with calcium carbonate. Five of 17 (29%) patients were withdrawn from calcium ketoglutarate therapy within 1 to 2 weeks due to intolerance (anorexia, vomiting, diarrhea, general uneasiness), whereas the remaining 12 patients did not experience any side effects at all. The five patients with calcium ketoglutarate intolerance all had pre-existing gastrointestinal symptoms; four of them had received treatment with cimetidine or omeprazol before inclusion into the study. Calculations based on median doses after 12 weeks showed that the cost of the therapy in Denmark was 10 times higher for calcium ketoglutarate compared with calcium

  9. Analytical methods to determine the comparative DNA binding studies of curcumin-Cu(II) complexes

    Science.gov (United States)

    Rajesh, Jegathalaprathaban; Rajasekaran, Marichamy; Rajagopal, Gurusamy; Athappan, Periakaruppan

    2012-11-01

    DNA interaction studies of two mononuclear [1:1(1); 1:2(2)] copper(II) complexes of curcumin have been studied. The interaction of these complexes with CT-DNA has been explored by physical methods to propose modes of DNA binding of the complexes. Absorption spectral titrations of complex 1 with CT-DNA shows a red-shift of 3 nm with the DNA binding affinity of Kb, 5.21 × 104 M-1 that are higher than that obtained for 2 (red-shift, 2 nm; Kb, 1.73 × 104 M-1) reveal that the binding occurs in grooves as a result of the interaction is via exterior phosphates. The CD spectra of these Cu(II) complexes show a red shift of 3-10 nm in the positive band with increase in intensities. This spectral change of induced CD due to the hydrophobic interaction of copper complexes with DNA is the characteristic of B to A conformational change. The EB displacement assay also reveals the same trend as observed in UV-Vis spectral titration. The addition of complexes 1 and 2 to the DNA bound ethidium bromide (EB) solutions causes an obvious reduction in emission intensities indicating that these complexes competitively bind to DNA with EB. The positive shift of both the Epc and E0' accompanied by reduction of peak currents in differential pulse voltammogram (DPV), upon adding different concentrations of DNA to the metal complexes, are obviously in favor of strong binding to DNA. The super coiled plasmid pUC18 DNA cleavage ability of Cu(II) complexes in the presence of reducing agent reveals the single strand DNA cleavage (ssDNA) is observed. The hydroxyl radical (HOrad ) and the singlet oxygen are believed to be the reactive species responsible for the cleavage.

  10. Stat2 binding to the interferon-alpha receptor 2 subunit is not required for interferon-alpha signaling.

    Science.gov (United States)

    Nguyen, Vinh-Phúc; Saleh, Abu Z M; Arch, Allison E; Yan, Hai; Piazza, Flavia; Kim, John; Krolewski, John J

    2002-03-22

    The interferon-alpha (IFNalpha) receptor consists of two subunits, the IFNalpha receptor 1 (IFNaR1) and 2 (IFNaR2) chains. Following ligand binding, IFNaR1 is phosphorylated on tyrosine 466, and this site recruits Stat2 via its SH2 domain. In contrast, IFNaR2 binds Stat2 constitutively. In this study we have characterized the Stat2-IFNaR2 interaction and examined its role in IFNalpha signaling. Stat2 binds the major IFNaR2 protein but not a variant containing a shorter cytoplasmic domain. The interaction does not require a STAT SH2 domain. Both tyrosine-phosphorylated and non-phosphorylated Stat2 bind IFNaR2 in vitro; however, relatively little phosphorylated Stat2 associates with IFNaR2 in vivo. In vitro binding assays defined IFNaR2 residues 418-444 as the minimal interaction domain and site-specific mutation of conserved acidic residues within this domain disrupted in vitro and in vivo binding. An IFNaR2 construct carrying these mutations was either (i) overexpressed in 293T cells or (ii) used to complement IFNaR2-deficient U5A cells. Unexpectedly, the activity of an IFNalpha-dependent reporter gene was not reduced but, instead, was enhanced up to 2-fold. This suggests that this particular IFNaR2-Stat2 interaction is not required for IFNalpha signaling, but might act to negatively inhibit signaling. Finally, a doubly truncated recombinant fragment of Stat2, spanning residues 136-702, associated with IFNaR2 in vitro, indicating that the interaction with IFNaR2 is direct and occurs in a central region of Stat2 marked by a hydrophobic core.

  11. A Comparative Reverse Docking Strategy to Identify Potential Antineoplastic Targets of Tea Functional Components and Binding Mode

    Directory of Open Access Journals (Sweden)

    Rong Zheng

    2011-08-01

    Full Text Available The main functional components of green tea, such as epigallocatechin gallate (EGCG, epigallocatechin (EGC, epicatechin gallate (ECG and epicatechin (EC, are found to have a broad antineoplastic activity. The discovery of their targets plays an important role in revealing the antineoplastic mechanism. Therefore, to identify potential target proteins for tea polyphenols, we have taken a comparative virtual screening approach using two reverse docking systems, one based on Autodock software and the other on Tarfisdock. Two separate in silico workflows were implemented to derive a set of target proteins related to human diseases and ranked by the binding energy score. Several conventional clinically important proteins with anti-tumor effects are screened out from the PDTD protein database as the potential receptors by both procedures. To further analyze the validity of docking results, we study the binding mode of EGCG and the potential target protein Leukotriene A4 hydrolase in detail. We indicate that interactions mediated by electrostatic and hydrogen bond play a key role in ligand binding. EGCG binds to the enzyme with certain orientation and conformation that is suitable for nucleophilic attacks by several electrical residues inside the enzyme’s activity cavity. This study provides useful information for studying the antitumor mechanism of tea’s functional components. The comparative reverse docking strategy presented generates a tractable set of antineoplastic proteins for future experimental validation as drug targets against tumors.

  12. SPIC: A novel similarity metric for comparing transcription factor binding site motifs based on information contents

    OpenAIRE

    Zhang, Shaoqiang; Zhou, Xiguo; Du, Chuanbin; Su, Zhengchang

    2013-01-01

    Background Discovering transcription factor binding sites (TFBS) is one of primary challenges to decipher complex gene regulatory networks encrypted in a genome. A set of short DNA sequences identified by a transcription factor (TF) is known as a motif, which can be expressed accurately in matrix form such as a position-specific scoring matrix (PSSM) and a position frequency matrix. Very frequently, we need to query a motif in a database of motifs by seeking its similar motifs, merge similar ...

  13. Cyanide binding to human plasma heme-hemopexin: A comparative study

    Energy Technology Data Exchange (ETDEWEB)

    Ascenzi, Paolo, E-mail: ascenzi@uniroma3.it [Laboratorio Interdipartimentale di Microscopia Elettronica, Universita Roma Tre, Roma (Italy); Istituto Nazionale di Biostrutture e Biosistemi, Roma (Italy); Leboffe, Loris [Istituto Nazionale di Biostrutture e Biosistemi, Roma (Italy); Polticelli, Fabio [Dipartimento di Biologia, Universita Roma Tre, Roma (Italy)

    2012-11-16

    Highlights: Black-Right-Pointing-Pointer Cyanide binding to ferric HHPX-heme-Fe. Black-Right-Pointing-Pointer Cyanide binding to ferrous HHPX-heme-Fe. Black-Right-Pointing-Pointer Dithionite-mediated reduction of ferric HHPX-heme-Fe-cyanide. Black-Right-Pointing-Pointer Cyanide binding to HHPX-heme-Fe is limited by ligand deprotonation. Black-Right-Pointing-Pointer Cyanide dissociation from HHPX-heme-Fe-cyanide is limited by ligand protonation. -- Abstract: Hemopexin (HPX) displays a pivotal role in heme scavenging and delivery to the liver. In turn, heme-Fe-hemopexin (HPX-heme-Fe) displays heme-based spectroscopic and reactivity properties. Here, kinetics and thermodynamics of cyanide binding to ferric and ferrous hexa-coordinate human plasma HPX-heme-Fe (HHPX-heme-Fe(III) and HHPX-heme-Fe(II), respectively), and for the dithionite-mediated reduction of the HHPX-heme-Fe(III)-cyanide complex, at pH 7.4 and 20.0 Degree-Sign C, are reported. Values of thermodynamic and kinetic parameters for cyanide binding to HHPX-heme-Fe(III) and HHPX-heme-Fe(II) are K = (4.1 {+-} 0.4) Multiplication-Sign 10{sup -6} M, k{sub on} = (6.9 {+-} 0.5) Multiplication-Sign 10{sup 1} M{sup -1} s{sup -1}, and k{sub off} = 2.8 Multiplication-Sign 10{sup -4} s{sup -1}; and H = (6 {+-} 1) Multiplication-Sign 10{sup -1} M, h{sub on} = 1.2 Multiplication-Sign 10{sup -1} M{sup -1} s{sup -1}, and h{sub off} = (7.1 {+-} 0.8) Multiplication-Sign 10{sup -2} s{sup -1}, respectively. The value of the rate constant for the dithionite-mediated reduction of the HHPX-heme-Fe(III)-cyanide complex is l = 8.9 {+-} 0.8 M{sup -1/2} s{sup -1}. HHPX-heme-Fe reactivity is modulated by proton acceptor/donor amino acid residue(s) (e.g., His236) assisting the deprotonation and protonation of the incoming and outgoing ligand, respectively.

  14. CREB binding protein is required for both short-term and long-term memory formation

    NARCIS (Netherlands)

    Chen, G.; Zou, X.; Watanabe, H.; Deursen, J.M.A. van; Shen, J.

    2010-01-01

    CREB binding protein (CBP) is a transcriptional coactivator with histone acetyltransferase activity. Our prior study suggested that CBP might be a key target of presenilins in the regulation of memory formation and neuronal survival. To elucidate the role of CBP in the adult brain, we generated cond

  15. Binding of more than one Tva800 molecule is required for ASLV-A entry

    Directory of Open Access Journals (Sweden)

    Gray Eleanor R

    2011-11-01

    Full Text Available Abstract Background Understanding the mechanism by which viruses enter their target cell is an essential part of understanding their infectious cycle. Previous studies have focussed on the multiplicity of viral envelope proteins that need to bind to their cognate receptor to initiate entry. Avian sarcoma and leukosis virus Envelope protein (ASLV Env mediates entry via a receptor, Tva, which can be attached to the cell surface either by a phospholipid anchor (Tva800 or a transmembrane domain (Tva950. In these studies, we have now investigated the number of target receptors necessary for entry of ASLV Env-pseudotyped virions. Results Using titration and modelling experiments we provide evidence that binding of more than one receptor, probably two, is needed for entry of virions via Tva800. However, binding of just one Tva950 receptor is sufficient for successful entry. Conclusions The different modes of attachment of Tva800 and Tva950 to the cell membrane have important implications for the utilisation of these proteins as receptors for viral binding and/or uptake.

  16. The acyl-CoA binding protein is required for normal epidermal barrier function in mice

    DEFF Research Database (Denmark)

    Bloksgaard, Maria; Bek, Signe; Marcher, Ann-Britt;

    2012-01-01

    The acyl-CoA binding protein (ACBP) is a 10 kDa intracellular protein expressed in all eukaryotic species. Mice with targeted disruption of Acbp (ACBP(-/-) mice) are viable and fertile but present a visible skin and fur phenotype characterized by greasy fur and development of alopecia and scaling...

  17. Repellent Taxis in Response to Nickel Ion Requires neither Ni2+ Transport nor the Periplasmic NikA Binding Protein▿

    OpenAIRE

    Englert, Derek L.; Adase, Christopher A.; Jayaraman, Arul; Manson, Michael D.

    2010-01-01

    Ni2+ and Co2+ are sensed as repellents by the Escherichia coli Tar chemoreceptor. The periplasmic Ni2+ binding protein, NikA, has been suggested to sense Ni2+. We show here that neither NikA nor the membrane-bound NikB and NikC proteins of the Ni2+ transport system are required for repellent taxis in response to Ni2+.

  18. Comparative genome analysis of cortactin and HS1: the significance of the F-actin binding repeat domain

    Directory of Open Access Journals (Sweden)

    Seggelen Vera

    2005-02-01

    Full Text Available Abstract Background In human carcinomas, overexpression of cortactin correlates with poor prognosis. Cortactin is an F-actin-binding protein involved in cytoskeletal rearrangements and cell migration by promoting actin-related protein (Arp2/3 mediated actin polymerization. It shares a high amino acid sequence and structural similarity to hematopoietic lineage cell-specific protein 1 (HS1 although their functions differ considerable. In this manuscript we describe the genomic organization of these two genes in a variety of species by a combination of cloning and database searches. Based on our analysis, we predict the genesis of the actin-binding repeat domain during evolution. Results Cortactin homologues exist in sponges, worms, shrimps, insects, urochordates, fishes, amphibians, birds and mammalians, whereas HS1 exists in vertebrates only, suggesting that both genes have been derived from an ancestor cortactin gene by duplication. In agreement with this, comparative genome analysis revealed very similar exon-intron structures and sequence homologies, especially over the regions that encode the characteristic highly conserved F-actin-binding repeat domain. Cortactin splice variants affecting this F-actin-binding domain were identified not only in mammalians, but also in amphibians, fishes and birds. In mammalians, cortactin is ubiquitously expressed except in hematopoietic cells, whereas HS1 is mainly expressed in hematopoietic cells. In accordance with their distinct tissue specificity, the putative promoter region of cortactin is different from HS1. Conclusions Comparative analysis of the genomic organization and amino acid sequences of cortactin and HS1 provides inside into their origin and evolution. Our analysis shows that both genes originated from a gene duplication event and subsequently HS1 lost two repeats, whereas cortactin gained one repeat. Our analysis genetically underscores the significance of the F-actin binding domain in

  19. Comparative covalent protein binding of 2,5-hexanedione and 3-acetyl-2,5-hexanedione in the rat.

    Science.gov (United States)

    DeCaprio, Anthony P; Kinney, Elizabeth A; LoPachin, Richard M

    2009-01-01

    2,5-Hexanedione (HD) is the metabolite implicated in n-hexane neurotoxicity. This gamma-diketone reacts with protein lysine amines to form 2,5-dimethylpyrrole adducts. Pyrrole adduction of neurofilaments (NF) and/or other axonal proteins was proposed as a critical step in the neuropathy. While pyrrole adduction is widely accepted as necessary, subsequent pyrrole oxidation, which may result in protein cross-linking, was alternatively postulated as the critical mechanistic step. Previous studies have indicated that 3-acetyl-2,5-HD (AcHD), an analogue that forms pyrroles that do not oxidize, was not neurotoxic in rats. However, relative levels of pyrrole adduction of NF or other axonal proteins were not reported. In the present study, groups of 6 male Wistar rats were given saline, [1,6-(14)C]-HD (3 mmol/kg/d), or [5-(14)C]-AcHD (0.1 mmol/kg/d), i.p. for 21 d. HD- and AcHD-treated rats lost 10% and gained 14% body weight, respectively, compared to a 22% gain for control rats. At termination, HD- and AcHD-treated rats exhibited mean scores of 3.5 and 1.4, respectively, for hindlimb weakness (0-5 scale). Incorporation of radiolabel from HD was 27.8 +/- 3.9, 13.9 +/- 2.6, and 7.8 +/- 0.6 nmol/mg in plasma protein, purified globin, and axonal cytoskeletal proteins, respectively, compared to 0.6 +/- 0.1, 1.6 +/- 0.5, and 1.0 +/- 0.1 for AcHD. Binding of HD to the NF-L, -M, and -H subunit proteins from treated animals was 4-, 24-, and 13-fold higher, respectively, that that of AcHD, indicating differing stoichiometry and patterns of NF adduction for the two diketones. Sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) analysis of globin and NF proteins did not demonstrate protein cross-linking for either diketone at the dose levels and time period examined. These results indicate that that the lack of neurotoxicity previously reported for AcHD may reflect differences in adduct levels at critical axonal target sites rather than an inability to form cross

  20. Mycobacterium tuberculosis universal stress protein Rv2623 regulates bacillary growth by ATP-Binding: requirement for establishing chronic persistent infection.

    Directory of Open Access Journals (Sweden)

    Joshua E Drumm

    2009-05-01

    Full Text Available Tuberculous latency and reactivation play a significant role in the pathogenesis of tuberculosis, yet the mechanisms that regulate these processes remain unclear. The Mycobacterium tuberculosisuniversal stress protein (USP homolog, rv2623, is among the most highly induced genes when the tubercle bacillus is subjected to hypoxia and nitrosative stress, conditions thought to promote latency. Induction of rv2623 also occurs when M. tuberculosis encounters conditions associated with growth arrest, such as the intracellular milieu of macrophages and in the lungs of mice with chronic tuberculosis. Therefore, we tested the hypothesis that Rv2623 regulates tuberculosis latency. We observed that an Rv2623-deficient mutant fails to establish chronic tuberculous infection in guinea pigs and mice, exhibiting a hypervirulence phenotype associated with increased bacterial burden and mortality. Consistent with this in vivo growth-regulatory role, constitutive overexpression of rv2623 attenuates mycobacterial growth in vitro. Biochemical analysis of purified Rv2623 suggested that this mycobacterial USP binds ATP, and the 2.9-A-resolution crystal structure revealed that Rv2623 engages ATP in a novel nucleotide-binding pocket. Structure-guided mutagenesis yielded Rv2623 mutants with reduced ATP-binding capacity. Analysis of mycobacteria overexpressing these mutants revealed that the in vitro growth-inhibitory property of Rv2623 correlates with its ability to bind ATP. Together, the results indicate that i M. tuberculosis Rv2623 regulates mycobacterial growth in vitro and in vivo, and ii Rv2623 is required for the entry of the tubercle bacillus into the chronic phase of infection in the host; in addition, iii Rv2623 binds ATP; and iv the growth-regulatory attribute of this USP is dependent on its ATP-binding activity. We propose that Rv2623 may function as an ATP-dependent signaling intermediate in a pathway that promotes persistent infection.

  1. A viral nuclear noncoding RNA binds re-localized poly(A binding protein and is required for late KSHV gene expression.

    Directory of Open Access Journals (Sweden)

    Sumit Borah

    2011-10-01

    Full Text Available During the lytic phase of infection, the gamma herpesvirus Kaposi's Sarcoma-Associated Herpesvirus (KSHV expresses a highly abundant, 1.1 kb nuclear noncoding RNA of unknown function. We observe that this polyadenylated nuclear (PAN RNA avidly binds host poly(A-binding protein C1 (PABPC1, which normally functions in the cytoplasm to bind the poly(A tails of mRNAs, regulating mRNA stability and translation efficiency. During the lytic phase of KSHV infection, PABPC1 is re-localized to the nucleus as a consequence of expression of the viral shutoff exonuclease (SOX protein; SOX also mediates the host shutoff effect in which host mRNAs are downregulated while viral mRNAs are selectively expressed. We show that whereas PAN RNA is not required for the host shutoff effect or for PABPC1 re-localization, SOX strongly upregulates the levels of PAN RNA in transient transfection experiments. This upregulation is destroyed by the same SOX mutation that ablates the host shutoff effect and PABPC1 nuclear re-localization or by removal of the poly(A tail of PAN. In cells induced into the KSHV lytic phase, depletion of PAN RNA using RNase H-targeting antisense oligonucleotides reveals that it is necessary for the production of late viral proteins from mRNAs that are themselves polyadenylated. Our results add to the repertoire of functions ascribed to long noncoding RNAs and suggest a mechanism of action for nuclear noncoding RNAs in gamma herpesvirus infection.

  2. Transcriptional Activation of sclA by Mga Requires a Distal Binding Site in Streptococcus pyogenes

    OpenAIRE

    Almengor, Audry C.; McIver, Kevin S.

    2004-01-01

    Streptococcus pyogenes (the group A streptococcus [GAS]) is a medically significant pathogen of humans, causing a range of diseases from pharyngitis to necrotizing fasciitis. Several important GAS virulence genes are under the control of a pleiotropic regulator called Mga, or the multiple gene regulator of GAS, including the gene encoding the streptococcal collagen-like protein, or sclA. Analysis of the genome sequence upstream of sclA revealed two potential Mga-binding sites with homology to...

  3. Proteolytic activation of human pancreatitis associated protein is required for peptidoglycan binding and bacterial aggregation

    OpenAIRE

    Medveczky, Péter; Szmola, Richárd; Sahin-Tóth, Miklós

    2009-01-01

    Pancreatitis associated protein (PAP) is a 16 kDa lectin-like protein, which becomes robustly upregulated in the pancreatic juice during acute pancreatitis. Trypsin cleaves the N terminus of PAP, which in turn forms insoluble fibrils. PAP and its paralog the pancreatic stone protein induce bacterial aggregation and, more recently, PAP was shown to bind to the peptidoglycan of Gram positive bacteria and exert a direct bactericidal effect. However, the role of N-terminal processing in the antib...

  4. 78 FR 78910 - Comparability Determination for Japan: Certain Entity-Level Requirements

    Science.gov (United States)

    2013-12-27

    ... COMMISSION Comparability Determination for Japan: Certain Entity-Level Requirements AGENCY: Commodity Futures... substituted compliance determination.\\3\\ \\2\\ 78 FR 43785 (July 22, 2013). \\3\\ The Entity-Level Requirements... of Japan. SUMMARY: The following is the analysis and determination of the Commodity Futures...

  5. Chicken avidin-related proteins show altered biotin-binding and physico-chemical properties as compared with avidin.

    Science.gov (United States)

    Laitinen, Olli H; Hytönen, Vesa P; Ahlroth, Mervi K; Pentikäinen, Olli T; Gallagher, Ciara; Nordlund, Henri R; Ovod, Vladimir; Marttila, Ari T; Porkka, Eevaleena; Heino, Sanna; Johnson, Mark S; Airenne, Kari J; Kulomaa, Markku S

    2002-01-01

    Chicken avidin and bacterial streptavidin are proteins familiar from their use in various (strept)avidin-biotin technological applications. Avidin binds the vitamin biotin with the highest affinity known for non-covalent interactions found in nature. The gene encoding avidin (AVD) has homologues in chicken, named avidin-related genes (AVRs). In the present study we used the AVR genes to produce recombinant AVR proteins (AVRs 1, 2, 3, 4/5, 6 and 7) in insect cell cultures and characterized their biotin-binding affinity and biochemical properties. Amino acid sequence analysis and molecular modelling were also used to predict and explain the properties of the AVRs. We found that the AVR proteins are very similar to avidin, both structurally and functionally. Despite the numerous amino acid substitutions in the subunit interface regions, the AVRs form extremely stable tetramers similar to those of avidin. Differences were found in some physico-chemical properties of the AVRs as compared with avidin, including lowered pI, increased glycosylation and, most notably, reversible biotin binding for two AVRs (AVR1 and AVR2). Molecular modelling showed how the replacement Lys(111)-->isoleucine in AVR2 alters the shape of the biotin-binding pocket and thus results in reversible binding. Both modelling and biochemical analyses showed that disulphide bonds can form and link monomers in AVR4/5, a property not found in avidin. These, together with the other properties of the AVRs described in the present paper, may offer advantages over avidin and streptavidin, making the AVRs applicable for improved avidin-biotin technological applications. PMID:11964162

  6. Comparative mapping of the actin-binding protein 280 genes in human and mouse

    Energy Technology Data Exchange (ETDEWEB)

    Gariboldi, M.; Canzian, F.; Manenti, G.; De Gregorio, L. (Istituto Nazionale Tumori, Milan (Italy)); Maestrini, E.; Rivella, S. (Istituto di Genetica Biochimica ed Evoluzionistica, Pavia (Italy)); Chatterjee, A.; Herman, G.E. (Universita di Bari (Italy)); Archidiacono, N.; Antonacci, R. (Institute for Molecular Genetics, Houston, TX (United States)) (and others)

    1994-05-15

    Two genes encode actin-binding protein 280 isoforms. ABP-280 or filamin (FLN1) is present in the cytoskeleton of many cell types, whereas expression of FLN2 is limited to skeletal muscle and heart. FLN1 maps to human chromosome Xq28, and, by physical mapping in YAC clones, the authors have mapped the homologous murine locus (Fln1) to mouse chromosome X, in a region of syntenic homology with human chromosome X. They mapped FLN2 to human chromosome 7q32-q35 by analysis of somatic cell hybrids containing portions of chromosome 7, and, by using a mapping panel from an interspecific murine cross, they mapped the corresponding murine locus (Fln2) to murine chromosome 6 in a region homologous to human chromosome 7. 21 refs., 1 fig., 1 tab.

  7. 78 FR 78890 - Comparability Determination for Japan: Certain Transaction-Level Requirements

    Science.gov (United States)

    2013-12-27

    ... COMMISSION Comparability Determination for Japan: Certain Transaction-Level Requirements AGENCY: Commodity... in the Japan provide a sufficient basis for an affirmative finding of comparability with respect to.... \\1\\ 78 FR 45292 (July 26, 2013). The Commission originally published proposed and further...

  8. 34 CFR Appendix B to Part 403 - Examples for 34 CFR 403.194-Comparability Requirements

    Science.gov (United States)

    2010-07-01

    ... TECHNOLOGY EDUCATION PROGRAM Pt. 403, App. B Appendix B to Part 403—Examples for 34 CFR 403.194—Comparability... 34 Education 3 2010-07-01 2010-07-01 false Examples for 34 CFR 403.194-Comparability Requirements B Appendix B to Part 403 Education Regulations of the Offices of the Department of...

  9. Minimal sequence requirements of a functional human immunodeficiency virus type 1 primer binding site.

    OpenAIRE

    Wakefield, J K; Rhim, H; Morrow, C D

    1994-01-01

    The initiation of human immunodeficiency virus type 1 (HIV-1) reverse transcription occurs by the extension of a tRNA(3Lys) primer bound near the 5' end of the genomic RNA at a position termed the primer binding site (PBS). The PBS is an 18-nucleotide sequence of the HIV-1 genome which is complementary to the 3'-terminal 18 nucleotides of the tRNA(3Lys). To investigate the sequence specificity of the interaction between tRNA(3Lys) and the PBS, we have constructed proviral genomes containing m...

  10. Binding of ADAM12, a marker of skeletal muscle regeneration, to the muscle-specific actin-binding protein, alpha -actinin-2, is required for myoblast fusion

    DEFF Research Database (Denmark)

    Galliano, M F; Huet, C; Frygelius, J;

    2000-01-01

    differentiation. Using the yeast two-hybrid screen, we found that the muscle-specific alpha-actinin-2 strongly binds to the cytoplasmic tail of ADAM12. In vitro binding assays with GST fusion proteins confirmed the specific interaction. The major binding site for alpha-actinin-2 was mapped to a short sequence in...

  11. The Rho-GTPase binding protein IQGAP2 is required for the glomerular filtration barrier.

    Science.gov (United States)

    Sugano, Yuya; Lindenmeyer, Maja T; Auberger, Ines; Ziegler, Urs; Segerer, Stephan; Cohen, Clemens D; Neuhauss, Stephan C F; Loffing, Johannes

    2015-11-01

    Podocyte dysfunction impairs the size selectivity of the glomerular filter, leading to proteinuria, hypoalbuminuria, and edema, clinically defined as nephrotic syndrome. Hereditary forms of nephrotic syndrome are linked to mutations in podocyte-specific genes. To identify genes contributing to podocyte dysfunction in acquired nephrotic syndrome, we studied human glomerular gene expression data sets for glomerular-enriched gene transcripts differentially regulated between pretransplant biopsy samples and biopsies from patients with nephrotic syndrome. Candidate genes were screened by in situ hybridization for expression in the zebrafish pronephros, an easy-to-use in vivo assay system to assess podocyte function. One glomerulus-enriched product was the Rho-GTPase binding protein, IQGAP2. Immunohistochemistry found a strong presence of IQGAP2 in normal human and zebrafish podocytes. In zebrafish larvae, morpholino-based knockdown of iqgap2 caused a mild foot process effacement of zebrafish podocytes and a cystic dilation of the urinary space of Bowman's capsule upon onset of urinary filtration. Moreover, the glomerulus of zebrafish morphants showed a glomerular permeability for injected high-molecular-weight dextrans, indicating an impaired size selectivity of the glomerular filter. Thus, IQGAP2 is a Rho-GTPase binding protein, highly abundant in human and zebrafish podocytes, which controls normal podocyte structure and function as evidenced in the zebrafish pronephros. PMID:26154927

  12. Somitogenesis clock-wave initiation requires differential decay and multiple binding sites for clock protein.

    Directory of Open Access Journals (Sweden)

    Mark Campanelli

    2010-04-01

    Full Text Available Somitogenesis is a process common to all vertebrate embryos in which repeated blocks of cells arise from the presomitic mesoderm (PSM to lay a foundational pattern for trunk and tail development. Somites form in the wake of passing waves of periodic gene expression that originate in the tailbud and sweep posteriorly across the PSM. Previous work has suggested that the waves result from a spatiotemporally graded control protein that affects the oscillation rate of clock-gene expression. With a minimally constructed mathematical model, we study the contribution of two control mechanisms to the initial formation of this gene-expression wave. We test four biologically motivated model scenarios with either one or two clock protein transcription binding sites, and with or without differential decay rates for clock protein monomers and dimers. We examine the sensitivity of wave formation with respect to multiple model parameters and robustness to heterogeneity in cell population. We find that only a model with both multiple binding sites and differential decay rates is able to reproduce experimentally observed waveforms. Our results show that the experimentally observed characteristics of somitogenesis wave initiation constrain the underlying genetic control mechanisms.

  13. Predicting transcription factor binding sites using local over-representation and comparative genomics

    Directory of Open Access Journals (Sweden)

    Touzet Hélène

    2006-08-01

    Full Text Available Abstract Background Identifying cis-regulatory elements is crucial to understanding gene expression, which highlights the importance of the computational detection of overrepresented transcription factor binding sites (TFBSs in coexpressed or coregulated genes. However, this is a challenging problem, especially when considering higher eukaryotic organisms. Results We have developed a method, named TFM-Explorer, that searches for locally overrepresented TFBSs in a set of coregulated genes, which are modeled by profiles provided by a database of position weight matrices. The novelty of the method is that it takes advantage of spatial conservation in the sequence and supports multiple species. The efficiency of the underlying algorithm and its robustness to noise allow weak regulatory signals to be detected in large heterogeneous data sets. Conclusion TFM-Explorer provides an efficient way to predict TFBS overrepresentation in related sequences. Promising results were obtained in a variety of examples in human, mouse, and rat genomes. The software is publicly available at http://bioinfo.lifl.fr/TFM-Explorer.

  14. Complement-mediated neutralization of dengue virus requires mannose-binding lectin

    DEFF Research Database (Denmark)

    Avirutnan, Panisadee; Hauhart, Richard E; Marovich, Mary A;

    2011-01-01

    -dependent activation of the complement cascade neutralized insect cell-derived West Nile virus (WNV) in cell culture and restricted pathogenesis in mice. Here, we investigated the antiviral activity of MBL in infection by dengue virus (DENV), a related flavivirus. Using a panel of naïve sera from mouse strains...... with lower levels. Our studies suggest that allelic variation of MBL in humans may impact complement-dependent control of DENV pathogenesis. IMPORTANCE Dengue virus (DENV) is a mosquito-transmitted virus that causes a spectrum of clinical disease in humans ranging from subclinical infection to dengue...... hemorrhagic fever and dengue shock syndrome. Four serotypes of DENV exist, and severe illness is usually associated with secondary infection by a different serotype. Here, we show that mannose-binding lectin (MBL), a pattern recognition molecule that initiates the lectin pathway of complement activation...

  15. Stem-loop binding protein is required for retinal cell proliferation, neurogenesis, and intraretinal axon pathfinding in zebrafish.

    Science.gov (United States)

    Imai, Fumiyasu; Yoshizawa, Asuka; Matsuzaki, Ayako; Oguri, Eri; Araragi, Masato; Nishiwaki, Yuko; Masai, Ichiro

    2014-10-01

    In the developing retina, neurogenesis and cell differentiation are coupled with cell proliferation. However, molecular mechanisms that coordinate cell proliferation and differentiation are not fully understood. In this study, we found that retinal neurogenesis is severely delayed in the zebrafish stem-loop binding protein (slbp) mutant. SLBP binds to a stem-loop structure at the 3'-end of histone mRNAs, and regulates a replication-dependent synthesis and degradation of histone proteins. Retinal cell proliferation becomes slower in the slbp1 mutant, resulting in cessation of retinal stem cell proliferation. Although retinal stem cells cease proliferation by 2 days postfertilization (dpf) in the slbp mutant, retinal progenitor cells in the central retina continue to proliferate and generate neurons until at least 5dpf. We found that this progenitor proliferation depends on Notch signaling, suggesting that Notch signaling maintains retinal progenitor proliferation when faced with reduced SLBP activity. Thus, SLBP is required for retinal stem cell maintenance. SLBP and Notch signaling are required for retinal progenitor cell proliferation and subsequent neurogenesis. We also show that SLBP1 is required for intraretinal axon pathfinding, probably through morphogenesis of the optic stalk, which expresses attractant cues. Taken together, these data indicate important roles of SLBP in retinal development.

  16. The ChIP-seq-defined networks of Bcl-3 gene binding support its required role in skeletal muscle atrophy.

    Directory of Open Access Journals (Sweden)

    Robert W Jackman

    Full Text Available NF-kappaB transcriptional activation is required for skeletal muscle disuse atrophy. We are continuing to study how the activation of NF-kB regulates the genes that encode the protein products that cause atrophy. Using ChIP-sequencing we found that Bcl-3, an NF-kB transcriptional activator required for atrophy, binds to the promoters of a number of genes whose collective function describes two major aspects of muscle wasting. By means of bioinformatics analysis of ChIP-sequencing data we found Bcl-3 to be directing transcription networks of proteolysis and energy metabolism. The proteolytic arm of the Bcl-3 networks includes many E3 ligases associated with proteasomal protein degradation, including that of the N-end rule pathway. The metabolic arm appears to be involved in organizing the change from oxidative phosphorylation to glycolysis in atrophying muscle. For one gene, MuRF1, ChIP-sequencing data identified the location of Bcl-3 and p50 binding in the promoter region which directed the creation of deletant and base-substitution mutations of MuRF1 promoter constructs to determine the effect on gene transcription. The results provide the first direct confirmation that the NF-kB binding site is involved in the muscle unloading regulation of MuRF1. Finally, we have combined the ChIP-sequencing results with gene expression microarray data from unloaded muscle to map several direct targets of Bcl-3 that are transcription factors whose own targets describe a set of indirect targets for NF-kB in atrophy. ChIP-sequencing provides the first molecular explanation for the finding that Bcl3 knockout mice are resistant to disuse muscle atrophy. Mapping the transcriptional regulation of muscle atrophy requires an unbiased analysis of the whole genome, which we show is now possible with ChIP-sequencing.

  17. Export of malaria proteins requires co-translational processing of the PEXEL motif independent of phosphatidylinositol-3-phosphate binding.

    Science.gov (United States)

    Boddey, Justin A; O'Neill, Matthew T; Lopaticki, Sash; Carvalho, Teresa G; Hodder, Anthony N; Nebl, Thomas; Wawra, Stephan; van West, Pieter; Ebrahimzadeh, Zeinab; Richard, Dave; Flemming, Sven; Spielmann, Tobias; Przyborski, Jude; Babon, Jeff J; Cowman, Alan F

    2016-01-01

    Plasmodium falciparum exports proteins into erythrocytes using the Plasmodium export element (PEXEL) motif, which is cleaved in the endoplasmic reticulum (ER) by plasmepsin V (PMV). A recent study reported that phosphatidylinositol-3-phosphate (PI(3)P) concentrated in the ER binds to PEXEL motifs and is required for export independent of PMV, and that PEXEL motifs are functionally interchangeable with RxLR motifs of oomycete effectors. Here we show that the PEXEL does not bind PI(3)P, and that this lipid is not concentrated in the ER. We find that RxLR motifs cannot mediate export in P. falciparum. Parasites expressing a mutated version of KAHRP, with the PEXEL motif repositioned near the signal sequence, prevented PMV cleavage. This mutant possessed the putative PI(3)P-binding residues but is not exported. Reinstatement of PEXEL to its original location restores processing by PMV and export. These results challenge the PI(3)P hypothesis and provide evidence that PEXEL position is conserved for co-translational processing and export. PMID:26832821

  18. Non-cysteine linked MUC1 cytoplasmic dimers are required for Src recruitment and ICAM-1 binding induced cell invasion

    Directory of Open Access Journals (Sweden)

    Gunasekara Nirosha

    2011-07-01

    Full Text Available Abstract Background The mucin MUC1, a type I transmembrane glycoprotein, is overexpressed in breast cancer and has been correlated with increased metastasis. We were the first to report binding between MUC1 and Intercellular adhesion molecule-1 (ICAM-1, which is expressed on stromal and endothelial cells throughout the migratory tract of a metastasizing breast cancer cell. Subsequently, we found that MUC1/ICAM-1 binding results in pro-migratory calcium oscillations, cytoskeletal reorganization, and simulated transendothelial migration. These events were found to involve Src kinase, a non-receptor tyrosine kinase also implicated in breast cancer initiation and progression. Here, we further investigated the mechanism of MUC1/ICAM-1 signalling, focusing on the role of MUC1 dimerization in Src recruitment and pro-metastatic signalling. Methods To assay MUC1 dimerization, we used a chemical crosslinker which allowed for the detection of dimers on SDS-PAGE. We then generated MUC1 constructs containing an engineered domain which allowed for manipulation of dimerization status through the addition of ligands to the engineered domain. Following manipulation of dimerization, we immunoprecipitated MUC1 to investigate recruitment of Src, or assayed for our previously observed ICAM-1 binding induced events. To investigate the nature of MUC1 dimers, we used both non-reducing SDS-PAGE and generated a mutant construct lacking cysteine residues. Results We first demonstrate that the previously observed MUC1/ICAM-1signalling events are dependent on the activity of Src kinase. We then report that MUC1 forms constitutive cytoplasmic domain dimers which are necessary for Src recruitment, ICAM-1 induced calcium oscillations and simulated transendothelial migration. The dimers are not covalently linked constitutively or following ICAM-1 binding. In contrast to previously published reports, we found that membrane proximal cysteine residues were not involved in

  19. Spectrophotometric determination of total proteins in blood plasma: a comparative study among dye-binding methods

    OpenAIRE

    Dimas Augusto Morozin Zaia; Fábio Rangel Marques; Cássia Thaïs Bussamra Vieira Zaia

    2005-01-01

    A comparative study between the biuret method (standard method for total proteins) and spectrophotometric methods using dyes (Bradford, 3',3",5',5"-tetrabromophenolphthalein ethyl ester-TBPEE, and erythrosin-B) was carried out for the determination of total proteins in blood plasma from rats. Bradford method showed the highest sensitivity for proteins and biuret method showed the lowest. For all the methods, the absorbance for different proteins (BSA, casein, and egg albumin) was measured and...

  20. Comparative Analysis of QSAR-based vs. Chemical Similarity Based Predictors of GPCRs Binding Affinity.

    Science.gov (United States)

    Luo, Man; Wang, Xiang S; Tropsha, Alexander

    2016-01-01

    Ligand based virtual screening (LBVS) approaches could be broadly divided into those relying on chemical similarity searches and those employing Quantitative Structure-Activity Relationship (QSAR) models. We have compared the predictive power of these approaches using some datasets of compounds tested against several G-Protein Coupled Receptors (GPCRs). The k-Nearest Neighbors (kNN) QSAR models were built for known ligands of each GPCR target independently, with a fraction of tested ligands for each target set aside as a validation set. The prediction accuracies of QSAR models for making active/inactive calls for compounds in both training and validation sets were compared to those achieved by the Prediction of Activity Spectra for Substances' (PASS) and the Similarity Ensemble Approach (SEA) tools both available online. Models developed with the kNN QSAR method showed the highest predictive power for almost all tested GPCR datasets. The PASS software, which incorporates multiple end-point specific QSAR models demonstrated a moderate predictive power, while SEA, a chemical similarity based approach, had the lowest prediction power. Our studies suggest that when sufficient amount of data is available to develop and rigorously validate QSAR models such models should be chosen as the preferred virtual screening tool in ligand-based computational drug discovery as compared to chemical similarity based approaches. PMID:27491652

  1. The Actin-Binding Protein α-Adducin Is Required for Maintaining Axon Diameter

    Directory of Open Access Journals (Sweden)

    Sérgio Carvalho Leite

    2016-04-01

    Full Text Available The actin-binding protein adducin was recently identified as a component of the neuronal subcortical cytoskeleton. Here, we analyzed mice lacking adducin to uncover the function of this protein in actin rings. α-adducin knockout mice presented progressive axon enlargement in the spinal cord and optic and sciatic nerves, followed by axon degeneration and loss. Using stimulated emission depletion super-resolution microscopy, we show that a periodic subcortical actin cytoskeleton is assembled in every neuron type inspected including retinal ganglion cells and dorsal root ganglia neurons. In neurons devoid of adducin, the actin ring diameter increased, although the inter-ring periodicity was maintained. In vitro, the actin ring diameter adjusted as axons grew, suggesting the lattice is dynamic. Our data support a model in which adducin activity is not essential for actin ring assembly and periodicity but is necessary to control the diameter of both actin rings and axons and actin filament growth within rings.

  2. Electrostatically induced recruitment of membrane peptides into clusters requires ligand binding at both interfaces.

    Directory of Open Access Journals (Sweden)

    Yuri N Antonenko

    Full Text Available Protein recruitment to specific membrane locations may be governed or facilitated by electrostatic attraction, which originates from a multivalent ligand. Here we explored the energetics of a model system in which this simple electrostatic recruitment mechanism failed. That is, basic poly-L-lysine binding to one leaflet of a planar lipid bilayer did not recruit the triply-charged peptide (O-Pyromellitylgramicidin. Clustering was only observed in cases where PLL was bound to both channel ends. Clustering was indicated (i by the decreased diffusional PLL mobility D(PLL and (ii by an increased lifetime τ(PLL of the clustered channels. In contrast, if PLL was bound to only one leaflet, neither D(PLL nor τ(P changed. Simple calculations suggest that electrostatic repulsion of the unbound ends prevented neighboring OPg dimers from approaching each other. We believe that a similar mechanism may also operate in cell signaling and that it may e.g. contribute to the controversial results obtained for the ligand driven dimerization of G protein-coupled receptors.

  3. Minimal domain of bacterial phytochrome required for chromophore binding and fluorescence

    Science.gov (United States)

    Rumyantsev, Konstantin A.; Shcherbakova, Daria M.; Zakharova, Natalia I.; Emelyanov, Alexander V.; Turoverov, Konstantin K.; Verkhusha, Vladislav V.

    2015-12-01

    Fluorescent proteins (FP) are used to study various biological processes. Recently, a series of near-infrared (NIR) FPs based on bacterial phytochromes was developed. Finding ways to improve NIR FPs is becoming progressively important. By applying rational design and molecular evolution we have engineered R. palustris bacterial phytochrome into a single-domain NIR FP of 19.6 kDa, termed GAF-FP, which is 2-fold and 1.4-fold smaller than bacterial phytochrome-based NIR FPs and GFP-like proteins, respectively. Engineering of GAF-FP involved a substitution of 15% of its amino acids and a deletion of the knot structure. GAF-FP covalently binds two tetrapyrrole chromophores, biliverdin (BV) and phycocyanobilin (PCB). With the BV chromophore GAF-FP absorbs at 635 nm and fluoresces at 670 nm. With the PCB chromophore GAF-FP becomes blue-shifted and absorbs at 625 nm and fluoresces at 657 nm. The GAF-FP structure has a high tolerance to small peptide insertions. The small size of GAF-FP and its additional absorbance band in the violet range has allowed for designing a chimeric protein with Renilla luciferase. The chimera exhibits efficient non-radiative energy transfer from luciferase to GAF-FP, resulting in NIR bioluminescence. This study opens the way for engineering of small NIR FPs and NIR luciferases from bacterial phytochromes.

  4. Testosterone is required for corticosteroid-binding globulin upregulation by morphine to be fully manifested.

    Science.gov (United States)

    Nock, B; Wich, M; Cicero, T J; O'Connor, L H

    2000-09-01

    We previously reported that morphine increases the concentration of corticosteroid-binding globulin (CBG) in blood of male, but not female, rats. This pronounced sexual dimorphism suggested that CBG upregulation by morphine might be androgen-dependent. In the current studies, we found that castration, whether performed just before or just after puberty or in adulthood, increased the concentration of CBG in adult male rats. Naltrexone did not prevent this increase and, therefore, it does not appear to be attributable to the release of endogenous opioids. Exposure to morphine for 1 week in adulthood increased ( approximately 100%) the concentration of CBG in intact, i.e., sham-castrated, males. The CBG levels of castrated rats treated with morphine did not differ from those of intact rats treated with morphine. However, because castration increased the concentration of CBG, the difference between the placebo and morphine groups decreased with time after castration. At 4 weeks after castration, the difference between the morphine and placebo groups (19%) was no longer statistically significant. Testosterone replacement prevented the rise in CBG levels following castration and maintained the magnitude of the difference between placebo and morphine-treated rats within the normal range. Thus, testosterone appears necessary for morphine effects on CBG to be fully manifested. PMID:11113500

  5. 49 CFR 37.121 - Requirement for comparable complementary paratransit service.

    Science.gov (United States)

    2010-10-01

    ... paratransit service. 37.121 Section 37.121 Transportation Office of the Secretary of Transportation TRANSPORTATION SERVICES FOR INDIVIDUALS WITH DISABILITIES (ADA) Paratransit as a Complement to Fixed Route Service § 37.121 Requirement for comparable complementary paratransit service. (a) Except as provided...

  6. The ligand binding domain of GCNF is not required for repression of pluripotency genes in mouse fetal ovarian germ cells.

    Directory of Open Access Journals (Sweden)

    Leah M Okumura

    Full Text Available In mice, successful development and reproduction require that all cells, including germ cells, transition from a pluripotent to a differentiated state. This transition is associated with silencing of the pluripotency genes Oct4 and Nanog. Interestingly, these genes are repressed at different developmental timepoints in germ and somatic cells. Ovarian germ cells maintain their expression until about embryonic day (E 14.5, whereas somatic cells silence them much earlier, at about E8.0. In both somatic cells and embryonic stem cells, silencing of Oct4 and Nanog requires the nuclear receptor GCNF. However, expression of the Gcnf gene has not been investigated in fetal ovarian germ cells, and whether it is required for silencing Oct4 and Nanog in that context is not known. Here we demonstrate that Gcnf is expressed in fetal ovarian germ cells, peaking at E14.5, when Oct4 and Nanog are silenced. However, conditional ablation of the ligand-binding domain of Gcnf using a ubiquitous, tamoxifen-inducible Cre indicates that Gcnf is not required for the down-regulation of pluripotency genes in fetal ovarian germ cells, nor is it required for initiation of meiosis and oogenesis. These results suggest that the silencing of Oct4 and Nanog in germ cells occurs via a different mechanism from that operating in somatic cells during gastrulation.

  7. The H-loop in the Second Nucleotide-binding Domain of the Cystic Fibrosis Transmembrane Conductance Regulator is Required for Efficient Chloride Channel Closing

    OpenAIRE

    Kloch, Monika; Milewski, Michał; Nurowska, Ewa; Dworakowska, Beata; Cutting, Garry R; Dołowy, Krzysztof

    2010-01-01

    The cystic fibrosis transmembrane conductance regulator (CFTR) is an ATP-binding cassette (ABC) transporter that functions as a cAMP-activated chloride channel. The recent model of CFTR gating predicts that the ATP binding to both nucleotide-binding domains (NBD1 and NBD2) of CFTR is required for the opening of the channel, while the ATP hydrolysis at NBD2 induces subsequent channel closing. In most ABC proteins, efficient hydrolysis of ATP requires the presence of the invariant histidine res...

  8. Sperm surface protein PH-20 is bifunctional: one activity is a hyaluronidase and a second, distinct activity is required in secondary sperm-zona binding.

    Science.gov (United States)

    Hunnicutt, G R; Primakoff, P; Myles, D G

    1996-07-01

    In previous studies, we have found that the sperm membrane protein PH-20 acts during two different stages of fertilization. On acrosome-intact sperm, PH-20 has a hyaluronidase activity that is required for sperm penetration through the cumulus cell layer that surrounds the oocyte. On acrosome-reacted sperm, PH-20 has a required function in sperm-zona binding (secondary binding). Because hyaluronic acid (HA) has been detected in the zona pellucida, secondary sperm-zona adhesion could depend on repetitive binding and hydrolysis of HA by PH-20 acting as a hyaluronidase. Alternatively, PH-20 may be bifunctional and have a second, different activity required for secondary binding. To distinguish between these two possibilities, in this study we used reagents that inhibit either PH-20's function in sperm-zona binding or its hyaluronidase activity. We found that an anti-PH-20 monoclonal antibody that inhibited sperm-zona binding (approximately 90%) had no effect on hyaluronidase activity. Conversely, apigenin, a hyaluronidase inhibitor, blocked PH-20 hyaluronidase activity 93% without inhibiting sperm-zona binding. Similarly, another anti-PH-20 monoclonal antibody that inhibited hyaluronidase activity 95% only partially inhibited sperm-zona binding (approximately 45%). We also extensively pretreated oocytes with hyaluronidase to remove all accessible HA on or in the zona pellucida and found little or no effect on secondary sperm-zona binding. Our results suggest that PH-20 is bifunctional and has two activities: a hyaluronidase activity and a second, separate activity required for secondary sperm-zona binding. PMID:8793062

  9. Spectrophotometric determination of total proteins in blood plasma: a comparative study among dye-binding methods

    Directory of Open Access Journals (Sweden)

    Dimas Augusto Morozin Zaia

    2005-05-01

    Full Text Available A comparative study between the biuret method (standard method for total proteins and spectrophotometric methods using dyes (Bradford, 3',3",5',5"-tetrabromophenolphthalein ethyl ester-TBPEE, and erythrosin-B was carried out for the determination of total proteins in blood plasma from rats. Bradford method showed the highest sensitivity for proteins and biuret method showed the lowest. For all the methods, the absorbance for different proteins (BSA, casein, and egg albumin was measured and Bradford method showed the lowest variation of absorbance. The concentration of total protein obtained by using Bradford method was not statistically different (p>0.05 from concentration of total protein obtained by the biuret method. But in regard to erythrosin-B and TBPEE methods the concentrations of total protein were statistically different (pA determinação de proteínas totais em plasma sangüíneo é importante em diversas áreas de pesquisa. Um estudo comparativo entre o método de biureto (método padrão para proteínas totais e diversos métodos que utilizam corantes (Bradford, tetrabromofenolftaleína etil éster-TBPEE, e eritrosina-B foi realizado para a determinação de proteínas totais em plasma sangüíneo de ratos. O método de Bradford mostrou a maior sensibilidade para proteínas e o de biureto a menor. Para todos os métodos, as absorbâncias para diferentes proteínas (BSA, caseína, e ovoalbumina foram medidas e o método de Bradford mostrou a menor variação da absorbância. Utilizando o método de Bradford a concentração de proteínas totais obtida não foi estatisticamente diferente (p>0.05 daquela obtida pelo método do biureto. Porém, para os métodos da eritrosina-B e TBPEE as concentrações de proteínas totais foram estatisticamente diferentes (p<0.05 da obtida pelo método de biureto. Portanto o método de Bradford pode ser utilizado no lugar do método de biureto para a determinação de proteínas totais em plasma sangüíneo.

  10. Comparative study of somatostatin-human serum albumin fusion proteins and natural somatostatin on receptor binding, internalization and activation.

    Directory of Open Access Journals (Sweden)

    Ying Peng

    Full Text Available Albumin fusion technology, the combination of small molecular proteins or peptides with human serum albumin (HSA, is an effective method for improving the medicinal values of natural small molecular proteins or peptides. However, comparative studies between HSA-fusion proteins or peptides and the parent small molecules in biological and molecular mechanisms are less reported. In this study, we examined the binding property of two novel somatostatin-HSA fusion proteins, (SST142-HSA and (SST282-HSA, to human SSTRs in stably expressing SSTR1-5 HEK 293 cells; observed the regulation of receptor internalization and internalized receptor recycling; and detected the receptors activation of HSA fusion proteins in stably expressing SSTR2- and SSTR3-EGFP cells. We showed that both somatostatin-HSA fusion proteins had high affinity to all five SSTRs, stimulated the ERK1/2 phosphorylation and persistently inhibited the accumulation of forskolin-stimulated cAMP in SSTR2- and SSTR3-expressing cells; but were less potent than the synthetic somatostatin-14 (SST-14. Our experiments also showed that somatostatin-HSA fusion proteins did not induce the receptors internalization; rather, they accelerated the recycling of the internalized receptors induced by SST-14 to the plasma membrane. Our results indicated that somatostatin-HSA fusion proteins, different from SST-14, exhibit some particular properties in binding, regulating, and activating somatostatin receptors.

  11. Sterol Regulatory Element Binding Protein (Srb1) Is Required for Hypoxic Adaptation and Virulence in the Dimorphic Fungus Histoplasma capsulatum

    Science.gov (United States)

    DuBois, Juwen C.; Smulian, A. George

    2016-01-01

    The Histoplasma capsulatum sterol regulatory element binding protein (SREBP), Srb1 is a member of the basic helix-loop-helix (bHLH), leucine zipper DNA binding protein family of transcription factors that possess a unique tyrosine (Y) residue instead of an arginine (R) residue in the bHLH region. We have determined that Srb1 message levels increase in a time dependent manner during growth under oxygen deprivation (hypoxia). To further understand the role of Srb1 during infection and hypoxia, we silenced the gene encoding Srb1 using RNA interference (RNAi); characterized the resulting phenotype, determined its response to hypoxia, and its ability to cause disease within an infected host. Silencing of Srb1 resulted in a strain of H. capsulatum that is incapable of surviving in vitro hypoxia. We found that without complete Srb1 expression, H. capsulatum is killed by murine macrophages and avirulent in mice given a lethal dose of yeasts. Additionally, silencing Srb1 inhibited the hypoxic upregulation of other known H. capsulatum hypoxia-responsive genes (HRG), and genes that encode ergosterol biosynthetic enzymes. Consistent with these regulatory functions, Srb1 silenced H. capsulatum cells were hypersensitive to the antifungal azole drug itraconazole. These data support the theory that the H. capsulatum SREBP is critical for hypoxic adaptation and is required for H. capsulatum virulence. PMID:27711233

  12. A Novel C2-Domain Phospholipid-Binding Protein,OsPBP1.Is Required for Pollen Fertility in Rice

    Institute of Scientific and Technical Information of China (English)

    Wen-Qiang Yang; Ying Lai; Mei-Na Li; Wen-Ying Xu; Yong-Biao Xue

    2008-01-01

    Pollen fertility is a crucial factor for successful pollination and essential for seed formation.Recent studies have suggested that a diverse range of internal and external factors,signaling components and their related pathways are likely involved in pollen fertility.Here,we reporta single C2-domain containing protein.OsPBPl.initially identified through cDNA microarray analysis.OsP8P1 is a single copy gene and preferentially expressed in pistil and pollen but downregulated by pollination.OsPBP1 had a calcium concentration-dependent phospholipid-binding activity and was localized mainly in cytoplasm and nucleus,but translocated onto the plasma membrane in response to an intracellular Ca2+increase.Pollen grains of antisense OsPBP1 transgenic Iines were largely nonviable.germinated poorly in vitro and of low fertility,OsPBP1 protein was localized in a region peripheral to pollen wall and vesicles of elongating pollen tube.and its repressed expression reduced substantially this association and led to alteration of microfilament polymerization during pollen germination.Taken together,these results indicate that OsPBP1 is a novel functional C2-domain phosphoIipids-binding protein that is required for pollen fertility likely by regulating Ca2+ and phospholipid signaling pathways.

  13. THE NEW EU ACCOUNTING DIRECTIVE – A COMPARATION OF REPORTING REQUIREMENTS

    Directory of Open Access Journals (Sweden)

    MARIUS DEAC

    2014-05-01

    Full Text Available The adoption of Directive 2013/34/EU constitutes the materialization of a modernization process of the Directive 78/660/EEC and Directive 83/349/EEC that has started in 2009. The new accounting directive replaces the two accounting directives and sets the requirements for both individual and consolidated financial statements. It also introduces a new category of entities “micro-undertakings” in order to implement the requirements of Directive 2012/6/EU. The paper tries to summarize and compare the typologies of enterprises and groups defined in the old and new accounting directives as well as in the Romanian accounting system (RAS. It also compares the requirements of both the new and the old accounting directives as well as RAS regarding the structure and presentation of the annual financial statements. European Union (EU wide, about 99% of the enterprises fall into micro and small categories and might benefit from the simplifications of preparation and publication requirements of the annual financial statements. They will also be exempted from the requirements of auditing their financial statements. Only 1.21% of the enterprises in Europe will be classified as medium and large and will be required to prepare and publish a full set of annual financial statements and to audit their financial statements. If Romania will opt for reducing the administrative burdens for micro entities and implement simplifications for micro-entities as they were defined in the EU directive 2013/34/EU, the thresholds would have to be dramatically increased and many more enterprises will be reclassified as micro instead of small or medium enterprises.

  14. Switch control pocket inhibitors of p38-MAP kinase. Durable type II inhibitors that do not require binding into the canonical ATP hinge region

    Energy Technology Data Exchange (ETDEWEB)

    Ahn, Yu Mi; Clare, Michael; Ensinger, Carol L.; Hood, Molly M.; Lord, John W.; Lu, Wei-Ping; Miller, David F.; Patt, William C.; Smith, Bryan D.; Vogeti, Lakshminarayana; Kaufman, Michael D.; Petillo, Peter A.; Wise, Scott C.; Abendroth, Jan; Chun, Lawrence; Clark, Robin; Feese, Michael; Kim, Hidong; Stewart, Lance; Flynn, Daniel L. (Deciphera); (Emerald); (Cocrystal)

    2012-01-20

    Switch control pocket inhibitors of p38-alpha kinase are described. Durable type II inhibitors were designed which bind to arginines (Arg67 or Arg70) that function as key residues for mediating phospho-threonine 180 dependant conformational fluxing of p38-alpha from an inactive type II state to an active type I state. Binding to Arg70 in particular led to potent inhibitors, exemplified by DP-802, which also exhibited high kinase selectivity. Binding to Arg70 obviated the requirement for binding into the ATP Hinge region. X-ray crystallography revealed that DP-802 and analogs induce an enhanced type II conformation upon binding to either the unphosphorylated or the doubly phosphorylated form of p38-alpha kinase.

  15. Switch control pocket inhibitors of p38-MAP kinase. Durable type II inhibitors that do not require binding into the canonical ATP hinge region.

    Science.gov (United States)

    Ahn, Yu Mi; Clare, Michael; Ensinger, Carol L; Hood, Molly M; Lord, John W; Lu, Wei-Ping; Miller, David F; Patt, William C; Smith, Bryan D; Vogeti, Lakshminarayana; Kaufman, Michael D; Petillo, Peter A; Wise, Scott C; Abendroth, Jan; Chun, Lawrence; Clark, Robin; Feese, Michael; Kim, Hidong; Stewart, Lance; Flynn, Daniel L

    2010-10-01

    Switch control pocket inhibitors of p38-alpha kinase are described. Durable type II inhibitors were designed which bind to arginines (Arg67 or Arg70) that function as key residues for mediating phospho-threonine 180 dependant conformational fluxing of p38-alpha from an inactive type II state to an active type I state. Binding to Arg70 in particular led to potent inhibitors, exemplified by DP-802, which also exhibited high kinase selectivity. Binding to Arg70 obviated the requirement for binding into the ATP Hinge region. X-ray crystallography revealed that DP-802 and analogs induce an enhanced type II conformation upon binding to either the unphosphorylated or the doubly phosphorylated form of p38-alpha kinase.

  16. Moving the mountain: analysis of the effort required to transform comparative anatomy into computable anatomy.

    Science.gov (United States)

    Dahdul, Wasila; Dececchi, T Alexander; Ibrahim, Nizar; Lapp, Hilmar; Mabee, Paula

    2015-01-01

    The diverse phenotypes of living organisms have been described for centuries, and though they may be digitized, they are not readily available in a computable form. Using over 100 morphological studies, the Phenoscape project has demonstrated that by annotating characters with community ontology terms, links between novel species anatomy and the genes that may underlie them can be made. But given the enormity of the legacy literature, how can this largely unexploited wealth of descriptive data be rendered amenable to large-scale computation? To identify the bottlenecks, we quantified the time involved in the major aspects of phenotype curation as we annotated characters from the vertebrate phylogenetic systematics literature. This involves attaching fully computable logical expressions consisting of ontology terms to the descriptions in character-by-taxon matrices. The workflow consists of: (i) data preparation, (ii) phenotype annotation, (iii) ontology development and (iv) curation team discussions and software development feedback. Our results showed that the completion of this work required two person-years by a team of two post-docs, a lead data curator, and students. Manual data preparation required close to 13% of the effort. This part in particular could be reduced substantially with better community data practices, such as depositing fully populated matrices in public repositories. Phenotype annotation required ∼40% of the effort. We are working to make this more efficient with Natural Language Processing tools. Ontology development (40%), however, remains a highly manual task requiring domain (anatomical) expertise and use of specialized software. The large overhead required for data preparation and ontology development contributed to a low annotation rate of approximately two characters per hour, compared with 14 characters per hour when activity was restricted to character annotation. Unlocking the potential of the vast stores of morphological

  17. Developing a 3D Road Cadastral System: Comparing Legal Requirements and User Needs

    Science.gov (United States)

    Gristina, S.; Ellul, C.; Scianna, A.

    2016-10-01

    Road transport has always played an important role in a country's growth and, in order to manage road networks and ensure a high standard of road performance (e.g. durability, efficiency and safety), both public and private road inventories have been implemented using databases and Geographical Information Systems. They enable registering and managing significant amounts of different road information, but to date do not focus on 3D road information, data integration and interoperability. In an increasingly complex 3D urban environment, and in the age of smart cities, however, applications including intelligent transport systems, mobility and traffic management, road maintenance and safety require digital data infrastructures to manage road data: thus new inventories based on integrated 3D road models (queryable, updateable and shareable on line) are required. This paper outlines the first step towards the implementation of 3D GIS-based road inventories. Focusing on the case study of the "Road Cadastre" (the Italian road inventory as established by law), it investigates current limitations and required improvements, and also compares the required data structure imposed by cadastral legislation with real road users' needs. The study aims to: a) determine whether 3D GIS would improve road cadastre (for better management of data through the complete life-cycle infrastructure projects); b) define a conceptual model for a 3D road cadastre for Italy (whose general principles may be extended also to other countries).

  18. The Specific Direction Requirement for Aiding and Abetting: A Call for Revisiting Comparative Criminal Law

    DEFF Research Database (Denmark)

    Aksenova, Marina

    2015-01-01

    . The saga started with unexpected acquittals of Perišić, Stanišić, and Simatović due to a lack of specific direction in their aid and assistance towards specific offences. More specifically, the tribunal found that the traditional test - the provision of aid with the awareness that it would have...... a substantial effect on the crimes committed in the context of war - was insufficient to create individual criminal responsibility in these cases. The response to this new and heightened interpretation of aiding and abetting followed quickly, as the Šainović et al. appeal judgment rejected the novel requirement...... that the innovative element of the specific direction lacks a proper foundation in international law. Second, the article discusses several conceptual difficulties with the specific direction requirement, some of which go beyond the issues of accomplice liability. This article concludes by finding that comparative...

  19. Mgm101p is a novel component of the mitochondrial nucleoid that binds DNA and is required for the repair of oxidatively damaged mitochondrial DNA

    International Nuclear Information System (INIS)

    Maintenance of mitochondrial DNA (mtDNA) during cell division is required for progeny to be respiratory competent. Maintenance involves the replication, repair, assembly, segregation, and partitioning of the mitochondrial nucleoid. MGM101 has been identified as a gene essential for mtDNA maintenance in S. cerevisiae, but its role is unknown. Using liquid chromatography coupled with tandem mass spectrometry, we identified Mgm101p as a component of highly enriched nucleoids, suggesting that it plays a nucleoid-specific role in maintenance. Subcellular fractionation, indirect immunofluorescence and GFP tagging show that Mgm101p is exclusively associated with the mitochondrial nucleoid structure in cells. Furthermore, DNA affinity chromatography of nucleoid extracts indicates that Mgm101p binds to DNA, suggesting that its nucleoid localization is in part due to this activity. Phenotypic analysis of cells containing a temperature sensitive mgm101 allele suggests that Mgm101p is not involved in mtDNA packaging, segregation, partitioning or required for ongoing mtDNA replication. We examined Mgm101p's role in mtDNA repair. As compared with wild-type cells, mgm101 cells were more sensitive to mtDNA damage induced by UV irradiation and were hypersensitive to mtDNA damage induced by gamma rays and H2O2 treatment. Thus, we propose that Mgm101p performs an essential function in the repair of oxidatively damaged mtDNA that is required for the maintenance of the mitochondrial genome. (author)

  20. The MCM-binding protein ETG1 aids sister chromatid cohesion required for postreplicative homologous recombination repair.

    Directory of Open Access Journals (Sweden)

    Naoki Takahashi

    2010-01-01

    Full Text Available The DNA replication process represents a source of DNA stress that causes potentially spontaneous genome damage. This effect might be strengthened by mutations in crucial replication factors, requiring the activation of DNA damage checkpoints to enable DNA repair before anaphase onset. Here, we demonstrate that depletion of the evolutionarily conserved minichromosome maintenance helicase-binding protein ETG1 of Arabidopsis thaliana resulted in a stringent late G2 cell cycle arrest. This arrest correlated with a partial loss of sister chromatid cohesion. The lack-of-cohesion phenotype was intensified in plants without functional CTF18, a replication fork factor needed for cohesion establishment. The synergistic effect of the etg1 and ctf18 mutants on sister chromatid cohesion strengthened the impact on plant growth of the replication stress caused by ETG1 deficiency because of inefficient DNA repair. We conclude that the ETG1 replication factor is required for efficient cohesion and that cohesion establishment is essential for proper development of plants suffering from endogenous DNA stress. Cohesion defects observed upon knockdown of its human counterpart suggest an equally important developmental role for the orthologous mammalian ETG1 protein.

  1. Curcumin binds in silico to anti-cancer drug target enzyme MMP-3 (human stromelysin-1) with affinity comparable to two known inhibitors of the enzyme

    OpenAIRE

    Jerah, Ahmed; Hobani, Yahya; Kumar, B. Vinod; Bidwai, Anil

    2015-01-01

    In silico interaction of curcumin with the enzyme MMP-3 (human stromelysin-1) was studied by molecular docking using AutoDock 4.2 as the docking software application. AutoDock 4.2 software serves as a valid and acceptable docking application to study the interactions of small compounds with proteins. Interactions of curcumin with MMP-3 were compared to those of two known inhibitors of the enzyme, PBSA and MPPT. The calculated free energy of binding (ΔG binding) shows that curcumin binds with ...

  2. A Comparative Study of Software Requirement, Elicitation, Prioritization and Decision Making.

    OpenAIRE

    Pradeep Kumar. G; Sandhiya .R; Prof. Manjula R

    2016-01-01

    The failure of many software systems are mainly due to the lack of the requirement engineering. Where software requirement play a very vital role in the field of software engineering. The main task of the requirement engineering are eliciting the requirements from the customer and to prioritize those requirements to make decisions in the software design. Prioritization of the software requirement is very much useful in giving priority within the set of requirements. Requirement pr...

  3. A C-terminal PDZ domain binding sequence is required for striatal distribution of the dopamine transporter

    OpenAIRE

    Rickhag, Mattias; Hansen, Freja Herborg; Sørensen, Gunnar; Strandfelt, Kristine Nørgaard; Andresen, Bjørn; Gotfryd, Kamil; Madsen, Kenneth L; Vestergaard-Klewe, Ib; Ammendrup-Johnsen, Ina; Eriksen, Jacob; Füchtbauer, Ernst-Martin; Gomeza, Jesus; Woldbye, David P.D.; Wörtwein, Gitta; Gether, Ulrik

    2013-01-01

    The dopamine transporter (DAT) mediates reuptake of dopamine from the synaptic cleft. The cellular mechanisms controlling DAT levels in striatal nerve terminals remain poorly understood. DAT contains a C-terminal PDZ (PSD-95/Discs-large/ZO-1) domain binding sequence believed to bind synaptic scaffolding proteins, but its functional significance is uncertain. Here we demonstrate that two different DAT knock-in mice with disrupted PDZ-binding motifs (DAT-AAA and DAT+Ala) are characterized by dr...

  4. Osteopontin binding to the alpha 4 integrin requires highest affinity integrin conformation, but is independent of post-translational modifications of osteopontin

    DEFF Research Database (Denmark)

    Hui, Tommy; Sørensen, Esben Skipper; Rittling, Susan R.

    2015-01-01

    Osteopontin (OPN) is a ligand for the α4 integrin, but the physiological importance of this binding is not well understood. Here, we have assessed the effect of posttranslational modifications on OPN binding to the α4 integrin on cultured human leukocyte cell lines, and compared OPN interaction...... affinity forms of this integrin. Together, the results suggest OPN has very low affinity for the α4 integrin on human leukocytes under physiological conditions....

  5. A comparative study of polyethylene glycol hydrogels derivatized with the RGD peptide and the cell-binding domain of fibronectin.

    Science.gov (United States)

    Zhang, Chen; Hekmatfer, Sogol; Karuri, Nancy W

    2014-01-01

    The goal of our study was to compare the biological responses of cells cultured on polyethylene glycol (PEG) hydrogels functionalized with varying concentrations of the widely used adhesion peptide, RGD, and the cell-binding domain of fibronectin, III(9-10). We used Michael addition chemistry to covalently link cysteines in GRGDSPC and glutathione S-transferase (GST) tagged III(9-10) (GST-III(9-10)), to the acrylate groups in PEG diacrylate (PEGDA). Conjugation of GST-III(9-10) to PEGDA occurred through cysteine residues in GST. Ellman's reagent and immunoblotting studies demonstrated an efficiency of 90% or more for PEG conjugation of 1 μM GST-III(9-10) or GRDGSPC in 10% (wt/vol) PEGDA at 37°C for 1 h. Circular dichroism and limited proteolysis demonstrated that conjugating PEGDA to GST-III(9-10) did not significantly perturb its native secondary structure. Sodium dodecyl sulfate polyacrylamide gel electrophoresis characterization of the wash solution of PEG hydrogels after photopolymerization demonstrated that >95% of the 1 μM GST-III(9-10) was incorporated into the PEG hydrogels after cross-linking. PEG hydrogels derivatized with 1 μM GST-III(9-10) had significantly higher cell adhesion and spreading than PEG hydrogels with 1 μM GRGDSPC. A comparable adhesion response between GRGDSPC and GST-III(9-10) was obtained when the former was at millimolar and the latter at micromolar concentration. The amount and type of conjugate in the PEG hydrogel derivative was statistically more significant than hydrogel rigidity in stimulating the biological responses observed. This report presents new evidence of the robustness of III(9-10) in mediating cell adhesion and spreading on PEG hydrogels.

  6. TNPO3 is required for HIV-1 replication after nuclear import but prior to integration and binds the HIV-1 core.

    OpenAIRE

    Valle-Casuso, Jose Carlos; Di Nunzio, Francesca; Yang, Yang; Reszka, Natalia; Lienlaf, Maritza; Arhel, Nathalie; Perez, Patricio; Brass, Abraham L.; Diaz-Griffero, Felipe

    2012-01-01

    International audience TNPO3 is a nuclear importer required for HIV-1 infection. Here, we show that depletion of TNPO3 leads to an HIV-1 block after nuclear import but prior to integration. To investigate the mechanistic requirement of TNPO3 in HIV-1 infection, we tested the binding of TNPO3 to the HIV-1 core and found that TNPO3 binds to the HIV-1 core. Overall, this work suggests that TNPO3 interacts with the incoming HIV-1 core in the cytoplasm to assist a process that is important for ...

  7. Polymerase (Pol) III TATA Box-Binding Protein (TBP)-Associated Factor Brf Binds to a Surface on TBP Also Required for Activated Pol II Transcription

    OpenAIRE

    Shen, Yuhong; Kassavetis, George A.; Bryant, Gene O.; Berk, Arnold J.

    1998-01-01

    The TATA box-binding protein (TBP) plays an essential role in transcription by all three eukaryotic nuclear RNA polymerases, polymerases (Pol) I, II, and III. In each case, TBP interacts with class-specific TBP-associated factors (TAFs) to form class-specific transcription initiation factors. For yeast Pol III transcription, TBP associates with Brf (from TFIIB-related factor) and B", two Pol III TAFs, to form Pol III transcription factor TFIIIB. Here, we identify TBP surface residues that are...

  8. Effects of temperature on the p53-DNA binding interactions and their dynamical behavior: comparing the wild type to the R248Q mutant.

    Directory of Open Access Journals (Sweden)

    Khaled Barakat

    Full Text Available BACKGROUND: The protein p53 plays an active role in the regulation of cell cycle. In about half of human cancers, the protein is inactivated by mutations located primarily in its DNA-binding domain. Interestingly, a number of these mutations possess temperature-induced DNA-binding characteristics. A striking example is the mutation of Arg248 into glutamine or tryptophan. These mutants are defective for binding to DNA at 310 K although they have been shown to bind specifically to several p53 response elements at sub-physiological temperatures (298-306 K. METHODOLOGY/PRINCIPAL FINDINGS: This important experimental finding motivated us to examine the effects of temperature on the structure and configuration of R248Q mutant and compare it to the wild type protein. Our aim is to determine how and where structural changes of mutant variants take place due to temperature changes. To answer these questions, we compared the mutant to the wild-type proteins from two different aspects. First, we investigated the systems at the atomistic level through their DNA-binding affinity, hydrogen bond networks and spatial distribution of water molecules. Next, we assessed changes in their long-lived conformational motions at the coarse-grained level through the collective dynamics of their side-chain and backbone atoms separately. CONCLUSIONS: The experimentally observed effect of temperature on the DNA-binding properties of p53 is reproduced. Analysis of atomistic and coarse-grained data reveal that changes in binding are determined by a few key residues and provide a rationale for the mutant-loss of binding at physiological temperatures. The findings can potentially enable a rescue strategy for the mutant structure.

  9. Binding Procurement

    Science.gov (United States)

    Rao, Gopalakrishna M.; Vaidyanathan, Hari

    2007-01-01

    This viewgraph presentation reviews the use of the binding procurement process in purchasing Aerospace Flight Battery Systems. NASA Engineering and Safety Center (NESC) requested NASA Aerospace Flight Battery Systems Working Group to develop a set of guideline requirements document for Binding Procurement Contracts.

  10. Organizational requirements of the SaeR binding sites for a functional P1 promoter of the sae operon in Staphylococcus aureus.

    Science.gov (United States)

    Cho, Hoonsik; Jeong, Do-Won; Li, Chunling; Bae, Taeok

    2012-06-01

    In Staphylococcus aureus, the SaeRS two-component system controls the expression of multiple virulence factors. Of the two promoters in the sae operon, P1 is autoinduced and has two binding sites for the response regulator SaeR. In this study, we examined the organizational requirements of the SaeR binding sites in P1 for transcription activation. Mutational studies showed that both binding sites are essential for binding to phosphorylated SaeR (P-SaeR) and transcription activation. When the 21-bp distance between the centers of the two SaeR binding sites was altered to 26 bp, 31 bp, 36 bp, or 41 bp, only the 31-bp mutant retained approximately 40% of the original promoter activity. When the -1-bp spacing (i.e.,1-bp overlap) between the primary SaeR binding site and the -35 promoter region was altered, all mutant P1 promoters failed to initiate transcription; however, when the first nucleotide of the -35 region was changed from A to T, the mutants with 0-bp or 22-bp spacing showed detectable promoter activity. Although P-SaeR was essential for the binding of RNA polymerase to P1, it was not essential for the binding of the enzyme to the alpha-hemolysin promoter. When the nonoptimal spacing between promoter elements in P1 or the coagulase promoter was altered to the optimal spacing of 17 bp, both promoters failed to initiate transcription. These results suggest that SaeR binding sites are under rather strict organizational restrictions and provide clues for understanding the molecular mechanism of sae-mediated transcription activation.

  11. Guanine nucleotide-binding protein (Gα) endocytosis by a cascade of ubiquitin binding domain proteins is required for sustained morphogenesis and proper mating in yeast.

    Science.gov (United States)

    Dixit, Gauri; Baker, Rachael; Sacks, Carly M; Torres, Matthew P; Dohlman, Henrik G

    2014-05-23

    Heterotrimeric G proteins are well known to transmit signals from cell surface receptors to intracellular effector proteins. There is growing appreciation that G proteins are also present at endomembrane compartments, where they can potentially interact with a distinct set of signaling proteins. Here, we examine the cellular trafficking function of the G protein α subunit in yeast, Gpa1. Gpa1 contains a unique 109-amino acid insert within the α-helical domain that undergoes a variety of posttranslational modifications. Among these is monoubiquitination, catalyzed by the NEDD4 family ubiquitin ligase Rsp5. Using a newly optimized method for G protein purification together with biophysical measures of structure and function, we show that the ubiquitination domain does not influence enzyme activity. By screening a panel of 39 gene deletion mutants, each lacking a different ubiquitin binding domain protein, we identify seven that are necessary to deliver Gpa1 to the vacuole compartment including four proteins (Ede1, Bul1, Ddi1, and Rup1) previously not known to be involved in this process. Finally, we show that proper endocytosis of the G protein is needed for sustained cellular morphogenesis and mating in response to pheromone stimulation. We conclude that a cascade of ubiquitin-binding proteins serves to deliver the G protein to its final destination within the cell. In this instance and in contrast to the previously characterized visual system, endocytosis from the plasma membrane is needed for proper signal transduction rather than for signal desensitization.

  12. Transcriptional activation requires protection of the TATA-binding protein Tbp1 by the ubiquitin-specific protease Ubp3.

    Science.gov (United States)

    Chew, Boon Shang; Siew, Wee Leng; Xiao, Benjamin; Lehming, Norbert

    2010-11-01

    Tbp1, the TATA-binding protein, is essential for transcriptional activation, and Gal4 and Gcn4 are unable to fully activate transcription in a Saccharomyces cerevisiae TBP1E86D mutant strain. In the present study we have shown that the Tbp1E186D mutant protein is proteolytically instable, and we have isolated intragenic and extragenic suppressors of the transcription defects of the TBP1E186D mutant strain. The TBP1R6S mutation stabilizes the Tbp1E186D mutant protein and suppresses the defects of the TBP1E186D mutant strain. Furthermore, we found that the overexpression of the de-ubiquitinating enzyme Ubp3 (ubiquitin-specific protease 3) also stabilizes the Tbp1E186D mutant protein and suppresses of the defects of the TBP1E186D mutant strain. Importantly, the deletion of UBP3 and its cofactor BRE5 lead to increased degradation of wild-type Tbp1 protein and to defects in transcriptional activation by Gal4 and Gcn4. Purified GST (glutathione transferase)-Ubp3 reversed Tbp1 ubiquitination, and the deletion of UBP3 lead to the accumulation of poly-ubiquitinated species of Tbp1 in a proteaseome-deficient genetic background, demonstrating that Ubp3 reverses ubiquitination of Tbp1 in vitro and in vivo. Chromatin immunoprecipitation showed that Ubp3 was recruited to the GAL1 and HIS3 promoters upon the induction of the respective gene, indicating that protection of promoter-bound Tbp1 by Ubp3 is required for transcriptional activation.

  13. A conserved acidic patch in the Myb domain is required for activation of an endogenous target gene and for chromatin binding

    Directory of Open Access Journals (Sweden)

    Chen Carolyn

    2008-10-01

    Full Text Available Abstract The c-Myb protein is a transcriptional regulator initially identified by homology to the v-Myb oncoprotein, and has since been implicated in human cancer. The most highly conserved portion of the c-Myb protein is the DNA-binding domain which consists of three imperfect repeats. Many other proteins contain one or more Myb-related domains, including a number of proteins that do not bind directly to DNA. We performed a phylogenetic analysis of diverse classes of Myb-related domains and discovered a highly conserved patch of acidic residues common to all Myb-related domains. These acidic residues are positioned in the first of three alpha-helices within each of the three repeats that comprise the c-Myb DNA-binding domain. Interestingly, these conserved acidic residues are present on a surface of the protein which is distinct from that which binds to DNA. Alanine mutagenesis revealed that the acidic patch of the third c-Myb repeat is essential for transcriptional activity, but neither for nuclear localization nor DNA-binding. Instead, these acidic residues are required for efficient chromatin binding and interaction with the histone H4 N-terminal tail.

  14. A Comparative Study of Software Requirement, Elicitation, Prioritization and Decision Making.

    Directory of Open Access Journals (Sweden)

    Pradeep Kumar. G

    2016-04-01

    Full Text Available The failure of many software systems are mainly due to the lack of the requirement engineering. Where software requirement play a very vital role in the field of software engineering. The main task of the requirement engineering are eliciting the requirements from the customer and to prioritize those requirements to make decisions in the software design. Prioritization of the software requirement is very much useful in giving priority within the set of requirements. Requirement prioritization is very much important when there are strict constraints on schedule and the resources, then the software engineer must take some decisions on neglecting or to give prioritization to some of the requirements that are to be added to the project which makes it successful. This paper is the frame work of comparison of various techniques and to propose a most competent method among them

  15. Comparative analyses of lipoprotein lipase, hepatic lipase, and endothelial lipase, and their binding properties with known inhibitors.

    Directory of Open Access Journals (Sweden)

    Ziyun Wang

    Full Text Available The triglyceride lipase gene subfamily plays a central role in lipid and lipoprotein metabolism. There are three members of this subfamily: lipoprotein lipase, hepatic lipase, and endothelial lipase. Although these lipases are implicated in the pathophysiology of hyperlipidemia and atherosclerosis, their structures have not been fully solved. In the current study, we established homology models of these three lipases, and carried out analysis of their activity sites. In addition, we investigated the kinetic characteristics for the catalytic residues using a molecular dynamics simulation strategy. To elucidate the molecular interactions and determine potential key residues involved in the binding to lipase inhibitors, we analyzed the binding pockets and binding poses of known inhibitors of the three lipases. We identified the spatial consensus catalytic triad "Ser-Asp-His", a characteristic motif in all three lipases. Furthermore, we found that the spatial characteristics of the binding pockets of the lipase molecules play a key role in ligand recognition, binding poses, and affinities. To the best of our knowledge, this is the first report that systematically builds homology models of all the triglyceride lipase gene subfamily members. Our data provide novel insights into the molecular structures of lipases and their structure-function relationship, and thus provides groundwork for functional probe design towards lipase-based therapeutic inhibitors for the treatment of hyperlipidemia and atherosclerosis.

  16. Curcumin binds in silico to anti-cancer drug target enzyme MMP-3 (human stromelysin-1) with affinity comparable to two known inhibitors of the enzyme.

    Science.gov (United States)

    Jerah, Ahmed; Hobani, Yahya; Kumar, B Vinod; Bidwai, Anil

    2015-01-01

    In silico interaction of curcumin with the enzyme MMP-3 (human stromelysin-1) was studied by molecular docking using AutoDock 4.2 as the docking software application. AutoDock 4.2 software serves as a valid and acceptable docking application to study the interactions of small compounds with proteins. Interactions of curcumin with MMP-3 were compared to those of two known inhibitors of the enzyme, PBSA and MPPT. The calculated free energy of binding (ΔG binding) shows that curcumin binds with affinity comparable to or better than the two known inhibitors. Binding interactions of curcumin with active site residues of the enzyme are also predicted. Curcumin appears to bind in an extendended conformation making extensive VDW contacts in the active site of the enzyme. Hydrogen bonding and pi-pi interactions with key active site residues is also observed. Thus, curcumin can be considered as a good lead compound in the development of new inhibitors of MMP-3 which is a potential target of anticancer drugs. The results of these studies can serve as a starting point for further computational and experimental studies. PMID:26420919

  17. Comparative Study for Selection of Open Source Hypervisors and Cloud Architectures under Variant Requirements

    Directory of Open Access Journals (Sweden)

    BELA SHRIMALI

    2016-04-01

    Full Text Available Cloud computing has been emerging as one of the prominent technologies to the end users which offers pay-asyou- go model with the help of various underlying technologies. One of such technologies is virtualization that forms the foundation for Cloud environment. Virtualization makes it possible to run multiple computing resources (in form of Virtual Machines - VMs on the same (physical server at same time, concurrently. Hypervisors, also known as Virtual Machine Monitors (VMM, create, manage and run virtual machines on physical machines. There are many open source hypervisors available viz. KVM, Xen, OpenVZ, VirtualBox, Lguest, LXD etc. In addition, there are many Cloud-computing software platforms available that manage the provisioning of VMs for the Cloud provider. Compatibility issues of these VMM and Cloud architectures are required to be handled for smooth functioning of the Cloud. This research aims to compare and analyze the most frequently used open source hypervisors and cloud architectures, and it would be useful to novice researchers who intend to plunge into the field of Cloud computing with an aim to use open source options for implementation of their Cloud setup.

  18. The HhH2/NDD domain of the Drosophila Nod chromokinesin-like protein is required for binding to chromosomes in the oocyte nucleus.

    Science.gov (United States)

    Cui, Wei; Hawley, R Scott

    2005-12-01

    Nod is a chromokinesin-like protein that plays a critical role in segregating achiasmate chromosomes during female meiosis. The C-terminal half of the Nod protein contains two putative DNA-binding domains. The first of these domains, known as the HMGN domain, consists of three tandemly repeated high-mobility group N motifs. This domain was previously shown to be both necessary and sufficient for binding of the C-terminal half of Nod to mitotic chromosomes in embryos. The second putative DNA-binding domain, denoted HhH(2)/NDD, is a helix-hairpin-helix(2)/Nod-like DNA-binding domain. Although the HhH(2)/NDD domain is not required or sufficient for chromosome binding in embryos, several well-characterized nod mutations have been mapped in this domain. To characterize the role of the HhH(2)/NDD domain in mediating Nod function, we created a series of UAS-driven transgene constructs capable of expressing either a wild-type Nod-GFP fusion protein or proteins in which the HhH(2)/NDD domain had been altered by site-directed mutagenesis. Although wild-type Nod-GFP localizes to the oocyte chromosomes and rescues the segregation defect in nod mutant oocytes, two of three proteins carrying mutants in the HhH(2)/NDD domain fail to either rescue the nod mutant phenotype or bind to oocyte chromosomes. However, these mutant proteins do bind to the polytene chromosomes in nurse-cell nuclei and enter the oocyte nucleus. Thus, even though the HhH(2)/NDD domain is not essential for chromosome binding in other cell types, it is required for chromosome binding in the oocyte. These HhH(2)/NDD mutants also block the localization of Nod to the posterior pole of stage 9-10A oocytes, a process that is thought to facilitate the interaction of Nod with the plus ends of microtubules (Cui et al. 2005). This observation suggests that the Nod HhH2/NDD domain may play other roles in addition to binding Nod to meiotic chromosomes.

  19. The wing in yeast heat shock transcription factor (HSF) DNA-binding domain is required for full activity

    OpenAIRE

    Cicero, Marco P.; T. Hubl, Susan; Harrison, Celia J.; Littlefield, Otis; Hardy, Jeanne A.; Nelson, Hillary C. M.

    2001-01-01

    The yeast heat shock transcription factor (HSF) belongs to the winged helix family of proteins. HSF binds DNA as a trimer, and additional trimers can bind DNA co-operatively. Unlike other winged helix–turn–helix proteins, HSF’s wing does not appear to contact DNA, as based on a previously solved crystal structure. Instead, the structure implies that the wing is involved in protein–protein interactions, possibly within a trimer or between adjacent trimers. To unders...

  20. CYB5D2 requires heme-binding to regulate HeLa cell growth and confer survival from chemotherapeutic agents.

    Directory of Open Access Journals (Sweden)

    Anthony Bruce

    Full Text Available The cytochrome b5 domain containing 2 (CYB5D2; Neuferricin protein has been reported to bind heme, however, the critical residues responsible for heme-binding are undefined. Furthermore, the relationship between heme-binding and CYB5D2-mediated intracellular functions remains unknown. Previous studies examining heme-binding in two cytochrome b5 heme-binding domain-containing proteins, damage-associated protein 1 (Dap1; Saccharomyces cerevisiae and human progesterone receptor membrane component 1 (PGRMC1, have revealed that conserved tyrosine (Y 73, Y79, aspartic acid (D 86, and Y127 residues present in human CYB5D2 may be involved in heme-binding. CYB5D2 binds to type b heme, however, only the substitution of glycine (G at D86 (D86G within its cytochrome b5 heme-binding (cyt-b5 domain abolished its heme-binding ability. Both CYB5D2 and CYB5D2(D86G localize to the endoplasmic reticulum. Ectopic CYB5D2 expression inhibited cell proliferation and anchorage-independent colony growth of HeLa cells. Conversely, CYB5D2 knockdown and ectopic CYB5D2(D86G expression increased cell proliferation and colony growth. As PGRMC1 has been reported to regulate the expression and activities of cytochrome P450 proteins (CYPs, we examined the role of CYB5D2 in regulating the activities of CYPs involved in sterol synthesis (CYP51A1 and drug metabolism (CYP3A4. CYB5D2 co-localizes with cytochrome P450 reductase (CYPOR, while CYB5D2 knockdown reduced lanosterol demethylase (CYP51A1 levels and rendered HeLa cells sensitive to mevalonate. Additionally, knockdown of CYB5D2 reduced CYP3A4 activity. Lastly, CYB5D2 expression conferred HeLa cell survival from chemotherapeutic agents (paclitaxel, cisplatin and doxorubicin, with its ability to promote survival being dependent on its heme-binding ability. Taken together, this study provides evidence that heme-binding is critical for CYB5D2 in regulating HeLa cell growth and survival, with endogenous CYB5D2 being required to

  1. Comparative genome analysis of cortactin and HSI : the significance of the F-actin binding repeat domain

    NARCIS (Netherlands)

    van Rossum, AGSH; Schuuring-Scholtes, E; Seggelen, VV; Kluin, PM; Schuuring, E

    2005-01-01

    Background: In human carcinomas, overexpression of cortactin correlates with poor prognosis. Cortactin is an F-actin-binding protein involved in cytoskeletal rearrangements and cell migration by promoting actin-related protein (Arp)2/3 mediated actin polymerization. It shares a high amino acid seque

  2. Calmodulin binding to glutamate decarboxylase is required for regulation of glutamate and GABA metabolism and normal development in plants.

    Science.gov (United States)

    Baum, G; Lev-Yadun, S; Fridmann, Y; Arazi, T; Katsnelson, H; Zik, M; Fromm, H

    1996-06-17

    Glutamate decarboxylase (GAD) catalyzes the decarboxylation of glutamate to CO2 and gamma-aminobutyrate (GABA). GAD is ubiquitous in prokaryotes and eukaryotes, but only plant GAD has been shown to bind calmodulin (CaM). Here, we assess the role of the GAD CaM-binding domain in vivo. Transgenic tobacco plants expressing a mutant petunia GAD lacking the CaM-binding domain (GADdeltaC plants) exhibit severe morphological abnormalities, such as short stems, in which cortex parenchyma cells fail to elongate, associated with extremely high GABA and low glutamate levels. The morphology of transgenic plants expressing the full-length GAD (GAD plants) is indistinguishable from that of wild-type (WT) plants. In WT and GAD plant extracts, GAD activity is inhibited by EGTA and by the CaM antagonist trifluoperazine, and is associated with a CaM-containing protein complex of approximately 500 kDa. In contrast, GADdeltaC plants lack normal GAD complexes, and GAD activity in their extracts is not affected by EGTA and trifluoperazine. We conclude that CaM binding to GAD is essential for the regulation of GABA and glutamate metabolism, and that regulation of GAD activity is necessary for normal plant development. This study is the first to demonstrate an in vivo function for CaM binding to a target protein in plants.

  3. Comparative Analysis of DNA-Binding Selectivity of Hairpin and Cyclic Pyrrole-Imidazole Polyamides Based on Next-Generation Sequencing.

    Science.gov (United States)

    Kashiwazaki, Gengo; Chandran, Anandhakumar; Asamitsu, Sefan; Kawase, Takashi; Kawamoto, Yusuke; Sawatani, Yoshito; Hashiya, Kaori; Bando, Toshikazu; Sugiyama, Hiroshi

    2016-09-15

    Many long pyrrole-imidazole polyamides (PIPs) have been synthesized in the search for higher specificity, with the aim of realizing the great potential of such compounds in biological and clinical areas. Among several types of PIPs, we designed and synthesized hairpin and cyclic PIPs targeting identical sequences. Bind-n-Seq analysis revealed that both bound to the intended sequences. However, adenines in the data analyzed by the previously reported Bind-n-Seq method appeared to be significantly higher in the motif ratio than thymines, even though the PIPs were not expected to distinguish A from T. We therefore examined the experimental protocol and analysis pipeline in detail and developed a new method based on Bind-n-Seq motif identification with a reference sequence (Bind-n-Seq-MR). High-throughput sequence analysis of the PIP-enriched DNA data by Bind-n-Seq-MR presented A and T comparably. Surface plasmon resonance assays were performed to validate the new method. PMID:27387250

  4. Thyroid hormone receptor binds to a site in the rat growth hormone promoter required for induction by thyroid hormone

    International Nuclear Information System (INIS)

    Transcription of the rat growth hormone (rGH) gene in pituitary cells is increased by addition of thyroid hormone (T3). This induction is dependent on the presence of specific sequences just upstream of the rGH promoter. The authors have partially purified T3 receptor from rat liver and examined its interaction with these rGH sequences. They show here that T3 receptor binds specifically to a site just upstream of the basal rGH promoter. This binding site includes two copies of a 7-base-pair direct repeat, the centers of which are separated by 10 base pairs. Deletions that specifically remove the T3 receptor binding site drastically reduce response to T3 in transient transfection experiments. These results demonstrate that T3 receptor can recognize specific DNA sequences and suggest that it can act directly as a positive transcriptional regulatory factor

  5. Targeted deletion of multiple CTCF-binding elements in the human C-MYC gene reveals a requirement for CTCF in C-MYC expression.

    Directory of Open Access Journals (Sweden)

    Wendy M Gombert

    Full Text Available BACKGROUND: Insulators and domain boundaries both shield genes from adjacent enhancers and inhibit intrusion of heterochromatin into transgenes. Previous studies examined the functional mechanism of the MYC insulator element MINE and its CTCF binding sites in the context of transgenes that were randomly inserted into the genome by transfection. However, the contribution of CTCF binding sites to both gene regulation and maintenance of chromatin has not been tested at the endogenous MYC gene. METHODOLOGY/PRINCIPAL FINDINGS: To determine the impact of CTCF binding on MYC expression, a series of mutant human chromosomal alleles was prepared in homologous recombination-efficient DT40 cells and individually transferred by microcell fusion into murine cells. Functional tests reported here reveal that deletion of CTCF binding elements within the MINE does not impact the capacity of this locus to correctly organize an 'accessible' open chromatin domain, suggesting that these sites are not essential for the formation of a competent, transcriptionally active locus. Moreover, deletion of the CTCF site at the MYC P2 promoter reduces transcription but does not affect promoter acetylation or serum-inducible transcription. Importantly, removal of either CTCF site leads to DNA methylation of flanking sequences, thereby contributing to progressive loss of transcriptional activity. CONCLUSIONS: These findings collectively demonstrate that CTCF-binding at the human MYC locus does not repress transcriptional activity but is required for protection from DNA methylation.

  6. Stringent integrity requirements for both trans-activation and DNA-binding in a trans-activator, Oct3.

    OpenAIRE

    Imagawa, M; Miyamoto, A.; Shirakawa, M.; Hamada, H; Muramatsu, M

    1991-01-01

    POU-specific and POU-homeo domains of Oct3 were produced in Echerichia coli for characterization of DNA binding to the octamer sequence. POU domain protein including A, B and H domains could bind to the octamer sequence efficiently and specifically, and DNase I footprint analysis gave an indistinguishable protection pattern between recombinant POU protein of Oct3 and native Oct3 from undifferentiated P19 cells. Truncated mutants, which contained B-specific and H domains or the H domain only, ...

  7. A prospective cohort study comparing early opioid requirement between Chinese from Hong Kong and Caucasian Australians after major abdominal surgery

    DEFF Research Database (Denmark)

    Konstantatos, A H; Imberger, G; Angliss, M;

    2012-01-01

    The relationship between ethnicity and early opioid consumption is not well understood. Our prospective cohort study tested whether Chinese patients in Hong Kong require less opioid after major abdominal surgery compared with Caucasian patients in Australia.......The relationship between ethnicity and early opioid consumption is not well understood. Our prospective cohort study tested whether Chinese patients in Hong Kong require less opioid after major abdominal surgery compared with Caucasian patients in Australia....

  8. A protein binding AT-rich sequence in the soybean leghemoglobin c3 promoter is a general cis element that requires proximal DNA elements to stimulate transcription

    DEFF Research Database (Denmark)

    Laursen, N B; Larsen, K; Knudsen, J Y;

    1994-01-01

    A nodule nuclear factor, NAT2, interacts with two AT-rich binding sites (NAT2 BS1 and NAT2 BS2) in the soybean leghemoglobin (lb) c3 promoter. In transgenic Lotus corniculatus nodules, an oligonucleotide containing NAT2 BS1 activated an inactive -159 lbc3 promoter when placed immediately upstream...... of the promoter. The activation was independent of the orientation of NAT2 BS1 but was dependent on its position in the promoter. The abilities of different mutated binding sites to activate expression in vivo were correlated to their respective in vitro affinities for binding NAT2. This suggested......(s) is required for the function of NAT2 BS1. Thus, the -102 lbc3 promoter lacking the organ-specific element (-139 to -102) was not reactivated by the presence of the binding site, and the rbcS-8B promoter required sequences between -312 and -257 to be activated by NAT2 BS1. This implies that NAT2 has to work...

  9. Vaccinia protein F12 has structural similarity to kinesin light chain and contains a motor binding motif required for virion export.

    Directory of Open Access Journals (Sweden)

    Gareth W Morgan

    2010-02-01

    Full Text Available Vaccinia virus (VACV uses microtubules for export of virions to the cell surface and this process requires the viral protein F12. Here we show that F12 has structural similarity to kinesin light chain (KLC, a subunit of the kinesin-1 motor that binds cargo. F12 and KLC share similar size, pI, hydropathy and cargo-binding tetratricopeptide repeats (TPRs. Moreover, molecular modeling of F12 TPRs upon the crystal structure of KLC2 TPRs showed a striking conservation of structure. We also identified multiple TPRs in VACV proteins E2 and A36. Data presented demonstrate that F12 is critical for recruitment of kinesin-1 to virions and that a conserved tryptophan and aspartic acid (WD motif, which is conserved in the kinesin-1-binding sequence (KBS of the neuronal protein calsyntenin/alcadein and several other cellular kinesin-1 binding proteins, is essential for kinesin-1 recruitment and virion transport. In contrast, mutation of WD motifs in protein A36 revealed they were not required for kinesin-1 recruitment or IEV transport. This report of a viral KLC-like protein containing a KBS that is conserved in several cellular proteins advances our understanding of how VACV recruits the kinesin motor to virions, and exemplifies how viruses use molecular mimicry of cellular components to their advantage.

  10. Fatty Acid-binding Proteins Interact with Comparative Gene Identification-58 Linking Lipolysis with Lipid Ligand Shuttling*

    OpenAIRE

    Hofer, Peter; Boeszoermenyi, Andras; Jaeger, Doris; Feiler, Ursula; Arthanari, Haribabu; Mayer, Nicole; Zehender, Fabian; Rechberger, Gerald; Oberer, Monika; Zimmermann, Robert; Lass, Achim; Haemmerle, Guenter; Breinbauer, Rolf; Zechner, Rudolf; Preiss-Landl, Karina

    2015-01-01

    Background: A multiprotein complex designated as lipolysome degrades intracellular triglycerides and contains proteins such as adipose triglyceride lipase (Atgl) and its co-activator Cgi-58. Results: Cgi-58 interacts with fatty acid-binding proteins (Fabps), which impact Atgl-mediated lipolysis and lipid signaling. Conclusion: Fabps modulate Atgl-mediated TG hydrolysis and link lipolysis with intracellular lipid ligand shuttling. Significance: Novel mechanistic insights into the regulation of...

  11. Artificial tissue binding models : development and comparative evaluation of high - throughput lipophilicity assays and their use for PET tracer optimization

    OpenAIRE

    Assmus, Frauke

    2015-01-01

    The purpose of this thesis was to increase the efficiency of the Positron Emission Tomography (PET) tracer development process. Since many neuroimaging agents fail due to undesirably high non-specific binding (NSB) to brain tissue, we aimed at estimating the extent of NSB as early as possible, preferably before radioactive labeling and extensive animal testing. To this purpose we have developed, optimized and evaluated several in vitro assays with respect to their ability to predict brain tis...

  12. The nucleolar GTP-binding proteins Gnl2 and nucleostemin are required for retinal neurogenesis in developing zebrafish

    NARCIS (Netherlands)

    Paridaen, J.T.M.; Janson, E.; Utami, K.H.; Pereboom, T.C.; Essers, P.; van Rooijen, C.R.; Zivkovic, D.; MacInnes, A.W.

    2011-01-01

    Nucleostemin (NS), a member of a family of nucleolar GTP-binding proteins, is highly expressed in proliferating cells such as stem and cancer cells and is involved in the control of cell cycle progression. Both depletion and overexpression of NS result in stabilization of the tumor suppressor p53 pr

  13. Mannose-Binding Lectin Is Required for the Effective Clearance of Apoptotic Cells by Adipose Tissue Macrophages During Obesity

    NARCIS (Netherlands)

    Stienstra, R.; Dijk, W.; Beek, van L.; Jansen, H.; Heemskerk, M.; Houtkooper, R.H.; Denis, S.; Harmelen, van V.; Willems van Dijk, K.; Tack, C.J.; Kersten, A.H.

    2014-01-01

    Obesity is accompanied by the presence of chronic low-grade inflammation manifested by infiltration of macrophages into adipose tissue. Mannose-binding lectin (MBL), a soluble mediator of innate immunity, promotes phagocytosis and alters macrophage function. To assess the function of MBL in the deve

  14. A C-terminal PDZ domain-binding sequence is required for striatal distribution of the dopamine transporter

    DEFF Research Database (Denmark)

    Rickhag, Karl Mattias; Hansen, Freja Herborg; Sørensen, Gunnar;

    2013-01-01

    The dopamine transporter mediates reuptake of dopamine from the synaptic cleft. The cellular mechanisms controlling dopamine transporter levels in striatal nerve terminals remain poorly understood. The dopamine transporters contain a C-terminal PDZ (PSD-95/Discs-large/ZO-1) domain-binding sequenc...

  15. Is It Reliable to Use Common Molecular Docking Methods for Comparing the Binding Affinities of Enantiomer Pairs for Their Protein Target?

    Science.gov (United States)

    Ramírez, David; Caballero, Julio

    2016-01-01

    Molecular docking is a computational chemistry method which has become essential for the rational drug design process. In this context, it has had great impact as a successful tool for the study of ligand-receptor interaction modes, and for the exploration of large chemical datasets through virtual screening experiments. Despite their unquestionable merits, docking methods are not reliable for predicting binding energies due to the simple scoring functions they use. However, comparisons between two or three complexes using the predicted binding energies as a criterion are commonly found in the literature. In the present work we tested how wise is it to trust the docking energies when two complexes between a target protein and enantiomer pairs are compared. For this purpose, a ligand library composed by 141 enantiomeric pairs was used, including compounds with biological activities reported against seven protein targets. Docking results using the software Glide (considering extra precision (XP), standard precision (SP), and high-throughput virtual screening (HTVS) modes) and AutoDock Vina were compared with the reported biological activities using a classification scheme. Our test failed for all modes and targets, demonstrating that an accurate prediction when binding energies of enantiomers are compared using docking may be due to chance. We also compared pairs of compounds with different molecular weights and found the same results. PMID:27104528

  16. Unbiased mutagenesis of MHV68 LANA reveals a DNA-binding domain required for LANA function in vitro and in vivo.

    Directory of Open Access Journals (Sweden)

    Clinton R Paden

    2012-09-01

    Full Text Available The Latency-Associated Nuclear Antigen (LANA, encoded by ORF73, is a conserved gene among the γ2-herpesviruses (rhadinoviruses. The Kaposi's Sarcoma-Associated Herpesvirus (KSHV LANA is consistently expressed in KSHV-associated malignancies. In the case of the rodent γ2-herpesvirus, murine gammaherpesvirus 68 (MHV68, the LANA homolog (mLANA is required for efficient virus replication, reactivation from latency and immortalization of murine fetal liver-derived B cells. To gain insights into mLANA function(s, knowing that KSHV LANA binds DNA and can modulate transcription of a variety of promoters, we sought out and identified a mLANA-responsive promoter which maps to the terminal repeat (TR of MHV68. Notably, mLANA strongly repressed activity from this promoter. We extended these analyses to demonstrate direct, sequence-specific binding of recombinant mLANA to TR DNA by DNase I footprinting. To assess whether the DNA-binding and/or transcription modulating function is important in the known mLANA phenotypes, we generated an unbiased library of mLANA point mutants using error-prone PCR, and screened a large panel of mutants for repression of the mLANA-responsive promoter to identify loss of function mutants. Notably, among the mutant mLANA proteins recovered, many of the mutations are in a predicted EBNA-1-like DNA-binding domain. Consistent with this prediction, those tested displayed loss of DNA binding activity. We engineered six of these mLANA mutants into the MHV68 genome and tested the resulting mutant viruses for: (i replication fitness; (ii efficiency of latency establishment; and (iii reactivation from latency. Interestingly, each of these mLANA-mutant viruses exhibited phenotypes similar to the mLANA-null mutant virus, indicating that DNA-binding is critical for mLANA function.

  17. IGD motifs, which are required for migration stimulatory activity of fibronectin type I modules, do not mediate binding in matrix assembly.

    Directory of Open Access Journals (Sweden)

    Lisa M Maurer

    Full Text Available Picomolar concentrations of proteins comprising only the N-terminal 70-kDa region (70K of fibronectin (FN stimulate cell migration into collagen gels. The Ile-Gly-Asp (IGD motifs in four of the nine FN type 1 (FNI modules in 70K are important for such migratory stimulating activity. The 70K region mediates binding of nanomolar concentrations of intact FN to cell-surface sites where FN is assembled. Using baculovirus, we expressed wildtype 70K and 70K with Ile-to-Ala mutations in (3FNI and (5FNI; (7FNI and (9FNI; or (3FNI, (5FNI, (7FNI, and (9FNI. Wildtype 70K and 70K with Ile-to-Ala mutations were equally active in binding to assembly sites of FN-null fibroblasts. This finding indicates that IGD motifs do not mediate the interaction between 70K and the cell-surface that is important for FN assembly. Further, FN fragment N-(3FNIII, which does not stimulate migration, binds to assembly sites on FN-null fibroblast. The Ile-to-Ala mutations had effects on the structure of FNI modules as evidenced by decreases in abilities of 70K with Ile-to-Ala mutations to bind to monoclonal antibody 5C3, which recognizes an epitope in (9FNI, or to bind to FUD, a polypeptide based on the F1 adhesin of Streptococcus pyogenes that interacts with 70K by the β-zipper mechanism. These results suggest that the picomolar interactions of 70K with cells that stimulate cell migration require different conformations of FNI modules than the nanomolar interactions required for assembly.

  18. Idas, a novel phylogenetically conserved geminin-related protein, binds to geminin and is required for cell cycle progression.

    Science.gov (United States)

    Pefani, Dafni-Eleutheria; Dimaki, Maria; Spella, Magda; Karantzelis, Nickolas; Mitsiki, Eirini; Kyrousi, Christina; Symeonidou, Ioanna-Eleni; Perrakis, Anastassis; Taraviras, Stavros; Lygerou, Zoi

    2011-07-01

    Development and homeostasis of multicellular organisms relies on an intricate balance between cell proliferation and differentiation. Geminin regulates the cell cycle by directly binding and inhibiting the DNA replication licensing factor Cdt1. Geminin also interacts with transcriptional regulators of differentiation and chromatin remodelling factors, and its balanced interactions are implicated in proliferation-differentiation decisions during development. Here, we describe Idas (Idas being a cousin of the Gemini in Ancient Greek Mythology), a previously uncharacterised coiled-coil protein related to Geminin. We show that human Idas localizes to the nucleus, forms a complex with Geminin both in cells and in vitro through coiled-coil mediated interactions, and can change Geminin subcellular localization. Idas does not associate with Cdt1 and prevents Geminin from binding to Cdt1 in vitro. Idas depletion from cells affects cell cycle progression; cells accumulate in S phase and are unable to efficiently progress to mitosis. Idas protein levels decrease in anaphase, whereas its overexpression causes mitotic defects. During development, we show that Idas exhibits high level expression in the choroid plexus and the cortical hem of the mouse telencephalon. Our data highlight Idas as a novel Geminin binding partner, implicated in cell cycle progression, and a putative regulator of proliferation-differentiation decisions during development. PMID:21543332

  19. Correlation between oxytocin neuronal sensitivity and oxytocin receptor binding: An electrophysiological and autoradiographical study comparing rat and guinea pig hippocampus

    Energy Technology Data Exchange (ETDEWEB)

    Raggenbass, M.; Tribollet, E.; Dubois-Dauphin, M.; Dreifuss, J.J. (Univ. Medical Center, Geneva (Switzerland))

    1989-01-01

    In transverse hippocampal slices from rat and guinea pig brains, the authors obtained unitary extracellular recordings from nonpyramidal neurones located in or near the stratum pyramidale in the CA1 field and in the transition region between the CA1 and the subiculum. In rats, these neurones responded to oxytocin at 50-1,000 nM by a reversible increase in firing rate. The oxytocin-induced excitation was suppressed by a synthetic structural analogue that acts as a potent, selective antioxytocic on peripheral receptors. Nonpyramidal neurones were also excited by carbachol at 0.5-10 {mu}M. The effect of this compound was postsynaptic and was blocked by the muscarinic antagonist atropine. In guinea pigs, by contrast, nonpyramidal neurones were unaffected by oxytocin, although they were excited by carbachol. Light microscopic autoradiography, carried out using a radioiodinated selective antioxytocic as a ligand, revealed labeling in the subiculum and in the CA1 area of the hippocampus of rats, whereas no oxytocin-binding sites were detected in the hippocampus of guinea pigs. The results indicate (i) that a hippocampal action of oxytocin is species-dependent and (ii) that a positive correlation exists between neuronal responsiveness to oxytocin and the presence in the hippocampus of high-affinity binding sites for this peptide.

  20. 78 FR 78839 - Comparability Determination for Canada: Certain Entity-Level Requirements

    Science.gov (United States)

    2013-12-27

    ... issues, and remedial action plans. In addition, the OSFI Corporate Governance Guideline of Federally Regulated Financial Institutions (``OSFI Corporate Governance Guideline'') states that the bank's board of... regulation Sec. 23.600. The OSFI Corporate Governance Guideline requires that each bank establish a...

  1. 78 FR 78852 - Comparability Determination for Hong Kong: Certain Entity-Level Requirements

    Science.gov (United States)

    2013-12-27

    ... reports from the AIs. AIs are required to submit quarterly capital adequacy ratio returns to the HKMA that... Kong. This notice also does not address capital adequacy because the Commission has not yet finalized... regulations promulgated thereunder. \\1\\ 78 FR 45292 (July 26, 2013). The Commission originally...

  2. 78 FR 78878 - Comparability Determination for the European Union: Certain Transaction-Level Requirements

    Science.gov (United States)

    2013-12-27

    ... hedging definition both portfolio and macro hedging are allowed. \\19\\ See EMIR Article 10 and RTS Article... requirements of the CEA and the Commission's regulations promulgated thereunder. \\1\\ 78 FR 45292 (July 26, 2013... Exchange Act, 77 FR 41214 (July 12, 2012) and Further Proposed Guidance Regarding Compliance with...

  3. Vsx2 controls eye organogenesis and retinal progenitor identity via homeodomain and non-homeodomain residues required for high affinity DNA binding.

    Directory of Open Access Journals (Sweden)

    Changjiang Zou

    2012-09-01

    Full Text Available The homeodomain and adjacent CVC domain in the visual system homeobox (VSX proteins are conserved from nematodes to humans. Humans with missense mutations in these regions of VSX2 have microphthalmia, suggesting both regions are critical for function. To assess this, we generated the corresponding mutations in mouse Vsx2. The homeodomain mutant protein lacked DNA binding activity and the knock-in mutant phenocopied the null mutant, ocular retardation J. The CVC mutant protein exhibited weakened DNA binding; and, although the corresponding knock-in allele was recessive, it unexpectedly caused the strongest phenotype, as indicated by severe microphthalmia and hyperpigmentation of the neural retina. This occurred through a cryptic transcriptional feedback loop involving the transcription factors Mitf and Otx1 and the Cdk inhibitor p27(Kip1. Our data suggest that the phenotypic severity of the CVC mutant depends on the weakened DNA binding activity elicited by the CVC mutation and a previously unknown protein interaction between Vsx2 and its regulatory target Mitf. Our data also suggest that an essential function of the CVC domain is to assist the homeodomain in high-affinity DNA binding, which is required for eye organogenesis and unhindered execution of the retinal progenitor program in mammals. Finally, the genetic and phenotypic behaviors of the CVC mutation suggest it has the characteristics of a recessive neomorph, a rare type of genetic allele.

  4. Compared Binding Properties between Resveratrol and Other Polyphenols to Plasmatic Albumin: Consequences for the Health Protecting Effect of Dietary Plant Microcomponents

    Directory of Open Access Journals (Sweden)

    Norbert Latruffe

    2014-10-01

    Full Text Available Phytophenols are considered to have beneficial effects towards human physiology. They are food microcomponents with potent chemopreventive properties towards the most three frequent contemporary human diseases, e.g., cardiovascular alterations, cancer and neurodegenerative pathologies. Related to this, the plasmatic form and plasmatic level of plant polyphenols in the body circulation are crucial for their efficiency. Thus, determinations of the binding process of resveratrol and of common flavonoids produced by major edible plants, berries and fruits to plasma proteins are essential. The interactions between resveratrol and albumin, a major plasma protein, were compared with those already published, involving curcumin, genistein, quercetin and other well-known food-containing polyphenols. The approaches used are usually intrinsic fluorescence intensity changes, quenching of protein intrinsic fluorescence and infrared spectroscopy. It appears that: (1 all of the studied polyphenols interact with albumin; (2 while most of the studied polyphenols interact at one albumin binding site, there are two different types of resveratrol binding sites for bovine serum albumin, one with the highest affinity (apparent KD of 4 µM with a stoichiometry of one per monomer and a second with a lower affinity (apparent KD of 20 µM with also a stoichiometry of one per monomer; (3 at least one binding site is in the vicinity of one tryptophanyl residue of bovine serum albumin; and (4 resveratrol binding to bovine serum albumin produces a very small structural conformation change of the polypeptide chain. These results support a role played by polyphenols-albumin interactions in the plasma for the bio-activities of these food microcomponents in the body.

  5. Comparative Analysis Between US NRC Requirements and US DOE Orders - 13402

    International Nuclear Information System (INIS)

    Small modular reactor (SMR) is a nuclear reactor design approach that is expected to herald in a new era of clean energy in the U.S. These reactors are less than one-third the size of conventional large nuclear power reactors, and have factory-fabricated components that may be transported by rail or truck to a site selected to house a small nuclear reactor. To facilitate the licensing of these smaller nuclear reactor designs, the Nuclear Regulatory Commission (NRC) is in the process of developing a regulatory infrastructure to support licensing review of these unique reactor designs. As part of these activities, the NRC has been meeting with the Department of Energy (DOE) and with individual SMR designers to discuss potential policy, licensing, and key technical differences in SMR designs. It is anticipated by the NRC that such licensing interaction and guidance early in the design process will contribute towards minimizing complexity while adding stability and predictability in the licensing and subsequent regulation of new reactor designs such as SMRs. In conjunction with the current NRC initiative of developing the SMR licensing process, early communication and collaboration in the identification and resolution of any potential technical and licensing differences between NRC requirements and similar requirements applicable at DOE sites would help to expedite demonstration and implementation of SMR technology in the US. In order to foster such early communication, Savannah River Nuclear Solutions (SRNS) has begun taking the first steps in identifying and evaluating potential licensing gaps that may exist between NRC and DOE requirements in siting SMRs at DOE sites. A comparison between the existing NRC regulations for Early Site Permits and the DOE Orders was undertaken to establish the degree of correlation between NRC requirements and compliance methods in place at DOE sites. The ability to use existing data and information to expedite the development of the

  6. DIFFERENT REQUIREMENTS OF THE KINASE AND UHM DOMAINS OF KIS FOR ITS NUCLEAR LOCALIZATION AND BINDING TO SPLICING FACTORS

    OpenAIRE

    Manceau, Valérie; Kielkopf, Clara L.; Sobel, André; Maucuer, Alexandre

    2008-01-01

    The protein kinase KIS is made by the juxtaposition of a unique kinase domain and a C-terminal domain with a U2AF Homology Motif (UHM), a sequence motif for protein interaction initially identified in the heterodimeric pre-mRNA splicing factor U2AF. This domain of KIS is closely related to the C-terminal UHM domain of the U2AF large subunit, U2AF65. KIS phosphorylates the splicing factor SF1, which in turn enhances SF1 binding to U2AF65 and the 3′ splice site, an event known to take place at ...

  7. The extracytoplasmic domain of the Mycobacterium tuberculosis Ser/Thr kinase PknB binds specific muropeptides and is required for PknB localization.

    Directory of Open Access Journals (Sweden)

    Mushtaq Mir

    2011-07-01

    Full Text Available The Mycobacterium tuberculosis Ser/Thr kinase PknB has been implicated in the regulation of cell growth and morphology in this organism. The extracytoplasmic domain of this membrane protein comprises four penicillin binding protein and Ser/Thr kinase associated (PASTA domains, which are predicted to bind stem peptides of peptidoglycan. Using a comprehensive library of synthetic muropeptides, we demonstrate that the extracytoplasmic domain of PknB binds muropeptides in a manner dependent on the presence of specific amino acids at the second and third positions of the stem peptide, and on the presence of the sugar moiety N-acetylmuramic acid linked to the peptide. We further show that PknB localizes strongly to the mid-cell and also to the cell poles, and that the extracytoplasmic domain is required for PknB localization. In contrast to strong growth stimulation by conditioned medium, we observe no growth stimulation of M. tuberculosis by a synthetic muropeptide with high affinity for the PknB PASTAs. We do find a moderate effect of a high affinity peptide on resuscitation of dormant cells. While the PASTA domains of PknB may play a role in stimulating growth by binding exogenous peptidoglycan fragments, our data indicate that a major function of these domains is for proper PknB localization, likely through binding of peptidoglycan fragments produced locally at the mid-cell and the cell poles. These data suggest a model in which PknB is targeted to the sites of peptidoglycan turnover to regulate cell growth and cell division.

  8. Subunit architecture of the Golgi Dsc E3 ligase required for sterol regulatory element-binding protein (SREBP) cleavage in fission yeast.

    Science.gov (United States)

    Lloyd, S Julie-Ann; Raychaudhuri, Sumana; Espenshade, Peter J

    2013-07-19

    The membrane-bound sterol regulatory element-binding protein (SREBP) transcription factors regulate lipogenesis in mammalian cells and are activated through sequential cleavage by the Golgi-localized Site-1 and Site-2 proteases. The mechanism of fission yeast SREBP cleavage is less well defined and, in contrast, requires the Golgi-localized Dsc E3 ligase complex. The Dsc E3 ligase consists of five integral membrane subunits, Dsc1 through Dsc5, and resembles membrane E3 ligases that function in endoplasmic reticulum-associated degradation. Using immunoprecipitation assays and blue native electrophoresis, we determined the subunit architecture for the complex of Dsc1 through Dsc5, showing that the Dsc proteins form subcomplexes and display defined connectivity. Dsc2 is a rhomboid pseudoprotease family member homologous to mammalian UBAC2 and a central component of the Dsc E3 ligase. We identified conservation in the architecture of the Dsc E3 ligase and the multisubunit E3 ligase gp78 in mammals. Specifically, Dsc1-Dsc2-Dsc5 forms a complex resembling gp78-UBAC2-UBXD8. Further characterization of Dsc2 revealed that its C-terminal UBA domain can bind to ubiquitin chains but that the Dsc2 UBA domain is not essential for yeast SREBP cleavage. Based on the ability of rhomboid superfamily members to bind transmembrane proteins, we speculate that Dsc2 functions in SREBP recognition and binding. Homologs of Dsc1 through Dsc4 are required for SREBP cleavage and virulence in the human opportunistic pathogen Aspergillus fumigatus. Thus, these studies advance our organizational understanding of multisubunit E3 ligases involved in endoplasmic reticulum-associated degradation and fungal pathogenesis.

  9. Comparing credentialing requirements of substance abuse treatment staff by funding source.

    Science.gov (United States)

    Kubiak, Sheryl Pimlott; Arfken, Cynthia L

    2008-07-01

    Studies have found that clinicians with higher education and/or attainment of national certification have a more favorable outlook regarding the adoption of evidence-based practices. However, staff hiring decisions may be based on a multitude of factors, including available resources and demands stemming from different funders. Using a mixed-methods case study approach with 34 agencies within one state, we assessed administrators' perspectives of the most important funding source, views on clinical hiring practices, and current staffing. We found that funding source predicted views and actual staff level of credentialing and education. Those agencies citing a criminal justice entity as the most important funder had the lowest requirements for credentialing and education. As the substance abuse treatment delivery system evolves and expands, we must ensure that vulnerable groups have access to more highly--rather than less--skilled workers to assess and facilitate recovery.

  10. Comparative study of the binding pockets of mammalian proprotein convertases and its implications for the design of specific small molecule inhibitors

    Directory of Open Access Journals (Sweden)

    Sun Tian, Wu Jianhua

    2010-01-01

    Full Text Available Proprotein convertases are enzymes that proteolytically cleave protein precursors in the secretory pathway to yield functional proteins. Seven mammalian subtilisin/Kex2p-like proprotein convertases have been identified: furin, PC1, PC2, PC4, PACE4, PC5 and PC7. The binding pockets of all seven proprotein convertases are evolutionarily conserved and highly similar. Among the seven proprotein convertases, the furin cleavage site motif has recently been characterized as a 20-residue motif that includes one core region P6-P2´ inside the furin binding pocket. This study extended this information by examining the 3D structural environment of the furin binding pocket surrounding the core region P6-P2´ of furin substrates. The physical properties of mutations in the binding pockets of the other six mammalian proprotein convertases were compared. The results suggest that: 1 mutations at two positions, Glu230 and Glu257, change the overall density of the negative charge of the binding pockets, and govern the substrate specificities of mammalian proprotein convertases; 2 two proprotein convertases (PC1 and PC2 may have reduced sensitivity for positively charged residues at substrate position P5 or P6, whereas the substrate specificities of three proprotein convertases (furin, PACE4, and PC5 are similar to each other. This finding led to a novel design of a short peptide pattern for small molecule inhibitors: [K/R]-X-V-X-K-R. Compared with the widely used small molecule dec-RVKR-cmk that inhibits all seven proprotein convertases, a finely-tuned derivative of the short peptide pattern [K/R]-X-V-X-K-R may have the potential to more effectively inhibit five of the proprotein convertases (furin, PC4, PACE4, PC5 and PC7 compared to the remaining two (PC1 and PC2. The results not only provide insights into the molecular evolution of enzyme function in the proprotein convertase family, but will also aid the study of the functional redundancy of proprotein

  11. Requirements for capsid-binding and an effector function in TRIMCyp-mediated restriction of HIV-1

    International Nuclear Information System (INIS)

    In owl monkeys, a retrotransposition event replaced the gene encoding the retroviral restriction factor TRIM5α with one encoding TRIMCyp, a fusion between the RING, B-box 2 and coiled-coil domains of TRIM5 and cyclophilin A. TRIMCyp restricts human immunodeficiency virus (HIV-1) infection by a mechanism dependent on the interaction of the cyclophilin A moiety and the HIV-1 capsid protein. Here, we show that infection by retroviruses other than HIV-1 can be restricted by TRIMCyp, providing an explanation for the evolutionary retention of the TRIMCyp gene in owl monkey lineages. The TRIMCyp-mediated block to HIV-1 infection occurs before the earliest step of reverse transcription. TRIMCyp-mediated restriction involves at least two functions: (1) capsid binding, which occurs most efficiently for trimeric TRIMCyp proteins that retain the coiled-coil and cyclophilin A domains, and (2) an effector function that depends upon the B-box 2 domain

  12. Binding Affinity, Specificity and Comparative Biodistribution of the Parental Murine Monoclonal Antibody MX35 (Anti-NaPi2b and Its Humanized Version Rebmab200.

    Directory of Open Access Journals (Sweden)

    Sture Lindegren

    Full Text Available The aim of this preclinical study was to evaluate the characteristics of the monoclonal antibody Rebmab200, which is a humanized version of the ovarian-specific murine antibody MX35. This investigation contributes to the foundation for future clinical α-radioimmunotherapy of minimal residual ovarian cancer with 211At-Rebmab200. Here, the biodistribution of 211At-Rebmab200 was evaluated, as was the utility of 99mTc-Rebmab200 for bioimaging. Rebmab200 was directly compared with its murine counterpart MX35 in terms of its in-vitro capacity for binding the immobilized NaPi2B epitope and live cells; we also assessed its biodistribution in nude mice carrying subcutaneous OVCAR-3 tumors. Tumor antigen and cell binding were similar between Rebmab200 and murine MX35, as was biodistribution, including normal tissue uptake and in-vivo tumor binding. We also demonstrated that 99mTc-Rebmab200 can be used for single-photon emission computed tomography of subcutaneous ovarian carcinomas in tumor-bearing mice. Taken together, our data support the further development of Rebmab200 for radioimmunotherapy and diagnostics.

  13. The Agrobacterium tumefaciens chaperone-like protein, VirE1, interacts with VirE2 at domains required for single-stranded DNA binding and cooperative interaction.

    Science.gov (United States)

    Sundberg, C D; Ream, W

    1999-11-01

    Agrobacterium tumefaciens transfers single-stranded DNA (ssDNA) into plants. Efficient tumorigenesis requires VirE1-dependent export of ssDNA-binding (SSB) protein VirE2. VirE1 binds VirE2 domains involved in SSB and self-association, and VirE1 may facilitate VirE2 export by preventing VirE2 aggregation and the premature binding of VirE2 to ssDNA. PMID:10542192

  14. A combined in silico/in vitro approach unveils common molecular requirements for efficient BVDV RdRp binding of linear aromatic N-polycyclic systems.

    Science.gov (United States)

    Carta, A; Briguglio, I; Piras, S; Corona, P; Ibba, R; Laurini, E; Fermeglia, M; Pricl, S; Desideri, N; Atzori, E M; La Colla, P; Collu, G; Delogu, I; Loddo, R

    2016-07-19

    In this work, we present and discuss a comprehensive set of both newly and previously synthesized compounds belonging to 5 distinct molecular classes of linear aromatic N-polycyclic systems that efficiently inhibits bovine viral diarrhea virus (BVDV) infection. A coupled in silico/in vitro investigation was employed to formulate a molecular rationale explaining the notable affinity of all molecules to BVDV RNA dependent RNA polymerase (RdRp) NS5B. We initially developed a three-dimensional common-feature pharmacophore model according to which two hydrogen bond acceptors and one hydrophobic aromatic feature are shared by all molecular series in binding the viral polymerase. The pharmacophoric information was used to retrieve a putative binding site on the surface of the BVDV RdRp and to guide compound docking within the protein binding site. The affinity of all compounds towards the enzyme was scored via molecular dynamics-based simulations, showing high correlation with in vitro EC50 data. The determination of the interaction spectra of the protein residues involved in inhibitor binding highlighted amino acids R295 and Y674 as the two fundamental H-bond donors, while two hydrophobic cavities HC1 (residues A221, I261, I287, and Y289) and HC2 (residues V216, Y303, V306, K307, P408, and A412) fulfill the third pharmacophoric requirement. Three RdRp (K263, R295 and Y674) residues critical for drug binding were selected and mutagenized, both in silico and in vitro, into alanine, and the affinity of a set of selected compounds towards the mutant RdRp isoforms was determined accordingly. The agreement between predicted and experimental data confirmed the proposed common molecular rationale shared by molecules characterized by different chemical scaffolds in binding to the BVDV RdRp, ultimately yielding compound 6b (EC50 = 0.3 μM; IC50 = 0.48 μM) as a new, potent inhibitor of this Pestivirus. PMID:27161176

  15. Comparative distribution of relaxin-3 inputs and calcium-binding protein-positive neurons in rat amygdala

    Directory of Open Access Journals (Sweden)

    Fabio N Santos

    2016-04-01

    Full Text Available The neural circuits involved in mediating complex behaviors are being rapidly elucidated using various newly developed and powerful anatomical and molecular techniques, providing insights into the neural basis for anxiety disorders, depression, addiction, and dysfunctional social behaviors. Many of these behaviors and associated physiological processes involve the activation of the amygdala in conjunction with cortical and hippocampal circuits. Ascending subcortical projections provide modulatory inputs to the extended amygdala and its related nodes (or ‘hubs’ within these key circuits. One such input arises from the nucleus incertus (NI in the tegmentum, which sends amino acid- and peptide-containing projections throughout the forebrain. Notably, a distinct population of GABAergic NI neurons expresses the highly-conserved neuropeptide, relaxin-3, and relaxin-3 signaling has been implicated in the modulation of reward/motivation and anxiety- and depressive-like behaviors in rodents via actions within the extended amygdala. Thus, a detailed description of the relaxin-3 innervation of the extended amygdala would provide an anatomical framework for an improved understanding of NI and relaxin-3 modulation of these and other specific amygdala-related functions. Therefore, in this study, we examined the distribution of NI projections and relaxin-3-positive elements (axons/fibers/terminals within the amygdala, relative to the distribution of neurons expressing the calcium-binding proteins, parvalbumin, calretinin and/or calbindin. Anterograde tracer injections into the NI revealed a topographic distribution of NI efferents within the amygdala that was near identical to the distribution of relaxin-3-immunoreactive fibers. Highest densities of anterogradely-labeled elements and relaxin-3-immunoreactive fibers were observed in the medial nucleus of the amygdala, medial divisions of the bed nucleus of the stria terminalis (BST and in the endopiriform

  16. Comparative Distribution of Relaxin-3 Inputs and Calcium-Binding Protein-Positive Neurons in Rat Amygdala.

    Science.gov (United States)

    Santos, Fabio N; Pereira, Celia W; Sánchez-Pérez, Ana M; Otero-García, Marcos; Ma, Sherie; Gundlach, Andrew L; Olucha-Bordonau, Francisco E

    2016-01-01

    The neural circuits involved in mediating complex behaviors are being rapidly elucidated using various newly developed and powerful anatomical and molecular techniques, providing insights into the neural basis for anxiety disorders, depression, addiction, and dysfunctional social behaviors. Many of these behaviors and associated physiological processes involve the activation of the amygdala in conjunction with cortical and hippocampal circuits. Ascending subcortical projections provide modulatory inputs to the extended amygdala and its related nodes (or "hubs") within these key circuits. One such input arises from the nucleus incertus (NI) in the tegmentum, which sends amino acid- and peptide-containing projections throughout the forebrain. Notably, a distinct population of GABAergic NI neurons expresses the highly-conserved neuropeptide, relaxin-3, and relaxin-3 signaling has been implicated in the modulation of reward/motivation and anxiety- and depressive-like behaviors in rodents via actions within the extended amygdala. Thus, a detailed description of the relaxin-3 innervation of the extended amygdala would provide an anatomical framework for an improved understanding of NI and relaxin-3 modulation of these and other specific amygdala-related functions. Therefore, in this study, we examined the distribution of NI projections and relaxin-3-positive elements (axons/fibers/terminals) within the amygdala, relative to the distribution of neurons expressing the calcium-binding proteins, parvalbumin (PV), calretinin (CR) and/or calbindin. Anterograde tracer injections into the NI revealed a topographic distribution of NI efferents within the amygdala that was near identical to the distribution of relaxin-3-immunoreactive fibers. Highest densities of anterogradely-labeled elements and relaxin-3-immunoreactive fibers were observed in the medial nucleus of the amygdala, medial divisions of the bed nucleus of the stria terminalis (BST) and in the endopiriform nucleus. In

  17. Cellular adhesion responses to the heparin-binding (HepII) domain of fibronectin require heparan sulfate with specific properties

    DEFF Research Database (Denmark)

    Mahalingam, Yashithra; Gallagher, John T; Couchman, John R

    2006-01-01

    Cell surface heparan sulfate (HS) proteoglycans are required in development and postnatal repair. Important classes of ligands for HS include growth factors and extracellular matrix macromolecules. For example, the focal adhesion component syndecan-4 interacts with the III(12-14) region of fibron......Cell surface heparan sulfate (HS) proteoglycans are required in development and postnatal repair. Important classes of ligands for HS include growth factors and extracellular matrix macromolecules. For example, the focal adhesion component syndecan-4 interacts with the III(12-14) region...

  18. The ATP-binding Cassette Transporter OsABCG15 is Required for Anther Development and Pollen Fertility in Rice

    Institute of Scientific and Technical Information of China (English)

    Bai-Xiao Niu; Fu-Rong He; Ming He; Ding Ren; Le-Tian Chen; Yao-Guang Liu

    2013-01-01

    Plant male reproductive development is a complex biological process,but the underlying mechanism is not well understood.Here,we characterized a rice (Oryza sativa L.) male sterile mutant.Based on mapbased cloning and sequence analysis,we identified a 1,459-bp deletion in an adenosine triphosphate (ATP)-binding cassette (ABC) transporter gene,OsABCG15,causing abnormal anthers and male sterility.Therefore,we named this mutant osabcg15.Expression analysis showed that OsABCG15 is expressed specifically in developmental anthers from stage 8 (meiosis Ⅱ stage) to stage 10 (late microspore stage).Two genes CYP704B2 and WDA1,involved in the biosynthesis of very-long-chain fatty acids for the establishment of the anther cuticle and pollen exine,were downregulated in osabcg15 mutant,suggesting that OsABCG15 may play a key function in the processes related to sporopollenin biosynthesis or sporopollenin transfer from tapetal cells to anther locules.Consistently,histological analysis showed that osabcg15 mutants developed obvious abnormality in postmeiotic tapetum degeneration,leading to rapid degredation of young microspores.The results suggest that OsABCG15 plays a critical role in exine formation and pollen development,similar to the homologous gene of AtABCG26 in Arabidopsis.This work is helpful to understand the regulatory network in rice anther development.

  19. The docking domain of histone H2A is required for H1 binding and RSC-mediated nucleosome remodeling.

    Science.gov (United States)

    Shukla, Manu Shubhdarshan; Syed, Sajad Hussain; Goutte-Gattat, Damien; Richard, John Lalith Charles; Montel, Fabien; Hamiche, Ali; Travers, Andrew; Faivre-Moskalenko, Cendrine; Bednar, Jan; Hayes, Jeffrey J; Angelov, Dimitar; Dimitrov, Stefan

    2011-04-01

    Histone variants within the H2A family show high divergences in their C-terminal regions. In this work, we have studied how these divergences and in particular, how a part of the H2A COOH-terminus, the docking domain, is implicated in both structural and functional properties of the nucleosome. Using biochemical methods in combination with Atomic Force Microscopy and Electron Cryo-Microscopy, we show that the H2A-docking domain is a key structural feature within the nucleosome. Deletion of this domain or replacement with the incomplete docking domain from the variant H2A.Bbd results in significant structural alterations in the nucleosome, including an increase in overall accessibility to nucleases, un-wrapping of ∼10 bp of DNA from each end of the nucleosome and associated changes in the entry/exit angle of DNA ends. These structural alterations are associated with a reduced ability of the chromatin remodeler RSC to both remodel and mobilize the nucleosomes. Linker histone H1 binding is also abrogated in nucleosomes containing the incomplete docking domain of H2A.Bbd. Our data illustrate the unique role of the H2A-docking domain in coordinating the structural-functional aspects of the nucleosome properties. Moreover, our data suggest that incorporation of a 'defective' docking domain may be a primary structural role of H2A.Bbd in chromatin.

  20. Rice Stomatal Closure Requires Guard Cell Plasma Membrane ATP-Binding Cassette Transporter RCN1/OsABCG5.

    Science.gov (United States)

    Matsuda, Shuichi; Takano, Sho; Sato, Moeko; Furukawa, Kaoru; Nagasawa, Hidetaka; Yoshikawa, Shoko; Kasuga, Jun; Tokuji, Yoshihiko; Yazaki, Kazufumi; Nakazono, Mikio; Takamure, Itsuro; Kato, Kiyoaki

    2016-03-01

    Water stress is one of the major environmental stresses that affect agricultural production worldwide. Water loss from plants occurs primarily through stomatal pores. Here, we report that an Oryza sativa half-size ATP-binding cassette (ABC) subfamily G protein, RCN1/OsABCG5, is involved in stomatal closure mediated by phytohormone abscisic acid (ABA) accumulation in guard cells. We found that the GFP-RCN1/OsABCG5-fusion protein was localized at the plasma membrane in guard cells. The percentage of guard cell pairs containing both ABA and GFP-RCN1/OsABCG5 increased after exogenous ABA treatment, whereas they were co-localized in guard cell pairs regardless of whether exogenous ABA was applied. ABA application resulted in a smaller increase in the percentage of guard cell pairs containing ABA in rcn1 mutant (A684P) and RCN1-RNAi than in wild-type plants. Furthermore, polyethylene glycol (drought stress)-inducible ABA accumulation in guard cells did not occur in rcn1 mutants. Stomata closure mediated by exogenous ABA application was strongly reduced in rcn1 mutants. Finally, rcn1 mutant plants had more rapid water loss from detached leaves than the wild-type plants. These results indicate that in response to drought stress, RCN1/OsABCG5 is involved in accumulation of ABA in guard cells, which is indispensable for stomatal closure. PMID:26708605

  1. A Fungal Effector With Host Nuclear Localization and DNA-Binding Properties Is Required for Maize Anthracnose Development.

    Science.gov (United States)

    Vargas, Walter A; Sanz-Martín, José M; Rech, Gabriel E; Armijos-Jaramillo, Vinicio D; Rivera, Lina P; Echeverria, María Mercedes; Díaz-Mínguez, José M; Thon, Michael R; Sukno, Serenella A

    2016-02-01

    Plant pathogens have the capacity to manipulate the host immune system through the secretion of effectors. We identified 27 putative effector proteins encoded in the genome of the maize anthracnose pathogen Colletotrichum graminicola that are likely to target the host's nucleus, as they simultaneously contain sequence signatures for secretion and nuclear localization. We functionally characterized one protein, identified as CgEP1. This protein is synthesized during the early stages of disease development and is necessary for anthracnose development in maize leaves, stems, and roots. Genetic, molecular, and biochemical studies confirmed that this effector targets the host's nucleus and defines a novel class of double-stranded DNA-binding protein. We show that CgEP1 arose from a gene duplication in an ancestor of a lineage of monocot-infecting Colletotrichum spp. and has undergone an intense evolution process, with evidence for episodes of positive selection. We detected CgEP1 homologs in several species of a grass-infecting lineage of Colletotrichum spp., suggesting that its function may be conserved across a large number of anthracnose pathogens. Our results demonstrate that effectors targeted to the host nucleus may be key elements for disease development and aid in the understanding of the genetic basis of anthracnose development in maize plants.

  2. Unique carbohydrate-carbohydrate interactions are required for high affinity binding between FcgammaRIII and antibodies lacking core fucose.

    Science.gov (United States)

    Ferrara, Claudia; Grau, Sandra; Jäger, Christiane; Sondermann, Peter; Brünker, Peter; Waldhauer, Inja; Hennig, Michael; Ruf, Armin; Rufer, Arne Christian; Stihle, Martine; Umaña, Pablo; Benz, Jörg

    2011-08-01

    Antibody-mediated cellular cytotoxicity (ADCC), a key immune effector mechanism, relies on the binding of antigen-antibody complexes to Fcγ receptors expressed on immune cells. Antibodies lacking core fucosylation show a large increase in affinity for FcγRIIIa leading to an improved receptor-mediated effector function. Although afucosylated IgGs exist naturally, a next generation of recombinant therapeutic, glycoenginereed antibodies is currently being developed to exploit this finding. In this study, the crystal structures of a glycosylated Fcγ receptor complexed with either afucosylated or fucosylated Fc were determined allowing a detailed, molecular understanding of the regulatory role of Fc-oligosaccharide core fucosylation in improving ADCC. The structures reveal a unique type of interface consisting of carbohydrate-carbohydrate interactions between glycans of the receptor and the afucosylated Fc. In contrast, in the complex structure with fucosylated Fc, these contacts are weakened or nonexistent, explaining the decreased affinity for the receptor. These findings allow us to understand the higher efficacy of therapeutic antibodies lacking the core fucose and also suggest a unique mechanism by which the immune system can regulate antibody-mediated effector functions.

  3. Characterization of a highly conserved binding site of Mlh1 required for exonuclease I-dependent mismatch repair

    DEFF Research Database (Denmark)

    Dherin, Claudine; Gueneau, Emeric; Francin, Mathilde;

    2009-01-01

    Mlh1 is an essential factor of mismatch repair (MMR) and meiotic recombination. It interacts through its C-terminal region with MutL homologs and proteins involved in DNA repair and replication. In this study, we identified the site of yeast Mlh1 critical for the interaction with Exo1, Ntg2...... protein box. Direct and specific interactions between yeast Mlh1 and peptides derived from Exo1, Ntg2, and Sgs1 and between human MLH1 and peptide derived from EXO1 and BLM were measured with K(d) values ranging from 8.1 to 17.4 microM. In Saccharomyces cerevisiae, a mutant of Mlh1 targeted at site S2......, and Sgs1 proteins, designated as site S2 by reference to the Mlh1/Pms1 heterodimerization site S1. We show that site S2 is also involved in the interaction between human MLH1 and EXO1 or BLM. Binding at this site involves a common motif on Mlh1 partners that we called the MIP-box for the Mlh1 interacting...

  4. The Calmodulin-Binding Transcription Activator CAMTA1 Is Required for Long-Term Memory Formation in Mice

    Science.gov (United States)

    Bas-Orth, Carlos; Tan, Yan-Wei; Oliveira, Ana M. M.; Bengtson, C. Peter; Bading, Hilmar

    2016-01-01

    The formation of long-term memory requires signaling from the synapse to the nucleus to mediate neuronal activity-dependent gene transcription. Synapse-to-nucleus communication is initiated by influx of calcium ions through synaptic NMDA receptors and/or L-type voltage-gated calcium channels and involves the activation of transcription factors by…

  5. Identification of the promoter region required for human adiponectin gene transcription: Association with CCAAT/enhancer binding protein-β and tumor necrosis factor-α

    International Nuclear Information System (INIS)

    Adiponectin, an adipose tissue-specific plasma protein, is involved in insulin sensitizing and has anti-atherosclerotic properties. Plasma levels of adiponectin are decreased in obese individuals and patients with type 2 diabetes with insulin resistance. Tumor necrosis factor-α (TNF-α) decreases the expression of adiponectin in adipocytes. The aims of the present study were: (1) to identify the promoter region responsible for basal transcription of the human adiponectin gene, and (2) to investigate the mechanism by which adiponectin was regulated by TNF-α. The human adiponectin promoter (2.1 kb) was isolated and used for luciferase reporter analysis by transient transfection into 3T3-L1 adipocytes. Deletion analysis demonstrated that the promoter region from -676 to +41 was sufficient for basal transcriptional activity. Mutation analysis of putative response elements for sterol regulatory element binding protein (SREBP) (-431 to -423) and CCAAT/enhancer binding protein (C/EBP) (-230 to -224) showed that both elements were required for basal promoter activity. Adiponectin transcription was increased 3-fold in cells that over-expressed constitutively active C/EBP-β. Electrophoretic mobility shift assay, using nuclear extract from 3T3-L1 cells and the -258 to -199 region as a probe, demonstrated specific DNA-protein binding, which was abolished by TNF-α treatment. The present data indicate that the putative response elements for SREBP and C/EBP are required for human adiponectin promoter activity, and that suppression by TNF-α may, at least in part, be associated with inactivation of C/EBP-β

  6. TATA-binding protein (TBP)-like protein is required for p53-dependent transcriptional activation of upstream promoter of p21Waf1/Cip1 gene.

    Science.gov (United States)

    Suzuki, Hidefumi; Ito, Ryo; Ikeda, Kaori; Tamura, Taka-Aki

    2012-06-01

    TATA-binding protein-like protein (TLP) is involved in development, checkpoint, and apoptosis through potentiation of gene expression. TLP-overexpressing human cells, especially p53-containing cells, exhibited a decreased growth rate and increased proportion of G(1) phase cells. TLP stimulated expression of several growth-related genes including p21 (p21(Waf1/Cip1)). TLP-mediated activation of the p21 upstream promoter in cells was shown by a promoter-luciferase reporter assay. The p53-binding sequence located in the p21 upstream promoter and p53 itself are required for TLP-mediated transcriptional activation. TLP and p53 bound to each other and synergistically enhanced activity of the upstream promoter. TLP specifically activated transcription from the endogenous upstream promoter, and p53 was required for this activation. Etoposide treatment also resulted in activation of the upstream promoter as well as nuclear accumulation of TLP and p53. Moreover, the upstream promoter was associated with endogenous p53 and TLP, and the p53 recruitment was enhanced by TLP. The results of the present study suggest that TLP mediates p53-governed transcriptional activation of the p21 upstream promoter.

  7. cAMP response element binding protein is required for differentiation of respiratory epithelium during murine development.

    Directory of Open Access Journals (Sweden)

    A Daniel Bird

    Full Text Available The cAMP response element binding protein 1 (Creb1 transcription factor regulates cellular gene expression in response to elevated levels of intracellular cAMP. Creb1(-/- fetal mice are phenotypically smaller than wildtype littermates, predominantly die in utero and do not survive after birth due to respiratory failure. We have further investigated the respiratory defect of Creb1(-/- fetal mice during development. Lungs of Creb1(-/- fetal mice were pale in colour and smaller than wildtype controls in proportion to their reduced body size. Creb1(-/- lungs also did not mature morphologically beyond E16.5 with little or no expansion of airway luminal spaces, a phenotype also observed with the Creb1(-/- lung on a Crem(-/- genetic background. Creb1 was highly expressed throughout the lung at all stages examined, however activation of Creb1 was detected primarily in distal lung epithelium. Cell differentiation of E17.5 Creb1(-/- lung distal epithelium was analysed by electron microscopy and showed markedly reduced numbers of type-I and type-II alveolar epithelial cells. Furthermore, immunomarkers for specific lineages of proximal epithelium including ciliated, non-ciliated (Clara, and neuroendocrine cells showed delayed onset of expression in the Creb1(-/- lung. Finally, gene expression analyses of the E17.5 Creb1(-/- lung using whole genome microarray and qPCR collectively identified respiratory marker gene profiles and provide potential novel Creb1-regulated genes. Together, these results demonstrate a crucial role for Creb1 activity for the development and differentiation of the conducting and distal lung epithelium.

  8. Job requirements compared to medical school education: differences between graduates from problem-based learning and conventional curricula

    Directory of Open Access Journals (Sweden)

    Federkeil Gero

    2010-01-01

    Full Text Available Abstract Background Problem-based Learning (PBL has been suggested as a key educational method of knowledge acquisition to improve medical education. We sought to evaluate the differences in medical school education between graduates from PBL-based and conventional curricula and to what extent these curricula fit job requirements. Methods Graduates from all German medical schools who graduated between 1996 and 2002 were eligible for this study. Graduates self-assessed nine competencies as required at their day-to-day work and as taught in medical school on a 6-point Likert scale. Results were compared between graduates from a PBL-based curriculum (University Witten/Herdecke and conventional curricula. Results Three schools were excluded because of low response rates. Baseline demographics between graduates of the PBL-based curriculum (n = 101, 49% female and the conventional curricula (n = 4720, 49% female were similar. No major differences were observed regarding job requirements with priorities for "Independent learning/working" and "Practical medical skills". All competencies were rated to be better taught in PBL-based curriculum compared to the conventional curricula (all p Conclusion Among medical graduates in Germany, PBL demonstrated benefits with regard to competencies which were highly required in the job of physicians. Research and business competence deserve closer attention in future curricular development.

  9. Cytoplasmic Localization of Human cdc25C during Interphase Requires an Intact 14-3-3 Binding Site

    OpenAIRE

    Dalal, Sorab N.; Schweitzer, Colleen M.; Gan, Jianmin; DeCaprio, James A.

    1999-01-01

    cdc25C induces mitosis by activating the cdc2-cyclin B complex. The intracellular localization of cyclin B1 is regulated in a cell cycle-specific manner, and its entry into the nucleus may be required for the initiation of mitosis. To determine the cellular localization of cdc25C, monoclonal antibodies specific for cdc25C were developed and used to demonstrate that in human cells, cdc25C is retained in the cytoplasm during interphase. A deletion analysis identified a 58-amino-acid region (ami...

  10. Lower fetuin-A, retinol binding protein 4 and several metabolites after gastric bypass compared to sleeve gastrectomy in patients with type 2 diabetes.

    Directory of Open Access Journals (Sweden)

    Mia Jüllig

    Full Text Available BACKGROUND: Bypass of foregut secreted factors promoting insulin resistance is hypothesized to be one of the mechanisms by which resolution of type 2 diabetes (T2D follows roux-en-y gastric bypass (GBP surgery. AIM: To identify insulin resistance-associated proteins and metabolites which decrease more after GBP than after sleeve gastrectomy (SG prior to diabetes remission. METHODS: Fasting plasma from 15 subjects with T2D undergoing GBP or SG was analyzed by proteomic and metabolomic methods 3 days before and 3 days after surgery. Subjects were matched for age, BMI, metformin therapy and glycemic control. Insulin resistance was calculated using homeostasis model assessment (HOMA-IR. For proteomics, samples were depleted of abundant plasma proteins, digested with trypsin and labeled with iTRAQ isobaric tags prior to liquid chromatography-tandem mass spectrometry analysis. Metabolomic analysis was performed using gas chromatography-mass spectrometry. The effect of the respective bariatric surgery on identified proteins and metabolites was evaluated using two-way analysis of variance and appropriate post-hoc tests. RESULTS: HOMA-IR improved, albeit not significantly, in both groups after surgery. Proteomic analysis yielded seven proteins which decreased significantly after GBP only, including Fetuin-A and Retinol binding protein 4, both previously linked to insulin resistance. Significant decrease in Fetuin-A and Retinol binding protein 4 after GBP was confirmed using ELISA and immunoassay. Metabolomic analysis identified significant decrease of citrate, proline, histidine and decanoic acid specifically after GBP. CONCLUSION: Greater early decrease was seen for Fetuin-A, Retinol binding protein 4, and several metabolites after GBP compared to SG, preceding significant weight loss. This may contribute to enhanced T2D remission observed following foregut bypass procedures.

  11. A COMPARATIVE EVALUATION OF GABAPENTIN AND CLONIDINE PREMEDICATION ON POST OPERATIVE ANALGESIA REQUIREMENT FOLLOWING ABDOMINAL SURGERIES UNDER GENERAL ANAESTHESIA

    Directory of Open Access Journals (Sweden)

    Ashish

    2014-08-01

    Full Text Available AIM: Aim of our study was to compare the relative effectiveness of gabapentin and clonidine premedication on patients undergoing elective abdominal surgeries under G.A. OBJECTIVE: gabapentine and clonidine have anti-nociceptive properties .This study assess their efficacy in prolonging the analgesic effect intra-operative and postoperative analgesic requirement. MATERIAL AND METHOD: 225 patients of either sex of age between 20-60 years, ASA grade I & II, patient admitted to Hamidia hospital for elective abdominal surgeries under general anaesthesia were included in the study. The patients were randomly allocated into three groups 75 each group I : Control group (patients received placebo tablet at 90 min before the surgery,group II Gabapentin 300 mg tablet orally 90 min before surgery ,groupIII:clonidine150µg tablet orally given 90 min before surgery. Duration of postoperative analgesia, Degree of postoperative pain (VAS scoreand added rescue analgesia required in 24 hrs were recorded postoperatively. RESULT: Analysis reveled that there was no difference in the HR, SBP among the three group during the study. Duration of postoperative analgesia, observed from time of reversal to first demand of analgesia in the recovery room was more in group II compared to group I and group III (p-value <0.001, highly significant. Pain perception was highly blunted in groups II compared to group I & group III. Total rescue analgesic requirement during the postoperative 24hrs period was much lower in group II inj Diclofenac compared to group I and group III . ( p-value < 0.001, highly significant.CONCLUSION: Given 90 min before induction of GA oral gabapentin(300 mg or clonidine(150 µg preoperatively was effective in lowering postoperative VAS pain score and consumption of analgesics, it was also shows that gabapentin significantly decreases postoperative pain intensity and analgesic consumption after abdominal surgeries.

  12. A conserved motif in the linker domain of STAT1 transcription factor is required for both recognition and release from high-affinity DNA-binding sites.

    Directory of Open Access Journals (Sweden)

    Bettina Hüntelmann

    Full Text Available Binding to specific palindromic sequences termed gamma-activated sites (GAS is a hallmark of gene activation by members of the STAT (signal transducer and activator of transcription family of cytokine-inducible transcription factors. However, the precise molecular mechanisms involved in the signal-dependent finding of target genes by STAT dimers have not yet been very well studied. In this study, we have characterized a sequence motif in the STAT1 linker domain which is highly conserved among the seven human STAT proteins and includes surface-exposed residues in close proximity to the bound DNA. Using site-directed mutagenesis, we have demonstrated that a lysine residue in position 567 of the full-length molecule is required for GAS recognition. The substitution of alanine for this residue completely abolished both binding to high-affinity GAS elements and transcriptional activation of endogenous target genes in cells stimulated with interferon-γ (IFNγ, while the time course of transient nuclear accumulation and tyrosine phosphorylation were virtually unchanged. In contrast, two glutamic acid residues (E559 and E563 on each monomer are important for the dissociation of dimeric STAT1 from DNA and, when mutated to alanine, result in elevated levels of tyrosine-phosphorylated STAT1 as well as prolonged IFNγ-stimulated nuclear accumulation. In conclusion, our data indicate that the kinetics of signal-dependent GAS binding is determined by an array of glutamic acid residues located at the interior surface of the STAT1 dimer. These negatively charged residues appear to align the long axis of the STAT1 dimer in a position perpendicular to the DNA, thereby facilitating the interaction between lysine 567 and the phosphodiester backbone of a bound GAS element, which is a prerequisite for transient gene induction.

  13. Human Telomerase Reverse Transcriptase (hTERT) Transcription Requires Sp1/Sp3 Binding to the Promoter and a Permissive Chromatin Environment.

    Science.gov (United States)

    Cheng, De; Zhao, Yuanjun; Wang, Shuwen; Jia, Wenwen; Kang, Jiuhong; Zhu, Jiyue

    2015-12-11

    The transcription of human telomerase gene hTERT is regulated by transcription factors (TFs), including Sp1 family proteins, and its chromatin environment. To understand its regulation in a relevant chromatin context, we employed bacterial artificial chromosome reporters containing 160 kb of human genomic sequence containing the hTERT gene. Upon chromosomal integration, the bacterial artificial chromosomes recapitulated endogenous hTERT expression, contrary to transient reporters. Sp1/Sp3 expression did not correlate with hTERT promoter activity, and these TFs bound to the hTERT promoters in both telomerase-positive and telomerase-negative cells. Mutation of the proximal GC-box resulted in a dramatic decrease of hTERT promoter activity, and mutations of all five GC-boxes eliminated its transcriptional activity. Neither mutations of GC-boxes nor knockdown of endogenous Sp1 impacted promoter binding by other TFs, including E-box-binding proteins, and histone acetylation and trimethylation of histone H3K9 at the hTERT promoter in telomerase-positive and -negative cells. The result indicated that promoter binding by Sp1/Sp3 was essential, but not a limiting step, for hTERT transcription. hTERT transcription required a permissive chromatin environment. Importantly, our data also revealed different functions of GC-boxes and E-boxes in hTERT regulation; although GC-boxes were essential for promoter activity, factors bound to the E-boxes functioned to de-repress hTERT promoter.

  14. Comparative metabolism of [14C]benzene to excretable products and bioactivation to DNA-binding derivatives in maternal and neonatal mice

    International Nuclear Information System (INIS)

    Lactating adult female mice treated with a single dose of 880 mg/kg i.p. [14C]benzene, and their 2-day-old sucklings similarly treated or nursed by their treated dams were compared in terms of their ability to metabolize benzene to urinary products or reactive intermediates as assessed by covalently-bound benzene derivatives in whole blood or liver DNA. Six metabolite fractions were identified in the urine of sucklings by high performance liquid chromatographic (HPLC) analysis at 5 h following intraperitoneal (direct) treatment with benzene. Three of the metabolite fractions co-chromatographed with authentic phenol, phenyl glucuronide, and muconic acid, and contributed 11, 6.9 and 0.6%, respectively, to the total urinary benzene metabolites. Two of the fractions were unidentified. The sixth and most polar fraction consisted of multiple metabolites, 21% of which were conjugates, and accounted for 72% of the total urinary metabolites. A similar metabolite profile was observed in 24-h urine samples from treated dams with the exception that one of the unidentified fractions in the sucklings was absent and levels of the metabolites were quantitatively higher than those observed in sucklings 5 h following their treatment with benzene. Furthermore, 78% of the most polar fraction from the dams consisted of conjugates compared with 21% of that from the sucklings. The metabolite pattern in urine of sucklings nursed by treated dams was qualitatively similar to, but quantitatively different from the pattern in treated dams. Five hours following intraperitoneal treatment with benzene, covalent binding of the compound to DNA (expressed as pmol benzene equivalents/mg DNA) in sucklings was slightly higher in whole blood (1.15±0.07) than in liver (0.77±0.07), whereas in the dam, it was slightly lower in whole blood (0.88±0.48) than in liver (1.63±0.61). Twenty four hours following benzene exposure in sucklings of benzene-treated dams, DNA binding by the compound in whole blood

  15. Sequence characteristics required for cooperative binding and efficient in vivo titration of the replication initiator protein DnaA in E. coli

    DEFF Research Database (Denmark)

    Hansen, Flemming G.; Christensen, Bjarke Bak; Atlung, Tove

    2007-01-01

    Plasmids carrying the mioC promoter region, which contains two DnaA boxes, R5 and R6 with one misfit to the consensus TT(A)/(T)TNCACA, are as efficient in in vivo titration of the DnaA protein as plasmids carrying a replication-inactivated oriC region with its eight DnaA boxes. Three additional Dna......A boxes around the promoter proximal R5 DnaA box were identified and shown by mutational analysis to be necessary for the cooperative binding of DnaA required for titration. These four DnaA boxes are located in the same orientation and with a spacing of two or three base-pairs. The cooperative binding...... was eliminated by insertion of half a helical turn between any of the DnaA boxes. Titration strongly depends on the presence and orientation of the promoter distal R6 DnaA box located 104 bp upstream of the R5 box as well as neighbouring sequences downstream of R6. Titration depends on the integrity of a 43 bp...

  16. A NASP (N1/N2)-related protein, Sim3, binds CENP-A and is required for its deposition at fission yeast centromeres.

    Science.gov (United States)

    Dunleavy, Elaine M; Pidoux, Alison L; Monet, Marie; Bonilla, Carolina; Richardson, William; Hamilton, Georgina L; Ekwall, Karl; McLaughlin, Paul J; Allshire, Robin C

    2007-12-28

    A defining feature of centromeres is the presence of the histone H3 variant CENP-A(Cnp1). It is not known how CENP-A(Cnp1) is specifically delivered to, and assembled into, centromeric chromatin. Through a screen for factors involved in kinetochore integrity in fission yeast, we identified Sim3. Sim3 is homologous to known histone binding proteins NASP(Human) and N1/N2(Xenopus) and aligns with Hif1(S. cerevisiae), defining the SHNi-TPR family. Sim3 is distributed throughout the nucleoplasm, yet it associates with CENP-A(Cnp1) and also binds H3. Cells defective in Sim3 function have reduced levels of CENP-A(Cnp1) at centromeres (and increased H3) and display chromosome segregation defects. Sim3 is required to allow newly synthesized CENP-A(Cnp1) to accumulate at centromeres in S and G2 phase-arrested cells in a replication-independent mechanism. We propose that one function of Sim3 is to act as an escort that hands off CENP-A(Cnp1) to chromatin assembly factors, allowing its incorporation into centromeric chromatin. PMID:18158900

  17. Interaction between Escherichia coli DNA polymerase IV and single-stranded DNA-binding protein is required for DNA synthesis on SSB-coated DNA.

    Science.gov (United States)

    Furukohri, Asako; Nishikawa, Yoshito; Akiyama, Masahiro Tatsumi; Maki, Hisaji

    2012-07-01

    DNA polymerase IV (Pol IV) is one of three translesion polymerases in Escherichia coli. A mass spectrometry study revealed that single-stranded DNA-binding protein (SSB) in lysates prepared from exponentially-growing cells has a strong affinity for column-immobilized Pol IV. We found that purified SSB binds directly to Pol IV in a pull-down assay, whereas SSBΔC8, a mutant protein lacking the C-terminal tail, failed to interact with Pol IV. These results show that the interaction between Pol IV and SSB is mediated by the C-terminal tail of SSB. When polymerase activity was tested on an SSBΔC8-coated template, we observed a strong inhibition of Pol IV activity. Competition experiments using a synthetic peptide containing the amino acid sequence of SSB tail revealed that the chain-elongating capacity of Pol IV was greatly impaired when the interaction between Pol IV and SSB tail was inhibited. These results demonstrate that Pol IV requires the interaction with the C-terminal tail of SSB to replicate DNA efficiently when the template ssDNA is covered with SSB. We speculate that at the primer/template junction, Pol IV interacts with the tail of the nearest SSB tetramer on the template, and that this interaction allows the polymerase to travel along the template while disassembling SSB.

  18. Talpid3-binding centrosomal protein Cep120 is required for centriole duplication and proliferation of cerebellar granule neuron progenitors.

    Directory of Open Access Journals (Sweden)

    Chuanqing Wu

    Full Text Available Granule neuron progenitors (GNPs are the most abundant neuronal type in the cerebellum. GNP proliferation and thus cerebellar development require Sonic hedgehog (Shh secreted from Purkinje cells. Shh signaling occurs in primary cilia originating from the mother centriole. Centrioles replicate only once during a typical cell cycle and are responsible for mitotic spindle assembly and organization. Recent studies have linked cilia function to cerebellar morphogenesis, but the role of centriole duplication in cerebellar development is not known. Here we show that centrosomal protein Cep120 is asymmetrically localized to the daughter centriole through its interaction with Talpid3 (Ta3, another centrosomal protein. Cep120 null mutant mice die in early gestation with abnormal heart looping. Inactivation of Cep120 in the central nervous system leads to both hydrocephalus, due to the loss of cilia on ependymal cells, and severe cerebellar hypoplasia, due to the failed proliferation of GNPs. The mutant GNPs lack Hedgehog pathway activity. Cell biological studies show that the loss of Cep120 results in failed centriole duplication and consequently ciliogenesis, which together underlie Cep120 mutant cerebellar hypoplasia. Thus, our study for the first time links a centrosomal protein necessary for centriole duplication to cerebellar morphogenesis.

  19. Expression of FK506 binding protein 65 (FKBP65) is decreased in epithelial ovarian cancer cells compared to benign tumor cells and to ovarian epithelium

    DEFF Research Database (Denmark)

    Henriksen, Rudi; Sørensen, Flemming Brandt; Orntoft, Torben Falck;

    2011-01-01

    FK506 binding protein 65 (FKBP65) belongs to a group of proteins termed immunophilins that have a high binding affinity to immunosuppressant drugs as FK506 (tacrolimus) and rapamycin (sirolimus). Treatment of female premenopausal women with tacrolimus, which binds to FKBP65, has been reported...

  20. Betaglycan has two independent domains required for high affinity TGF-β binding: proteolytic cleavage separates the domains and inactivates the neutralizing activity of the soluble receptor

    Science.gov (United States)

    Mendoza, Valentín; Vilchis-Landeros, M. Magdalena; Mendoza-Hernández, Guillermo; Huang, Tao; Villarreal, Maria M.; Hinck, Andrew P.; López-Casillas, Fernando; Montiel, Jose-Luis

    2009-01-01

    Summary Betaglycan is a co-receptor for members of the TGF-β superfamily. Mutagenesis has identified two ligand binding regions, one at the membrane-distal and the other at the membrane-proximal half of the betaglycan ectodomain. Here we show that partial plasmin digestion of soluble betaglycan produces two proteolysis-resistant fragments of 45 and 55 kDa, consistent with the predicted secondary structure, which indicates an intervening non-structured linker region separating the highly structured N- and C-terminal domains. Amino terminal sequencing indicates that the 45 and 55 kDa fragments correspond, respectively, to the membrane-distal and -proximal regions. Plasmin treatment of membrane betaglycan results in the production of equivalent proteolysis-resistant fragments. The 45 and 55 kDa fragments, as well as their recombinant soluble counterparts, Sol Δ10 and Sol Δ11, bind TGF-β, nonetheless, compared to intact soluble betaglycan, have severely diminished ability to block TGF-β activity. Surface plasmon resonance (SPR) analysis indicates that soluble betaglycan has Kds in the low nanomolar range for the three TGF-β isoforms, while those for Sol Δ10 and Sol Δ11 are 1 – 2 orders of magnitude higher. SPR analysis further shows that the Kds of Sol Δ11 are not changed in the presence of Sol Δ10, indicating that the high affinity of soluble betaglycan is a consequence of tethering of the domains together. Overall, these results, suggest that betaglycan ectodomain exhibits a bi-lobular structure in which each lobule folds independently, binds TGF-β through distinct non-overlapping interfaces, and that linker modification may be an approach to improve soluble betaglycan’s TGF-β neutralizing activity. PMID:19842711

  1. Calcium-binding protein-containing neuronal populations in mammalian visual cortex: a comparative study in whales, insectivores, bats, rodents, and primates.

    Science.gov (United States)

    Glezer, I I; Hof, P R; Leranth, C; Morgane, P J

    1993-01-01

    This study is focused on comparative analysis of gamma-aminobutyric acid-positive (GABAergic) neuronal populations in primary visual cortex of totally aquatic toothed whales and select terrestrial mammals with different evolutionary histories and various ecological adaptations. The distribution of neuronal populations containing the calcium-binding proteins calbindin and parvalbumin, which are recognized markers for the GABAergic neurons in cerebral cortex, is compared in five species of toothed whales and in representatives (one species each) of insectivores, bats, rodents, and primates. Computerized image analysis has shown that overall quantitative characteristics of GABAergic cortical neurons in toothed whales are similar to those in other mammalian orders. Thus, GABA-positive neurons represent 26% of the total population of cortical neurons in the visual cortex of whales. Some 97% of GABA-positive cells contain calcium-binding proteins, which is numerically similar to these parameters found in primates and other mammals. On the other hand, the typology and laminar distribution of calcium-binding protein-containing neurons in the primary visual cortex of five whale species (Delphinapterus leucas, Globicephala melaena, Phocoena phocoena, Stenella coeruleoalba, and Tursiops truncatus) differ significantly from those of primates (Macaca mulatta) and rodents (Rattus rattus) and are similar to those found in insectivorous bats (Eptesicus fuscus) and hedgehogs (Erinaceus europaeus). In whales, bats, and hedgehogs a significant concentration of calbindin-positive, vertically oriented bipolar and bitufted neurons was found in layers I, II, and IIIc/V with their axons arranged in a three-dimensional network. In primates and rodents they are distributed evenly across all cortical layers and are predominantly multipolar or bitufted neurons found in all cortical layers with their axons oriented along the vertical axis of the cortical plate. The parvalbumin-positive neurons

  2. The ileal lipid binding protein is required for efficient absorption and transport of bile acids in the distal portion of the murine small intestine.

    Science.gov (United States)

    Praslickova, Dana; Torchia, Enrique C; Sugiyama, Michael G; Magrane, Elijah J; Zwicker, Brittnee L; Kolodzieyski, Lev; Agellon, Luis B

    2012-01-01

    The ileal lipid binding protein (ilbp) is a cytoplasmic protein that binds bile acids with high affinity. However evidence demonstrating the role of this protein in bile acid transport and homeostasis is missing. We created a mouse strain lacking ilbp (Fabp6(-/-) mice) and assessed the impact of ilbp deficiency on bile acid homeostasis and transport in vivo. Elimination of ilbp increased fecal bile acid excretion (54.2%, Pexcreted by 24 h after oral administration was 102% (Psmall and large intestines was increased by 22% (P<0.02) and decreased by 62.7% (P<0.01), respectively, in male Fabp6(-/-) mice relative wild-type mice, whereas no changes were seen in female Fabp6(-/-) mice. Mucosal to serosal bile acid transport using everted distal gut sacs was decreased by 74% (P<0.03) in both sexes of Fabp6(-/-) mice as compared to wild-type mice. The results demonstrate that ilbp is involved in the apical to basolateral transport of bile acids in ileal enterocytes, and is vital for the maintenance of bile acid homeostasis in the enterohepatic circulation (EHC) in mice.

  3. Cell cycle requirements for transduction by foamy virus vectors compared to those of oncovirus and lentivirus vectors.

    Science.gov (United States)

    Trobridge, Grant; Russell, David W

    2004-03-01

    Retroviral vectors based on foamy viruses (FV) are efficient gene delivery vehicles for therapeutic and research applications. While previous studies have shown that FV vectors transduce quiescent cell cultures more efficiently than oncoviral vectors, their specific cell cycle requirements have not been determined. Here we compare the transduction frequencies of FV vectors with those of onco- and lentiviral vectors in nondividing and dividing normal human fibroblasts by several methods. FV vectors transduced serum-deprived fibroblast cultures more efficiently than oncoretroviral vectors and at rates comparable to those of lentiviral vectors. However, in these cultures FV vectors only transduced a subpopulation of proliferating cells, as determined by bromodeoxyuridine staining for DNA synthesis. In contrast to lentiviral vectors, FV vectors were unable to transduce human fibroblasts arrested by aphidicolin (G(1)/S phase) or gamma-irradiation (G(2) phase), and a partial cell cycle that included mitosis but not DNA synthesis was required. We could not determine if mitosis facilitated nuclear entry of FV vectors, since cell-free vector preparations contained long terminal repeat circles, precluding their use as nuclear markers. In contrast to oncoviral vectors, both FV and lentiviral vectors efficiently transduced G(0) fibroblasts that were later stimulated to divide. In the case of FV vectors, this was due to the persistence of a stable transduction intermediate in quiescent cells. Our findings support the use of FV vectors as a safe and effective alternative to lentiviral vectors for ex vivo transduction of stem cells that are quiescent during culture but divide following transplantation.

  4. Expanding comparative-advantage biological market models: contingency of mutualism on partners' resource requirements and acquisition trade-offs.

    Science.gov (United States)

    Hoeksema, Jason D; Schwartz, Mark W

    2003-05-01

    We expand the comparative-advantage biological market-modelling framework to show how differences between partners, both in their abilities to acquire two resources and in their requirements for those resources, can affect the net benefit of participating in interspecific resource exchange. In addition, the benefits derived from resource trading depend strongly on the nature of the trade-off between the acquisition of one resource and the acquisition of another, described here by the shape (linear, convex or concave) of the resource acquisition constraints of the individuals involved. Combined with previous results, these analyses provide a suite of predictions about whether or not resource exchange is beneficial for two heterospecific individuals relative to a strategy of non-interaction. The benefit derived from resource exchange depends on three factors: (i) relative differences between the partners in their resource acquisition abilities; (ii) relative differences between the partners in their resource requirements; and (iii) variation in the shape of resource acquisition trade-offs. We find that such an explicit consideration of resource requirements and acquisition abilities can provide useful and sometimes non-intuitive predictions about the benefits of resource exchange, and also which resources should be traded by which species.

  5. Comparative genomics analyses on EPS biosynthesis genes required for floc formation of Zoogloea resiniphila and other activated sludge bacteria.

    Science.gov (United States)

    An, Weixing; Guo, Feng; Song, Yulong; Gao, Na; Bai, Shijie; Dai, Jingcheng; Wei, Hehong; Zhang, Liping; Yu, Dianzhen; Xia, Ming; Yu, Ying; Qi, Ming; Tian, Chunyuan; Chen, Haofeng; Wu, Zhenbin; Zhang, Tong; Qiu, Dongru

    2016-10-01

    Activated sludge (AS) process has been widely utilized for municipal sewage and industrial wastewater treatment. Zoolgoea and its related floc-forming bacteria are required for formation of AS flocs which is the key to gravitational effluent-and-sludge separation and AS recycling. However, little is known about the genetics, biochemistry and physiology of Zoogloea and its related bacteria. This report deals with the comparative genomic analyses on two Zoogloea resiniphila draft genomes and the closely related proteobacterial species commonly found in AS. In particular, the metabolic processes involved in removal of organic matters, nitrogen and phosphorus were analyzed. Furthermore, it is revealed that a large gene cluster, encoding eight glycosyltransferases and other proteins involved in biosynthesis and export of extracellular polysaccharides (EPS), was required for floc formation. One of the two asparagine synthase paralogues, associated with this EPS biosynthesis gene cluster, was required for floc formation in Zoogloea. Similar EPS biosynthesis gene cluster(s) were identified in the genome of other AS proteobacteria including polyphosphate-accumulating Candidatus Accumulibacter phosphatis (CAP) and nitrifying Nitrosopira and Nitrosomonas bacteria, but the gene composition varies interspecifically and intraspecifically. Our results indicate that floc formation of desired AS bacteria, including CAP strains, facilitate their recruitment into AS and gradual enrichment via repeated AS settling and recycling processes. PMID:27403872

  6. The ileal lipid binding protein is required for efficient absorption and transport of bile acids in the distal portion of the murine small intestine.

    Directory of Open Access Journals (Sweden)

    Dana Praslickova

    Full Text Available The ileal lipid binding protein (ilbp is a cytoplasmic protein that binds bile acids with high affinity. However evidence demonstrating the role of this protein in bile acid transport and homeostasis is missing. We created a mouse strain lacking ilbp (Fabp6(-/- mice and assessed the impact of ilbp deficiency on bile acid homeostasis and transport in vivo. Elimination of ilbp increased fecal bile acid excretion (54.2%, P<0.05 in female but not male Fabp6(-/- mice. The activity of cholesterol 7α-hydroxylase (cyp7a1, the rate-controlling enzyme of the classical bile acid biosynthetic pathway, was significantly increased in female (63.5%, P<0.05 but not in male Fabp6(-/- mice. The amount of [(3H]taurocholic acid (TCA excreted by 24 h after oral administration was 102% (P<0.025 higher for female Fabp6(-/- mice whereas it was 57.3% (P<0.01 lower for male Fabp6(-/- mice, compared to wild-type mice. The retained fraction of the [(3H]TCA localized in the small and large intestines was increased by 22% (P<0.02 and decreased by 62.7% (P<0.01, respectively, in male Fabp6(-/- mice relative wild-type mice, whereas no changes were seen in female Fabp6(-/- mice. Mucosal to serosal bile acid transport using everted distal gut sacs was decreased by 74% (P<0.03 in both sexes of Fabp6(-/- mice as compared to wild-type mice. The results demonstrate that ilbp is involved in the apical to basolateral transport of bile acids in ileal enterocytes, and is vital for the maintenance of bile acid homeostasis in the enterohepatic circulation (EHC in mice.

  7. Epidermal Fatty Acid Binding Protein (E-FABP) Is Not Required for the Generation or Maintenance of Effector and Memory T Cells following Infection with Listeria monocytogenes.

    Science.gov (United States)

    Li, Bing; Schmidt, Nathan W

    2016-01-01

    Following activation of naïve T cells there are dynamic changes in the metabolic pathways used by T cells to support both the energetic needs of the cell and the macromolecules required for growth and proliferation. Among other changes, lipid metabolism undergoes dynamic transitions between fatty acid oxidation and fatty acid synthesis as cells progress from naïve to effector and effector to memory T cells. The hydrophobic nature of lipids requires that they be bound to protein chaperones within a cell. Fatty acid binding proteins (FABPs) represent a large class of lipid chaperones, with epidermal FABP (E-FABP) expressed in T cells. The objective of this study was to determine the contribution of E-FABP in antigen-specific T cell responses. Following infection with Listeria monocytogenes, we observed similar clonal expansion, contraction and formation of memory CD8 T cells in WT and E-FABP-/- mice, which also exhibited similar phenotypic and functional characteristics. Analysis of Listeria-specific CD4 T cells also revealed no defect in the expansion, contraction, and formation of memory CD4 T cells in E-FABP-/- mice. These data demonstrate that E-FABP is dispensable for antigen-specific T cell responses following a bacterial infection. PMID:27588422

  8. Genome-Wide Comparative Analyses Reveal the Dynamic Evolution of Nucleotide-Binding Leucine-Rich Repeat Gene Family among Solanaceae Plants

    Science.gov (United States)

    Seo, Eunyoung; Kim, Seungill; Yeom, Seon-In; Choi, Doil

    2016-01-01

    Plants have evolved an elaborate innate immune system against invading pathogens. Within this system, intracellular nucleotide-binding leucine-rich repeat (NLR) immune receptors are known play critical roles in effector-triggered immunity (ETI) plant defense. We performed genome-wide identification and classification of NLR-coding sequences from the genomes of pepper, tomato, and potato using fixed criteria. We then compared genomic duplication and evolution features. We identified intact 267, 443, and 755 NLR-encoding genes in tomato, potato, and pepper genomes, respectively. Phylogenetic analysis and classification of Solanaceae NLRs revealed that the majority of NLR super family members fell into 14 subgroups, including a TIR-NLR (TNL) subgroup and 13 non-TNL subgroups. Specific subgroups have expanded in each genome, with the expansion in pepper showing subgroup-specific physical clusters. Comparative analysis of duplications showed distinct duplication patterns within pepper and among Solanaceae plants suggesting subgroup- or species-specific gene duplication events after speciation, resulting in divergent evolution. Taken together, genome-wide analysis of NLR family members provide insights into their evolutionary history in Solanaceae. These findings also provide important foundational knowledge for understanding NLR evolution and will empower broader characterization of disease resistance genes to be used for crop breeding.

  9. JC virus small T antigen binds phosphatase PP2A and Rb family proteins and is required for efficient viral DNA replication activity.

    Directory of Open Access Journals (Sweden)

    Brigitte Bollag

    Full Text Available BACKGROUND: The human polyomavirus, JC virus (JCV produces five tumor proteins encoded by transcripts alternatively spliced from one precursor messenger RNA. Significant attention has been given to replication and transforming activities of JCV's large tumor antigen (TAg and three T' proteins, but little is known about small tumor antigen (tAg functions. Amino-terminal sequences of tAg overlap with those of the other tumor proteins, but the carboxy half of tAg is unique. These latter sequences are the least conserved among the early coding regions of primate polyomaviruses. METHODOLOGY AND FINDINGS: We investigated the ability of wild type and mutant forms of JCV tAg to interact with cellular proteins involved in regulating cell proliferation and survival. The JCV P99A tAg is mutated at a conserved proline, which in the SV40 tAg is required for efficient interaction with protein phosphatase 2A (PP2A, and the C157A mutant tAg is altered at one of two newly recognized LxCxE motifs. Relative to wild type and C157A tAgs, P99A tAg interacts inefficiently with PP2A in vivo. Unlike SV40 tAg, JCV tAg binds to the Rb family of tumor suppressor proteins. Viral DNAs expressing mutant t proteins replicated less efficiently than did the intact JCV genome. A JCV construct incapable of expressing tAg was replication-incompetent, a defect not complemented in trans using a tAg-expressing vector. CONCLUSIONS: JCV tAg possesses unique properties among the polyomavirus small t proteins. It contributes significantly to viral DNA replication in vivo; a tAg null mutant failed to display detectable DNA replication activity, and a tAg substitution mutant, reduced in PP2A binding, was replication-defective. Our observation that JCV tAg binds Rb proteins, indicates all five JCV tumor proteins have the potential to influence cell cycle progression in infected and transformed cells. It remains unclear how these proteins coordinate their unique and overlapping functions.

  10. Intravital Imaging of Vascular Transmigration by the Lyme Spirochete: Requirement for the Integrin Binding Residues of the B. burgdorferi P66 Protein.

    Directory of Open Access Journals (Sweden)

    Devender Kumar

    2015-12-01

    Full Text Available Vascular extravasation, a key step in systemic infection by hematogenous microbial pathogens, is poorly understood, but has been postulated to encompass features similar to vascular transmigration by leukocytes. The Lyme disease spirochete can cause a variety of clinical manifestations, including arthritis, upon hematogenous dissemination. This pathogen encodes numerous surface adhesive proteins (adhesins that may promote extravasation, but none have yet been implicated in this process. In this work we report the novel use of intravital microscopy of the peripheral knee vasculature to study transmigration of the Lyme spirochete in living Cd1d-/-mice. In the absence of iNKT cells, major immune modulators in the mouse joint, spirochetes that have extravasated into joint-proximal tissue remain in the local milieu and can be enumerated accurately. We show that BBK32, a fibronectin and glycosaminoglycan adhesin of B. burgdorferi involved in early steps of endothelial adhesion, is not required for extravasation from the peripheral knee vasculature. In contrast, almost no transmigration occurs in the absence of P66, an outer membrane protein that has porin and integrin adhesin functions. Importantly, P66 mutants specifically defective in integrin binding were incapable of promoting extravasation. P66 itself does not promote detectable microvascular interactions, suggesting that vascular adhesion of B. burgdorferi mediated by other adhesins, sets the stage for P66-integrin interactions leading to transmigration. Although integrin-binding proteins with diverse functions are encoded by a variety of bacterial pathogens, P66 is the first to have a documented and direct role in vascular transmigration. The emerging picture of vascular escape by the Lyme spirochete shows similarities, but distinct differences from leukocyte transmigration.

  11. Conserved aspartate and lysine residues of RcsB are required for amylovoran biosynthesis, virulence, and DNA binding in Erwinia amylovora.

    Science.gov (United States)

    Ancona, Veronica; Chatnaparat, Tiyakhon; Zhao, Youfu

    2015-08-01

    In Erwinia amylovora, the Rcs phosphorelay system is essential for amylovoran production and virulence. To further understand the role of conserved aspartate residue (D56) in the phosphor receiver (PR) domain and lysine (K180) residue in the function domain of RcsB, amino acid substitutions of RcsB mutant alleles were generated by site-directed mutagenesis and complementation of various rcs mutants were performed. A D56E substitution of RcsB, which mimics the phosphorylation state of RcsB, complemented the rcsB mutant, resulting in increased amylovoran production and gene expression, reduced swarming motility, and restored pathogenicity. In contrast, D56N and K180A or K180Q substitutions of RcsB did not complement the rcsB mutant. Electrophoresis mobility shift assays showed that D56E, but not D56N, K180Q and K180A substitutions of RcsB bound to promoters of amsG and flhD, indicating that both D56 and K180 are required for DNA binding. Interestingly, the RcsBD56E allele could also complement rcsAB, rcsBC and rcsABCD mutants with restored virulence and increased amylovoran production, indicating that RcsB phosphorylation is essential for virulence of E. amylovora. In addition, mutations of T904 and A905, but not phosphorylation mimic mutation of D876 in the PR domain of RcsC, constitutively activate the Rcs system, suggesting that phosphor transfer is required for activating the Rcs system and indicating both A905 and T904 are required for the phosphatase activity of RcsC. Our results demonstrated that RcsB phosphorylation and dephosphorylation, phosphor transfer from RcsC are essential for the function of the Rcs system, and also suggested that constitutive activation of the Rcs system could reduce the fitness of E. amylovora.

  12. DEXMEDETOMIDINE DECREASES PROPOFOL DOSE REQUIREMENT FOR INDUCTION OF ANAESTHESIA: A COMPARATIVE STUDY CONDUCTED ON PATIENTS UNDERGOING LAPAROSCOPIC CHOLECYSTECTOMY

    Directory of Open Access Journals (Sweden)

    Farhana

    2015-04-01

    Full Text Available Dexmedetomidine is a highly selective α - 2 agonist with properties of sedation, analgesia and anxiolysis. We conducted this study on dexmedetomidine to evaluate its effect in reducing dose of propofol for induction of anaesthesia. A prospective, double blind, placebo controlled study was conducted on 100 patients of ASA I and II statu s of both sexes in the age group of 20 - 60 years. Patients were randomly allocated to two groups: Group A(n=50 that received dexmedetomidine loading dose of 1μ g /kg wt.(50ml over 10 minutes that was given 15 minutes prior to induction of anaesthesia and Group B(n=50 received same volume (50 ml of 0.9% normal saline(NS as placebo. Dose requirement of propofol was calculated at induction maintaining BIS of 40 - 60. It was observed that mean requirement of propofol for induction of anaesthesia was reduced to 50.6% in group A patients as compared to group B patients. CONCLUSION: Induction dose of propofol is significantly decreased after administration of dexmedetomidine.

  13. Phenotype-associated lectin-binding profiles of normal and transformed blood cells: a comparative analysis of mannose- and galactose-binding lectins from plants and human serum/placenta.

    Science.gov (United States)

    Mann, K K; André, S; Gabius, H J; Sharp, J G

    1994-10-01

    Surface glycoconjugates of normal and transformed blood cells are commonly characterized by plant lectins. To infer physiological significance of protein-carbohydrate interactions, mammalian lectins are obviously preferable as research tools. So far, human serum lectins have not been used to assess their binding to immunophenotyped human normal or transformed blood cells. Thus, our study combines two groups of lectins with different specificity from plant and human sources. Besides concanavalin A (ConA) we have isolated the mannose-binding protein and serum amyloid P component from human serum. Especially the mannose-binding protein is believed to play a role in host defence against bacteria and yeast cells with unknown impact on normal and tumor cells. These three lectins establish the first group. In addition to the immunomodulatory mistletoe lectin, whose binding can elicit enhanced cytokine secretion from mononuclear blood cells, we included the beta-galactoside-binding lectin (14 kDa) from human placenta in the second group. The initial series of measurements was undertaken using two-color flow cytometry to determine the phenotype-associated binding (based on cluster designation; CD) of the lectins to blood and bone marrow cells from normal donors and the cell line CEM (T-lymphoblastoid), KG1-A (primitive myeloid leukemia) and Croco II (B-lymphoblastoid). Heterogeneity was apparent for each lectin in the CD-defined cell populations. Significant differences in binding were noted between Viscum album agglutinin (VAA) and other lectins for CD4+ cells from blood and between mannose-binding protein (MBP) and VAA versus 14 kDa, ConA and serum amyloid P component (SAP) for CD19+ cells from bone marrow.(ABSTRACT TRUNCATED AT 250 WORDS)

  14. Sm and DNA Binding by dual reactive B cells requires distinct V{sub H}, V{sub {kappa}}, and V{sub H} CDR3 structures

    Energy Technology Data Exchange (ETDEWEB)

    Retter, M.W.; Eisenberg, R.A.; Cohen, P.L. [Univ. of North Carolina, Chapel Hill, NC (United States)] [and others

    1995-08-15

    We have previously demonstrated an overlap of the anti-Sm and anti-DNA responses in MRL/Mp-Ipr/Ipr mice. The Ab produced by many anti-Sm hybridomas bind DNA and are encoded by Ig V genes used by anti-DNA hybridomas. In addition, some anti-Sm Ab that bind DNA have acquired mutations that improve DNA binding, indicating that DNA is a selecting Ag in the anti-Sm response. To gain insight into the basis for the dual binding ability of these Ab, we coexpressed the H chain from the anti-Sm hybridoma 2-12 with nine different L chains. Hybridoma 2-12 binds Sm but not DNA, yet expresses the same J558 V{sub H} gene as three anti-Sm hybridomas that bind ssDNA and at least one anti-DNA hybridoma that does not bind Sm. We found that most of the transfectoma Ab bind Sm, but their avidities vary over more than 3 orders of magnitude. Five of the nine transfectoma Ab bind ssDNA, and none bind dsDNA. In general, the ability to bind each Ag follows the binding ability of the hybridoma from which the L chain is derived. H Chain swapping experiments indicate that the H chain, V{sub H} CDR3 in particular contributes to the binding of both Sm and DNA. We conclude that Sm and DNA select for distinct features of V{sub H}, V{sub {kappa}}, and V{sub H} CDR3, suggesting selection by both Ag in the anti-Sm response.

  15. VgrG C terminus confers the type VI effector transport specificity and is required for binding with PAAR and adaptor-effector complex.

    Science.gov (United States)

    Bondage, Devanand D; Lin, Jer-Sheng; Ma, Lay-Sun; Kuo, Chih-Horng; Lai, Erh-Min

    2016-07-01

    Type VI secretion system (T6SS) is a macromolecular machine used by many Gram-negative bacteria to inject effectors/toxins into eukaryotic hosts or prokaryotic competitors for survival and fitness. To date, our knowledge of the molecular determinants and mechanisms underlying the transport of these effectors remains limited. Here, we report that two T6SS encoded valine-glycine repeat protein G (VgrG) paralogs in Agrobacterium tumefaciens C58 specifically control the secretion and interbacterial competition activity of the type VI DNase toxins Tde1 and Tde2. Deletion and domain-swapping analysis identified that the C-terminal extension of VgrG1 specifically confers Tde1 secretion and Tde1-dependent interbacterial competition activity in planta, and the C-terminal variable region of VgrG2 governs this specificity for Tde2. Functional studies of VgrG1 and VgrG2 variants with stepwise deletion of the C terminus revealed that the C-terminal 31 aa (C31) of VgrG1 and 8 aa (C8) of VgrG2 are the molecular determinants specifically required for delivery of each cognate Tde toxin. Further in-depth studies on Tde toxin delivery mechanisms revealed that VgrG1 interacts with the adaptor/chaperone-effector complex (Tap-1-Tde1) in the absence of proline-alanine-alanine-arginine (PAAR) and the VgrG1-PAAR complex forms independent of Tap-1 and Tde1. Importantly, we identified the regions involved in these interactions. Although the entire C31 segment is required for binding with the Tap-1-Tde1 complex, only the first 15 aa of this region are necessary for PAAR binding. These results suggest that the VgrG1 C terminus interacts sequentially or simultaneously with the Tap-1-Tde1 complex and PAAR to govern Tde1 translocation across bacterial membranes and delivery into target cells for antibacterial activity. PMID:27313214

  16. VgrG C terminus confers the type VI effector transport specificity and is required for binding with PAAR and adaptor–effector complex

    Science.gov (United States)

    Bondage, Devanand D.; Lin, Jer-Sheng; Ma, Lay-Sun; Kuo, Chih-Horng; Lai, Erh-Min

    2016-01-01

    Type VI secretion system (T6SS) is a macromolecular machine used by many Gram-negative bacteria to inject effectors/toxins into eukaryotic hosts or prokaryotic competitors for survival and fitness. To date, our knowledge of the molecular determinants and mechanisms underlying the transport of these effectors remains limited. Here, we report that two T6SS encoded valine-glycine repeat protein G (VgrG) paralogs in Agrobacterium tumefaciens C58 specifically control the secretion and interbacterial competition activity of the type VI DNase toxins Tde1 and Tde2. Deletion and domain-swapping analysis identified that the C-terminal extension of VgrG1 specifically confers Tde1 secretion and Tde1-dependent interbacterial competition activity in planta, and the C-terminal variable region of VgrG2 governs this specificity for Tde2. Functional studies of VgrG1 and VgrG2 variants with stepwise deletion of the C terminus revealed that the C-terminal 31 aa (C31) of VgrG1 and 8 aa (C8) of VgrG2 are the molecular determinants specifically required for delivery of each cognate Tde toxin. Further in-depth studies on Tde toxin delivery mechanisms revealed that VgrG1 interacts with the adaptor/chaperone–effector complex (Tap-1–Tde1) in the absence of proline-alanine-alanine-arginine (PAAR) and the VgrG1–PAAR complex forms independent of Tap-1 and Tde1. Importantly, we identified the regions involved in these interactions. Although the entire C31 segment is required for binding with the Tap-1–Tde1 complex, only the first 15 aa of this region are necessary for PAAR binding. These results suggest that the VgrG1 C terminus interacts sequentially or simultaneously with the Tap-1–Tde1 complex and PAAR to govern Tde1 translocation across bacterial membranes and delivery into target cells for antibacterial activity. PMID:27313214

  17. Comparative Analysis of Certain Requirements of Food Legislation in the European Union and the Customs Union of Russia, Belarus, and Kazakhstan

    OpenAIRE

    International Finance Corporation

    2015-01-01

    This report presents a comparative analysis of the food legislation requirements of the European Union (EU) and the Customs Union. Its purpose is to guide food business operators and public authorities engaged in reforming national food safety systems in the peculiarities of EU and Customs Union legal requirements and help them evaluate their capabilities in meeting those requirements. Thi...

  18. Independent regions of adenovirus E1A are required for binding to and dissociation of E2F-protein complexes

    DEFF Research Database (Denmark)

    Fattaey, A R; Harlow, E; Helin, K

    1993-01-01

    The transcription factor E2F is present in independent complexes with the product of the retinoblastoma susceptibility gene, pRB, and a related gene product, p107, in association with the cyclin A-cdk2 or the cyclin E-cdk2 kinase complex. pRB and p107 can negatively regulate E2F activity, since...... overexpression of pRB or p107 in cells lacking a functional pRB leads to the repression of E2F activity. The products of the adenovirus E1A gene can disrupt E2F complexes and result in free and presumably active E2F transcription factor. The regions of E1A required for this function are also essential...... for binding to a number of cellular proteins, including pRB and p107. Through the use of a number of glutathione S-transferase fusion proteins representing different regions of E1A, as well as in vivo expression of E1A proteins containing deletions of either conserved region 1 (CR1) or CR2, we find that CR2...

  19. The galactophilic lectin (PA-IL, gene LecA) from Pseudomonas aeruginosa. Its binding requirements and the localization of lectin receptors in various mouse tissues

    DEFF Research Database (Denmark)

    Kirkeby, Svend; Hansen, Axel K; d'Apice, Anthony;

    2006-01-01

    The opportunistic pathogen Pseudomonas aeruginosa contains lectins of which one of them, PA-IL (gene lecA), shows preference for alpha-galactosylated glycans. The bacterial lectin is probably important in the carbohydrate-mediated adhesion of the microorganism to endothelia and epithelia and ther......The opportunistic pathogen Pseudomonas aeruginosa contains lectins of which one of them, PA-IL (gene lecA), shows preference for alpha-galactosylated glycans. The bacterial lectin is probably important in the carbohydrate-mediated adhesion of the microorganism to endothelia and epithelia....... It is concluded that the carbohydrate recognizing site of the lectin can have a binding requirement of only one saccharide. Lectin histochemistry was performed on sections from wild type mice and from knock-out mice, which lack function of the alpha.1,3-galactosyltransferase gene. All assays with the P......, kidney and adrenal gland while none of the two lectins were able to detect capillaries in the pancreas. This could indicate a differential glycosylation with respect to endothelial cell Gal alpha. epitopes among different organs. Further, since no PA-IL bindingto the endothelial cells in the KO mouse...

  20. Comparative expression profiling of insulin-like growth factor binding protein-5 in milk of Bos indicus and Bubalus bubalis during lactation.

    Science.gov (United States)

    Mohapatra, S K; Singh, S; Kumar, S; Dang, A K; Datta, T K; Das, S K; Mohanty, T K; Kaushik, J K; Mohanty, A K

    2015-04-01

    Insulin-like growth factor binding protein-5 (IGFBP-5) is a key molecule in mammary gland development, which facilitates the removal of mammary epithelial cells (MECs) by apoptosis that takes place during remodeling of the mammary gland during involution. IGFBP-5 binds with IGFs for their bioavailability. IGFBP-5 has been reported to perform pleiotropic roles such as cellular apoptosis, proliferation and differentiation. To understand the role of IGFBP-5 during lactation and clinical mastitis, expression profiling of IGFBP-5 at the protein level was performed in both indigenous cows (Bos indicus) and buffaloes (Bubalus bubalis) belonging to two different breeds - Sahiwal cows and Murrah buffaloes. Reverse-transcriptase PCR (RT-PCR) of IGFBP-5 mRNA confirmed its expression in milk somatic cells and MECs of Sahiwal cows. ELISA was performed for quantitative measurement of IGFBP-5 concentrations in milk during different days (0, 50, 100, 150, 200, 250 and 300) of lactation, during the involution period and in animals exhibiting short lactation and clinical mastitis. The highest concentration of IGFBP-5 in milk was observed during the involution period followed by colostrum, late and early lactation, respectively, in both cattle and buffaloes. No significant difference in the concentration of IGFBP-5 was observed during the first 150 days of lactation between cows and buffaloes. However, higher concentration of IGFBP-5 was observed in cows during late lactation (200 to 300 days) in comparison with buffaloes. To validate the ELISA data, quantitative real-time PCR was performed in MECs of Sahiwal cows. The relative mRNA abundance of IGFBP-5 was found to be significantly (Pcows. Highest mRNA expression of IGFBP-5 was observed around 300 days of lactation followed by 200 and 250 days (Pmilk as compared with Sahiwal cows during lactation in ELISA. Animals having history of short lactation length (short lactating animals) showed higher levels of IGFBP-5 expression (at

  1. The subtilisin-like protease AprV2 is required for virulence and uses a novel disulphide-tethered exosite to bind substrates.

    Directory of Open Access Journals (Sweden)

    Ruth M Kennan

    Full Text Available Many bacterial pathogens produce extracellular proteases that degrade the extracellular matrix of the host and therefore are involved in disease pathogenesis. Dichelobacter nodosus is the causative agent of ovine footrot, a highly contagious disease that is characterized by the separation of the hoof from the underlying tissue. D. nodosus secretes three subtilisin-like proteases whose analysis forms the basis of diagnostic tests that differentiate between virulent and benign strains and have been postulated to play a role in virulence. We have constructed protease mutants of D. nodosus; their analysis in a sheep virulence model revealed that one of these enzymes, AprV2, was required for virulence. These studies challenge the previous hypothesis that the elastase activity of AprV2 is important for disease progression, since aprV2 mutants were virulent when complemented with aprB2, which encodes a variant that has impaired elastase activity. We have determined the crystal structures of both AprV2 and AprB2 and characterized the biological activity of these enzymes. These data reveal that an unusual extended disulphide-tethered loop functions as an exosite, mediating effective enzyme-substrate interactions. The disulphide bond and Tyr92, which was located at the exposed end of the loop, were functionally important. Bioinformatic analyses suggested that other pathogenic bacteria may have proteases that utilize a similar mechanism. In conclusion, we have used an integrated multidisciplinary combination of bacterial genetics, whole animal virulence trials in the original host, biochemical studies, and comprehensive analysis of crystal structures to provide the first definitive evidence that the extracellular secreted proteases produced by D. nodosus are required for virulence and to elucidate the molecular mechanism by which these proteases bind to their natural substrates. We postulate that this exosite mechanism may be used by proteases produced by

  2. Comparative Genomic Analysis of Drechmeria coniospora Reveals Core and Specific Genetic Requirements for Fungal Endoparasitism of Nematodes

    Science.gov (United States)

    Thakur, Nishant; Arguel, Marie-Jeanne; Polanowska, Jolanta; Henrissat, Bernard; Record, Eric; Magdelenat, Ghislaine; Barbe, Valérie; Raffaele, Sylvain; Barbry, Pascal

    2016-01-01

    Drechmeria coniospora is an obligate fungal pathogen that infects nematodes via the adhesion of specialized spores to the host cuticle. D. coniospora is frequently found associated with Caenorhabditis elegans in environmental samples. It is used in the study of the nematode’s response to fungal infection. Full understanding of this bi-partite interaction requires knowledge of the pathogen’s genome, analysis of its gene expression program and a capacity for genetic engineering. The acquisition of all three is reported here. A phylogenetic analysis placed D. coniospora close to the truffle parasite Tolypocladium ophioglossoides, and Hirsutella minnesotensis, another nematophagous fungus. Ascomycete nematopathogenicity is polyphyletic; D. coniospora represents a branch that has not been molecularly characterized. A detailed in silico functional analysis, comparing D. coniospora to 11 fungal species, revealed genes and gene families potentially involved in virulence and showed it to be a highly specialized pathogen. A targeted comparison with nematophagous fungi highlighted D. coniospora-specific genes and a core set of genes associated with nematode parasitism. A comparative gene expression analysis of samples from fungal spores and mycelia, and infected C. elegans, gave a molecular view of the different stages of the D. coniospora lifecycle. Transformation of D. coniospora allowed targeted gene knock-out and the production of fungus that expresses fluorescent reporter genes. It also permitted the initial characterisation of a potential fungal counter-defensive strategy, involving interference with a host antimicrobial mechanism. This high-quality annotated genome for D. coniospora gives insights into the evolution and virulence of nematode-destroying fungi. Coupled with genetic transformation, it opens the way for molecular dissection of D. coniospora physiology, and will allow both sides of the interaction between D. coniospora and C. elegans, as well as the

  3. Simultaneous optimal experimental design for in vitro binding parameter estimation.

    Science.gov (United States)

    Ernest, C Steven; Karlsson, Mats O; Hooker, Andrew C

    2013-10-01

    Simultaneous optimization of in vitro ligand binding studies using an optimal design software package that can incorporate multiple design variables through non-linear mixed effect models and provide a general optimized design regardless of the binding site capacity and relative binding rates for a two binding system. Experimental design optimization was employed with D- and ED-optimality using PopED 2.8 including commonly encountered factors during experimentation (residual error, between experiment variability and non-specific binding) for in vitro ligand binding experiments: association, dissociation, equilibrium and non-specific binding experiments. Moreover, a method for optimizing several design parameters (ligand concentrations, measurement times and total number of samples) was examined. With changes in relative binding site density and relative binding rates, different measurement times and ligand concentrations were needed to provide precise estimation of binding parameters. However, using optimized design variables, significant reductions in number of samples provided as good or better precision of the parameter estimates compared to the original extensive sampling design. Employing ED-optimality led to a general experimental design regardless of the relative binding site density and relative binding rates. Precision of the parameter estimates were as good as the extensive sampling design for most parameters and better for the poorly estimated parameters. Optimized designs for in vitro ligand binding studies provided robust parameter estimation while allowing more efficient and cost effective experimentation by reducing the measurement times and separate ligand concentrations required and in some cases, the total number of samples. PMID:23943088

  4. Analysis of Imatinib and Sorafenib Binding to p38 Compared with c-Abl and b-Raf Provides Structural Insights for Understanding the Selectivity of Inhibitors Targeting the DFG-Out Form of Protein Kinases

    Energy Technology Data Exchange (ETDEWEB)

    Namboodiri, H.; Bukhtiyarova, M; Ramcharan, J; Karpusas, M; Lee, Y; Springman, E

    2010-01-01

    Protein kinases c-Abl, b-Raf, and p38{alpha} are recognized as important targets for therapeutic intervention. c-Abl and b-Raf are major targets of marketed oncology drugs Imatinib (Gleevec) and Sorafenib (Nexavar), respectively, and BIRB-796 is a p38{alpha} inhibitor that reached Phase II clinical trials. A shared feature of these drugs is the fact that they bind to the DFG-out forms of their kinase targets. Although the discovery of this class of kinase inhibitors has increased the level of emphasis on the design of DFG-out inhibitors, the structural determinants for their binding and stabilization of the DFG-out conformation remain unclear. To improve our understanding of these determinants, we determined cocrystal structures of Imatinib and Sorafenib with p38{alpha}. We also conducted a detailed analysis of Imatinib and Sorafenib binding to p38{alpha} in comparison with BIRB-796, including binding kinetics, binding interactions, the solvent accessible surface area (SASA) of the ligands, and stabilization of key structural elements of the protein upon ligand binding. Our results yield an improved understanding of the structural requirements for stabilizing the DFG-out form and a rationale for understanding the genesis of ligand selectivity among DFG-out inhibitors of protein kinases.

  5. The PDZ-binding motif of Yes-associated protein is required for its co-activation of TEAD-mediated CTGF transcription and oncogenic cell transforming activity

    Energy Technology Data Exchange (ETDEWEB)

    Shimomura, Tadanori; Miyamura, Norio; Hata, Shoji; Miura, Ryota; Hirayama, Jun, E-mail: hirayama.dbio@mri.tmd.ac.jp; Nishina, Hiroshi, E-mail: nishina.dbio@mri.tmd.ac.jp

    2014-01-17

    Highlights: •Loss of the PDZ-binding motif inhibits constitutively active YAP (5SA)-induced oncogenic cell transformation. •The PDZ-binding motif of YAP promotes its nuclear localization in cultured cells and mouse liver. •Loss of the PDZ-binding motif inhibits YAP (5SA)-induced CTGF transcription in cultured cells and mouse liver. -- Abstract: YAP is a transcriptional co-activator that acts downstream of the Hippo signaling pathway and regulates multiple cellular processes, including proliferation. Hippo pathway-dependent phosphorylation of YAP negatively regulates its function. Conversely, attenuation of Hippo-mediated phosphorylation of YAP increases its ability to stimulate proliferation and eventually induces oncogenic transformation. The C-terminus of YAP contains a highly conserved PDZ-binding motif that regulates YAP’s functions in multiple ways. However, to date, the importance of the PDZ-binding motif to the oncogenic cell transforming activity of YAP has not been determined. In this study, we disrupted the PDZ-binding motif in the YAP (5SA) protein, in which the sites normally targeted by Hippo pathway-dependent phosphorylation are mutated. We found that loss of the PDZ-binding motif significantly inhibited the oncogenic transformation of cultured cells induced by YAP (5SA). In addition, the increased nuclear localization of YAP (5SA) and its enhanced activation of TEAD-dependent transcription of the cell proliferation gene CTGF were strongly reduced when the PDZ-binding motif was deleted. Similarly, in mouse liver, deletion of the PDZ-binding motif suppressed nuclear localization of YAP (5SA) and YAP (5SA)-induced CTGF expression. Taken together, our results indicate that the PDZ-binding motif of YAP is critical for YAP-mediated oncogenesis, and that this effect is mediated by YAP’s co-activation of TEAD-mediated CTGF transcription.

  6. A novel sterol regulatory element-binding protein gene (sreA) identified in penicillium digitatum is required for prochloraz resistance, full virulence and erg11 (cyp51) regulation.

    Science.gov (United States)

    Liu, Jing; Yuan, Yongze; Wu, Zhi; Li, Na; Chen, Yuanlei; Qin, Tingting; Geng, Hui; Xiong, Li; Liu, Deli

    2015-01-01

    Penicillium digitatum is the most destructive postharvest pathogen of citrus fruits, causing fruit decay and economic loss. Additionally, control of the disease is further complicated by the emergence of drug-resistant strains due to the extensive use of triazole antifungal drugs. In this work, an orthologus gene encoding a putative sterol regulatory element-binding protein (SREBP) was identified in the genome of P. digitatum and named sreA. The putative SreA protein contains a conserved domain of unknown function (DUF2014) at its carboxyl terminus and a helix-loop-helix (HLH) leucine zipper DNA binding domain at its amino terminus, domains that are functionally associated with SREBP transcription factors. The deletion of sreA (ΔsreA) in a prochloraz-resistant strain (PdHS-F6) by Agrobacterium tumefaciens-mediated transformation led to increased susceptibility to prochloraz and a significantly lower EC50 value compared with the HS-F6 wild-type or complementation strain (COsreA). A virulence assay showed that the ΔsreA strain was defective in virulence towards citrus fruits, while the complementation of sreA could restore the virulence to a large extent. Further analysis by quantitative real-time PCR demonstrated that prochloraz-induced expression of cyp51A and cyp51B in PdHS-F6 was completely abolished in the ΔsreA strain. These results demonstrate that sreA is a critical transcription factor gene required for prochloraz resistance and full virulence in P. digitatum and is involved in the regulation of cyp51 expression.

  7. A novel sterol regulatory element-binding protein gene (sreA) identified in penicillium digitatum is required for prochloraz resistance, full virulence and erg11 (cyp51) regulation.

    Science.gov (United States)

    Liu, Jing; Yuan, Yongze; Wu, Zhi; Li, Na; Chen, Yuanlei; Qin, Tingting; Geng, Hui; Xiong, Li; Liu, Deli

    2015-01-01

    Penicillium digitatum is the most destructive postharvest pathogen of citrus fruits, causing fruit decay and economic loss. Additionally, control of the disease is further complicated by the emergence of drug-resistant strains due to the extensive use of triazole antifungal drugs. In this work, an orthologus gene encoding a putative sterol regulatory element-binding protein (SREBP) was identified in the genome of P. digitatum and named sreA. The putative SreA protein contains a conserved domain of unknown function (DUF2014) at its carboxyl terminus and a helix-loop-helix (HLH) leucine zipper DNA binding domain at its amino terminus, domains that are functionally associated with SREBP transcription factors. The deletion of sreA (ΔsreA) in a prochloraz-resistant strain (PdHS-F6) by Agrobacterium tumefaciens-mediated transformation led to increased susceptibility to prochloraz and a significantly lower EC50 value compared with the HS-F6 wild-type or complementation strain (COsreA). A virulence assay showed that the ΔsreA strain was defective in virulence towards citrus fruits, while the complementation of sreA could restore the virulence to a large extent. Further analysis by quantitative real-time PCR demonstrated that prochloraz-induced expression of cyp51A and cyp51B in PdHS-F6 was completely abolished in the ΔsreA strain. These results demonstrate that sreA is a critical transcription factor gene required for prochloraz resistance and full virulence in P. digitatum and is involved in the regulation of cyp51 expression. PMID:25699519

  8. A novel sterol regulatory element-binding protein gene (sreA identified in penicillium digitatum is required for prochloraz resistance, full virulence and erg11 (cyp51 regulation.

    Directory of Open Access Journals (Sweden)

    Jing Liu

    Full Text Available Penicillium digitatum is the most destructive postharvest pathogen of citrus fruits, causing fruit decay and economic loss. Additionally, control of the disease is further complicated by the emergence of drug-resistant strains due to the extensive use of triazole antifungal drugs. In this work, an orthologus gene encoding a putative sterol regulatory element-binding protein (SREBP was identified in the genome of P. digitatum and named sreA. The putative SreA protein contains a conserved domain of unknown function (DUF2014 at its carboxyl terminus and a helix-loop-helix (HLH leucine zipper DNA binding domain at its amino terminus, domains that are functionally associated with SREBP transcription factors. The deletion of sreA (ΔsreA in a prochloraz-resistant strain (PdHS-F6 by Agrobacterium tumefaciens-mediated transformation led to increased susceptibility to prochloraz and a significantly lower EC50 value compared with the HS-F6 wild-type or complementation strain (COsreA. A virulence assay showed that the ΔsreA strain was defective in virulence towards citrus fruits, while the complementation of sreA could restore the virulence to a large extent. Further analysis by quantitative real-time PCR demonstrated that prochloraz-induced expression of cyp51A and cyp51B in PdHS-F6 was completely abolished in the ΔsreA strain. These results demonstrate that sreA is a critical transcription factor gene required for prochloraz resistance and full virulence in P. digitatum and is involved in the regulation of cyp51 expression.

  9. Potentiation of growth factor signaling by insulin-like growth factor-binding protein-3 in breast epithelial cells requires sphingosine kinase activity.

    Science.gov (United States)

    Martin, Janet L; Lin, Mike Z; McGowan, Eileen M; Baxter, Robert C

    2009-09-18

    We have investigated the mechanism underlying potentiation of epidermal growth factor receptor (EGFR) and type 1 insulin-like growth factor receptor (IGFR1) signaling by IGF-binding protein-3 (IGFBP-3) in MCF-10A breast epithelial cells, focusing on a possible involvement of the sphingosine kinase (SphK) system. IGFBP-3 potentiated EGF-stimulated EGF receptor activation and DNA synthesis, and this was blocked by inhibitors of SphK activity or small interference RNA-mediated silencing of SphK1, but not SphK2, expression. Similarly, IGFR1 phosphorylation and DNA synthesis stimulated by LR3-IGF-I (an IGF-I analog not bound by IGFBP-3), were enhanced by IGFBP-3, and this was blocked by SphK1 silencing. SphK1 expression and activity were stimulated by IGFBP-3 approximately 2-fold over 24 h. Silencing of sphingosine 1-phosphate receptor 1 (S1P1) or S1P3, but not S1P2, abolished the effect of IGFBP-3 on EGF-stimulated EGFR activation. The effects of IGFBP-3 could be reproduced with exogenous S1P or medium conditioned by cells treated with IGFBP-3, and this was also blocked by inhibition of S1P1 and S1P3. These data indicate that potentiation of growth factor signaling by IGFBP-3 in MCF-10A cells requires SphK1 activity and S1P1/S1P3, suggesting that S1P, the product of SphK activity and ligand for S1P1 and S1P3, is the "missing link" mediating IGF and EGFR transactivation and cell growth stimulation by IGFBP-3. PMID:19633297

  10. Drosophila E-cadherin and its binding partner Armadillo/ beta-catenin are required for axonal pathway choices in the developing larval brain.

    Science.gov (United States)

    Fung, Siaumin; Wang, Fay; Spindler, Shana R; Hartenstein, Volker

    2009-08-15

    The fly brain is formed by approximately hundred paired lineages of neurons, each lineage derived from one neuroblast. Embryonic neuroblasts undergo a small number of divisions and produce the primary neurons that form the functioning larval brain. In the larva, neuroblasts produce the secondary lineages that make up the bulk of the adult brain. Axons of a given secondary lineage fasciculate with each other and form a discrete bundle, the secondary axon tract (SAT). Secondary axon tracts prefigure the long axon connections of the adult brain, and therefore pathway choices of SATs made in the larva determine adult brain circuitry. Drosophila Shotgun/E-cadherin (DE-cad) and its binding partner Armadillo/beta-catenin (beta-cat) are expressed in newly born secondary neurons and their axons. The fact that the highly diverse, yet invariant pattern of secondary lineages and SATs has been recently mapped in the wild-type brain enabled us to investigate the role of DE-cad and beta-cat with the help of MARCM clones. Clones were validated by their absence of DE-cad immuno-reactivity. The most significant phenotype consists in the defasciculation and an increased amount of branching of SATs at the neuropile-cortex boundary, as well as subtle changes in the trajectory of SATs within the neuropile. In general, only a fraction of mutant clones in a given lineage showed structural abnormalities. Furthermore, although they all globally express DE-cad and beta-cat, lineages differ in their requirement for DE-cad function. Some lineages never showed morphological abnormalities in MARCM clones, whereas others reacted with abnormal branching and changes in SAT trajectory at a high frequency. We conclude that DE-cad/beta-cat form part of the mechanism that control branching and trajectory of axon tracts in the larval brain. PMID:19520071

  11. Drosophila E-cadherin and its binding partner Armadillo/ β-catenin are required for axonal pathway choices in the developing larval brain

    Science.gov (United States)

    Fung, Siaumin; Wang, Fay; Spindler, Shana R; Hartenstein, Volker

    2009-01-01

    The fly brain is formed by approximately hundred paired lineages of neurons, each lineage derived from one neuroblast. Embryonic neuroblasts undergo a small number of divisions and produce the primary neurons that form the functioning larval brain. In the larva, neuroblasts produce the secondary lineages that make up the bulk of the adult brain. Axons of a given secondary lineage fasciculate with each other and form a discrete bundle, the secondary axon tract (SAT). Secondary axon tracts prefigure the long axon connections of the adult brain, and therefore pathway choices of SATs made in the larva determine adult brain circuitry. Drosophila Shotgun/E-cadherin (DE-cad) and its binding partner Armadillo/β-catenin (β-cat) are expressed in newly born secondary neurons and their axons. The fact that the highly diverse, yet invariant pattern of secondary lineages and SATs has been recently mapped in the wild-type brain enabled us to investigate the role of DE-cad and β-cat with the help of MARCM clones. Clones were validated by their absence of DE-cad immuno-reactivity. The most significant phenotype consists in the defasciculation and an increased amount of branching of SATs at the neuropile-cortex boundary, as well as subtle changes in the trajectory of SATs within the neuropile. In general, only a fraction of mutant clones in a given lineage showed structural abnormalities. Furthermore, although they all globally express DE-cad and β-cat, lineages differ in their requirement for DE-cad function. Some lineages never showed morphological abnormalities in MARCM clones, whereas others reacted with abnormal branching and changes in SAT trajectory at a high frequency. We conclude that DE-cad/β-cat form part of the mechanism that control branching and trajectory of axon tracts in the larval brain. PMID:19520071

  12. Potentiation of growth factor signaling by insulin-like growth factor-binding protein-3 in breast epithelial cells requires sphingosine kinase activity.

    Science.gov (United States)

    Martin, Janet L; Lin, Mike Z; McGowan, Eileen M; Baxter, Robert C

    2009-09-18

    We have investigated the mechanism underlying potentiation of epidermal growth factor receptor (EGFR) and type 1 insulin-like growth factor receptor (IGFR1) signaling by IGF-binding protein-3 (IGFBP-3) in MCF-10A breast epithelial cells, focusing on a possible involvement of the sphingosine kinase (SphK) system. IGFBP-3 potentiated EGF-stimulated EGF receptor activation and DNA synthesis, and this was blocked by inhibitors of SphK activity or small interference RNA-mediated silencing of SphK1, but not SphK2, expression. Similarly, IGFR1 phosphorylation and DNA synthesis stimulated by LR3-IGF-I (an IGF-I analog not bound by IGFBP-3), were enhanced by IGFBP-3, and this was blocked by SphK1 silencing. SphK1 expression and activity were stimulated by IGFBP-3 approximately 2-fold over 24 h. Silencing of sphingosine 1-phosphate receptor 1 (S1P1) or S1P3, but not S1P2, abolished the effect of IGFBP-3 on EGF-stimulated EGFR activation. The effects of IGFBP-3 could be reproduced with exogenous S1P or medium conditioned by cells treated with IGFBP-3, and this was also blocked by inhibition of S1P1 and S1P3. These data indicate that potentiation of growth factor signaling by IGFBP-3 in MCF-10A cells requires SphK1 activity and S1P1/S1P3, suggesting that S1P, the product of SphK activity and ligand for S1P1 and S1P3, is the "missing link" mediating IGF and EGFR transactivation and cell growth stimulation by IGFBP-3.

  13. Comparative CYP-dependent binding of the adrenocortical toxicants 3-methylsulfonyl-DDE and o,p'-DDD in Y-1 adrenal cells.

    Science.gov (United States)

    Hermansson, Veronica; Asp, Vendela; Bergman, Ake; Bergström, Ulrika; Brandt, Ingvar

    2007-11-01

    The environmental pollutant 3-MeSO(2)-DDE [2-(3-methylsulfonyl-4-chlorophenyl)-2-(4-chlorophenyl)-1,1-dichloroethene] is an adrenocortical toxicant in mice, specifically in the glucocorticoid-producing zona fasciculata, due to a cytochrome P450 11B1 (CYP11B1)-catalysed bioactivation and formation of covalently bound protein adducts. o,p'-DDD [2-(2-chlorophenyl)-2-(4-chlorophenyl)-1,1-dichloroethane] is toxic and inhibits steroidogenesis in the human adrenal cortex after bioactivation by unidentified CYPs, but does not exert any toxic effects on the mouse adrenal. As a step towards determining in vitro/in vivo relationships for the CYP-catalysed binding and toxicity of 3-MeSO(2)-DDE and o,p'-DDD, we have investigated the irreversible protein binding of these two toxicants in the murine adrenocortical cell line Y-1. The irreversible binding of 3-MeSO(2)-DDE previously demonstrated in vivo was successfully reproduced and could be inhibited by the CYP-inhibitors etomidate, ketoconazole and metyrapone. Surprisingly, o,p'-DDD reached similar levels of binding as 3-MeSO(2)-DDE. The binding of o,p'-DDD was sensitive to etomidate and ketoconazole, but not to metyrapone. Moreover, GSH depletion increased the binding of 3-MeSO(2)-DDE, but not of o,p'-DDD, indicating an important role of GSH conjugation in the detoxification of the 3-MeSO(2)-DDE-derived reactive metabolite. In addition, the specificity of CYP11B1 in activating 3-MeSO(2)-DDE was investigated using structurally analogous compounds. None of the analogues produced histopathological lesions in the mouse adrenal in vivo following a single i.p. injection of 100 mg/kg body weight, but two of the compounds were able to decrease the irreversible binding of 3-MeSO(2)-DDE to Y-1 cells. These results indicate that the bioactivation of 3-MeSO(2)-DDE by CYP11B1 is highly structure-dependent. In conclusion, both 3-MeSO(2)-DDE and o,p'-DDD bind irreversibly to Y-1 cells despite differences in binding and adrenotoxicity in mice

  14. Zygotic Expression of the Double-Stranded RNA Binding Motif Protein Drb2p Is Required for DNA Elimination in the Ciliate Tetrahymena thermophila ▿

    OpenAIRE

    Motl, Jason A.; Chalker, Douglas L.

    2011-01-01

    Double-stranded RNA binding motif (DSRM)-containing proteins play many roles in the regulation of gene transcription and translation, including some with tandem DSRMs that act in small RNA biogenesis. We report the characterization of the genes for double-stranded RNA binding proteins 1 and 2 (DRB1 and DRB2), two genes encoding nuclear proteins with tandem DSRMs in the ciliate Tetrahymena thermophila. Both proteins are expressed throughout growth and development but exhibit distinct peaks of ...

  15. The Anti-sigma Factor RsiV Is a Bacterial Receptor for Lysozyme: Co-crystal Structure Determination and Demonstration That Binding of Lysozyme to RsiV Is Required for σV Activation.

    Science.gov (United States)

    Hastie, Jessica L; Williams, Kyle B; Bohr, Lindsey L; Houtman, Jon C; Gakhar, Lokesh; Ellermeier, Craig D

    2016-09-01

    σ factors provide RNA polymerase with promoter specificity in bacteria. Some σ factors require activation in order to interact with RNA polymerase and transcribe target genes. The Extra-Cytoplasmic Function (ECF) σ factor, σV, is encoded by several Gram-positive bacteria and is specifically activated by lysozyme. This activation requires the proteolytic destruction of the anti-σ factor RsiV via a process of regulated intramembrane proteolysis (RIP). In many cases proteases that cleave at site-1 are thought to directly sense a signal and initiate the RIP process. We previously suggested binding of lysozyme to RsiV initiated the proteolytic destruction of RsiV and activation of σV. Here we determined the X-ray crystal structure of the RsiV-lysozyme complex at 2.3 Å which revealed that RsiV and lysozyme make extensive contacts. We constructed RsiV mutants with altered abilities to bind lysozyme. We find that mutants that are unable to bind lysozyme block site-1 cleavage of RsiV and σV activation in response to lysozyme. Taken together these data demonstrate that RsiV is a receptor for lysozyme and binding of RsiV to lysozyme is required for σV activation. In addition, the co-structure revealed that RsiV binds to the lysozyme active site pocket. We provide evidence that in addition to acting as a sensor for the presence of lysozyme, RsiV also inhibits lysozyme activity. Thus we have demonstrated that RsiV is a protein with multiple functions. RsiV inhibits σV activity in the absence of lysozyme, RsiV binds lysozyme triggering σV activation and RsiV inhibits the enzymatic activity of lysozyme. PMID:27602573

  16. Flavodoxin cofactor binding induces structural changes that are required for protein-protein interactions with NADP(+) oxidoreductase and pyruvate formate-lyase activating enzyme.

    Science.gov (United States)

    Crain, Adam V; Broderick, Joan B

    2013-12-01

    Flavodoxin (Fld) conformational changes, thermal stability, and cofactor binding were studied using circular dichroism (CD), isothermal titration calorimetry (ITC), and limited proteolysis. Thermodynamics of apo and holo-Fld folding were examined to discern the features of this important electron transfer protein and to provide data on apo-Fld. With the exception of fluorescence and UV-vis binding experiments with its cofactor flavin mononucleotide (FMN), apo-Fld is almost completely uncharacterized in Escherichia coli. Fld is more structured when the FMN cofactor is bound; the association is tight and driven by enthalpy of binding. Surface plasmon resonance binding experiments were carried out under anaerobic conditions for both apo- and holo-Fld and demonstrate the importance of structure and conformation for the interaction with binding partners. Holo-Fld is capable of associating with NADP(+)-dependent flavodoxin oxidoreductase (FNR) and pyruvate formate-lyase activating enzyme (PFL-AE) whereas there is no detectable interaction between apo-Fld and either protein. Limited proteolysis experiments were analyzed by LC-MS to identify the regions in Fld that are involved in conformation changes upon cofactor binding. Docking software was used to model the Fld/PFL-AE complex to understand the interactions between these two proteins and gain insight into electron transfer reactions from Fld to PFL-AE.

  17. Acyl-coenzyme A-binding protein regulates Beta-oxidation required for growth and survival of non-small cell lung cancer.

    Science.gov (United States)

    Harris, Fredrick T; Rahman, S M Jamshedur; Hassanein, Mohamed; Qian, Jun; Hoeksema, Megan D; Chen, Heidi; Eisenberg, Rosana; Chaurand, Pierre; Caprioli, Richard M; Shiota, Masakazu; Massion, Pierre P

    2014-07-01

    We identified acyl-coenzyme A-binding protein (ACBP) as part of a proteomic signature predicting the risk of having lung cancer. Because ACBP is known to regulate β-oxidation, which in turn controls cellular proliferation, we hypothesized that ACBP contributes to regulation of cellular proliferation and survival of non-small cell lung cancer (NSCLC) by modulating β-oxidation. We used matrix-assisted laser desorption/ionization-imaging mass spectrometry (MALDI-IMS) and immunohistochemistry (IHC) to confirm the tissue localization of ABCP in pre-invasive and invasive NSCLCs. We correlated ACBP gene expression levels in NSCLCs with clinical outcomes. In loss-of-function studies, we tested the effect of the downregulation of ACBP on cellular proliferation and apoptosis in normal bronchial and NSCLC cell lines. Using tritiated-palmitate ((3)H-palmitate), we measured β-oxidation levels and tested the effect of etomoxir, a β-oxidation inhibitor, on proliferation and apoptosis. MALDI-IMS and IHC analysis confirmed that ACBP is overexpressed in pre-invasive and invasive lung cancers. High ACBP gene expression levels in NSCLCs correlated with worse survival (HR = 1.73). We observed a 40% decrease in β-oxidation and concordant decreases in proliferation and increases in apoptosis in ACBP-depleted NSCLC cells as compared with bronchial airway epithelial cells. Inhibition of β-oxidation by etomoxir in ACBP-overexpressing cells produced dose-dependent decrease in proliferation and increase in apoptosis (P = 0.01 and P oxidation.

  18. Comparative study of the fatty acid binding process of a new FABP from Cherax quadricarinatus by fluorescence intensity, lifetime and anisotropy.

    Directory of Open Access Journals (Sweden)

    Jiayao Li

    Full Text Available Fatty acid-binding proteins (FABPs are small cytosolic proteins, largely distributed in invertebrates and vertebrates, which accomplish uptake and intracellular transport of hydrophobic ligands such as fatty acids. Although long chain fatty acids play multiple crucial roles in cellular functions (structural, energy metabolism, regulation of gene expression, the precise functions of FABPs, especially those of invertebrate species, remain elusive. Here, we have identified and characterized a novel FABP family member, Cq-FABP, from the hepatopancreas of red claw crayfish Cherax quadricarinatus. We report the characterization of fatty acid-binding affinity of Cq-FABP by four different competitive fluorescence-based assays. In the two first approaches, the fluorescent probe 8-Anilino-1-naphthalenesulfonate (ANS, a binder of internal cavities of protein, was used either by directly monitoring its fluorescence emission or by monitoring the fluorescence resonance energy transfer occurring between the single tryptophan residue of Cq-FABP and ANS. The third and the fourth approaches were based on the measurement of the fluorescence emission intensity of the naturally fluorescent cis-parinaric acid probe or the steady-state fluorescence anisotropy measurements of a fluorescently labeled fatty acid (BODIPY-C16, respectively. The four methodologies displayed consistent equilibrium constants for a given fatty acid but were not equivalent in terms of analysis. Indeed, the two first methods were complicated by the existence of non specific binding modes of ANS while BODIPY-C16 and cis-parinaric acid specifically targeted the fatty acid binding site. We found a relationship between the affinity and the length of the carbon chain, with the highest affinity obtained for the shortest fatty acid, suggesting that steric effects primarily influence the interaction of fatty acids in the binding cavity of Cq-FABP. Moreover, our results show that the binding affinities

  19. Comparative study of the fatty acid binding process of a new FABP from Cherax quadricarinatus by fluorescence intensity, lifetime and anisotropy.

    Science.gov (United States)

    Li, Jiayao; Henry, Etienne; Wang, Lanmei; Delelis, Olivier; Wang, Huan; Simon, Françoise; Tauc, Patrick; Brochon, Jean-Claude; Zhao, Yunlong; Deprez, Eric

    2012-01-01

    Fatty acid-binding proteins (FABPs) are small cytosolic proteins, largely distributed in invertebrates and vertebrates, which accomplish uptake and intracellular transport of hydrophobic ligands such as fatty acids. Although long chain fatty acids play multiple crucial roles in cellular functions (structural, energy metabolism, regulation of gene expression), the precise functions of FABPs, especially those of invertebrate species, remain elusive. Here, we have identified and characterized a novel FABP family member, Cq-FABP, from the hepatopancreas of red claw crayfish Cherax quadricarinatus. We report the characterization of fatty acid-binding affinity of Cq-FABP by four different competitive fluorescence-based assays. In the two first approaches, the fluorescent probe 8-Anilino-1-naphthalenesulfonate (ANS), a binder of internal cavities of protein, was used either by directly monitoring its fluorescence emission or by monitoring the fluorescence resonance energy transfer occurring between the single tryptophan residue of Cq-FABP and ANS. The third and the fourth approaches were based on the measurement of the fluorescence emission intensity of the naturally fluorescent cis-parinaric acid probe or the steady-state fluorescence anisotropy measurements of a fluorescently labeled fatty acid (BODIPY-C16), respectively. The four methodologies displayed consistent equilibrium constants for a given fatty acid but were not equivalent in terms of analysis. Indeed, the two first methods were complicated by the existence of non specific binding modes of ANS while BODIPY-C16 and cis-parinaric acid specifically targeted the fatty acid binding site. We found a relationship between the affinity and the length of the carbon chain, with the highest affinity obtained for the shortest fatty acid, suggesting that steric effects primarily influence the interaction of fatty acids in the binding cavity of Cq-FABP. Moreover, our results show that the binding affinities of several fatty

  20. I. A comparative study of ribo-, deoxyribo-, and hybrid oligonucleotide helices by nuclear magnetic resonance. II. Optical studies of ethidium binding to oligonucleotides

    Energy Technology Data Exchange (ETDEWEB)

    Pardi, A.

    1980-11-01

    The conformations and the helix-to-coil transitions in the following oligonucleotides: (I) a DNA duplex dCT/sub 5/G + dCA/sub 5/G; (II) an RNA duplex rCU/sub 5/G + rCA/sub 5/G; (III) a DNA-RNA hybrid duplex dCT/sub 5/G + rCA/sub 5/G; and (IV) a DNA-RNA hybrid triplex rCU/sub 5/G + dCA/sub 5/G were studied. The first three mixtures all form stable double helical structures at 5/sup 0/C, whereas IV forms a triple strand with a ratio of 2:1 rCU/sub 5/G:dCA/sub 5/G. The chemical shifts of the imino protons in the double strands indicate that I, II, and III have different conformations in solution. This implies a significant change in helical parameters. The chemical shift and sugar pucker data are consistent with helix I having B form geometry, whereas II and III have A (or A') geometry. The chemical shifts of the base protons in system I had transition midpoints of 28 to 30/sup 0/C indicating an all-or-none transition. The exchange lifetimes of the imino protons on helix I were a factor of two longer, for the interior A.T base pairs, than those on helix III. This reflects the greater stability of the DNA helix compared to the hybrid helix. The two terminal C.G base pairs in helix I were also found to have much different exchange rates, indicative of a sequence dependence for these exchange rates. The thermodynamic properties of ethidium binding to several oligonucleotides were investigated. The order of the stability for the 2:1 oligonucleotide:ethidium complexes was found to be rCpG > dCpG > rCpUpG approx. = rUpA > rGpUpG + rCpC. These complexes present models for a possible mechanism for frameshift mutagenesis by ethidium. A large positive induced circular dichroism (CD) has been observed for ethidium upon intercalation into nucleic acids. (ERB)

  1. The lectin domains of polypeptide GalNAc-transferases exhibit carbohydrate-binding specificity for GalNAc: lectin binding to GalNAc-glycopeptide substrates is required for high density GalNAc-O-glycosylation

    DEFF Research Database (Denmark)

    Wandall, Hans H; Irazoqui, Fernando; Tarp, Mads Agervig;

    2007-01-01

    to the enzyme. We have previously shown that the lectin domain of GalNAc-T4 modulates its substrate specificity to enable unique GalNAc-glycopeptide specificities and that this effect is selectively inhibitable by GalNAc; however, direct evidence of carbohydrate binding of GalNAc-transferase lectins has......-T2). Both lectins exhibited specificity for binding of free GalNAc. Kinetic and time-course analysis of GalNAc-T2 demonstrated that the lectin domain did not affect transfer to initial glycosylation sites, but selectively modulated velocity of transfer to subsequent sites and affected the number......Initiation of mucin-type O-glycosylation is controlled by a large family of UDP GalNAc:polypeptide N-acetylgalactosaminyltransferases (GalNAc-transferases). Most GalNAc-transferases contain a ricin-like lectin domain in the C-terminal end, which may confer GalNAc-glycopeptide substrate specificity...

  2. Genome-wide comparative analysis reveals possible common ancestors of nucleotide-binding sites domain containing genes in hybrid Citrus sinensis genome and original Citrus clementina genome

    Science.gov (United States)

    We identified and re-annotated candidate disease resistance (R) genes with nucleotide-binding sites (NBS) domain from a Citrus clementina genome and two complete Citrus sinensis genome sequences (one from the USA and one from China). We found similar numbers of NBS genes from three citrus genomes, r...

  3. Power requirements of biogas upgrading by water scrubbing and biomethane compression: comparative analysis of various plant configurations

    OpenAIRE

    Budzianowski, Wojciech; Wylock, Christophe; Marciniak, Przemysław

    2016-01-01

    Biogas upgrading by water scrubbing followed by biomethane compression is an environmentally benign process. It may be achieved using various plant configurations characterised by various power requirements with associated effects on biomethane sustainability. Therefore, the current study has been undertaken to systematically investigate the power requirements of a range of water scrubbing options. Two groups of water scrubbing are analysed: (1) high pressure water scrubbing (HPWS) and (2) ne...

  4. Solvent-exposed serines in the Gal4 DNA-binding domain are required for promoter occupancy and transcriptional activation in vivo.

    Science.gov (United States)

    Jeličić, Branka; Nemet, Josipa; Traven, Ana; Sopta, Mary

    2014-03-01

    The yeast transcriptional activator Gal4 has long been the prototype for studies of eukaryotic transcription. Gal4 is phosphorylated in the DNA-binding domain (DBD); however, the molecular details and functional significance of this remain unknown. We mutagenized seven potential phosphoserines that lie on the solvent-exposed face of the DBD structure and assessed them for transcriptional activity and DNA binding in vivo. Serine to alanine mutants at positions 22, 47, and 85 show the greatest reduction in promoter occupancy and transcriptional activity at the MEL1 promoter containing a single UASGAL . Substitutions with the phosphomimetic aspartate restored DNA-binding and transcriptional activity at serines 22 and 85, suggesting that they are potential sites of Gal4 phosphorylation in vivo. In contrast, the serine to alanine mutants, except serine 22, were fully proficient for binding to the GAL1-10 promoter, containing multiple UASGAL sites, although they had a reduced ability to activate transcription. Collectively, these data show that at the GAL1-10 promoter, functions of the DBD in transcriptional activation can be uncoupled from roles in promoter binding. We suggest that the serines in the DBD mediate protein-protein contacts with the transcription machinery, leading to stabilization of Gal4 at promoters.

  5. Binding of cellular export factor REF/Aly by Kaposi's sarcoma-associated herpesvirus (KSHV) ORF57 protein is not required for efficient KSHV lytic replication.

    Science.gov (United States)

    Li, Da-Jiang; Verma, Dinesh; Swaminathan, Sankar

    2012-09-01

    Kaposi's sarcoma-associated herpesvirus (KSHV) ORF57 protein is expressed early during lytic KSHV replication, enhances expression of many KSHV genes, and is essential for virus production. ORF57 is a member of a family of proteins conserved among all human and many animal herpesviruses that are multifunctional regulators of gene expression and act posttranscriptionally to increase accumulation of their target mRNAs. The mechanism of ORF57 action is complex and may involve effects on mRNA transcription, stability, and export. ORF57 directly binds to REF/Aly, a cellular RNA-binding protein component of the TREX complex that mediates RNA transcription and export. We analyzed the effects of an ORF57 mutation known to abrogate REF/Aly binding and demonstrate that the REF-binding mutant is impaired in activation of viral mRNAs and noncoding RNAs confined to the nucleus. Although the inability to bind REF leads to decreased ORF57 activity in enhancing gene expression, there is no demonstrable effect on nuclear export of viral mRNA or the ability of ORF57 to support KSHV replication and virus production. These data indicate that REF/Aly-ORF57 interaction is not essential for KSHV lytic replication but may contribute to target RNA stability independent of effects on RNA export, suggesting a novel role for REF/Aly in viral RNA metabolism.

  6. Requirements for Vibrio cholerae HapR Binding and Transcriptional Repression at the hapR Promoter Are Distinct from Those at the aphA Promoter

    OpenAIRE

    Lin, Wei; Kovacikova, Gabriela; Skorupski, Karen

    2005-01-01

    Virulence gene expression in certain strains of Vibrio cholerae is regulated in response to cell density by a quorum-sensing cascade that influences the levels of the LuxR homolog HapR through small regulatory RNAs that control the stability of its message. At high cell density, HapR represses the expression of the gene encoding the virulence gene activator AphA by binding to a site between −85 and −58 in the aphA promoter. We show here that a second binding site for HapR lies within the hapR...

  7. Insulin-like growth factor binding protein-3 is required for the regulation of rat oval cell proliferation and differentiation in the 2AAF/PHX model

    Directory of Open Access Journals (Sweden)

    Nicole C Steiger-Luther

    2010-02-01

    Full Text Available Nicole C Steiger-Luther1, Houda Darwiche1, Seh-Hoon Oh1, Jennifer M Williams1, Bryon E Petersen1,21Department of Pathology, Immunology and Laboratory Medicine, 2Program in Stem Cell Biology and Regenerative Medicine, College of Medicine, University of Florida, Gainesville, FL, USAAbstract: Oval cell-mediated liver regeneration is a highly complex process that involves the coordination of several signaling factors, chemokines and cytokines to allow for proper maintenance of the liver architecture. When hepatocyte proliferation is inhibited, an hepatic stem cell population, often referred to as “oval cells”, is activated to aid in liver regeneration. The function of insulin-like growth factor binding protein-3 (IGFBP-3 during this process of oval cell activation is of particular interest because it is produced in liver and has been shown to induce migration and differentiation of other stem cell populations both in vitro and in vivo. Additionally, IGFBP-3 production has been linked to the transforming growth factor-β (TGF-β superfamily, a pathway known to be induced during oval cell proliferation. In this study, we set out to determine whether IGFBP-3 plays a role in oval cell proliferation, migration and differentiation during this specific type of regeneration. Through activation of the oval cell-mediated liver regeneration in a rat model, we found that IGFBP-3 is elevated in the liver and serum of animals during peak days of oval cell activation and proliferation. Furthermore, in vitro assays found that WB-344 cells, a liver stem cell line similar to oval cells, were induced to migrate in the presence of IGFBP-3. When expression of IGFBP-3 was knocked down during oval cell activation in vivo, we found that oval cell proliferation was increased and observed the appearance of numerous atypical ductular structures, which were OV-6 and Ki67-positive. Finally, quantitative realtime PCR analysis of liver tissue from IGFBP-3 small interfering

  8. The lectin domains of polypeptide GalNAc-transferases exhibit carbohydrate-binding specificity for GalNAc: lectin binding to GalNAc-glycopeptide substrates is required for high density GalNAc-O-glycosylation.

    Science.gov (United States)

    Wandall, Hans H; Irazoqui, Fernando; Tarp, Mads Agervig; Bennett, Eric P; Mandel, Ulla; Takeuchi, Hideyuki; Kato, Kentaro; Irimura, Tatsuro; Suryanarayanan, Ganesh; Hollingsworth, Michael A; Clausen, Henrik

    2007-04-01

    Initiation of mucin-type O-glycosylation is controlled by a large family of UDP GalNAc:polypeptide N-acetylgalactosaminyltransferases (GalNAc-transferases). Most GalNAc-transferases contain a ricin-like lectin domain in the C-terminal end, which may confer GalNAc-glycopeptide substrate specificity to the enzyme. We have previously shown that the lectin domain of GalNAc-T4 modulates its substrate specificity to enable unique GalNAc-glycopeptide specificities and that this effect is selectively inhibitable by GalNAc; however, direct evidence of carbohydrate binding of GalNAc-transferase lectins has not been previously presented. Here, we report the direct carbohydrate binding of two GalNAc-transferase lectin domains, GalNAc-T4 and GalNAc-T2, representing isoforms reported to have distinct glycopeptide activity (GalNAc-T4) and isoforms without apparent distinct GalNAc-glycopeptide specificity (GalNAc-T2). Both lectins exhibited specificity for binding of free GalNAc. Kinetic and time-course analysis of GalNAc-T2 demonstrated that the lectin domain did not affect transfer to initial glycosylation sites, but selectively modulated velocity of transfer to subsequent sites and affected the number of acceptor sites utilized. The results suggest that GalNAc-transferase lectins serve to modulate the kinetic properties of the enzymes in the late stages of the initiation process of O-glycosylation to accomplish dense or complete O-glycan occupancy.

  9. Recognition of galactose-deficient O-glycans in the hinge region of IgA1 by N-acetylgalactosamine-specific snail lectins: a comparative binding study.

    Science.gov (United States)

    Gomes, Michelle M; Suzuki, Hitoshi; Brooks, Monica T; Tomana, Milan; Moldoveanu, Zina; Mestecky, Jiri; Julian, Bruce A; Novak, Jan; Herr, Andrew B

    2010-07-13

    Aberrancies in IgA1 glycosylation have been linked to the pathogenesis of IgA nephropathy (IgAN), a kidney disease characterized by deposits of IgA1-containing immune complexes in the glomerular mesangium. IgA1 from IgAN patients is characterized by the presence of galactose (Gal)-deficient O-glycans in the hinge region that can act as epitopes for anti-glycan IgG or IgA1 antibodies. The resulting circulating immune complexes are trapped in the glomerular mesangium of the kidney where they trigger localized inflammatory responses by activating mesangial cells. Certain lectins recognize the terminal N-acetylgalactosamine (GalNAc)-containing O-glycans on Gal-deficient IgA1 and can be potentially used as diagnostic tools. To improve our understanding of GalNAc recognition by these lectins, we have conducted binding studies to assess the interaction of Helix aspersa agglutinin (HAA) and Helix pomatia agglutinin (HPA) with Gal-deficient IgA1. Surface plasmon resonance spectroscopy revealed that both HAA and HPA bind to a Gal-deficient synthetic hinge region glycopeptide (HR-GalNAc) as well as various aberrantly glycosylated IgA1 myeloma proteins. Despite having six binding sites, both HAA and HPA bind IgA1 in a functionally bivalent manner, with the apparent affinity for IgA1 related to the number of exposed GalNAc groups in the IgA1 hinge. Finally, HAA and HPA were shown to discriminate very effectively between the IgA1 secreted by cell lines derived from peripheral blood cells of patients with IgAN and that from cells of healthy controls. These studies provide insight into lectin recognition of the Gal-deficient IgA1 hinge region and lay the groundwork for the development of reliable diagnostic tools for IgAN.

  10. Identification of a system required for the functional surface localization of sugar binding proteins with class III signal peptides in Sulfolobus solfataricus

    NARCIS (Netherlands)

    Zolghadr, Behnam; Weber, Stefan; Szabo, Zalan; Driessen, Arnold J. M.; Albers, Sonja-Verena

    2007-01-01

    The hyperthermophilic archaeon Sulfolobus solfataricus contains an unusual large number of sugar binding proteins that are synthesized as precursors with a class III signal peptide. Such signal peptides are commonly used to direct archaeal flagellin subunits or bacterial (pseudo)pilins into extracel

  11. Domain interplay in the urokinase receptor. Requirement for the third domain in high affinity ligand binding and demonstration of ligand contact sites in distinct receptor domains

    DEFF Research Database (Denmark)

    Behrendt, N; Ronne, E; Dano, K

    1996-01-01

    . This result shows that in addition to D1, which has an established function in ligand binding (Behrendt, N., Ploug, M., Patthy, L., Houen, G., Blasi, F., and Dano, K. (1991) J. Biol. Chem. 266, 7842-7847), D3 has an important role in governing a high affinity in the intact receptor. Real-time biomolecular...

  12. The case for applying an early-lifecycle technology evaluation methodology to comparative evaluation of requirements engineering research

    Science.gov (United States)

    Feather, Martin S.

    2003-01-01

    The premise of this paper is taht there is a useful analogy between evaluation of proposed problem solutions and evaluation of requirements engineering research itself. Both of these application areas face the challenges of evaluation early in the lifecycle, of the need to consider a wide variety of factors, and of the need to combine inputs from multiple stakeholders in making thse evaluation and subsequent decisions.

  13. Erythromycin, roxithromycin, and clarithromycin: use of slow-binding kinetics to compare their in vitro interaction with a bacterial ribosomal complex active in peptide bond formation.

    Science.gov (United States)

    Dinos, George P; Connell, Sean R; Nierhaus, Knud H; Kalpaxis, Dimitrios L

    2003-03-01

    In a cell-free system derived from Escherichia coli, it is shown that clarithromycin and roxithromycin, like their parent compound erythromycin, do not inhibit the puromycin reaction (i.e., the peptide bond formation between puromycin and AcPhe-tRNA bound at the P-site of 70S ribosomes programmed with heteropolymeric mRNA). Nevertheless, all three antibiotics compete for binding on the ribosome with tylosin, a 16-membered ring macrolide that behaves as a slow-binding, slowly reversible inhibitor of peptidyltransferase. The mutually exclusive binding of these macrolides to ribosomes is also corroborated by the fact that they protect overlapping sites in domain V of 23S rRNA from chemical modification by dimethyl sulfate. From this competition effect, detailed kinetic analysis revealed that roxithromycin or clarithromycin (A), like erythromycin, reacts rapidly with AcPhe-tRNA.MF-mRNA x 70S ribosomal complex (C) to form the encounter complex CA which is then slowly isomerized to a more tight complex, termed C*A. The value of the overall dissociation constant, K, encompassing both steps of macrolide interaction with complex C, is 36 nM for erythromycin, 20 nM for roxithromycin, and 8 nM for clarithromycin. Because the off-rate constant of C*A complex does not significantly differ among the three macrolides, the superiority of clarithromycin as an inhibitor of translation in E. coli cells and many Gram-positive bacteria may be correlated with its greater rate of association with ribosomes. PMID:12606769

  14. Insulin-like growth factor binding protein-3 is required for the regulation of rat oval cell proliferation and differentiation in the 2AAF/PHX model

    OpenAIRE

    Steiger-Luther, Nicole C; Darwiche, Houda; Oh, Seh-Hoon; Williams, Jennifer M.; PETERSEN, BRYON E.

    2010-01-01

    Oval cell-mediated liver regeneration is a highly complex process that involves the coordination of several signaling factors, chemokines and cytokines to allow for proper maintenance of the liver architecture. When hepatocyte proliferation is inhibited, an hepatic stem cell population, often referred to as “oval cells”, is activated to aid in liver regeneration. The function of insulin-like growth factor binding protein-3 (IGFBP-3) during this process of oval cell activation is of particular...

  15. Chemotaxis to the Quorum-Sensing Signal AI-2 Requires the Tsr Chemoreceptor and the Periplasmic LsrB AI-2-Binding Protein▿

    OpenAIRE

    Hegde, Manjunath; Englert, Derek L.; Schrock, Shanna; Cohn, William B.; Vogt, Christian; Wood, Thomas K.; Manson, Michael D.; Jayaraman, Arul

    2010-01-01

    AI-2 is an autoinducer made by many bacteria. LsrB binds AI-2 in the periplasm, and Tsr is the l-serine chemoreceptor. We show that AI-2 strongly attracts Escherichia coli. Both LsrB and Tsr are necessary for sensing AI-2, but AI-2 uptake is not, suggesting that LsrB and Tsr interact directly in the periplasm.

  16. A Short Splice Form of Xin-Actin Binding Repeat Containing 2 (XIRP2) Lacking the Xin Repeats Is Required for Maintenance of Stereocilia Morphology and Hearing Function

    OpenAIRE

    Francis, Shimon P.; Krey, Jocelyn F.; Krystofiak, Evan S.; Cui, Runjia; Nanda, Sonali; Xu, Wenhao; Kachar, Bechara; Barr-Gillespie, Peter G.; Shin, Jung-Bum

    2015-01-01

    Approximately one-third of known deafness genes encode proteins located in the hair bundle, the sensory hair cell's mechanoreceptive organelle. In previous studies, we used mass spectrometry to characterize the hair bundle's proteome, resulting in the discovery of novel bundle proteins. One such protein is Xin-actin binding repeat containing 2 (XIRP2), an actin-cross-linking protein previously reported to be specifically expressed in striated muscle. Because mutations in other actin-cross-lin...

  17. Explaining the Gender Gap: Comparing Undergraduate and Graduate/Faculty Beliefs about Talent Required for Success in Academic Fields

    Science.gov (United States)

    Bailey, Kimberlyn; Nanthakumar, Ampalavanar; Preston, Scott; Ilie, Carolina C.

    Recent research has proposed that the gender gap in academia is caused by differing perceptions of how much talent is needed to succeed in various fields. It was found that, across the STEM/non-STEM divide, the more that graduate students and faculty see success in their own field as requiring as requiring talent, the fewer women participate in that field. This research examines whether undergraduate students share these attitudes. If these attitudes trickle down to the undergraduate population to influence students to choose different fields of study, then undergraduate beliefs should reflect those of graduate students and faculty. Using a large survey of undergraduates across the country, this study aims to characterize undergraduate attitudes and to determine variables that explain the differences between the attitudes of these two populations. Our findings suggest that the two populations have similar beliefs, but that undergraduate beliefs are strongly influenced by information about the gender ratio in each field and that this strong influence greatly differs between STEM and non-STEM fields. These findings seek to help direct future research to ask the right questions and propose plausible hypotheses about gender the imbalance in academia.

  18. The Src Homology 3 Domain Is Required for Junctional Adhesion Molecule Binding to the Third PDZ Domain of the Scaffolding Protein ZO-1

    Energy Technology Data Exchange (ETDEWEB)

    Nomme, Julian; Fanning, Alan S.; Caffrey, Michael; Lye, Ming F.; Anderson, James M.; Lavie, Arnon (NIH); (UNC); (UIC)

    2012-01-20

    Tight junctions are cell-cell contacts that regulate the paracellular flux of solutes and prevent pathogen entry across cell layers. The assembly and permeability of this barrier are dependent on the zonula occludens (ZO) membrane-associated guanylate kinase (MAGUK) proteins ZO-1, -2, and -3. MAGUK proteins are characterized by a core motif of protein-binding domains that include a PDZ domain, a Src homology 3 (SH3) domain, and a region of homology to guanylate kinase (GUK); the structure of this core motif has never been determined for any MAGUK. To better understand how ZO proteins organize the assembly of protein complexes we have crystallized the entire PDZ3-SH3-GUK core motif of ZO-1. We have also crystallized this core motif in complex with the cytoplasmic tail of the ZO-1 PDZ3 ligand, junctional adhesion molecule A (JAM-A) to determine how the activity of different domains is coordinated. Our study shows a new feature for PDZ class II ligand binding that implicates the two highly conserved Phe{sup -2} and Ser{sup -3} residues of JAM. Our x-ray structures and NMR experiments also show for the first time a role for adjacent domains in the binding of ligands to PDZ domains in the MAGUK proteins family.

  19. A comparative study and analysis of QA requirements for the establishment of a nuclear R and D QA system

    Energy Technology Data Exchange (ETDEWEB)

    Kim, Kwan Hyun

    2000-06-01

    This technical report provides recommendations on how to fulfill the requirements of the code in relation to QA activities for nuclear R and D field. This guide applies to the quality assurance (QA) programmes of the responsible organization, i.e. the organization having overall responsibility for the nuclear power plant, as well as to any other separate QA programmes in each stage of a nuclear R and D project. This guide covers QA work on items, services and processes impacting nuclear safety during siting, design, construction, commissioning, operation and decommissioning of nuclear power plants. The impact on safety may occur during the performance of the QA work, or owing to the application of the results of the QA. This guide may, with appropriate modification, also be usefully applied at nuclear installations other than nuclear R and D field.

  20. Comparative Method for Indirect Sensitivity Measurement of UHF RFID Reader with Respect to Interoperability and Conformance Requirements

    Directory of Open Access Journals (Sweden)

    Lukas Kypus

    2014-01-01

    Full Text Available There is never-ending race for the competitive advantage that forces RFID technology service integrators to focus more on used technology qualitative aspects and theirs impacts inside RFID ecosystem. This paper contributes to UHF RFID reader qualitative parameters evaluation and assessment problematic. It presents and describes in details indirect method and procedure of sensitivity measurement created for UHF RFID readers. We applied this method on RFID readers within prepared test environment and confirmed long term intention and recognized trend. Due to regulations limitations, there is not possible to increase output power over defined limits, but there are possibilities to influence reader sensitivity. Our proposal is to use customized comparative measurement method with insertion loss compensation for return link. Beside the main goal achievement, results show as well the qualitative status of development snapshot of reader. Method and following experiment helped us to gain an external view, current values of important parameters and motivation we want to follow up on as well as compared developed reader with its commercial competitors.

  1. The mRNA cap-binding protein Cbc1 is required for high and timely expression of genes by promoting the accumulation of gene-specific activators at promoters.

    Science.gov (United States)

    Li, Tianlu; De Clercq, Nikki; Medina, Daniel A; Garre, Elena; Sunnerhagen, Per; Pérez-Ortín, José E; Alepuz, Paula

    2016-02-01

    The highly conserved Saccharomyces cerevisiae cap-binding protein Cbc1/Sto1 binds mRNA co-transcriptionally and acts as a key coordinator of mRNA fate. Recently, Cbc1 has also been implicated in transcription elongation and pre-initiation complex (PIC) formation. Previously, we described Cbc1 to be required for cell growth under osmotic stress and to mediate osmostress-induced translation reprogramming. Here, we observe delayed global transcription kinetics in cbc1Δ during osmotic stress that correlates with delayed recruitment of TBP and RNA polymerase II to osmo-induced promoters. Interestingly, we detect an interaction between Cbc1 and the MAPK Hog1, which controls most gene expression changes during osmostress, and observe that deletion of CBC1 delays the accumulation of the activator complex Hot1-Hog1 at osmostress promoters. Additionally, CBC1 deletion specifically reduces transcription rates of highly transcribed genes under non-stress conditions, such as ribosomal protein (RP) genes, while having low impact on transcription of weakly expressed genes. For RP genes, we show that recruitment of the specific activator Rap1, and subsequently TBP, to promoters is Cbc1-dependent. Altogether, our results indicate that binding of Cbc1 to the capped mRNAs is necessary for the accumulation of specific activators as well as PIC components at the promoters of genes whose expression requires high and rapid transcription.

  2. Transcriptional activation is a conserved feature of the early embryonic factor Zelda that requires a cluster of four zinc fingers for DNA binding and a low-complexity activation domain.

    Science.gov (United States)

    Hamm, Danielle C; Bondra, Eliana R; Harrison, Melissa M

    2015-02-01

    Delayed transcriptional activation of the zygotic genome is a nearly universal phenomenon in metazoans. Immediately following fertilization, development is controlled by maternally deposited products, and it is not until later stages that widespread activation of the zygotic genome occurs. Although the mechanisms driving this genome activation are currently unknown, the transcriptional activator Zelda (ZLD) has been shown to be instrumental in driving this process in Drosophila melanogaster. Here we define functional domains of ZLD required for both DNA binding and transcriptional activation. We show that the C-terminal cluster of four zinc fingers mediates binding to TAGteam DNA elements in the promoters of early expressed genes. All four zinc fingers are required for this activity, and splice isoforms lacking three of the four zinc fingers fail to activate transcription. These truncated splice isoforms dominantly suppress activation by the full-length, embryonically expressed isoform. We map the transcriptional activation domain of ZLD to a central region characterized by low complexity. Despite relatively little sequence conservation within this domain, ZLD orthologs from Drosophila virilis, Anopheles gambiae, and Nasonia vitripennis activate transcription in D. melanogaster cells. Transcriptional activation by these ZLD orthologs suggests that ZLD functions through conserved interactions with a protein cofactor(s). We have identified distinct DNA-binding and activation domains within the critical transcription factor ZLD that controls the initial activation of the zygotic genome.

  3. Potentiation of Growth Factor Signaling by Insulin-like Growth Factor-binding Protein-3 in Breast Epithelial Cells Requires Sphingosine Kinase Activity*

    OpenAIRE

    Martin, Janet L; Mike Z. Lin; Eileen M. McGowan; Baxter, Robert C.

    2009-01-01

    We have investigated the mechanism underlying potentiation of epidermal growth factor receptor (EGFR) and type 1 insulin-like growth factor receptor (IGFR1) signaling by IGF-binding protein-3 (IGFBP-3) in MCF-10A breast epithelial cells, focusing on a possible involvement of the sphingosine kinase (SphK) system. IGFBP-3 potentiated EGF-stimulated EGF receptor activation and DNA synthesis, and this was blocked by inhibitors of SphK activity or small interference RNA-mediated silencing of SphK1...

  4. Acyl-CoA-binding protein, Acb1p, is required for normal vacuole function and ceramide synthesis in Saccharomyces cerevisiae

    DEFF Research Database (Denmark)

    Færgeman, Nils J.; Feddersen, Søren; Christiansen, Janne K;

    2004-01-01

    In the present study, we show that depletion of acyl-CoA-binding protein, Acb1p, in yeast affects ceramide levels, protein trafficking, vacuole fusion and structure. Vacuoles in Acb1p-depleted cells are multi-lobed, contain significantly less of the SNAREs (soluble N -ethylmaleimide......-sensitive fusion protein attachment protein receptors) Nyv1p, Vam3p and Vti1p, and are unable to fuse in vitro. Mass spectrometric analysis revealed a dramatic reduction in the content of ceramides in whole-cell lipids and in vacuoles isolated from Acb1p-depleted cells. Maturation of yeast aminopeptidase I and...

  5. A NASP (N1/N2)-related protein, Sim3, binds CENP-A and is required for its deposition at fission yeast centromeres

    OpenAIRE

    Dunleavy, Elaine M; Pidoux, Alison L.; Monet, Marie; Bonilla, Carolina; Richardson, William; Hamilton, Georgina L.; Ekwall, Karl; McLaughlin, Paul J.; Allshire, Robin C.

    2007-01-01

    A defining feature of centromeres is the presence of the histone H3 variant CENP-A(Cnp1). It is not known how CENP-A(Cnp1) is specifically delivered to, and assembled into, centromeric chromatin. Through a screen for factors involved in kinetochore integrity in fission yeast, we identified Sim3. Sim3 is homologous to known histone binding proteins NASP(Human) and N1/N2(Xenopus) and aligns with Hif1(S. cerevisiae), defining the SHNi-TPR family. Sim3 is distributed throughout the nucleoplasm, y...

  6. Heterodimerization of the transcription factors E2F-1 and DP-1 is required for binding to the adenovirus E4 (ORF6/7) protein

    DEFF Research Database (Denmark)

    Helin, K; Harlow, E

    1994-01-01

    Adenovirus infection leads to E1A-dependent activation of the transcription factor E2F. E2F has recently been identified in complexes with cellular proteins such as the retinoblastoma protein (pRB) and the two pRB family members p107 and p130. E1A dissociates E2F from these cellular proteins...... experiments also demonstrate that pRB binding to the E2F-1/DP-1 heterodimer prevents the formation of an E2F-1/DP-1/E4 complex....

  7. Multi-species comparative analysis of the equine ACE gene identifies a highly conserved potential transcription factor binding site in intron 16.

    Directory of Open Access Journals (Sweden)

    Natasha A Hamilton

    Full Text Available Angiotensin converting enzyme (ACE is essential for control of blood pressure. The human ACE gene contains an intronic Alu indel (I/D polymorphism that has been associated with variation in serum enzyme levels, although the functional mechanism has not been identified. The polymorphism has also been associated with cardiovascular disease, type II diabetes, renal disease and elite athleticism. We have characterized the ACE gene in horses of breeds selected for differing physical abilities. The equine gene has a similar structure to that of all known mammalian ACE genes. Nine common single nucleotide polymorphisms (SNPs discovered in pooled DNA were found to be inherited in nine haplotypes. Three of these SNPs were located in intron 16, homologous to that containing the Alu polymorphism in the human. A highly conserved 18 bp sequence, also within that intron, was identified as being a potential binding site for the transcription factors Oct-1, HFH-1 and HNF-3β, and lies within a larger area of higher than normal homology. This putative regulatory element may contribute to regulation of the documented inter-individual variation in human circulating enzyme levels, for which a functional mechanism is yet to be defined. Two equine SNPs occurred within the conserved area in intron 16, although neither of them disrupted the putative binding site. We propose a possible regulatory mechanism of the ACE gene in mammalian species which was previously unknown. This advance will allow further analysis leading to a better understanding of the mechanisms underpinning the associations seen between the human Alu polymorphism and enzyme levels, cardiovascular disease states and elite athleticism.

  8. Comparative study of tight-binding and ab initio electronic structure calculations focused on magnetic anisotropy in ordered CoPt alloy

    Energy Technology Data Exchange (ETDEWEB)

    Zemen, J., E-mail: zemen@fzu.cz [School of Physics and Astronomy, University of Nottingham, Nottingham NG7 2RD (United Kingdom); Institute of Physics ASCR, v. v. i., Cukrovarnická 10, 162 00 Praha 6 (Czech Republic); Mašek, J. [Institute of Physics ASCR, v. v. i., Na Slovance 2, 182 21 Praha 8 (Czech Republic); Kučera, J. [Institute of Physics ASCR, v. v. i., Cukrovarnická 10, 162 00 Praha 6 (Czech Republic); Mol, J.A. [Kavli Institute of Nanoscience, Delft University of Technology, Lorentzweg 1, 2628 CJ Delft (Netherlands); Institute of Physics ASCR, v. v. i., Cukrovarnická 10, 162 00 Praha 6 (Czech Republic); Motloch, P. [Institute of Physics ASCR, v. v. i., Cukrovarnická 10, 162 00 Praha 6 (Czech Republic); Jungwirth, T. [Institute of Physics ASCR, v. v. i., Cukrovarnická 10, 162 00 Praha 6 (Czech Republic); School of Physics and Astronomy, University of Nottingham, Nottingham NG7 2RD (United Kingdom)

    2014-04-01

    An empirical multiorbital (spd) tight binding (TB) model including magnetism and spin–orbit coupling is applied to calculations of magnetic anisotropy energy (MAE) in CoPt L1{sub 0} structure. A realistic Slater–Koster parametrisation for single-element transition metals is adapted for the ordered binary alloy. Spin magnetic moment and density of states are calculated using a full-potential linearised augmented plane-wave (LAPW) ab initio method and our TB code with different variants of the interatomic parameters. Detailed mutual comparison of this data allows for determination of a subset of the compound TB parameters tuning of which improves the agreement of the TB and LAPW results. MAE calculated as a function of band filling using the refined parameters is in broad agreement with ab initio data for all valence states and in quantitative agreement with ab initio and experimental data for the natural band filling. Our work provides a practical basis for further studies of relativistic magnetotransport anisotropies by means of local Green's function formalism which is directly compatible with our TB approach. - Highlights: • Calculations of electronic structure properties of bulk ordered CoPt alloy using tight-binding (TB) and density functional theory (DFT) approach. • Refinement of existing single-element TB parameters for a binary alloy based on a comparison of band structure and spin magnetic moment per atom to DFT results. • Quantitative agreement of magnetic anisotropy energy (MAE) obtained by TB and DFT on a range of band fillings. • Successful description of ground state spin–orbit coupling phenomena using an extended TB model suitable for subsequent magnetotransport simulations.

  9. Zygotic Expression of the Double-Stranded RNA Binding Motif Protein Drb2p Is Required for DNA Elimination in the Ciliate Tetrahymena thermophila ▿

    Science.gov (United States)

    Motl, Jason A.; Chalker, Douglas L.

    2011-01-01

    Double-stranded RNA binding motif (DSRM)-containing proteins play many roles in the regulation of gene transcription and translation, including some with tandem DSRMs that act in small RNA biogenesis. We report the characterization of the genes for double-stranded RNA binding proteins 1 and 2 (DRB1 and DRB2), two genes encoding nuclear proteins with tandem DSRMs in the ciliate Tetrahymena thermophila. Both proteins are expressed throughout growth and development but exhibit distinct peaks of expression, suggesting different biological roles. In support of this, we show that expression of DRB2 is essential for vegetative growth while DRB1 expression is not. During conjugation, Drb1p and Drb2p localize to distinct nuclear foci. Cells lacking all DRB1 copies are able to produce viable progeny, although at a reduced rate relative to wild-type cells. In contrast, cells lacking germ line DRB2 copies, which thus cannot express Drb2p zygotically, fail to produce progeny, arresting late into conjugation. This arrest phenotype is accompanied by a failure to organize the essential DNA rearrangement protein Pdd1p into DNA elimination bodies and execute DNA elimination and chromosome breakage. These results implicate zygotically expressed Drb2p in the maturation of these nuclear structures, which are necessary for reorganization of the somatic genome. PMID:22021239

  10. Dynein Light Chain LC8 Is Required for RNA Polymerase I-Mediated Transcription in Trypanosoma brucei, Facilitating Assembly and Promoter Binding of Class I Transcription Factor A.

    Science.gov (United States)

    Kirkham, Justin K; Park, Sung Hee; Nguyen, Tu N; Lee, Ju Huck; Günzl, Arthur

    2016-01-01

    Dynein light chain LC8 is highly conserved among eukaryotes and has both dynein-dependent and dynein-independent functions. Interestingly, LC8 was identified as a subunit of the class I transcription factor A (CITFA), which is essential for transcription by RNA polymerase I (Pol I) in the parasite Trypanosoma brucei. Given that LC8 has never been identified with a basal transcription factor and that T. brucei relies on RNA Pol I for expressing the variant surface glycoprotein (VSG), the key protein in antigenic variation, we investigated the CITFA-specific role of LC8. Depletion of LC8 from mammalian-infective bloodstream trypanosomes affected cell cycle progression, reduced the abundances of rRNA and VSG mRNA, and resulted in rapid cell death. Sedimentation analysis, coimmunoprecipitation of recombinant proteins, and bioinformatic analysis revealed an LC8 binding site near the N terminus of the subunit CITFA2. Mutation of this site prevented the formation of a CITFA2-LC8 heterotetramer and, in vivo, was lethal, affecting assembly of a functional CITFA complex. Gel shift assays and UV cross-linking experiments identified CITFA2 as a promoter-binding CITFA subunit. Accordingly, silencing of LC8 or CITFA2 resulted in a loss of CITFA from RNA Pol I promoters. Hence, we discovered an LC8 interaction that, unprecedentedly, has a basal function in transcription.

  11. An essential virulence protein of Agrobacterium tumefaciens, VirB4, requires an intact mononucleotide binding domain to function in transfer of T-DNA.

    Science.gov (United States)

    Fullner, K J; Stephens, K M; Nester, E W

    1994-12-15

    The 11 gene products of the Agrobacterium tumefaciens virB operon, together with the VirD4 protein, are proposed to form a membrane complex which mediates the transfer of T-DNA to plant cells. This study examined one putative component of that complex, VirB4. A deletion of the virB4 gene on the Ti plasmid pTiA6NC was constructed by replacing the virB4 gene with the kanamycin resistance-conferring nptII gene. The virB4 gene was found to be necessary for virulence on plants and for the transfer of IncQ plasmids to recipient cells of A. tumefaciens. Genetic complementation of the deletion strain by the virB4 gene under control of the virB promoter confirmed that the deletion was nonpolar on downstream virB genes. Genetic complementation was also achieved with the virB4 gene placed under control of the lac promoter, even though synthesis of the VirB4 protein from this promoter is far below wild-type levels. Having shown a role for the VirB4 protein in DNA transfer, lysine-439, found within the conserved mononucleotide binding domain of VirB4, was changed to a glutamic acid, methionine, or arginine by oligonucleotide-directed mutagenesis. virB4 genes bearing these mutations were unable to complement the virB4 deletion for either virulence or for IncQ transfer, showing that an intact mononucleotide binding site is necessary for the function of VirB4 in DNA transfer. The necessity of the VirB4 protein with an intact mononucleotide binding site for extracellular complementation of virE2 mutants was also shown. In merodiploid studies, lysine-439 mutations present in trans decreased IncQ plasmid transfer frequencies, suggesting that VirB4 functions within a complex to facilitate DNA transfer. PMID:7830718

  12. Uses of Phage Display in Agriculture: Sequence Analysis and Comparative Modeling of Late Embryogenesis Abundant Client Proteins Suggest Protein-Nucleic Acid Binding Functionality

    Directory of Open Access Journals (Sweden)

    Rekha Kushwaha

    2013-01-01

    Full Text Available A group of intrinsically disordered, hydrophilic proteins—Late Embryogenesis Abundant (LEA proteins—has been linked to survival in plants and animals in periods of stress, putatively through safeguarding enzymatic function and prevention of aggregation in times of dehydration/heat. Yet despite decades of effort, the molecular-level mechanisms defining this protective function remain unknown. A recent effort to understand LEA functionality began with the unique application of phage display, wherein phage display and biopanning over recombinant Seed Maturation Protein homologs from Arabidopsis thaliana and Glycine max were used to retrieve client proteins at two different temperatures, with one intended to represent heat stress. From this previous study, we identified 21 client proteins for which clones were recovered, sometimes repeatedly. Here, we use sequence analysis and homology modeling of the client proteins to ascertain common sequence and structural properties that may contribute to binding affinity with the protective LEA protein. Our methods uncover what appears to be a predilection for protein-nucleic acid interactions among LEA client proteins, which is suggestive of subcellular residence. The results from this initial computational study will guide future efforts to uncover the protein protective mechanisms during heat stress, potentially leading to phage-display-directed evolution of synthetic LEA molecules.

  13. A comparative cytogenetic study of Drosophila parasitoids (Hymenoptera, Figitidae) using DNA-binding fluorochromes and FISH with 45S rDNA probe.

    Science.gov (United States)

    Gokhman, Vladimir E; Bolsheva, Nadezhda L; Govind, Shubha; Muravenko, Olga V

    2016-06-01

    Karyotypes of Leptopilina boulardi (Barbotin, Carton et Keiner-Pillault, 1979) (n = 9), L. heterotoma (Thomson, 1862) (n = 10), L. victoriae Nordlander, 1980 (n = 10) and Ganaspis xanthopoda (Ashmead, 1896) (n = 9) (Hymenoptera, Figitidae) were studied using DNA-binding ligands with different base specificity [propidium iodide (PI), chromomycin A3 (CMA3) and 4',6-diamidino-2-phenylindole (DAPI)], and fluorescence in situ hybridization (FISH) with a 45S rDNA probe. Fluorochrome staining was similar between the different fluorochromes, except for a single CMA3- and PI-positive and DAPI-negative band per haploid karyotype of each species. FISH with 45S rDNA probe detected a single rDNA site in place of the bright CMA3-positive band, thus identifying the nucleolus organizing region (NOR). Chromosomal locations of NORs were similar for both L. heterotoma and L. victoriae, but strongly differed in L. boulardi as well as in G. xanthopoda. Phylogenetic aspects of NOR localization in all studied species are briefly discussed.

  14. Comparative study of tight-binding and ab initio electronic structure calculations focused on magnetic anisotropy in ordered CoPt alloy

    Science.gov (United States)

    Zemen, J.; Mašek, J.; Kučera, J.; Mol, J. A.; Motloch, P.; Jungwirth, T.

    2014-04-01

    An empirical multiorbital (spd) tight binding (TB) model including magnetism and spin-orbit coupling is applied to calculations of magnetic anisotropy energy (MAE) in CoPt L10 structure. A realistic Slater-Koster parametrisation for single-element transition metals is adapted for the ordered binary alloy. Spin magnetic moment and density of states are calculated using a full-potential linearised augmented plane-wave (LAPW) ab initio method and our TB code with different variants of the interatomic parameters. Detailed mutual comparison of this data allows for determination of a subset of the compound TB parameters tuning of which improves the agreement of the TB and LAPW results. MAE calculated as a function of band filling using the refined parameters is in broad agreement with ab initio data for all valence states and in quantitative agreement with ab initio and experimental data for the natural band filling. Our work provides a practical basis for further studies of relativistic magnetotransport anisotropies by means of local Green's function formalism which is directly compatible with our TB approach.

  15. A comparative cytogenetic study of Drosophila parasitoids (Hymenoptera, Figitidae) using DNA-binding fluorochromes and FISH with 45S rDNA probe.

    Science.gov (United States)

    Gokhman, Vladimir E; Bolsheva, Nadezhda L; Govind, Shubha; Muravenko, Olga V

    2016-06-01

    Karyotypes of Leptopilina boulardi (Barbotin, Carton et Keiner-Pillault, 1979) (n = 9), L. heterotoma (Thomson, 1862) (n = 10), L. victoriae Nordlander, 1980 (n = 10) and Ganaspis xanthopoda (Ashmead, 1896) (n = 9) (Hymenoptera, Figitidae) were studied using DNA-binding ligands with different base specificity [propidium iodide (PI), chromomycin A3 (CMA3) and 4',6-diamidino-2-phenylindole (DAPI)], and fluorescence in situ hybridization (FISH) with a 45S rDNA probe. Fluorochrome staining was similar between the different fluorochromes, except for a single CMA3- and PI-positive and DAPI-negative band per haploid karyotype of each species. FISH with 45S rDNA probe detected a single rDNA site in place of the bright CMA3-positive band, thus identifying the nucleolus organizing region (NOR). Chromosomal locations of NORs were similar for both L. heterotoma and L. victoriae, but strongly differed in L. boulardi as well as in G. xanthopoda. Phylogenetic aspects of NOR localization in all studied species are briefly discussed. PMID:27150102

  16. The stress granule protein Vgl1 and poly(A)-binding protein Pab1 are required for doxorubicin resistance in the fission yeast Schizosaccharomyces pombe

    Energy Technology Data Exchange (ETDEWEB)

    Morita, Takahiro [Laboratory of Molecular Pharmacogenomics, School of Pharmaceutical Sciences, Kinki University, Kowakae 3-4-1, Higashi-Osaka 577-8502 (Japan); Satoh, Ryosuke [Laboratory of Molecular Pharmacogenomics, School of Pharmaceutical Sciences, Kinki University, Kowakae 3-4-1, Higashi-Osaka 577-8502 (Japan); Japan Society for the Promotion of Science, 1-8 Chiyoda-ku, Tokyo 102-8472 (Japan); Umeda, Nanae; Kita, Ayako [Laboratory of Molecular Pharmacogenomics, School of Pharmaceutical Sciences, Kinki University, Kowakae 3-4-1, Higashi-Osaka 577-8502 (Japan); Sugiura, Reiko, E-mail: sugiurar@phar.kindai.ac.jp [Laboratory of Molecular Pharmacogenomics, School of Pharmaceutical Sciences, Kinki University, Kowakae 3-4-1, Higashi-Osaka 577-8502 (Japan)

    2012-01-06

    Highlights: Black-Right-Pointing-Pointer Stress granules (SGs) as a mechanism of doxorubicin tolerance. Black-Right-Pointing-Pointer We characterize the role of stress granules in doxorubicin tolerance. Black-Right-Pointing-Pointer Deletion of components of SGs enhances doxorubicin sensitivity in fission yeast. Black-Right-Pointing-Pointer Doxorubicin promotes SG formation when combined with heat shock. Black-Right-Pointing-Pointer Doxorubicin regulates stress granule assembly independent of eIF2{alpha} phosphorylation. -- Abstract: Doxorubicin is an anthracycline antibiotic widely used for chemotherapy. Although doxorubicin is effective in the treatment of several cancers, including solid tumors and leukemias, the basis of its mechanism of action is not completely understood. Here, we describe the effects of doxorubicin and its relationship with stress granules formation in the fission yeast, Schizosaccharomyces pombe. We show that disruption of genes encoding the components of stress granules, including vgl1{sup +}, which encodes a multi-KH type RNA-binding protein, and pab1{sup +}, which encodes a poly(A)-binding protein, resulted in greater sensitivity to doxorubicin than seen in wild-type cells. Disruption of the vgl1{sup +} and pab1{sup +} genes did not confer sensitivity to other anti-cancer drugs such as cisplatin, 5-fluorouracil, and paclitaxel. We also showed that doxorubicin treatment promoted stress granule formation when combined with heat shock. Notably, doxorubicin treatment did not induce hyperphosphorylation of eIF2{alpha}, suggesting that doxorubicin is involved in stress granule assembly independent of eIF2{alpha} phosphorylation. Our results demonstrate the usefulness of fission yeast for elucidating the molecular targets of doxorubicin toxicity and suggest a novel drug-resistance mechanism involving stress granule assembly.

  17. Comparative analysis and molecular characterization of a gene BANF1 encoded a DNA-binding protein during mitosis from the Giant Panda and Black Bear.

    Science.gov (United States)

    Zeng, Yichun; Hou, Yi-Ling; Ding, Xiang; Hou, Wan-Ru; Li, Jian

    2014-01-01

    Barrier to autointegration factor 1 (BANF1) is a DNA-binding protein found in the nucleus and cytoplasm of eukaryotic cells that functions to establish nuclear architecture during mitosis. The cDNA and the genomic sequence of BANF1 were cloned from the Giant Panda (Ailuropoda melanoleuca) and Black Bear (Ursus thibetanus mupinensis) using RT-PCR technology and Touchdown-PCR, respectively. The cDNA of the BANF1 cloned from Giant Panda and Black Bear is 297 bp in size, containing an open reading frame of 270 bp encoding 89 amino acids. The length of the genomic sequence from Giant Panda is 521 bp, from Black Bear is 536 bp, which were found both to possess 2 exons. Alignment analysis indicated that the nucleotide sequence and the deduced amino acid sequence are highly conserved to some mammalian species studied. Topology prediction showed there is one Protein kinase C phosphorylation site, one Casein kinase II phosphorylation site, one Tyrosine kinase phosphorylation site, one N-myristoylation site, and one Amidation site in the BANF1 protein of the Giant Panda, and there is one Protein kinase C phosphorylation site, one Tyrosine kinase phosphorylation site, one N-myristoylation site, and one Amidation site in the BANF1 protein of the Black Bear. The BANF1 gene can be readily expressed in E. coli. Results showed that the protein BANF1 fusion with the N-terminally His-tagged form gave rise to the accumulation of an expected 14 kD polypeptide that formed inclusion bodies. The expression products obtained could be used to purify the proteins and study their function further. PMID:25009988

  18. Improved assay for measuring heparin binding to bull sperm

    International Nuclear Information System (INIS)

    The binding of heparin to sperm has been used to study capacitation and to rank relative fertility of bulls. Previous binding assays were laborious, used 107 sperm per assay point, and required large amounts of radiolabeled heparin. A modified heparin-binding assay is described that used only 5 x 104 cells per incubation well and required reduced amounts of [3H] heparin. The assay was performed in 96-well Millititer plates, enabling easy incubation and filtering. Dissociation constants and concentrations of binding sites did not differ if analyzed by Scatchard plots, Woolf plots, or by log-logit transformed weighted nonlinear least squares regression, except in the case of outliers. In such cases, Scatchard analysis was more sensitive to outliers. Nonspecific binding was insignificant using nonlinear logistic fit regression and a proportion graph. The effects were tested of multiple free-thawing of sperm in either a commercial egg yolk extender, 40 mM Tris buffer with 8% glycerol, or 40 mM Tris buffer without glycerol. Freeze-thawing in extender did not affect the dissociation constant or the concentration of binding sites. However, freeze-thawing three times in 40 mM Tris reduced the concentration of binding sites and lowered the dissociation constant (raised the affinity). The inclusion of glycerol in the 40 mM Tris did not significantly affect the estimated dissociation constant or the concentration of binding sites as compared to 40 mM Tris without glycerol

  19. The Microtubule Minus-End-Binding Protein Patronin/PTRN-1 Is Required for Axon Regeneration in C. elegans

    Directory of Open Access Journals (Sweden)

    Marian Chuang

    2014-11-01

    Full Text Available Precise regulation of microtubule (MT dynamics is increasingly recognized as a critical determinant of axon regeneration. In contrast to developing neurons, mature axons exhibit noncentrosomal microtubule nucleation. The factors regulating noncentrosomal MT architecture in axon regeneration remain poorly understood. We report that PTRN-1, the C. elegans member of the Patronin/Nezha/calmodulin- and spectrin-associated protein (CAMSAP family of microtubule minus-end-binding proteins, is critical for efficient axon regeneration in vivo. ptrn-1-null mutants display generally normal developmental axon outgrowth but significantly impaired regenerative regrowth after laser axotomy. Unexpectedly, mature axons in ptrn-1 mutants display elevated numbers of dynamic axonal MTs before and after injury, suggesting that PTRN-1 inhibits MT dynamics. The CKK domain of PTRN-1 is necessary and sufficient for its functions in axon regeneration and MT dynamics and appears to stabilize MTs independent of minus-end localization. Whereas in developing neurons, PTRN-1 inhibits activity of the DLK-1 mitogen-activated protein kinase (MAPK cascade, we find that, in regeneration, PTRN-1 and DLK-1 function together to promote axonal regrowth.

  20. Comparative Analysis Between Chinese & American Food Labeling Marked Requirement%中美食品标签标注要求比较分析

    Institute of Scientific and Technical Information of China (English)

    李小艳; 杨西安; 沈莉

    2013-01-01

    The differences of food labeling marked requirement between China and America were compared including product name , net content, burden sheet and etc. so as to provide the reference for the import and export food enterprises and supervision department.%比较分析了中美两国在食品标签标注要求上的差异,包括产品名称、净含量、配料表等内容,以期为中美进出口食品企业及监管部门提供参考。

  1. Yeast sterol regulatory element-binding protein (SREBP) cleavage requires Cdc48 and Dsc5, a ubiquitin regulatory X domain-containing subunit of the Golgi Dsc E3 ligase.

    Science.gov (United States)

    Stewart, Emerson V; Lloyd, S Julie-Ann; Burg, John S; Nwosu, Christine C; Lintner, Robert E; Daza, Riza; Russ, Carsten; Ponchner, Karen; Nusbaum, Chad; Espenshade, Peter J

    2012-01-01

    Schizosaccharomyces pombe Sre1 is a membrane-bound transcription factor that controls adaptation to hypoxia. Like its mammalian homolog, sterol regulatory element-binding protein (SREBP), Sre1 activation requires release from the membrane. However, in fission yeast, this release occurs through a strikingly different mechanism that requires the Golgi Dsc E3 ubiquitin ligase complex and the proteasome. The mechanistic details of Sre1 cleavage, including the link between the Dsc E3 ligase complex and proteasome, are not well understood. Here, we present results of a genetic selection designed to identify additional components required for Sre1 cleavage. From the selection, we identified two new components of the fission yeast SREBP pathway: Dsc5 and Cdc48. The AAA (ATPase associated with diverse cellular activities) ATPase Cdc48 and Dsc5, a ubiquitin regulatory X domain-containing protein, interact with known Dsc complex components and are required for SREBP cleavage. These findings provide a mechanistic link between the Dsc E3 ligase complex and the proteasome in SREBP cleavage and add to a growing list of similarities between the Dsc E3 ligase and membrane E3 ligases involved in endoplasmic reticulum-associated degradation.

  2. Functional connectivity supporting the selective maintenance of feature-location binding in visual working memory

    Directory of Open Access Journals (Sweden)

    Sachiko eTakahama

    2014-06-01

    Full Text Available Information on an object’s features bound to its location is very important for maintaining object representations in visual working memory. Interactions with dynamic multi-dimensional objects in an external environment require complex cognitive control, including the selective maintenance of feature-location binding. Here, we used event-related functional magnetic resonance imaging to investigate brain activity and functional connectivity related to the maintenance of complex feature-location binding. Participants were required to detect task-relevant changes in feature-location binding between objects defined by color, orientation, and location. We compared a complex binding task requiring complex feature-location binding (color-orientation-location with a simple binding task in which simple feature-location binding, such as color-location, was task-relevant and the other feature was task-irrelevant. Univariate analyses showed that the dorsolateral prefrontal cortex (DLPFC, hippocampus, and frontoparietal network were activated during the maintenance of complex feature-location binding. Functional connectivity analyses indicated cooperation between the inferior precentral sulcus (infPreCS, DLPFC, and hippocampus during the maintenance of complex feature-location binding. In contrast, the connectivity for the spatial updating of simple feature-location binding determined by reanalyzing the data from Takahama et al. (2010 demonstrated that the superior parietal lobule (SPL cooperated with the DLPFC and hippocampus. These results suggest that the connectivity for complex feature-location binding does not simply reflect general memory load and that the DLPFC and hippocampus flexibly modulate the dorsal frontoparietal network, depending on the task requirements, with the infPreCS involved in the maintenance of complex feature-location binding and the SPL involved in the spatial updating of simple feature-location binding.

  3. Functional connectivity supporting the selective maintenance of feature-location binding in visual working memory.

    Science.gov (United States)

    Takahama, Sachiko; Saiki, Jun

    2014-01-01

    Information on an object's features bound to its location is very important for maintaining object representations in visual working memory. Interactions with dynamic multi-dimensional objects in an external environment require complex cognitive control, including the selective maintenance of feature-location binding. Here, we used event-related functional magnetic resonance imaging to investigate brain activity and functional connectivity related to the maintenance of complex feature-location binding. Participants were required to detect task-relevant changes in feature-location binding between objects defined by color, orientation, and location. We compared a complex binding task requiring complex feature-location binding (color-orientation-location) with a simple binding task in which simple feature-location binding, such as color-location, was task-relevant and the other feature was task-irrelevant. Univariate analyses showed that the dorsolateral prefrontal cortex (DLPFC), hippocampus, and frontoparietal network were activated during the maintenance of complex feature-location binding. Functional connectivity analyses indicated cooperation between the inferior precentral sulcus (infPreCS), DLPFC, and hippocampus during the maintenance of complex feature-location binding. In contrast, the connectivity for the spatial updating of simple feature-location binding determined by reanalyzing the data from Takahama et al. (2010) demonstrated that the superior parietal lobule (SPL) cooperated with the DLPFC and hippocampus. These results suggest that the connectivity for complex feature-location binding does not simply reflect general memory load and that the DLPFC and hippocampus flexibly modulate the dorsal frontoparietal network, depending on the task requirements, with the infPreCS involved in the maintenance of complex feature-location binding and the SPL involved in the spatial updating of simple feature-location binding. PMID:24917833

  4. 40-Hz square-wave stimulation requires less energy to produce muscle contraction: compared with the TASER® X26 conducted energy weapon.

    Science.gov (United States)

    Comeaux, James A; Jauchem, James R; Cox, D Duane; Crane, Carrie C; D'Andrea, John A

    2013-07-01

    Conducted energy weapons (CEWs) (including the Advanced TASER(®) X26 model produced by TASER International, Inc.) incapacitate individuals by causing muscle contractions. In this study using anesthetized swine, the potential incapacitating effect of primarily monophasic, 19-Hz voltage imposed by the commercial CEW was compared with the effect of voltages imposed by a laboratory device that created 40-Hz square waves. Forces of muscle contraction were measured with the use of strain gauges. Stimulation with 40-Hz square waves required less pulse energy than stimulation with the commercial CEW to produce similar muscle contraction. The square-pulse stimulation, at the higher repetition rate, caused a more complete tetanus at a lower energy. Use of such a simple shape of waveform may be used to make future nonlethal weapon devices more efficient. PMID:23682682

  5. The Use of Online Pre-Lab Assessments Compared with Written Pre-Lab Assignments Requiring Experimental Result Prediction Shows No Difference in Student Performance

    Directory of Open Access Journals (Sweden)

    Erica L. Suchman

    2015-08-01

    Full Text Available Exam performance was compared for students who hand wrote questions designed to prepare them for daily lab activities in a senior level virology laboratory course versus those who answered questions created to mirror the written questions on-line.  No significant difference was noted in exam scores on any of the three midterms, written final exam, nor the practical exam.  Neither was there a significant difference in the quality of the laboratory reports turned in as evidenced by similar average scores over four years.  These results indicate that using online pre-labs to prepare students for the laboratory sessions leads to equivalent learning as answering handwritten pre-lab assignments.  Online pre-labs significantly reduced the amount of grading without reducing student learning, allowing a reduction in the number of teaching assistants required per section.

  6. Hereditary folate malabsorption: A positively charged amino acid at position 113 of the proton-coupled folate transporter (PCFT/SLC46A1) is required for folic acid binding

    Energy Technology Data Exchange (ETDEWEB)

    Lasry, Inbal; Berman, Bluma [The Fred Wyszkowski Cancer Research Laboratory, Dept. of Biology, Technion-Israel Institute of Technology, Haifa 32000 (Israel); Glaser, Fabian [Bioinformatics Knowledge Unit, The Lorry I. Lokey Interdisciplinary Center for Life Sciences and Engineering, Technion, Haifa 32000 (Israel); Jansen, Gerrit [Department of Rheumatology, VU University Medical Center, Amsterdam (Netherlands); Assaraf, Yehuda G., E-mail: assaraf@tx.technion.ac.il [The Fred Wyszkowski Cancer Research Laboratory, Dept. of Biology, Technion-Israel Institute of Technology, Haifa 32000 (Israel)

    2009-08-28

    The proton-coupled folate transporter (PCFT/SLC46A1) mediates intestinal folate uptake at acidic pH. Some loss of folic acid (FA) transport mutations in PCFT from hereditary folate malabsorption (HFM) patients cluster in R113, thereby suggesting a functional role for this residue. Herein, unlike non-conservative substitutions, an R113H mutant displayed 80-fold increase in the FA transport Km while retaining parental Vmax, hence indicating a major fall in folate substrate affinity. Furthermore, consistent with the preservation of 9% of parental transport activity, R113H transfectants displayed a substantial decrease in the FA growth requirement relative to mock transfectants. Homology modeling based on the crystal structures of the Escherichia coli transporter homologues EmrD and glycerol-3-phosphate transporter revealed that the R113H rotamer properly protrudes into the cytoplasmic face of the minor cleft normally occupied by R113. These findings constitute the first demonstration that a basic amino acid at position 113 is required for folate substrate binding.

  7. SCOWLP classification: Structural comparison and analysis of protein binding regions

    Directory of Open Access Journals (Sweden)

    Anders Gerd

    2008-01-01

    Full Text Available Abstract Background Detailed information about protein interactions is critical for our understanding of the principles governing protein recognition mechanisms. The structures of many proteins have been experimentally determined in complex with different ligands bound either in the same or different binding regions. Thus, the structural interactome requires the development of tools to classify protein binding regions. A proper classification may provide a general view of the regions that a protein uses to bind others and also facilitate a detailed comparative analysis of the interacting information for specific protein binding regions at atomic level. Such classification might be of potential use for deciphering protein interaction networks, understanding protein function, rational engineering and design. Description Protein binding regions (PBRs might be ideally described as well-defined separated regions that share no interacting residues one another. However, PBRs are often irregular, discontinuous and can share a wide range of interacting residues among them. The criteria to define an individual binding region can be often arbitrary and may differ from other binding regions within a protein family. Therefore, the rational behind protein interface classification should aim to fulfil the requirements of the analysis to be performed. We extract detailed interaction information of protein domains, peptides and interfacial solvent from the SCOWLP database and we classify the PBRs of each domain family. For this purpose, we define a similarity index based on the overlapping of interacting residues mapped in pair-wise structural alignments. We perform our classification with agglomerative hierarchical clustering using the complete-linkage method. Our classification is calculated at different similarity cut-offs to allow flexibility in the analysis of PBRs, feature especially interesting for those protein families with conflictive binding regions

  8. Comparative genomic analysis identifies divergent genomic features of pathogenic Enterococcus cecorum including a type IC CRISPR-Cas system, a capsule locus, an epa-like locus, and putative host tissue binding proteins.

    Science.gov (United States)

    Borst, Luke B; Suyemoto, M Mitsu; Scholl, Elizabeth H; Fuller, Fredrick J; Barnes, H John

    2015-01-01

    Enterococcus cecorum (EC) is the dominant enteric commensal of adult chickens and contributes to the gut consortia of many avian and mammalian species. While EC infection is an uncommon zoonosis, like other enterococcal species it can cause life-threating nosocomial infection in people. In contrast to other enterococci which are considered opportunistic pathogens, emerging pathogenic strains of EC cause outbreaks of musculoskeletal disease in broiler chickens. Typical morbidity and mortality is comparable to other important infectious diseases of poultry. In molecular epidemiologic studies, pathogenic EC strains were found to be genetically clonal. These findings suggested acquisition of specific virulence determinants by pathogenic EC. To identify divergent genomic features and acquired virulence determinants in pathogenic EC; comparative genomic analysis was performed on genomes of 3 pathogenic and 3 commensal strains of EC. Pathogenic isolates had smaller genomes with a higher GC content, and they demonstrated large regions of synteny compared to commensal isolates. A molecular phylogenetic analysis demonstrated sequence divergence in pathogenic EC genomes. At a threshold of 98% identity, 414 predicted proteins were identified that were highly conserved in pathogenic EC but not in commensal EC. Among these, divergent CRISPR-cas defense loci were observed. In commensal EC, the type IIA arrangement typical for enterococci was present; however, pathogenic EC had a type IC locus, which is novel in enterococci but commonly observed in streptococci. Potential mediators of virulence identified in this analysis included a polysaccharide capsular locus similar to that recently described for E. faecium, an epa-like locus, and cell wall associated proteins which may bind host extracellular matrix. This analysis identified specific genomic regions, coding sequences, and predicted proteins which may be related to the divergent evolution and increased virulence of emerging

  9. Sialic Acid Binding Properties of Soluble Coronavirus Spike (S1 Proteins: Differences between Infectious Bronchitis Virus and Transmissible Gastroenteritis Virus

    Directory of Open Access Journals (Sweden)

    Christine Winter

    2013-07-01

    Full Text Available The spike proteins of a number of coronaviruses are able to bind to sialic acids present on the cell surface. The importance of this sialic acid binding ability during infection is, however, quite different. We compared the spike protein of transmissible gastroenteritis virus (TGEV and the spike protein of infectious bronchitis virus (IBV. Whereas sialic acid is the only receptor determinant known so far for IBV, TGEV requires interaction with its receptor aminopeptidase N to initiate infection of cells. Binding tests with soluble spike proteins carrying an IgG Fc-tag revealed pronounced differences between these two viral proteins. Binding of the IBV spike protein to host cells was in all experiments sialic acid dependent, whereas the soluble TGEV spike showed binding to APN but had no detectable sialic acid binding activity. Our results underline the different ways in which binding to sialoglycoconjugates is mediated by coronavirus spike proteins.

  10. Physician Compare

    Data.gov (United States)

    U.S. Department of Health & Human Services — Physician Compare, which meets Affordable Care Act of 2010 requirements, helps you search for and select physicians and other healthcare professionals enrolled in...

  11. Reactive oxygen species decrease cAMP response element binding protein expression in cardiomyocytes via a protein kinase D1-dependent mechanism that does not require Ser133 phosphorylation.

    Science.gov (United States)

    Ozgen, Nazira; Guo, Jianfen; Gertsberg, Zoya; Danilo, Peter; Rosen, Michael R; Steinberg, Susan F

    2009-10-01

    Reactive oxygen species (ROS) exert pleiotropic effects on a wide array of signaling proteins that regulate cellular growth and apoptosis. This study shows that long-term treatment with a low concentration of H2O2 leads to the activation of signaling pathways involving extracellular signal-regulated kinase, ribosomal protein S6 kinase, and protein kinase D (PKD) that increase cAMP binding response element protein (CREB) phosphorylation at Ser(133) in cardiomyocytes. Although CREB-Ser(133) phosphorylation typically mediates cAMP-dependent increases in CREB target gene expression, the H2O2-dependent increase in CREB-Ser(133) phosphorylation is accompanied by a decrease in CREB protein abundance and no change in Cre-luciferase reporter activity. Mutagenesis studies indicate that H2O2 decreases CREB protein abundance via a mechanism that does not require CREB-Ser(133) phosphorylation. Rather, the H2O2-dependent decrease in CREB protein is prevented by the proteasome inhibitor lactacystin, by inhibitors of mitogen-activated protein kinase kinase or protein kinase C activity, or by adenoviral-mediated delivery of a small interfering RNA that decreases PKD1 expression. A PKD1-dependent mechanism that links oxidative stress to decreased CREB protein abundance is predicted to contribute to the pathogenesis of heart failure by influencing cardiac growth and apoptosis responses.

  12. Conserved region 2 of adenovirus E1A has a function distinct from pRb binding required to prevent cell cycle arrest by p16INK4a or p27Kip1.

    Science.gov (United States)

    Alevizopoulos, K; Sanchez, B; Amati, B

    2000-04-13

    Ectopic expression of the CDK inhibitors (CKIs) p16INK4a and p27Kip1 in Rat1 fibroblasts induces dephosphorylation and activation of Retinoblastoma-family proteins (pRb, p107 and p130), their association with E2F proteins, and cell cycle arrest in G1. The growth-inhibitory action of p16, in particular, is believed to be mediated essentially via pRb activation. The 12S E1A protein of human Adenovirus 5 associates with pRb-family proteins via residues in its Conserved Regions (CR) 1 and 2, in particular through the motif LXCXE in CR2. These interactions are required for E1A to prevent G1 arrest upon co-expression of CKIs. We show here that mutating either of two conserved motifs adjacent to LXCXE in CR2, GFP and SDDEDEE, also impairs the ability of E1A to overcome G1 arrest by p16 or p27. Strikingly, however, these mutations affect neither the association of E1A with pRb, p07 and p130, nor its ability to derepress E2F-1 transcriptional activity in transient transfection assays. One of the EIA mutants, however, is defective in derepressing several endogenous E2F target genes in the presence of p16 or p27. Thus, CR2 possesses an essential function besides pRb-binding. We speculate that this function might be required for the full derepression of E2F-regulated genes in their natural chromatin context. PMID:10803468

  13. The zinc binuclear cluster activator AlcR is able to bind to single sites but requires multiple repeated sites for synergistic activation of the alcA gene in Aspergillus nidulans.

    Science.gov (United States)

    Panozzo, C; Capuano, V; Fillinger, S; Felenbok, B

    1997-09-01

    The alcA gene which is part of the recently identified ethanol regulon, is one of the most strongly inducible genes in Aspergillus nidulans. Its transcriptional activation is mediated by the AlcR transactivator which contains a DNA-binding domain belonging to the C6 zinc binuclear cluster family. AlcR differs from the other members of this family by several features, the most striking characteristic being its binding to both symmetric and asymmetric DNA sites with the same apparent affinity. However, AlcR is also able to bind to a single site with high affinity, suggesting that unlike the other C6 proteins, AlcR binds as a monomer. In this report, we show that AlcR targets, to be functional in vivo, have to be organized as inverted or direct repeats. In addition, we show a strong synergistic activation of alcA transcription in which the number and the position of the AlcR-binding sites are crucial. The fact that the AlcR unit for in vitro binding is a single site whereas the in vivo functional unit is a repeat opens the question of the mechanism of the strong alcA transactivation. These results show that AlcR displays both in vitro and in vivo a new range of binding specificity and provides a novel example in the C6 zinc cluster protein family.

  14. Mutagenesis of the Agrobacterium VirE2 single-stranded DNA-binding protein identifies regions required for self-association and interaction with VirE1 and a permissive site for hybrid protein construction.

    Science.gov (United States)

    Zhou, X R; Christie, P J

    1999-07-01

    The VirE2 single-stranded DNA-binding protein (SSB) of Agrobacterium tumefaciens is required for delivery of T-DNA to the nuclei of susceptible plant cells. By yeast two-hybrid and immunoprecipitation analyses, VirE2 was shown to self-associate and to interact with VirE1. VirE2 mutants with small deletions or insertions of a 31-residue oligopeptide (i31) at the N or C terminus or with an i31 peptide insertion at Leu236 retained the capacity to form homomultimers. By contrast, VirE2 mutants with modifications outside a central region located between residues 320 and 390 retained the capacity to interact with VirE1. These findings suggest the tertiary structure of VirE2 is important for homomultimer formation whereas a central domain mediates formation of a complex with VirE1. The capacity of VirE2 mutants to interact with full-length VirE2 in the yeast Saccharomyces cerevisiae correlated with the abundance of the mutant proteins in A. tumefaciens, suggesting that VirE2 is stabilized by homomultimerization in the bacterium. We further characterized the promoter and N- and C-terminal sequence requirements for synthesis of functional VirE2. A PvirB::virE2 construct yielded functional VirE2 protein as defined by complementation of a virE2 null mutation. By contrast, PvirE or Plac promoter constructs yielded functional VirE2 only if virE1 was coexpressed with virE2. Deletion of 10 or 9 residues from the N or C terminus of VirE2, respectively, or addition of heterologous peptides or proteins to either terminus resulted in a loss of protein function. However, an i31 peptide insertion at Tyr39 had no effect on protein function as defined by the capacity of the mutant protein to (i) interact with native VirE2, (ii) interact with VirE1, (iii) accumulate at abundant levels in A. tumefaciens, and (iv) restore wild-type virulence to a virE2 null mutant. We propose that Tyr39 of VirE2 corresponds to a permissive site for insertion of heterologous peptides or proteins of interest

  15. Analyzing binding data.

    Science.gov (United States)

    Motulsky, Harvey J; Neubig, Richard R

    2010-07-01

    Measuring the rate and extent of radioligand binding provides information on the number of binding sites, and their affinity and accessibility of these binding sites for various drugs. This unit explains how to design and analyze such experiments.

  16. Genome-wide prediction, display and refinement of binding sites with information theory-based models

    Directory of Open Access Journals (Sweden)

    Leeder J Steven

    2003-09-01

    Full Text Available Abstract Background We present Delila-genome, a software system for identification, visualization and analysis of protein binding sites in complete genome sequences. Binding sites are predicted by scanning genomic sequences with information theory-based (or user-defined weight matrices. Matrices are refined by adding experimentally-defined binding sites to published binding sites. Delila-Genome was used to examine the accuracy of individual information contents of binding sites detected with refined matrices as a measure of the strengths of the corresponding protein-nucleic acid interactions. The software can then be used to predict novel sites by rescanning the genome with the refined matrices. Results Parameters for genome scans are entered using a Java-based GUI interface and backend scripts in Perl. Multi-processor CPU load-sharing minimized the average response time for scans of different chromosomes. Scans of human genome assemblies required 4–6 hours for transcription factor binding sites and 10–19 hours for splice sites, respectively, on 24- and 3-node Mosix and Beowulf clusters. Individual binding sites are displayed either as high-resolution sequence walkers or in low-resolution custom tracks in the UCSC genome browser. For large datasets, we applied a data reduction strategy that limited displays of binding sites exceeding a threshold information content to specific chromosomal regions within or adjacent to genes. An HTML document is produced listing binding sites ranked by binding site strength or chromosomal location hyperlinked to the UCSC custom track, other annotation databases and binding site sequences. Post-genome scan tools parse binding site annotations of selected chromosome intervals and compare the results of genome scans using different weight matrices. Comparisons of multiple genome scans can display binding sites that are unique to each scan and identify sites with significantly altered binding strengths

  17. 40 CFR Table C-5 to Subpart C of... - Summary of Comparability Field Testing Campaign Site and Seasonal Requirements for Class II and...

    Science.gov (United States)

    2010-07-01

    ... Campaign Site and Seasonal Requirements for Class II and III FEMs for PM10â2.5 and PM2.5 C Table C-5 to Subpart C of Part 53 Protection of Environment ENVIRONMENTAL PROTECTION AGENCY (CONTINUED) AIR PROGRAMS... Between Candidate Methods and Reference Methods Pt. 53, Subpt. C, Table C-5 Table C-5 to Subpart C of...

  18. Methods for Improving Aptamer Binding Affinity

    OpenAIRE

    Hijiri Hasegawa; Nasa Savory; Koichi Abe; Kazunori Ikebukuro

    2016-01-01

    Aptamers are single stranded oligonucleotides that bind a wide range of biological targets. Although aptamers can be isolated from pools of random sequence oligonucleotides using affinity-based selection, aptamers with high affinities are not always obtained. Therefore, further refinement of aptamers is required to achieve desired binding affinities. The optimization of primary sequences and stabilization of aptamer conformations are the main approaches to refining the binding properties of a...

  19. An Arabidopsis Ran-binding protein, AtRanBP1c, is a co-activator of Ran GTPase-activating protein and requires the C-terminus for its cytoplasmic localization

    Science.gov (United States)

    Kim, Soo-Hwan; Roux, Stanley J.

    2003-01-01

    Ran-binding proteins (RanBPs) are a group of proteins that bind to Ran (Ras-related nuclear small GTP-binding protein), and thus either control the GTP/GDP-bound states of Ran or help couple the Ran GTPase cycle to a cellular process. AtRanBP1c is a Ran-binding protein from Arabidopsis thaliana (L.) Heynh. that was recently shown to be critically involved in the regulation of auxin-induced mitotic progression [S.-H. Kim et al. (2001) Plant Cell 13:2619-2630]. Here we report that AtRanBP1c inhibits the EDTA-induced release of GTP from Ran and serves as a co-activator of Ran-GTPase-activating protein (RanGAP) in vitro. Transient expression of AtRanBP1c fused to a beta-glucuronidase (GUS) reporter reveals that the protein localizes primarily to the cytosol. Neither the N- nor C-terminus of AtRanBP1c, which flank the Ran-binding domain (RanBD), is necessary for the binding of PsRan1-GTP to the protein, but both are needed for the cytosolic localization of GUS-fused AtRanBP1c. These findings, together with a previous report that AtRanBP1c is critically involved in root growth and development, imply that the promotion of GTP hydrolysis by the Ran/RanGAP/AtRanBP1c complex in the cytoplasm, and the resulting concentration gradient of Ran-GDP to Ran-GTP across the nuclear membrane could be important in the regulation of auxin-induced mitotic progression in root tips of A. thaliana.

  20. A comparative analysis of the resources required for test and evaluation on Army-led weapon system programs, based upon program size and acquisition management complexity

    OpenAIRE

    Johnson, Arne A.; Philistine, John J.

    2007-01-01

    Joint Applied Project Test and Evaluation (TandE) is an integral part of every acquisition program, as such, it consumes considerable program resources. The Department of Defense (DoD) TandE program management requirements are written to meet the risk reduction needs of large acquisition programs, but do not provide the details needed to consistently scale TandE management efforts for smaller programs across DoD. This research study investigates ways that the TandE burden to programs d...

  1. Principles of a competitive binding assay for the study of the interactions of poorly water-soluble ligands with their soluble binding partners. Application to bilirubin with the use of Sephadex G-10 as a competitive adsorbent.

    Science.gov (United States)

    Meuwissen, J A; Kinnaert, M; Michiels, G; Heirwegh, K P

    1988-01-01

    1. A generally applicable method is described for obtaining experimental data on the interactions between a poorly water-soluble ligand and soluble binding factors, with the use of chemically inert solid adsorbent. The equilibrium distribution of the ligand between the liquid phase containing the soluble binders and the adsorbent must be measured and knowledge of the binding isotherm of the adsorbent is required. Procedures are given for the calculation of the binding parameters. 2. The method has been applied to quantify the interactions of bilirubin with serum and liver cytosol from the rat, Sephadex G-10 serving as the competing adsorbent. Reversible adsorption keeps the concentration of the free ligand low, thereby preventing formation of colloidal bilirubin. The sensitivity of the procedure accommodates the rather high binding affinities by which bilirubin generally interacts with its specific binding proteins. 3. The binding activities of serum and liver cytosol are of comparable magnitude. Binding of bilirubin by rat serum can be described by two independent binding sites, the affinities of which differ by two orders of magnitude. Only the site with the higher affinity appears to be of physiological importance. The major bilirubin-binding sites of rat liver cytosol seem to contribute equally to the overall binding activity of this preparation, provided that GSH is present. PMID:2449176

  2. A comparative evaluation of licensing requirements for dry storage of spent nuclear fuel in the United States, Germany, Canada and the Russian Federation

    International Nuclear Information System (INIS)

    Technical activities to support licensing of dry spent nuclear fuel storage facilities are complex, with policy and regulatory requirements often being influenced by politics. Moreover, the process is often convoluted, with numerous and diverse stake-holders making the licensing activity a difficult exercise in consensus-reaching. The objective of this evaluation is to present alternatives to assist the Republic of Kazakhstan (RK) in developing a licensing approach for a planned Dry Spent Fuel Storage Facility. Because the RK lacks experience in licensing a facility of this type, there is considerable interest in knowing more about the approval process in other countries so that an effective, non-redundant method of licensing can be established. This evaluation is limited to a comparison of approaches from the United States, Germany, Russia, and Canada. For each country considered, the following areas were addressed: siting; fuel handling and cask loading; dry fuel storage; and transportation of spent fuel. The regulatory requirements for each phase of the process are presented, and a licensing approach that would best serve the RK is recommended. (authors)

  3. Carboplatin binding to histidine

    Energy Technology Data Exchange (ETDEWEB)

    Tanley, Simon W. M. [University of Manchester, Brunswick Street, Manchester M13 9PL (United Kingdom); Diederichs, Kay [University of Konstanz, D-78457 Konstanz (Germany); Kroon-Batenburg, Loes M. J. [Utrecht University, Padualaan 8, 3584 CH Utrecht (Netherlands); Levy, Colin [University of Manchester, 131 Princess Street, Manchester M1 7DN (United Kingdom); Schreurs, Antoine M. M. [Utrecht University, Padualaan 8, 3584 CH Utrecht (Netherlands); Helliwell, John R., E-mail: john.helliwell@manchester.ac.uk [University of Manchester, Brunswick Street, Manchester M13 9PL (United Kingdom)

    2014-08-29

    An X-ray crystal structure showing the binding of purely carboplatin to histidine in a model protein has finally been obtained. This required extensive crystallization trials and various novel crystal structure analyses. Carboplatin is a second-generation platinum anticancer agent used for the treatment of a variety of cancers. Previous X-ray crystallographic studies of carboplatin binding to histidine (in hen egg-white lysozyme; HEWL) showed the partial conversion of carboplatin to cisplatin owing to the high NaCl concentration used in the crystallization conditions. HEWL co-crystallizations with carboplatin in NaBr conditions have now been carried out to confirm whether carboplatin converts to the bromine form and whether this takes place in a similar way to the partial conversion of carboplatin to cisplatin observed previously in NaCl conditions. Here, it is reported that a partial chemical transformation takes place but to a transplatin form. Thus, to attempt to resolve purely carboplatin binding at histidine, this study utilized co-crystallization of HEWL with carboplatin without NaCl to eliminate the partial chemical conversion of carboplatin. Tetragonal HEWL crystals co-crystallized with carboplatin were successfully obtained in four different conditions, each at a different pH value. The structural results obtained show carboplatin bound to either one or both of the N atoms of His15 of HEWL, and this particular variation was dependent on the concentration of anions in the crystallization mixture and the elapsed time, as well as the pH used. The structural details of the bound carboplatin molecule also differed between them. Overall, the most detailed crystal structure showed the majority of the carboplatin atoms bound to the platinum centre; however, the four-carbon ring structure of the cyclobutanedicarboxylate moiety (CBDC) remained elusive. The potential impact of the results for the administration of carboplatin as an anticancer agent are described.

  4. In Vitro Whole Genome DNA Binding Analysis of the Bacterial Replication Initiator and Transcription Factor DnaA.

    Directory of Open Access Journals (Sweden)

    Janet L Smith

    2015-05-01

    Full Text Available DnaA, the replication initiation protein in bacteria, is an AAA+ ATPase that binds and hydrolyzes ATP and exists in a heterogeneous population of ATP-DnaA and ADP-DnaA. DnaA binds cooperatively to the origin of replication and several other chromosomal regions, and functions as a transcription factor at some of these regions. We determined the binding properties of Bacillus subtilis DnaA to genomic DNA in vitro at single nucleotide resolution using in vitro DNA affinity purification and deep sequencing (IDAP-Seq. We used these data to identify 269 binding regions, refine the consensus sequence of the DnaA binding site, and compare the relative affinity of binding regions for ATP-DnaA and ADP-DnaA. Most sites had a slightly higher affinity for ATP-DnaA than ADP-DnaA, but a few had a strong preference for binding ATP-DnaA. Of the 269 sites, only the eight strongest binding ones have been observed to bind DnaA in vivo, suggesting that other cellular factors or the amount of available DnaA in vivo restricts DnaA binding to these additional sites. Conversely, we found several chromosomal regions that were bound by DnaA in vivo but not in vitro, and that the nucleoid-associated protein Rok was required for binding in vivo. Our in vitro characterization of the inherent ability of DnaA to bind the genome at single nucleotide resolution provides a backdrop for interpreting data on in vivo binding and regulation of DnaA, and is an approach that should be adaptable to many other DNA binding proteins.

  5. Distinct cytoplasmic domains of the growth hormone receptor are required for glucocorticoid- and phorbol ester-induced decreases in growth hormone (GH) binding. These domains are different from that reported for GH-induced receptor internalization

    DEFF Research Database (Denmark)

    King, A P; Tseng, M J; Logsdon, C D;

    1996-01-01

    GH binding are also observed in a Chinese hamster ovary (CHO) cell line stably transfected with a rat liver GHR cDNA, further arguing that DEX and PMA act post-translationally on GHR. Using mutant GHRs stably expressed in CHO cells, amino acids 455-506 and tyrosines 333 and/or 338 of GHR were shown...

  6. BIND – An algorithm for loss-less compression of nucleotide sequence data

    Indian Academy of Sciences (India)

    Tungadri Bose; Monzoorul Haque Mohammed; Anirban Dutta; Sharmila S Mande

    2012-09-01

    Recent advances in DNA sequencing technologies have enabled the current generation of life science researchers to probe deeper into the genomic blueprint. The amount of data generated by these technologies has been increasing exponentially since the last decade. Storage, archival and dissemination of such huge data sets require efficient solutions, both from the hardware as well as software perspective. The present paper describes BIND – an algorithm specialized for compressing nucleotide sequence data. By adopting a unique ‘block-length’ encoding for representing binary data (as a key step), BIND achieves significant compression gains as compared to the widely used general purpose compression algorithms (gzip, bzip2 and lzma). Moreover, in contrast to implementations of existing specialized genomic compression approaches, the implementation of BIND is enabled to handle non-ATGC and lowercase characters. This makes BIND a loss-less compression approach that is suitable for practical use. More importantly, validation results of BIND (with real-world data sets) indicate reasonable speeds of compression and decompression that can be achieved with minimal processor/memory usage. BIND is available for download at http://metagenomics.atc.tcs.com/compression/BIND. No license is required for academic or non-profit use.

  7. Transfusion requirements in septic shock (TRISS) trial - comparing the effects and safety of liberal versus restrictive red blood cell transfusion in septic shock patients in the ICU

    DEFF Research Database (Denmark)

    Holst, Lars B; Haase, Nicolai; Wetterslev, Jørn;

    2013-01-01

    BACKGROUND: Transfusion of red blood cells (RBC) is recommended in septic shock and the majority of these patients receive RBC transfusion in the intensive care unit (ICU). However, benefit and harm of RBCs have not been established in this group of high-risk patients. METHODS: The Transfusion...... Requirements in Septic Shock (TRISS) trial is a multicenter trial with assessor-blinded outcome assessment, randomising 1,000 patients with septic shock in 30 Scandinavian ICUs to receive transfusion with pre-storage leuko-depleted RBC suspended in saline-adenine-glucose and mannitol (SAGM) at haemoglobin...... and transfusion-related circulatory overload, and acute lung injury) and mortality at 28 days, 6 months and 1 year.The sample size will enable us to detect a 9% absolute difference in 90-day mortality assuming a 45% event rate with a type 1 error rate of 5% and power of 80%. An interim analysis will be performed...

  8. Comparative genome-wide analysis reveals that Burkholderia contaminans MS14 possesses multiple antimicrobial biosynthesis genes but not major genetic loci required for pathogenesis.

    Science.gov (United States)

    Deng, Peng; Wang, Xiaoqiang; Baird, Sonya M; Showmaker, Kurt C; Smith, Leif; Peterson, Daniel G; Lu, Shien

    2016-06-01

    Burkholderia contaminans MS14 shows significant antimicrobial activities against plant and animal pathogenic fungi and bacteria. The antifungal agent occidiofungin produced by MS14 has great potential for development of biopesticides and pharmaceutical drugs. However, the use of Burkholderia species as biocontrol agent in agriculture is restricted due to the difficulties in distinguishing between plant growth-promoting bacteria and the pathogenic bacteria. The complete MS14 genome was sequenced and analyzed to find what beneficial and virulence-related genes it harbors. The phylogenetic relatedness of B. contaminans MS14 and other 17 Burkholderia species was also analyzed. To research MS14's potential virulence, the gene regions related to the antibiotic production, antibiotic resistance, and virulence were compared between MS14 and other Burkholderia genomes. The genome of B. contaminans MS14 was sequenced and annotated. The genomic analyses reveal the presence of multiple gene sets for antimicrobial biosynthesis, which contribute to its antimicrobial activities. BLAST results indicate that the MS14 genome harbors a large number of unique regions. MS14 is closely related to another plant growth-promoting Burkholderia strain B. lata 383 according to the average nucleotide identity data. Moreover, according to the phylogenetic analysis, plant growth-promoting species isolated from soils and mammalian pathogenic species are clustered together, respectively. MS14 has multiple antimicrobial activity-related genes identified from the genome, but it lacks key virulence-related gene loci found in the pathogenic strains. Additionally, plant growth-promoting Burkholderia species have one or more antimicrobial biosynthesis genes in their genomes as compared with nonplant growth-promoting soil-isolated Burkholderia species. On the other hand, pathogenic species harbor multiple virulence-associated gene loci that are not present in nonpathogenic Burkholderia species. The MS14

  9. Comparative genomic analysis of slc39a12/ZIP12: insight into a zinc transporter required for vertebrate nervous system development.

    Directory of Open Access Journals (Sweden)

    Winyoo Chowanadisai

    Full Text Available The zinc transporter ZIP12, which is encoded by the gene slc39a12, has previously been shown to be important for neuronal differentiation in mouse Neuro-2a neuroblastoma cells and primary mouse neurons and necessary for neurulation during Xenopus tropicalis embryogenesis. However, relatively little is known about the biochemical properties, cellular regulation, or the physiological role of this gene. The hypothesis that ZIP12 is a zinc transporter important for nervous system function and development guided a comparative genetics approach to uncover the presence of ZIP12 in various genomes and identify conserved sequences and expression patterns associated with ZIP12. Ortholog detection of slc39a12 was conducted with reciprocal BLAST hits with the amino acid sequence of human ZIP12 in comparison to the human paralog ZIP4 and conserved local synteny between genomes. ZIP12 is present in the genomes of almost all vertebrates examined, from humans and other mammals to most teleost fish. However, ZIP12 appears to be absent from the zebrafish genome. The discrimination of ZIP12 compared to ZIP4 was unsuccessful or inconclusive in other invertebrate chordates and deuterostomes. Splice variation, due to the inclusion or exclusion of a conserved exon, is present in humans, rats, and cows and likely has biological significance. ZIP12 also possesses many putative di-leucine and tyrosine motifs often associated with intracellular trafficking, which may control cellular zinc uptake activity through the localization of ZIP12 within the cell. These findings highlight multiple aspects of ZIP12 at the biochemical, cellular, and physiological levels with likely biological significance. ZIP12 appears to have conserved function as a zinc uptake transporter in vertebrate nervous system development. Consequently, the role of ZIP12 may be an important link to reported congenital malformations in numerous animal models and humans that are caused by zinc deficiency.

  10. Comparative genomic analysis of slc39a12/ZIP12: insight into a zinc transporter required for vertebrate nervous system development.

    Science.gov (United States)

    Chowanadisai, Winyoo

    2014-01-01

    The zinc transporter ZIP12, which is encoded by the gene slc39a12, has previously been shown to be important for neuronal differentiation in mouse Neuro-2a neuroblastoma cells and primary mouse neurons and necessary for neurulation during Xenopus tropicalis embryogenesis. However, relatively little is known about the biochemical properties, cellular regulation, or the physiological role of this gene. The hypothesis that ZIP12 is a zinc transporter important for nervous system function and development guided a comparative genetics approach to uncover the presence of ZIP12 in various genomes and identify conserved sequences and expression patterns associated with ZIP12. Ortholog detection of slc39a12 was conducted with reciprocal BLAST hits with the amino acid sequence of human ZIP12 in comparison to the human paralog ZIP4 and conserved local synteny between genomes. ZIP12 is present in the genomes of almost all vertebrates examined, from humans and other mammals to most teleost fish. However, ZIP12 appears to be absent from the zebrafish genome. The discrimination of ZIP12 compared to ZIP4 was unsuccessful or inconclusive in other invertebrate chordates and deuterostomes. Splice variation, due to the inclusion or exclusion of a conserved exon, is present in humans, rats, and cows and likely has biological significance. ZIP12 also possesses many putative di-leucine and tyrosine motifs often associated with intracellular trafficking, which may control cellular zinc uptake activity through the localization of ZIP12 within the cell. These findings highlight multiple aspects of ZIP12 at the biochemical, cellular, and physiological levels with likely biological significance. ZIP12 appears to have conserved function as a zinc uptake transporter in vertebrate nervous system development. Consequently, the role of ZIP12 may be an important link to reported congenital malformations in numerous animal models and humans that are caused by zinc deficiency.

  11. Adaptive evolution of transcription factor binding sites

    Directory of Open Access Journals (Sweden)

    Berg Johannes

    2004-10-01

    Full Text Available Abstract Background The regulation of a gene depends on the binding of transcription factors to specific sites located in the regulatory region of the gene. The generation of these binding sites and of cooperativity between them are essential building blocks in the evolution of complex regulatory networks. We study a theoretical model for the sequence evolution of binding sites by point mutations. The approach is based on biophysical models for the binding of transcription factors to DNA. Hence we derive empirically grounded fitness landscapes, which enter a population genetics model including mutations, genetic drift, and selection. Results We show that the selection for factor binding generically leads to specific correlations between nucleotide frequencies at different positions of a binding site. We demonstrate the possibility of rapid adaptive evolution generating a new binding site for a given transcription factor by point mutations. The evolutionary time required is estimated in terms of the neutral (background mutation rate, the selection coefficient, and the effective population size. Conclusions The efficiency of binding site formation is seen to depend on two joint conditions: the binding site motif must be short enough and the promoter region must be long enough. These constraints on promoter architecture are indeed seen in eukaryotic systems. Furthermore, we analyse the adaptive evolution of genetic switches and of signal integration through binding cooperativity between different sites. Experimental tests of this picture involving the statistics of polymorphisms and phylogenies of sites are discussed.

  12. Comparative transcript profiling of Candida albicans and Candida dubliniensis identifies SFL2, a C. albicans gene required for virulence in a reconstituted epithelial infection model.

    LENUS (Irish Health Repository)

    Spiering, Martin J

    2010-02-01

    Candida albicans and Candida dubliniensis are closely related species displaying differences in virulence and genome content, therefore providing potential opportunities to identify novel C. albicans virulence genes. C. albicans gene arrays were used for comparative analysis of global gene expression in the two species in reconstituted human oral epithelium (RHE). C. albicans (SC5314) showed upregulation of hypha-specific and virulence genes within 30 min postinoculation, coinciding with rapid induction of filamentation and increased RHE damage. C. dubliniensis (CD36) showed no detectable upregulation of hypha-specific genes, grew as yeast, and caused limited RHE damage. Several genes absent or highly divergent in C. dubliniensis were upregulated in C. albicans. One such gene, SFL2 (orf19.3969), encoding a putative heat shock factor, was deleted in C. albicans. DeltaDeltasfl2 cells failed to filament under a range of hypha-inducing conditions and exhibited greatly reduced RHE damage, reversed by reintroduction of SFL2 into the DeltaDeltasfl2 strain. Moreover, SFL2 overexpression in C. albicans triggered hyphal morphogenesis. Although SFL2 deletion had no apparent effect on host survival in the murine model of systemic infection, DeltaDeltasfl2 strain-infected kidney tissues contained only yeast cells. These results suggest a role for SFL2 in morphogenesis and an indirect role in C. albicans pathogenesis in epithelial tissues.

  13. Comparative genomic analysis of magnetotactic bacteria from the Deltaproteobacteria provides new insights into magnetite and greigite magnetosome genes required for magnetotaxis.

    Science.gov (United States)

    Lefèvre, Christopher T; Trubitsyn, Denis; Abreu, Fernanda; Kolinko, Sebastian; Jogler, Christian; de Almeida, Luiz Gonzaga Paula; de Vasconcelos, Ana Tereza R; Kube, Michael; Reinhardt, Richard; Lins, Ulysses; Pignol, David; Schüler, Dirk; Bazylinski, Dennis A; Ginet, Nicolas

    2013-10-01

    Magnetotactic bacteria (MTB) represent a group of diverse motile prokaryotes that biomineralize magnetosomes, the organelles responsible for magnetotaxis. Magnetosomes consist of intracellular, membrane-bounded, tens-of-nanometre-sized crystals of the magnetic minerals magnetite (Fe3O4) or greigite (Fe3S4) and are usually organized as a chain within the cell acting like a compass needle. Most information regarding the biomineralization processes involved in magnetosome formation comes from studies involving Alphaproteobacteria species which biomineralize cuboctahedral and elongated prismatic crystals of magnetite. Many magnetosome genes, the mam genes, identified in these organisms are conserved in all known MTB. Here we present a comparative genomic analysis of magnetotactic Deltaproteobacteria that synthesize bullet-shaped crystals of magnetite and/or greigite. We show that in addition to mam genes, there is a conserved set of genes, designated mad genes, specific to the magnetotactic Deltaproteobacteria, some also being present in Candidatus Magnetobacterium bavaricum of the Nitrospirae phylum, but absent in the magnetotactic Alphaproteobacteria. Our results suggest that the number of genes associated with magnetotaxis in magnetotactic Deltaproteobacteria is larger than previously thought. We also demonstrate that the minimum set of mam genes necessary for magnetosome formation in Magnetospirillum is also conserved in magnetite-producing, magnetotactic Deltaproteobacteria. Some putative novel functions of mad genes are discussed. PMID:23607663

  14. A Novel Sterol Regulatory Element-Binding Protein Gene (sreA) Identified in Penicillium digitatum Is Required for Prochloraz Resistance, Full Virulence and erg11 (cyp51) Regulation

    OpenAIRE

    Jing Liu; Yongze Yuan; Zhi Wu; Na Li; Yuanlei Chen; Tingting Qin; Hui Geng; Li Xiong; Deli Liu

    2015-01-01

    Penicillium digitatum is the most destructive postharvest pathogen of citrus fruits, causing fruit decay and economic loss. Additionally, control of the disease is further complicated by the emergence of drug-resistant strains due to the extensive use of triazole antifungal drugs. In this work, an orthologus gene encoding a putative sterol regulatory element-binding protein (SREBP) was identified in the genome of P. digitatum and named sreA. The putative SreA protein contains a conserved doma...

  15. Comparative proteomic analysis of Salmonella enterica serovar Typhimurium ppGpp-deficient mutant to identify a novel virulence protein required for intracellular survival in macrophages

    Directory of Open Access Journals (Sweden)

    Kumagai Yoshinori

    2010-12-01

    Full Text Available Abstract Background The global ppGpp-mediated stringent response in pathogenic bacteria plays an important role in the pathogenesis of bacterial infections. In Salmonella enterica serovar Typhimurium (S. Typhimurium, several genes, including virulence genes, are regulated by ppGpp when bacteria are under the stringent response. To understand the control of virulence genes by ppGpp in S. Typhimurium, agarose 2-dimensional electrophoresis (2-DE combined with mass spectrometry was used and a comprehensive 2-DE reference map of amino acid-starved S. Typhimurium strain SH100, a derivative of ATCC 14028, was established. Results Of the 366 examined spots, 269 proteins were successfully identified. The comparative analysis of the wild-type and ppGpp0 mutant strains revealed 55 proteins, the expression patterns of which were affected by ppGpp. Using a mouse infection model, we further identified a novel virulence-associated factor, STM3169, from the ppGpp-regulated and Salmonella-specific proteins. In addition, Salmonella strains carrying mutations in the gene encoding STM3169 showed growth defects and impaired growth within macrophage-like RAW264.7 cells. Furthermore, we found that expression of stm3169 was controlled by ppGpp and SsrB, a response regulator of the two-component system located on Salmonella pathogenicity island 2. Conclusions A proteomic approach using a 2-DE reference map can prove a powerful tool for analyzing virulence factors and the regulatory network involved in Salmonella pathogenesis. Our results also provide evidence of a global response mediated by ppGpp in S. enterica.

  16. Localization-enhanced biexciton binding in semiconductors

    DEFF Research Database (Denmark)

    Langbein, Wolfgang Werner; Hvam, Jørn Märcher

    1999-01-01

    The influence of excitonic localization on the binding energy of biexcitons is investigated for quasi-three-dimensional and quasi-two-dimensional AlxGa1-xAs structures. An increase of the biexciton binding energy is observed for localization energies comparable to or larger than the free biexcito...

  17. Total iron binding capacity

    Science.gov (United States)

    ... page: //medlineplus.gov/ency/article/003489.htm Total iron binding capacity To use the sharing features on this page, please enable JavaScript. Total iron binding capacity (TIBC) is a blood test to ...

  18. Protein requirements of man: comparative nitrogen balance response within the submaintenance-to-maintenance range of intakes of wheat and beef proteins.

    Science.gov (United States)

    Young, V R; Fajardo, L; Murray, E; Rand, W M; Scrimshaw, N S

    1975-05-01

    Sixteen young male students participated in two studies designed to determine the nitrogen balance response to beef or stone-ground, whole wheat protein over the submaintenance-to-maintenance range of protein intake. The objective of the studies was to evaluate the relative capacities of these proteins to meet the minimum protein needs in young adult males. A modified Latin-square design was used to allocate subjects to the four 15-day metabolic balance diet periods in each study. The last 10 days were used for fecal nitrogen determination and the last 5 days for evaluation of urinary nitrogen excretion. The diet periods were separated by a 4-day break period and began with 1 day on a "protein-free" diet. The levels of protien (N times 6.25) intake tested were 0.2, 0.3, 0.4, and 0.5 g/kg body weight/day in the beef study. The results have been compared with those obtained in a previous similar study with egg protein. The regression of estimated "true" nitrogen balance (Y) (mg N/kg/day) on nitrogen intake (X) (mg N/kg/day) was: Y equals 0.51(plus or minus 0.08)X minus 41.9(plus or minus 4.6) for beef and Y equals 0.27(plus or minus 0.06)X minus 33.6(plus or minus 5.0) for whole wheat protein. The amounts of beef and wheat proteins estimated to be requried to support body nitrogen balance in 97.5% of the population supplied 96 and 178 mg N/kg/day, respectively. The relative protein value of beef and wheat proteins, in comparison with egg protein, was 78 plus or minus 12 and 41 plus or minus 10, respectively. It is concluded that the variations in dietary protein quality should be taken into account in assessing the protein adequacy of diets for individuals and population groups.

  19. Plant Hormone Binding Sites

    OpenAIRE

    Napier, Richard

    2004-01-01

    • Aims Receptors for plant hormones are becoming identified with increasing rapidity, although a frustrating number remain unknown. There have also been many more hormone‐binding proteins described than receptors. This Botanical Briefing summarizes what has been discovered about hormone binding sites, their discovery and descriptions, and will not dwell on receptor functions or activities except where these are relevant to understand binding.

  20. Analysis of binding heterogeneity.

    NARCIS (Netherlands)

    Nederlof, M.M.

    1992-01-01

    Binding heterogeneity, due to different functional groups on a reactive surface, plays an important role in the binding of small molecules or ions to many adsorbents, both in industrial processes and in natural environments. The binding heterogeneity is described by a distribution of affinity consta

  1. Stepwise bending of DNA by a single TATA box binding protein

    DEFF Research Database (Denmark)

    Tolic-Nørrelykke, Simon F; Rasmussen, Mette B; Pavone, Francesco S;

    2006-01-01

    bead is reduced compared to that of unbent DNA. We detected individual binding and dissocation events and derived kinetic parameters for the process. Dissociation was induced by increasing the salt concentration or by directly pulling on the tethered bead using optical tweezers. In addition to the well......The TATA-box binding protein (TBP) is required by all three eucaryotic RNA polymerases for the initiation of transcription from most promoters. TBP recognizes, binds to, and bends promoter sequences called "TATA-boxes" in the DNA. We present results from the study of individual Saccharomyces...... cerevisiae TBPs interacting with single DNA molecules containing a TATA-box. Using video microscopy, we observed the Brownian motion of the beads tethered by short surface-bound DNA. When TBP binds to and bends the DNA, the conformation of the DNA changes and the amplitude of Brownian motion of the tehtered...

  2. The helical structure of DNA facilitates binding

    Science.gov (United States)

    Berg, Otto G.; Mahmutovic, Anel; Marklund, Emil; Elf, Johan

    2016-09-01

    The helical structure of DNA imposes constraints on the rate of diffusion-limited protein binding. Here we solve the reaction-diffusion equations for DNA-like geometries and extend with simulations when necessary. We find that the helical structure can make binding to the DNA more than twice as fast compared to a case where DNA would be reactive only along one side. We also find that this rate advantage remains when the contributions from steric constraints and rotational diffusion of the DNA-binding protein are included. Furthermore, we find that the association rate is insensitive to changes in the steric constraints on the DNA in the helix geometry, while it is much more dependent on the steric constraints on the DNA-binding protein. We conclude that the helical structure of DNA facilitates the nonspecific binding of transcription factors and structural DNA-binding proteins in general.

  3. Function of the PEX19-binding site of human adrenoleukodystrophy protein as targeting motif in man and yeast. PMP targeting is evolutionarily conserved.

    Science.gov (United States)

    Halbach, André; Lorenzen, Stephan; Landgraf, Christiane; Volkmer-Engert, Rudolf; Erdmann, Ralf; Rottensteiner, Hanspeter

    2005-06-01

    We predicted in human peroxisomal membrane proteins (PMPs) the binding sites for PEX19, a key player in the topogenesis of PMPs, by virtue of an algorithm developed for yeast PMPs. The best scoring PEX19-binding site was found in the adrenoleukodystrophy protein (ALDP). The identified site was indeed bound by human PEX19 and was also recognized by the orthologous yeast PEX19 protein. Likewise, both human and yeast PEX19 bound with comparable affinities to the PEX19-binding site of the yeast PMP Pex13p. Interestingly, the identified PEX19-binding site of ALDP coincided with its previously determined targeting motif. We corroborated the requirement of the ALDP PEX19-binding site for peroxisomal targeting in human fibroblasts and showed that the minimal ALDP fragment targets correctly also in yeast, again in a PEX19-binding site-dependent manner. Furthermore, the human PEX19-binding site of ALDP proved interchangeable with that of yeast Pex13p in an in vivo targeting assay. Finally, we showed in vitro that most of the predicted binding sequences of human PMPs represent true binding sites for human PEX19, indicating that human PMPs harbor common PEX19-binding sites that do resemble those of yeast. Our data clearly revealed a role for PEX19-binding sites as PMP-targeting motifs across species, thereby demonstrating the evolutionary conservation of PMP signal sequences from yeast to man.

  4. Analyzing radioligand binding data.

    Science.gov (United States)

    Motulsky, Harvey; Neubig, Richard

    2002-08-01

    Radioligand binding experiments are easy to perform, and provide useful data in many fields. They can be used to study receptor regulation, discover new drugs by screening for compounds that compete with high affinity for radioligand binding to a particular receptor, investigate receptor localization in different organs or regions using autoradiography, categorize receptor subtypes, and probe mechanisms of receptor signaling, via measurements of agonist binding and its regulation by ions, nucleotides, and other allosteric modulators. This unit reviews the theory of receptor binding and explains how to analyze experimental data. Since binding data are usually best analyzed using nonlinear regression, this unit also explains the principles of curve fitting with nonlinear regression.

  5. Synthetic LPS-Binding Polymer Nanoparticles

    Science.gov (United States)

    Jiang, Tian

    Lipopolysaccharide (LPS), one of the principal components of most gram-negative bacteria's outer membrane, is a type of contaminant that can be frequently found in recombinant DNA products. Because of its strong and even lethal biological effects, selective LPS removal from bioproducts solution is of particular importance in the pharmaceutical and health care industries. In this thesis, for the first time, a proof-of-concept study on preparing LPS-binding hydrogel-like NPs through facile one-step free-radical polymerization was presented. With the incorporation of various hydrophobic (TBAm), cationic (APM, GUA) monomers and cross-linkers (BIS, PEG), a small library of NPs was constructed. Their FITC-LPS binding behaviors were investigated and compared with those of commercially available LPS-binding products. Moreover, the LPS binding selectivity of the NPs was also explored by studying the NPs-BSA interactions. The results showed that all NPs obtained generally presented higher FITC-LPS binding capacity in lower ionic strength buffer than higher ionic strength. However, unlike commercial poly-lysine cellulose and polymyxin B agarose beads' nearly linear increase of FITC-LPS binding with particle concentration, NPs exhibited serious aggregation and the binding quickly saturated or even decreased at high particle concentration. Among various types of NPs, higher FITC-LPS binding capacity was observed for those containing more hydrophobic monomers (TBAm). However, surprisingly, more cationic NPs with higher content of APM exhibited decreased FITC-LPS binding in high ionic strength conditions. Additionally, when new cationic monomer and cross-linker, GUA and PEG, were applied to replace APM and BIS, the obtained NPs showed improved FITC-LPS binding capacity at low NP concentration. But compared with APM- and BIS-containing NPs, the FITC-LPS binding capacity of GUA- and PEG-containing NPs saturated earlier. To investigate the NPs' binding to proteins, we tested the NPs

  6. Antimicrobial Peptide-Lipid Binding Interactions and Binding Selectivity

    OpenAIRE

    Lad, Mitaben D.; Birembaut, Fabrice; Clifton, Luke A.; Frazier, Richard A.; Webster, John R. P.; Green, Rebecca J.

    2007-01-01

    Surface pressure measurements, external reflection-Fourier transform infrared spectroscopy, and neutron reflectivity have been used to investigate the lipid-binding behavior of three antimicrobial peptides: melittin, magainin II, and cecropin P1. As expected, all three cationic peptides were shown to interact more strongly with the anionic lipid, 1,2 dihexadecanoyl-sn-glycerol-3-(phosphor-rac-(1-glycerol)) (DPPG), compared to the zwitterionic lipid, 1,2 dihexadecanoyl-sn-glycerol-3-phosphocho...

  7. LRRK2 kinase activity is dependent on LRRK2 GTP binding capacity but independent of LRRK2 GTP binding.

    Directory of Open Access Journals (Sweden)

    Jean-Marc Taymans

    Full Text Available Leucine rich repeat kinase 2 (LRRK2 is a Parkinson's disease (PD gene that encodes a large multidomain protein including both a GTPase and a kinase domain. GTPases often regulate kinases within signal transduction cascades, where GTPases act as molecular switches cycling between a GTP bound "on" state and a GDP bound "off" state. It has been proposed that LRRK2 kinase activity may be increased upon GTP binding at the LRRK2 Ras of complex proteins (ROC GTPase domain. Here we extensively test this hypothesis by measuring LRRK2 phosphorylation activity under influence of GDP, GTP or non-hydrolyzable GTP analogues GTPγS or GMPPCP. We show that autophosphorylation and lrrktide phosphorylation activity of recombinant LRRK2 protein is unaltered by guanine nucleotides, when co-incubated with LRRK2 during phosphorylation reactions. Also phosphorylation activity of LRRK2 is unchanged when the LRRK2 guanine nucleotide binding pocket is previously saturated with various nucleotides, in contrast to the greatly reduced activity measured for the guanine nucleotide binding site mutant T1348N. Interestingly, when nucleotides were incubated with cell lysates prior to purification of LRRK2, kinase activity was slightly enhanced by GTPγS or GMPPCP compared to GDP, pointing to an upstream guanine nucleotide binding protein that may activate LRRK2 in a GTP-dependent manner. Using metabolic labeling, we also found that cellular phosphorylation of LRRK2 was not significantly modulated by nucleotides, although labeling is significantly reduced by guanine nucleotide binding site mutants. We conclude that while kinase activity of LRRK2 requires an intact ROC-GTPase domain, it is independent of GDP or GTP binding to ROC.

  8. Metal binding stoichiometry and isotherm choice in biosorption

    Energy Technology Data Exchange (ETDEWEB)

    Schiewer, S.; Wong, M.H.

    1999-11-01

    Seaweeds that possess a high metal binding capacity may be used as biosorbents for the removal of toxic heavy metals from wastewater. The binding of Cu and Ni by three brown algae (Sargassum, Colpomenia, Petalonia) and one green alga (Ulva) was investigated at pH 4.0 and pH 3.0. The greater binding strength of Cu is reflected in a binding constant that is about 10 times as high as that of Ni. The extent of metal binding followed the order Petalonia {approximately} Sargassum > Colpomenia > Ulva. This was caused by a decreasing number of binding sites and by much lower metal binding constants for Ulva as compared to the brown algae. Three different stoichiometric assumptions are compared for describing the metal binding, which assume either that each metal ion M binds to one binding site B forming a BM complex or that a divalent metal ion M binds to two monovalent sites B forming BM{sub 0.5} or B{sub 2}M complexes, respectively. Stoichiometry plots are proposed as tools to discern the relevant binding stoichiometry. The pH effect in metal binding and the change in proton binding were well predicted for the B{sub 2}M or BM{sub 0.5} stoichiometries with the former being better for Cu and the latter preferable for Ni. Overall, the BM{sub 0.5} model is recommended because it avoids iterations.

  9. RNA-seq analysis of oil palm under cold stress reveals a different C-repeat binding factor (CBF) mediated gene expression pattern in Elaeis guineensis compared to other species.

    Science.gov (United States)

    Lei, Xintao; Xiao, Yong; Xia, Wei; Mason, Annaliese S; Yang, Yaodong; Ma, Zilong; Peng, Ming

    2014-01-01

    Elaeis guineensis as a tropical oil-crop is particularly sensitive to low temperature. Improvement of cold-tolerance may significantly increase the total cultivation area of this tropical oil-crop worldwide. We sequenced cold-treated and control (untreated) samples of Elaeis guineensis. De novo assembly generated 51,452 unigenes with an average length of 703 bp. Subsequently, these expressed sequences were functionally annotated. In the K category (transcription factors) of COG (Cluster of Orthologous Group) annotation, the largest proportion of genes induced and repressed at least two-fold under cold stress were from the AP2/ERE family, indicating that C-repeat binding factor, (CBFs, members of the AP2/ERE family) may play a central role in cold tolerance in Elaeis guineensis. Subsequently, the CBF-mediated signal transduction pathway was reconstructed based on transcriptome data and the gene expression profile involving the pathway was examined using real-time quantitative RT-PCR (qRT-PCR). CBFs reached maximum transcript level both at medium (4 h) and long period time points (7 days), contrary to the expression pattern of CBFs in Arabidopsis and rice. Moreover, the promoters of downstream Cold Responsive gene (CORs) regulated by CBFs were analyzed. Conservation, mutation and absence of the DRE core motif were detected in the promoters of six CORs. These mutations in DRE motifs suggest that CORs may not be induced via cold stress in Elaeis guineensis.

  10. RNA-seq analysis of oil palm under cold stress reveals a different C-repeat binding factor (CBF mediated gene expression pattern in Elaeis guineensis compared to other species.

    Directory of Open Access Journals (Sweden)

    Xintao Lei

    Full Text Available Elaeis guineensis as a tropical oil-crop is particularly sensitive to low temperature. Improvement of cold-tolerance may significantly increase the total cultivation area of this tropical oil-crop worldwide. We sequenced cold-treated and control (untreated samples of Elaeis guineensis. De novo assembly generated 51,452 unigenes with an average length of 703 bp. Subsequently, these expressed sequences were functionally annotated. In the K category (transcription factors of COG (Cluster of Orthologous Group annotation, the largest proportion of genes induced and repressed at least two-fold under cold stress were from the AP2/ERE family, indicating that C-repeat binding factor, (CBFs, members of the AP2/ERE family may play a central role in cold tolerance in Elaeis guineensis. Subsequently, the CBF-mediated signal transduction pathway was reconstructed based on transcriptome data and the gene expression profile involving the pathway was examined using real-time quantitative RT-PCR (qRT-PCR. CBFs reached maximum transcript level both at medium (4 h and long period time points (7 days, contrary to the expression pattern of CBFs in Arabidopsis and rice. Moreover, the promoters of downstream Cold Responsive gene (CORs regulated by CBFs were analyzed. Conservation, mutation and absence of the DRE core motif were detected in the promoters of six CORs. These mutations in DRE motifs suggest that CORs may not be induced via cold stress in Elaeis guineensis.

  11. How to Use SNP_TATA_Comparator to Find a Significant Change in Gene Expression Caused by the Regulatory SNP of This Gene’s Promoter via a Change in Affinity of the TATA-Binding Protein for This Promoter

    Directory of Open Access Journals (Sweden)

    Mikhail Ponomarenko

    2015-01-01

    Full Text Available The use of biomedical SNP markers of diseases can improve effectiveness of treatment. Genotyping of patients with subsequent searching for SNPs more frequent than in norm is the only commonly accepted method for identification of SNP markers within the framework of translational research. The bioinformatics applications aimed at millions of unannotated SNPs of the “1000 Genomes” can make this search for SNP markers more focused and less expensive. We used our Web service involving Fisher’s Z-score for candidate SNP markers to find a significant change in a gene’s expression. Here we analyzed the change caused by SNPs in the gene’s promoter via a change in affinity of the TATA-binding protein for this promoter. We provide examples and discuss how to use this bioinformatics application in the course of practical analysis of unannotated SNPs from the “1000 Genomes” project. Using known biomedical SNP markers, we identified 17 novel candidate SNP markers nearby: rs549858786 (rheumatoid arthritis; rs72661131 (cardiovascular events in rheumatoid arthritis; rs562962093 (stroke; rs563558831 (cyclophosphamide bioactivation; rs55878706 (malaria resistance, leukopenia, rs572527200 (asthma, systemic sclerosis, and psoriasis, rs371045754 (hemophilia B, rs587745372 (cardiovascular events; rs372329931, rs200209906, rs367732974, and rs549591993 (all four: cancer; rs17231520 and rs569033466 (both: atherosclerosis; rs63750953, rs281864525, and rs34166473 (all three: malaria resistance, thalassemia.

  12. RNA-seq analysis of oil palm under cold stress reveals a different C-repeat binding factor (CBF) mediated gene expression pattern in Elaeis guineensis compared to other species.

    Science.gov (United States)

    Lei, Xintao; Xiao, Yong; Xia, Wei; Mason, Annaliese S; Yang, Yaodong; Ma, Zilong; Peng, Ming

    2014-01-01

    Elaeis guineensis as a tropical oil-crop is particularly sensitive to low temperature. Improvement of cold-tolerance may significantly increase the total cultivation area of this tropical oil-crop worldwide. We sequenced cold-treated and control (untreated) samples of Elaeis guineensis. De novo assembly generated 51,452 unigenes with an average length of 703 bp. Subsequently, these expressed sequences were functionally annotated. In the K category (transcription factors) of COG (Cluster of Orthologous Group) annotation, the largest proportion of genes induced and repressed at least two-fold under cold stress were from the AP2/ERE family, indicating that C-repeat binding factor, (CBFs, members of the AP2/ERE family) may play a central role in cold tolerance in Elaeis guineensis. Subsequently, the CBF-mediated signal transduction pathway was reconstructed based on transcriptome data and the gene expression profile involving the pathway was examined using real-time quantitative RT-PCR (qRT-PCR). CBFs reached maximum transcript level both at medium (4 h) and long period time points (7 days), contrary to the expression pattern of CBFs in Arabidopsis and rice. Moreover, the promoters of downstream Cold Responsive gene (CORs) regulated by CBFs were analyzed. Conservation, mutation and absence of the DRE core motif were detected in the promoters of six CORs. These mutations in DRE motifs suggest that CORs may not be induced via cold stress in Elaeis guineensis. PMID:25479236

  13. Python bindings for libcloudph++

    OpenAIRE

    Jarecka, Dorota; Arabas, Sylwester; Del Vento, Davide

    2015-01-01

    This technical note introduces the Python bindings for libcloudph++. The libcloudph++ is a C++ library of algorithms for representing atmospheric cloud microphysics in numerical models. The bindings expose the complete functionality of the library to the Python users. The bindings are implemented using the Boost.Python C++ library and use NumPy arrays. This note includes listings with Python scripts exemplifying the use of selected library components. An example solution for using the Python ...

  14. Is there a link between selectivity and binding thermodynamics profiles?

    Science.gov (United States)

    Tarcsay, Ákos; Keserű, György M

    2015-01-01

    Thermodynamics of ligand binding is influenced by the interplay between enthalpy and entropy contributions of the binding event. The impact of these binding free energy components, however, is not limited to the primary target only. Here, we investigate the relationship between binding thermodynamics and selectivity profiles by combining publicly available data from broad off-target assay profiling and the corresponding thermodynamics measurements. Our analysis indicates that compounds binding their primary targets with higher entropy contributions tend to hit more off-targets compared with those ligands that demonstrated enthalpy-driven binding.

  15. DNS & Bind Cookbook

    CERN Document Server

    Liu, Cricket

    2011-01-01

    The DNS & BIND Cookbook presents solutions to the many problems faced by network administrators responsible for a name server. Following O'Reilly's popular problem-and-solution cookbook format, this title is an indispensable companion to DNS & BIND, 4th Edition, the definitive guide to the critical task of name server administration. The cookbook contains dozens of code recipes showing solutions to everyday problems, ranging from simple questions, like, "How do I get BIND?" to more advanced topics like providing name service for IPv6 addresses. It's full of BIND configuration files that yo

  16. Python bindings for libcloudph++

    CERN Document Server

    Jarecka, Dorota; Del Vento, Davide

    2015-01-01

    This technical note introduces the Python bindings for libcloudph++. The libcloudph++ is a C++ library of algorithms for representing atmospheric cloud microphysics in numerical models. The bindings expose the complete functionality of the library to the Python users. The bindings are implemented using the Boost.Python C++ library and use NumPy arrays. This note includes listings with Python scripts exemplifying the use of selected library components. An example solution for using the Python bindings to access libcloudph++ from Fortran is presented.

  17. Automatic Binding Time Analysis for a Typed Lambda-Calculus

    DEFF Research Database (Denmark)

    Nielson, Hanne Riis; Nielson, Flemming

    1988-01-01

    A binding time analysis imposes a distinction between the computations to be performed early (e.g. at compile-time) and those to be performed late (e.g. at run-time). For the lambda-calculus this distinction is formalized by a two-level lambda-calculus. The authors present an algorithm for static...... analysis of the binding times of a typed lambda-calculus with products, sums, lists and general recursive types. Given partial information about the binding times of some of the subexpressions it will complete that information such that (i) early bindings may be turned into late bindings but not vice versa......, (ii) the resulting two-level lambda-expression reflects our intuition about binding times, e.g. that early bindings are performed before late bindings, and (iii) as few changes as possible have been made compared with the initial binding information. The results can be applied in the implementation...

  18. Genomic plus-strand RNA synthesis by the brome mosaic virus (BMV) RNA replicase requires a sequence that is complementary to the binding site of the BMV helicase-like protein.

    Science.gov (United States)

    Sivakumaran, K; Kao, C C

    2000-11-01

    Summary Initiation of genomic plus-strand RNA synthesis by the brome mosaic virus (BMV) replicase in vitro requires a 26-nucleotide (nt) RNA sequence at the 3' end of the minus-strand RNA and a nontemplated nucleotide 3' of the initiation cytidylate [Sivakumaran, K. and Kao, C.C. (1999)J. Virol.64, 6415-6423]. At the 5' end of this RNA is a 9-nt sequence called the cB box, the complement of the previously defined B box. The cB box can not be functionally replaced by the B box and has specific positional and sequence requirements. The portion of the cB box that is required for RNA synthesis in vitro is well-conserved in species in the Bromoviridae family. An equivalent RNA from Cucumber mosaic virus was unable to direct efficient RNA synthesis by the BMV replicase until the cB box was positioned at the same site relative to the BMV RNA and guanylates were present at positions +6 and +7 from the initiation cytidylate. These results further define the elements required for the recognition and initiation of viral genomic plus-strand RNA synthesis and suggest that a sequence important for minus-strand RNA synthesis is also required for plus-strand RNA synthesis.

  19. A knowledge-guided strategy for improving the accuracy of scoring functions in binding affinity prediction

    Directory of Open Access Journals (Sweden)

    Wang Renxiao

    2010-04-01

    Full Text Available Abstract Background Current scoring functions are not very successful in protein-ligand binding affinity prediction albeit their popularity in structure-based drug designs. Here, we propose a general knowledge-guided scoring (KGS strategy to tackle this problem. Our KGS strategy computes the binding constant of a given protein-ligand complex based on the known binding constant of an appropriate reference complex. A good training set that includes a sufficient number of protein-ligand complexes with known binding data needs to be supplied for finding the reference complex. The reference complex is required to share a similar pattern of key protein-ligand interactions to that of the complex of interest. Thus, some uncertain factors in protein-ligand binding may cancel out, resulting in a more accurate prediction of absolute binding constants. Results In our study, an automatic algorithm was developed for summarizing key protein-ligand interactions as a pharmacophore model and identifying the reference complex with a maximal similarity to the query complex. Our KGS strategy was evaluated in combination with two scoring functions (X-Score and PLP on three test sets, containing 112 HIV protease complexes, 44 carbonic anhydrase complexes, and 73 trypsin complexes, respectively. Our results obtained on crystal structures as well as computer-generated docking poses indicated that application of the KGS strategy produced more accurate predictions especially when X-Score or PLP alone did not perform well. Conclusions Compared to other targeted scoring functions, our KGS strategy does not require any re-parameterization or modification on current scoring methods, and its application is not tied to certain systems. The effectiveness of our KGS strategy is in theory proportional to the ever-increasing knowledge of experimental protein-ligand binding data. Our KGS strategy may serve as a more practical remedy for current scoring functions to improve their

  20. Natural ligand binding and transfer from liver fatty acid binding protein (LFABP) to membranes.

    Science.gov (United States)

    De Gerónimo, Eduardo; Hagan, Robert M; Wilton, David C; Córsico, Betina

    2010-09-01

    Liver fatty acid-binding protein (LFABP) is distinctive among fatty acid-binding proteins because it binds more than one molecule of long-chain fatty acid and a variety of diverse ligands. Also, the transfer of fluorescent fatty acid analogues to model membranes under physiological ionic strength follows a different mechanism compared to most of the members of this family of intracellular lipid binding proteins. Tryptophan insertion mutants sensitive to ligand binding have allowed us to directly measure the binding affinity, ligand partitioning and transfer to model membranes of natural ligands. Binding of fatty acids shows a cooperative mechanism, while acyl-CoAs binding presents a hyperbolic behavior. Saturated fatty acids seem to have a stronger partition to protein vs. membranes, compared to unsaturated fatty acids. Natural ligand transfer rates are more than 200-fold higher compared to fluorescently-labeled analogues. Interestingly, oleoyl-CoA presents a markedly different transfer behavior compared to the rest of the ligands tested, probably indicating the possibility of specific targeting of ligands to different metabolic fates. PMID:20541621

  1. Melanin-binding radiopharmaceuticals

    Energy Technology Data Exchange (ETDEWEB)

    Packer, S; Fairchild, R G; Watts, K P; Greenberg, D; Hannon, S J

    1980-01-01

    The scope of this paper is limited to an analysis of the factors that are important to the relationship of radiopharmaceuticals to melanin. While the authors do not attempt to deal with differences between melanin-binding vs. melanoma-binding, a notable variance is assumed. (PSB)

  2. DNS BIND Server Configuration

    Directory of Open Access Journals (Sweden)

    Radu MARSANU

    2011-01-01

    Full Text Available After a brief presentation of the DNS and BIND standard for Unix platforms, the paper presents an application which has a principal objective, the configuring of the DNS BIND 9 server. The general objectives of the application are presented, follow by the description of the details of designing the program.

  3. To Bind or not to Bind: It’s in the Contract

    DEFF Research Database (Denmark)

    Tvarnø, Christina D.

    2016-01-01

    discusses, in a theoretical perspective, the legal reasoning behind the different partnering approaches, both from a historical and contract law perspective, and furthermore applies a game theoretical approach in evaluating binding versus non-binding partnering contracts. The analysis focuses on private......This article discusses the formalization of collaboration through partnering contracts in the construction industry in the USA, Great Britain and Denmark. The article compares the different types of collaborative partnering contracts in the three countries, and provides a conclusion on whether...... the collaborative partnering contract should be binding or non-binding, based on the three empirical contracts analyzed in this article. The partnering contracts in Great Britain and Denmark are legally binding, while in the USA the partnering agreements are non-binding charters or letters of intent. This article...

  4. Protein binding assay for hyaluronate

    Energy Technology Data Exchange (ETDEWEB)

    Lacy, B.E.; Underhill, C.B.

    1986-11-01

    A relatively quick and simple assay for hyaluronate was developed using the specific binding protein, hyaluronectin. The hyaluronectin was obtained by homogenizing the brains of Sprague-Dawley rats, and then centrifuging the homogenate. The resulting supernatant was used as a source of crude hyaluronectin. In the binding assay, the hyaluronectin was mixed with (/sup 3/H)hyaluronate, followed by an equal volume of saturated (NH/sub 4/)/sub 2/SO/sub 4/, which precipitated the hyaluronectin and any (/sup 3/H)hyaluronate associated with it, but left free (/sup 3/H)hyaluronate in solution. The mixture was then centrifuged, and the amount of bound (/sup 3/H)hyaluronate in the precipitate was determined. Using this assay, the authors found that hyaluronectin specifically bound hyaluronate, since other glycosaminoglycans failed to compete for the binding protein. In addition, the interaction between hyaluronectin and hyaluronate was of relatively high affinity, and the size of the hyaluronate did not appear to substantially alter the amount of binding. To determine the amount of hyaluronate in an unknown sample, they used a competition assay in which the binding of a set amount of (/sup 3/H)hyaluronate was blocked by the addition of unlabeled hyaluronate. By comparing the degree of competition of the unknown samples with that of known amounts of hyaluronate, it was possible to determine the amount of hyaluronate in the unknowns. They have found that this method is sensitive to 1 ..mu..g or less of hyaluronate, and is unaffected by the presence of proteins.

  5. Aging neuromodulation impairs associative binding: a neurocomputational account.

    Science.gov (United States)

    Li, Shu-Chen; Naveh-Benjamin, Moshe; Lindenberger, Ulman

    2005-06-01

    Relative to young adults, older adults are particularly impaired in episodic memory tasks requiring associative binding of separate components into compound episodes, such as tasks requiring item-context and item-item binding. This associative-binding deficit has been attributed to senescent changes in frontal-hippocampal circuitry but has not been formally linked to impaired neuromodulation involving this circuitry. Previous neurocomputational work showed that impaired neuromodulation could result in less distinct neurocognitive representations. Here we extend this computational principle to simulate aging-related deficits in associative binding. As expected, networks with simulated deficiency in neuromodulation resulted in less distinct internal representations than did networks simulating the processing and performance of young adults, and were also more impaired under task conditions that required associative binding. The findings suggest that senescent changes in neuromodulatory mechanisms may play a basic role in aging-related impairment in associative binding by reducing the efficacy of distributed conjunctive coding. PMID:15943670

  6. Anion binding in biological systems

    Science.gov (United States)

    Feiters, Martin C.; Meyer-Klaucke, Wolfram; Kostenko, Alexander V.; Soldatov, Alexander V.; Leblanc, Catherine; Michel, Gurvan; Potin, Philippe; Küpper, Frithjof C.; Hollenstein, Kaspar; Locher, Kaspar P.; Bevers, Loes E.; Hagedoorn, Peter-Leon; Hagen, Wilfred R.

    2009-11-01

    We compare aspects of biological X-ray absorption spectroscopy (XAS) studies of cations and anions, and report on some examples of anion binding in biological systems. Brown algae such as Laminaria digitata (oarweed) are effective accumulators of I from seawater, with tissue concentrations exceeding 50 mM, and the vanadate-containing enzyme haloperoxidase is implicated in halide accumulation. We have studied the chemical state of iodine and its biological role in Laminaria at the I K edge, and bromoperoxidase from Ascophyllum nodosum (knotted wrack) at the Br K edge. Mo is essential for many forms of life; W only for certain archaea, such as Archaeoglobus fulgidus and the hyperthermophilic archaeon Pyrococcus furiosus, and some bacteria. The metals are bound and transported as their oxo-anions, molybdate and tungstate, which are similar in size. The transport protein WtpA from P. furiosus binds tungstate more strongly than molybdate, and is related in sequence to Archaeoglobus fulgidus ModA, of which a crystal structure is known. We have measured A. fulgidus ModA with tungstate at the W L3 (2p3/2) edge, and compared the results with the refined crystal structure. XAS studies of anion binding are feasible even if only weak interactions are present, are biologically relevant, and give new insights in the spectroscopy.

  7. Anion binding in biological systems

    Energy Technology Data Exchange (ETDEWEB)

    Feiters, Martin C [Department of Organic Chemistry, Institute for Molecules and Materials, Faculty of Science, Radboud University Nijmegen, Heyendaalseweg 135, 6525 AJ Nijmegen (Netherlands); Meyer-Klaucke, Wolfram [EMBL Hamburg Outstation at DESY, Notkestrasse 85, D-22607 Hamburg (Germany); Kostenko, Alexander V; Soldatov, Alexander V [Faculty of Physics, Southern Federal University, Sorge 5, Rostov-na-Donu, 344090 (Russian Federation); Leblanc, Catherine; Michel, Gurvan; Potin, Philippe [Centre National de la Recherche Scientifique and Universite Pierre et Marie Curie Paris-VI, Station Biologique de Roscoff, Place Georges Teissier, BP 74, F-29682 Roscoff cedex, Bretagne (France); Kuepper, Frithjof C [Scottish Association for Marine Science, Dunstaffnage Marine Laboratory, Oban, Argyll PA37 1QA, Scotland (United Kingdom); Hollenstein, Kaspar; Locher, Kaspar P [Institute of Molecular Biology and Biophysics, ETH Zuerich, Schafmattstrasse 20, Zuerich, 8093 (Switzerland); Bevers, Loes E; Hagedoorn, Peter-Leon; Hagen, Wilfred R, E-mail: m.feiters@science.ru.n [Department of Biotechnology, Delft University of Technology, Julianalaan 67, 2628 BC Delft (Netherlands)

    2009-11-15

    We compare aspects of biological X-ray absorption spectroscopy (XAS) studies of cations and anions, and report on some examples of anion binding in biological systems. Brown algae such as Laminaria digitata (oarweed) are effective accumulators of I from seawater, with tissue concentrations exceeding 50 mM, and the vanadate-containing enzyme haloperoxidase is implicated in halide accumulation. We have studied the chemical state of iodine and its biological role in Laminaria at the I K edge, and bromoperoxidase from Ascophyllum nodosum (knotted wrack) at the Br K edge. Mo is essential for many forms of life; W only for certain archaea, such as Archaeoglobus fulgidus and the hyperthermophilic archaeon Pyrococcus furiosus, and some bacteria. The metals are bound and transported as their oxo-anions, molybdate and tungstate, which are similar in size. The transport protein WtpA from P. furiosus binds tungstate more strongly than molybdate, and is related in sequence to Archaeoglobus fulgidus ModA, of which a crystal structure is known. We have measured A. fulgidus ModA with tungstate at the W L{sub 3} (2p{sub 3/2}) edge, and compared the results with the refined crystal structure. XAS studies of anion binding are feasible even if only weak interactions are present, are biologically relevant, and give new insights in the spectroscopy.

  8. EHD3 Protein Is Required for Tubular Recycling Endosome Stabilization, and an Asparagine-Glutamic Acid Residue Pair within Its Eps15 Homology (EH) Domain Dictates Its Selective Binding to NPF Peptides.

    Science.gov (United States)

    Bahl, Kriti; Xie, Shuwei; Spagnol, Gaelle; Sorgen, Paul; Naslavsky, Naava; Caplan, Steve

    2016-06-24

    An elaborate network of dynamic lipid membranes, termed tubular recycling endosomes (TRE), coordinates the process of endocytic recycling in mammalian cells. The C-terminal Eps15 homology domain (EHD)-containing proteins have been implicated in the bending and fission of TRE, thus regulating endocytic recycling. EHD proteins have an EH domain that interacts with proteins containing an NPF motif. We found that NPF-containing EHD1 interaction partners such as molecules interacting with CasL-like1 (MICAL-L1) and Syndapin2 are essential for TRE biogenesis. Also crucial for TRE biogenesis is the generation of phosphatidic acid, an essential lipid component of TRE that serves as a docking point for MICAL-L1 and Syndapin2. EHD1 and EHD3 have 86% amino acid identity; they homo- and heterodimerize and partially co-localize to TRE. Despite their remarkable identity, they have distinct mechanistic functions. EHD1 induces membrane vesiculation, whereas EHD3 supports TRE biogenesis and/or stabilization by an unknown mechanism. While using phospholipase D inhibitors (which block the conversion of glycerophospholipids to phosphatidic acid) to deplete cellular TRE, we observed that, upon inhibitor washout, there was a rapid and dramatic regeneration of MICAL-L1-marked TRE. Using this "synchronized" TRE biogenesis system, we determined that EHD3 is involved in the stabilization of TRE rather than in their biogenesis. Moreover, we identify the residues Ala-519/Asp-520 of EHD1 and Asn-519/Glu-520 of EHD3 as defining the selectivity of these two paralogs for NPF-containing binding partners, and we present a model to explain the atomic mechanism and provide new insight for their differential roles in vesiculation and tubulation, respectively. PMID:27189942

  9. DNA Triplexes That Bind Several Cofactor Molecules.

    Science.gov (United States)

    Vollmer, Sven; Richert, Clemens

    2015-12-14

    Cofactors are critical for energy-consuming processes in the cell. Harnessing such processes for practical applications requires control over the concentration of cofactors. We have recently shown that DNA triplex motifs with a designed binding site can be used to capture and release nucleotides with low micromolar dissociation constants. In order to increase the storage capacity of such triplex motifs, we have explored the limits of ligand binding through designed cavities in the oligopurine tract. Oligonucleotides with up to six non-nucleotide bridges between purines were synthesized and their ability to bind ATP, cAMP or FAD was measured. Triplex motifs with several single-nucleotide binding sites were found to bind purines more tightly than triplexes with one large binding site. The optimized triplex consists of 59 residues and four C3-bridges. It can bind up to four equivalents of ligand with apparent Kd values of 52 µM for ATP, 9 µM for FAD, and 2 µM for cAMP. An immobilized version fuels bioluminescence via release of ATP at body temperature. These results show that motifs for high-density capture, storage and release of energy-rich biomolecules can be constructed from synthetic DNA. PMID:26561335

  10. The inhibition of assembly of HIV-1 virus-like particles by 3-O-(3',3'-dimethylsuccinyl betulinic acid (DSB is counteracted by Vif and requires its Zinc-binding domain

    Directory of Open Access Journals (Sweden)

    Bouaziz Serge

    2008-12-01

    Full Text Available Abstract Background DSB, the 3-O-(3',3'dimethylsuccinyl derivative of betulinic acid, blocks the last step of protease-mediated processing of HIV-1 Gag precursor (Pr55Gag, which leads to immature, noninfectious virions. When administered to Pr55Gag-expressing insect cells (Sf9, DSB inhibits the assembly and budding of membrane-enveloped virus-like particles (VLP. In order to explore the possibility that viral factors could modulate the susceptibility to DSB of the VLP assembly process, several viral proteins were coexpressed individually with Pr55Gag in DSB-treated cells, and VLP yields assayed in the extracellular medium. Results Wild-type Vif (Vifwt restored the VLP production in DSB-treated cells to levels observed in control, untreated cells. DSB-counteracting effect was also observed with Vif mutants defective in encapsidation into VLP, suggesting that packaging and anti-DSB effect were separate functions in Vif. The anti-DSB effect was abolished for VifC133S and VifS116V, two mutants which lacked the zinc binding domain (ZBD formed by the four H108C114C133H139 coordinates with a Zn atom. Electron microscopic analysis of cells coexpressing Pr55Gag and Vifwt showed that a large proportion of VLP budded into cytoplasmic vesicles and were released from Sf9 cells by exocytosis. However, in the presence of mutant VifC133S or VifS116V, most of the VLP assembled and budded at the plasma membrane, as in control cells expressing Pr55Gag alone. Conclusion The function of HIV-1 Vif protein which negated the DSB inhibition of VLP assembly was independent of its packaging capability, but depended on the integrity of ZBD. In the presence of Vifwt, but not with ZBD mutants VifC133S and VifS116V, VLP were redirected to a vesicular compartment and egressed via the exocytic pathway.

  11. Characterization of EhCaBP, a calcium-binding protein of Entamoeba histolytica and its binding proteins.

    Science.gov (United States)

    Yadava, N; Chandok, M R; Prasad, J; Bhattacharya, S; Sopory, S K; Bhattacharya, A

    1997-01-01

    A novel calcium-binding protein (EhCaBP) has been recently identified and characterized from the protozoan parasite Entamoeba histolytica. In order to decipher the function of this protein, a few basic properties were investigated and compared with the ubiquitous Ca(2+)-signal transducing protein calmodulin (CaM). Indirect immunofluorescence and immunoprecipitation analyses using specific antibodies against EhCaBP suggest that it is a soluble cytoplasmic protein with no major post-translational modification. EhCaBP did not stimulate cAMP-phosphodiesterase activity, differentiating it from all known CaMs. Affinity chromatography of [35S]methionine-labelled proteins of E. histolytica trophozoites using EhCaBP-sepharose column showed Ca(2+)-dependent binding of a group of proteins. Radiolabelled proteins from the same extract also bound to CaM-sepharose. However, the proteins bound to the two columns were different as revealed by sodium dodecyl sulphate polyacrylamide gel electrophoresis. At least one of the EhCaBP-binding proteins became phosphorylated as revealed by in vivo phosphorylation analysis. The binding-proteins could not be detected in E. invadens (a species that is pathogenic in reptiles) and E. moshkovskii (which is found in the human gut but is not pathogenic), two species in which EhCaBP-like protein has not been found. Two distinct Ca(2+)-dependent protein kinases, which get activated by EhCaBP and CaM respectively, were detected in E. histolytica. These kinases require different levels of Ca2+ for their maximal activities. Affinity chromatography also showed the binding of protein kinase(s) to EhCaBP in a Ca(2+)-dependent manner. Our data suggest that there may be novel Ca(2+)-signal transduction pathway in E. histolytica mediated by EhCaBP.

  12. Adjustment of legally binding local plans

    DEFF Research Database (Denmark)

    Hvingel, Line Træholt; Aunsborg, Christian; Christensen, Finn Kjær;

    2012-01-01

    Traditionally, and by law, new urban areas in Denmark are regulated and planned through legally binding local plans. Recently a tendency has occurred: The municipalities make the legally binding local plans quite open for future adjustment, and they are using a substantial amount of ‘empowerment......, which seem to be beyond the scope of the Danish Planning Act. This paper deals with this problem through case studies and a legal analysis of present law. If the combination of the legally binding local plan and subsequent added requirements is misused, it will weaken the legal rights of the citizens...... the considerations of legal rights, the extend of the legal use of empowerment provisions and the combination of the use of legal binding local plans and other legal instruments such as easements and sales agreements....

  13. SHBG (Sex Hormone Binding Globulin)

    Science.gov (United States)

    ... as: Testosterone-estrogen Binding Globulin; TeBG Formal name: Sex Hormone Binding Globulin Related tests: Testosterone , Free Testosterone, ... I should know? How is it used? The sex hormone binding globulin (SHBG) test may be used ...

  14. Binding site turnover produces pervasive quantitative changes in transcription factor binding between closely related Drosophila species.

    Directory of Open Access Journals (Sweden)

    Robert K Bradley

    2010-03-01

    Full Text Available Changes in gene expression play an important role in evolution, yet the molecular mechanisms underlying regulatory evolution are poorly understood. Here we compare genome-wide binding of the six transcription factors that initiate segmentation along the anterior-posterior axis in embryos of two closely related species: Drosophila melanogaster and Drosophila yakuba. Where we observe binding by a factor in one species, we almost always observe binding by that factor to the orthologous sequence in the other species. Levels of binding, however, vary considerably. The magnitude and direction of the interspecies differences in binding levels of all six factors are strongly correlated, suggesting a role for chromatin or other factor-independent forces in mediating the divergence of transcription factor binding. Nonetheless, factor-specific quantitative variation in binding is common, and we show that it is driven to a large extent by the gain and loss of cognate recognition sequences for the given factor. We find only a weak correlation between binding variation and regulatory function. These data provide the first genome-wide picture of how modest levels of sequence divergence between highly morphologically similar species affect a system of coordinately acting transcription factors during animal development, and highlight the dominant role of quantitative variation in transcription factor binding over short evolutionary distances.

  15. Discovery of widespread GTP-binding motifs in genomic DNA and RNA.

    Science.gov (United States)

    Curtis, Edward A; Liu, David R

    2013-04-18

    Biological RNAs that bind small molecules have been implicated in a variety of regulatory and catalytic processes. Inspired by these examples, we used in vitro selection to search a pool of genome-encoded RNA fragments for naturally occurring GTP aptamers. Several aptamer classes were identified, including one (the "G motif") with a G-quadruplex structure. Further analysis revealed that most RNA and DNA G-quadruplexes bind GTP. The G motif is abundant in eukaryotes, and the human genome contains ~75,000 examples with dissociation constants comparable to the GTP concentration of a eukaryotic cell (~300 μM). G-quadruplexes play roles in diverse cellular processes, and our findings raise the possibility that GTP may play a role in the function of these elements. Consistent with this possibility, the sequence requirements of several classes of regulatory G-quadruplexes parallel those of GTP binding.

  16. Comparative proteomics of uropathogenic Escherichia coli during growth in human urine identify UCA-like (UCL) fimbriae as an adherence factor involved in biofilm formation and binding to uroepithelial cells.

    Science.gov (United States)

    Wurpel, Daniël J; Totsika, Makrina; Allsopp, Luke P; Webb, Richard I; Moriel, Danilo G; Schembri, Mark A

    2016-01-10

    Uropathogenic Escherichia coli (UPEC) are the primary cause of urinary tract infection (UTI) in humans. For the successful colonisation of the human urinary tract, UPEC employ a diverse collection of secreted or surface-exposed virulence factors including toxins, iron acquisition systems and adhesins. In this study, a comparative proteomic approach was utilised to define the UPEC pan and core surface proteome following growth in pooled human urine. Identified proteins were investigated for subcellular origin, prevalence and homology to characterised virulence factors. Fourteen core surface proteins were identified, as well as eleven iron uptake receptor proteins and four distinct fimbrial types, including type 1, P, F1C/S and a previously uncharacterised fimbrial type, designated UCA-like (UCL) fimbriae in this study. These pathogenicity island (PAI)-associated fimbriae are related to UCA fimbriae of Proteus mirabilis, associated with UPEC and exclusively found in members of the E. coli B2 and D phylogroup. We further demonstrated that UCL fimbriae promote significant biofilm formation on abiotic surfaces and mediate specific attachment to exfoliated human uroepithelial cells. Combined, this study has defined the surface proteomic profiles and core surface proteome of UPEC during growth in human urine and identified a new type of fimbriae that may contribute to UTI.

  17. The cell-binding domain of intimin from enteropathogenic Escherichia coli binds to beta1 integrins.

    Science.gov (United States)

    Frankel, G; Lider, O; Hershkoviz, R; Mould, A P; Kachalsky, S G; Candy, D C; Cahalon, L; Humphries, M J; Dougan, G

    1996-08-23

    Bacteria interact with mammalian cells surface molecules, such as integrins, to colonize tissues and evade immunological detection. Herein, the ability of intimin, an outer membrane protein from enteropathogenic Escherichia coli, to bind beta1 integrins was investigated. Solid-phase binding assays revealed binding of the carboxyl-terminal 280 amino acids of intimin (Int280) to alpha4beta1 and alpha5beta1 integrins. The binding required divalent ions (in particular, it was enhanced by Mn2+) and was inhibited by an RGD-containing peptide. Nonderivatized Int280, but not Int280CS (like Int280 but with Cys-937 replaced by Ser) blocked the binding of biotinylated Int280 to integrins. Int280 did not efficiently inhibit beta1 integrin binding of invasin from Yersinia pseudotuberculosis. Both intimin and invasin, immobilized on plastic surfaces, mediated adherence of resting or phorbol 12-myristate 13-acetate-activated human CD4(+) T cells, whereas fibronectin mediated the adherence of only activated T cells. T cell binding to intimin and invasin was integrin mediated because it was specifically blocked by an RGD-containing peptide and by antibodies directed against the integrin subunits beta1, alpha4, and alpha5. These results demonstrate a specific integrin binding activity for intimin that is related to, but distinct from, that of invasin. PMID:8702771

  18. The readiness potential reflects intentional binding

    Directory of Open Access Journals (Sweden)

    Han-Gue eJo

    2014-06-01

    Full Text Available When a voluntary action is causally linked with a sensory outcome, the action and its consequent effect are perceived as being closer together in time. This effect is called intentional binding. Although many experiments were conducted on this phenomenon, the underlying neural mechanisms are not well understood. While intentional binding is specific to voluntary action, we presumed that preconscious brain activity (the readiness potential, RP, which occurs before an action is made, might play an important role in this binding effect. In this study, the brain dynamics were recorded with electroencephalography (EEG and analyzed in single-trials in order to estimate whether intentional binding is correlated with the early neural processes. Moreover, we were interested in different behavioral performance between meditators and non-meditators since meditators are expected to be able to keep attention more consistently on a task. Thus, we performed the intentional binding paradigm with twenty mindfulness meditators and compared them to matched controls. Although, we did not observe a group effect on either behavioral data or EEG recordings, we found that self-initiated movements following ongoing negative deflections of slow cortical potentials (SCPs result in a stronger binding effect compared to positive potentials, especially regarding the perceived time of the consequent effect. Our results provide the first direct evidence that the early neural activity within the range of SCPs affects perceived time of a sensory outcome that is caused by intentional action.

  19. The readiness potential reflects intentional binding.

    Science.gov (United States)

    Jo, Han-Gue; Wittmann, Marc; Hinterberger, Thilo; Schmidt, Stefan

    2014-01-01

    When a voluntary action is causally linked with a sensory outcome, the action and its consequent effect are perceived as being closer together in time. This effect is called intentional binding. Although many experiments were conducted on this phenomenon, the underlying neural mechanisms are not well understood. While intentional binding is specific to voluntary action, we presumed that preconscious brain activity (the readiness potential, RP), which occurs before an action is made, might play an important role in this binding effect. In this study, the brain dynamics were recorded with electroencephalography (EEG) and analyzed in single-trials in order to estimate whether intentional binding is correlated with the early neural processes. Moreover, we were interested in different behavioral performance between meditators and non-meditators since meditators are expected to be able to keep attention more consistently on a task. Thus, we performed the intentional binding paradigm with 20 mindfulness meditators and compared them to matched controls. Although, we did not observe a group effect on either behavioral data or EEG recordings, we found that self-initiated movements following ongoing negative deflections of slow cortical potentials (SCPs) result in a stronger binding effect compared to positive potentials, especially regarding the perceived time of the consequent effect. Our results provide the first direct evidence that the early neural activity within the range of SCPs affects perceived time of a sensory outcome that is caused by intentional action.

  20. Synthesis and spectroscopic studies on the Schiff base ligand derived from condensation of 2-furaldehyde and 3,3'-diaminobenzidene, L and its complexes with Co(II), Ni(II), Cu(II) and Zn(II): Comparative DNA binding studies of L and its Cu(II) and Zn(II) complexes

    Science.gov (United States)

    Shakir, Mohammad; Abbasi, Ambreen; Khan, Asad U.; Khan, Shahper N.

    2011-01-01

    The Schiff base ligand, N,N'-bis-(2-furancarboxaldimine)-3,3'-diaminobenzidene (L) obtained by condensation of 2-furaldehyde and 3,3'-diaminobenzidene, was used to synthesize the mononuclear complexes of the type, [M(L)](NO 3) 2 [M = Co(II), Ni(II), Cu(II) and Zn(II)]. The newly synthesized ligand, (L) and its complexes have been characterized on the basis of the results of the elemental analysis, molar conductance, magnetic susceptibility measurements and spectroscopic studies viz, FT-IR, 1H and 13C NMR, mass, UV-vis and EPR. EPR, UV-vis and magnetic moment data revealed a square planar geometry for the complexes with distortion in Cu(II) complex and conductivity data show a 1:2 electrolytic nature of the complexes. Absorption and fluorescence spectroscopic studies support that Schiff base ligand, L and its Cu(II) and Zn(II) complex exhibit significant binding to calf thymus DNA. The highest binding affinity in case of L may be due to the more open structure as compared to the metal coordinated complexes.

  1. Crystal Structure of the Botulinum Neurotoxin Type G Binding Domain: Insight into Cell Surface Binding

    Energy Technology Data Exchange (ETDEWEB)

    Stenmark, Pål; Dong, Min; Dupuy, Jérôme; Chapman, Edwin R.; Stevens, Raymond C. (Scripps); (UW)

    2011-11-02

    Botulinum neurotoxins (BoNTs) typically bind the neuronal cell surface via dual interactions with both protein receptors and gangliosides. We present here the 1.9-{angstrom} X-ray structure of the BoNT serotype G (BoNT/G) receptor binding domain (residues 868-1297) and a detailed view of protein receptor and ganglioside binding regions. The ganglioside binding motif (SxWY) has a conserved structure compared to the corresponding regions in BoNT serotype A and BoNT serotype B (BoNT/B), but several features of interactions with the hydrophilic face of the ganglioside are absent at the opposite side of the motif in the BoNT/G ganglioside binding cleft. This may significantly reduce the affinity between BoNT/G and gangliosides. BoNT/G and BoNT/B share the protein receptor synaptotagmin (Syt) I/II. The Syt binding site has a conserved hydrophobic plateau located centrally in the proposed protein receptor binding interface (Tyr1189, Phe1202, Ala1204, Pro1205, and Phe1212). Interestingly, only 5 of 14 residues that are important for binding between Syt-II and BoNT/B are conserved in BoNT/G, suggesting that the means by which BoNT/G and BoNT/B bind Syt diverges more than previously appreciated. Indeed, substitution of Syt-II Phe47 and Phe55 with alanine residues had little effect on the binding of BoNT/G, but strongly reduced the binding of BoNT/B. Furthermore, an extended solvent-exposed hydrophobic loop, located between the Syt binding site and the ganglioside binding cleft, may serve as a third membrane association and binding element to contribute to high-affinity binding to the neuronal membrane. While BoNT/G and BoNT/B are homologous to each other and both utilize Syt-I/Syt-II as their protein receptor, the precise means by which these two toxin serotypes bind to Syt appears surprisingly divergent.

  2. Glycolipid binding preferences of Shiga toxin variants.

    Directory of Open Access Journals (Sweden)

    Sayali S Karve

    Full Text Available The major virulence factor of Shiga toxin producing E. coli, is Shiga toxin (Stx, an AB5 toxin that consists of a ribosomal RNA-cleaving A-subunit surrounded by a pentamer of receptor-binding B subunits. The two major isoforms, Stx1 and Stx2, and Stx2 variants (Stx2a-h significantly differ in toxicity. The exact reason for this toxicity difference is unknown, however different receptor binding preferences are speculated to play a role. Previous studies used enzyme linked immunosorbent assay (ELISA to study binding of Stx1 and Stx2a toxoids to glycolipid receptors. Here, we studied binding of holotoxin and B-subunits of Stx1, Stx2a, Stx2b, Stx2c and Stx2d to glycolipid receptors globotriaosylceramide (Gb3 and globotetraosylceramide (Gb4 in the presence of cell membrane components such as phosphatidylcholine (PC, cholesterol (Ch and other neutral glycolipids. In the absence of PC and Ch, holotoxins of Stx2 variants bound to mixtures of Gb3 with other glycolipids but not to Gb3 or Gb4 alone. Binding of all Stx holotoxins significantly increased in the presence of PC and Ch. Previously, Stx2a has been shown to form a less stable B-pentamer compared to Stx1. However, its effect on glycolipid receptor binding is unknown. In this study, we showed that even in the absence of the A-subunit, the B-subunits of both Stx1 and Stx2a were able to bind to the glycolipids and the more stable B-pentamer formed by Stx1 bound better than the less stable pentamer of Stx2a. B-subunit mutant of Stx1 L41Q, which shows similar stability as Stx2a B-subunits, lacked glycolipid binding, suggesting that pentamerization is more critical for binding of Stx1 than Stx2a.

  3. Eligibility Requirements

    Science.gov (United States)

    ... Home > Donating Blood > Eligibility Requirements Printable Version Eligibility Requirements This page uses Javascript. Your browser either doesn' ... donors » Weigh at least 110 lbs. Additional weight requirements apply for donors 18-years-old and younger ...

  4. Isothermal titration calorimetry and surface plasmon resonance allow quantifying substrate binding to different binding sites of Bacillus subtilis xylanase

    DEFF Research Database (Denmark)

    Cuyvers, Sven; Dornez, Emmie; Abou Hachem, Maher;

    2012-01-01

    Isothermal titration calorimetry and surface plasmon resonance were tested for their ability to study substrate binding to the active site (AS) and to the secondary binding site (SBS) of Bacillus subtilis xylanase A separately. To this end, three enzyme variants were compared. The first was a cat......Isothermal titration calorimetry and surface plasmon resonance were tested for their ability to study substrate binding to the active site (AS) and to the secondary binding site (SBS) of Bacillus subtilis xylanase A separately. To this end, three enzyme variants were compared. The first...

  5. Binding energies of hypernuclei and hypernuclear interactions

    Energy Technology Data Exchange (ETDEWEB)

    Bodmer, A.R. [Argonne National Lab., IL (United States)]|[Univ. of Illinois, Chicago, IL (United States). Dept. of Physics; Murali, S.; Usmani, Q.N. [Jamia Millia Islamia, New Delhi (India). Dept. of Physics

    1996-05-01

    In part 1 the effect of nuclear core dynamics on the binding energies of {Lambda} hypernuclei is discussed in the framework of variational correlated wave functions. In particular, the authors discuss a new rearrangement energy contribution and its effect on the core polarization. In part 2 they consider the interpretation of the {Lambda} single-particle energy in terms of basic {Lambda}-nuclear interactions using a local density approximation based on a Fermi hypernetted chain calculation of the A binding to nuclear matter. To account for the data strongly repulsive 3-body {Lambda}NN forces are required. Also in this framework they discuss core polarization for medium and heavier hypernuclei.

  6. Binding biological motion and visual features in working memory.

    Science.gov (United States)

    Ding, Xiaowei; Zhao, Yangfan; Wu, Fan; Lu, Xiqian; Gao, Zaifeng; Shen, Mowei

    2015-06-01

    Working memory mechanisms for binding have been examined extensively in the last decade, yet few studies have explored bindings relating to human biological motion (BM). Human BM is the most salient and biologically significant kinetic information encountered in everyday life and is stored independently from other visual features (e.g., colors). The current study explored 3 critical issues of BM-related binding in working memory: (a) how many BM binding units can be retained in working memory, (b) whether involuntarily object-based binding occurs during BM binding, and (c) whether the maintenance of BM bindings in working memory requires attention above and beyond that needed to maintain the constituent dimensions. We isolated motion signals of human BM from non-BM sources by using point-light displays as to-be-memorized BM and presented the participants colored BM in a change detection task. We found that working memory capacity for BM-color bindings is rather low; only 1 or 2 BM-color bindings could be retained in working memory regardless of the presentation manners (Experiments 1-3). Furthermore, no object-based encoding took place for colored BM stimuli regardless of the processed dimensions (Experiments 4 and 5). Central executive attention contributes to the maintenance of BM-color bindings, yet maintaining BM bindings in working memory did not require more central attention than did maintaining the constituent dimensions in working memory (Experiment 6). Overall, these results suggest that keeping BM bindings in working memory is a fairly resource-demanding process, yet central executive attention does not play a special role in this cross-module binding. PMID:25893683

  7. Evolution of off-lattice model proteins under ligand binding constraints

    Science.gov (United States)

    Nelson, Erik D.; Grishin, Nick V.

    2016-08-01

    We investigate protein evolution using an off-lattice polymer model evolved to imitate the behavior of small enzymes. Model proteins evolve through mutations to nucleotide sequences (including insertions and deletions) and are selected to fold and maintain a specific binding site compatible with a model ligand. We show that this requirement is, in itself, sufficient to maintain an ordered folding domain, and we compare it to the requirement of folding an ordered (but otherwise unrestricted) domain. We measure rates of amino acid change as a function of local environment properties such as solvent exposure, packing density, and distance from the active site, as well as overall rates of sequence and structure change, both along and among model lineages in star phylogenies. The model recapitulates essentially all of the behavior found in protein phylogenetic analyses, and predicts that amino acid substitution rates vary linearly with distance from the binding site.

  8. 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.

  9. Sequential memory: Binding dynamics

    Science.gov (United States)

    Afraimovich, Valentin; Gong, Xue; Rabinovich, Mikhail

    2015-10-01

    Temporal order memories are critical for everyday animal and human functioning. Experiments and our own experience show that the binding or association of various features of an event together and the maintaining of multimodality events in sequential order are the key components of any sequential memories—episodic, semantic, working, etc. We study a robustness of binding sequential dynamics based on our previously introduced model in the form of generalized Lotka-Volterra equations. In the phase space of the model, there exists a multi-dimensional binding heteroclinic network consisting of saddle equilibrium points and heteroclinic trajectories joining them. We prove here the robustness of the binding sequential dynamics, i.e., the feasibility phenomenon for coupled heteroclinic networks: for each collection of successive heteroclinic trajectories inside the unified networks, there is an open set of initial points such that the trajectory going through each of them follows the prescribed collection staying in a small neighborhood of it. We show also that the symbolic complexity function of the system restricted to this neighborhood is a polynomial of degree L - 1, where L is the number of modalities.

  10. Lectin binding in meningiomas.

    Science.gov (United States)

    Kleinert, R; Radner, H

    1987-01-01

    Forty-two meningiomas of different morphological sub-type were examined to determine their pattern of binding to 11 different lectins which characterize cell surface components such as carbohydrate residues. Histiocytic and xanthoma cells within meningiomas could be demonstrated with six different lectins: wheat germ agglutinin (WGA), peanut agglutinin (PNA) Bauhinia purpurea agglutinin (BPA), Helix pomatia agglutinin (HPA), Vicia fava agglutinin (VFA) and Soyabean agglutinin (SBA). Vascular elements including endothelial cells and intimal cells, bound Ulex europaeus agglutinin type 1 (UEA 1), WGA and HPA. The fibrous stroma in fibrous and fibroblastic meningiomas bound PNA, Laburnum alpinum agglutinin (LAA) and SBA. Tumour cells in meningotheliomatous meningiomas and some areas of anaplastic meningiomas bound Concanavalin A, PNA, LAA and VFA whereas tumour cells in fibrous and fibroblastic meningiomas bound BPA, LAA and VFA. Lectin binding has proved to be of value in detecting histiocytic and xanthoma cells together with vascular elements within meningiomas. In addition, the different lectin binding patterns allow different histological sub-types of meningioma to be distinguished although the biological significance of the binding patterns is unclear. PMID:3658105

  11. 对比分析视黄醇结合蛋白在高血压肾病早期检测的临床价值%To compare and analyse the clinical significance of the early diagnosis of the Hypertensive Renal Disease by the Retinol binding protein testing

    Institute of Scientific and Technical Information of China (English)

    丁峰

    2015-01-01

    Objective To analyse the clinical significance of the early diagnosis of the Hypertensive Renal Disease by the Retinol binding protein testing, in order to provide reference for clinic.Methods Choosing 113 samples of patients with Hypertensive Renal Disease for the experimental group, according to the course of hypertension can be divided into the first group of 40 patients, the second group of 36 patients and the third group of 37 patients, and choosing 40 samples of healthy for the control group,then all of them were testing the Retinol binding protein and the Cystatin C, and statistical analysis of data.Results Compared to the control group, there was obviously increased of the concentration level of the Retinol binding protein and the Cystatin C(P0.05) . The detection rate of the one phase group can be significantly improved than the single detection(P<0.05).Conclusions There is a certain clinical value of the Retinol binding protein detection for the patient monitoring, it can be effectively reduce the rate of missed diagnosis by jointing detection of the Retinol binding protein and the Cystatin C.%目的:探讨分析高血压肾病早期检测视黄醇结合蛋白的临床价值,为临床提供参考依据。方法:选择113例确诊为高血压肾病的患者设为试验组,根据高血压病程可分为一期组40人,二期组36人和三期组37人,选择同期体检健康者40人设为对照组,同时检测患者和对照者的血清视黄醇结合蛋白浓度水平和胱抑素C浓度水平,统计分析结果。结果:试验三个小组的患者视黄醇结合蛋白和胱抑素C浓度水平均明显高于对照组,差异有显著性(p<0.05),随着患者病程的增长,试验三个小组间的患者视黄醇结合蛋白和胱抑素C浓度水平呈现正相关升高(r=0.9997和r=0.9947);各组患者的视黄醇结合蛋白阳性检出率分别与对应组患者的胱抑素C阳性检出率两两比

  12. Computational Design of DNA-Binding Proteins.

    Science.gov (United States)

    Thyme, Summer; Song, Yifan

    2016-01-01

    Predicting the outcome of engineered and naturally occurring sequence perturbations to protein-DNA interfaces requires accurate computational modeling technologies. It has been well established that computational design to accommodate small numbers of DNA target site substitutions is possible. This chapter details the basic method of design used in the Rosetta macromolecular modeling program that has been successfully used to modulate the specificity of DNA-binding proteins. More recently, combining computational design and directed evolution has become a common approach for increasing the success rate of protein engineering projects. The power of such high-throughput screening depends on computational methods producing multiple potential solutions. Therefore, this chapter describes several protocols for increasing the diversity of designed output. Lastly, we describe an approach for building comparative models of protein-DNA complexes in order to utilize information from homologous sequences. These models can be used to explore how nature modulates specificity of protein-DNA interfaces and potentially can even be used as starting templates for further engineering. PMID:27094297

  13. Detection of secondary binding sites in proteins using fragment screening.

    Science.gov (United States)

    Ludlow, R Frederick; Verdonk, Marcel L; Saini, Harpreet K; Tickle, Ian J; Jhoti, Harren

    2015-12-29

    Proteins need to be tightly regulated as they control biological processes in most normal cellular functions. The precise mechanisms of regulation are rarely completely understood but can involve binding of endogenous ligands and/or partner proteins at specific locations on a protein that can modulate function. Often, these additional secondary binding sites appear separate to the primary binding site, which, for example for an enzyme, may bind a substrate. In previous work, we have uncovered several examples in which secondary binding sites were discovered on proteins using fragment screening approaches. In each case, we were able to establish that the newly identified secondary binding site was biologically relevant as it was able to modulate function by the binding of a small molecule. In this study, we investigate how often secondary binding sites are located on proteins by analyzing 24 protein targets for which we have performed a fragment screen using X-ray crystallography. Our analysis shows that, surprisingly, the majority of proteins contain secondary binding sites based on their ability to bind fragments. Furthermore, sequence analysis of these previously unknown sites indicate high conservation, which suggests that they may have a biological function, perhaps via an allosteric mechanism. Comparing the physicochemical properties of the secondary sites with known primary ligand binding sites also shows broad similarities indicating that many of the secondary sites may be druggable in nature with small molecules that could provide new opportunities to modulate potential therapeutic targets.

  14. Binding in short-term visual memory.

    Science.gov (United States)

    Wheeler, Mary E; Treisman, Anne M

    2002-03-01

    The integration of complex information in working memory, and its effect on capacity, shape the limits of conscious cognition. The literature conflicts on whether short-term visual memory represents information as integrated objects. A change-detection paradigm using objects defined by color with location or shape was used to investigate binding in short-term visual memory. Results showed that features from the same dimension compete for capacity, whereas features from different dimensions can be stored in parallel. Binding between these features can occur, but focused attention is required to create and maintain the binding over time, and this integrated format is vulnerable to interference. In the proposed model, working memory capacity is limited both by the independent capacity of simple feature stores and by demands on attention networks that integrate this distributed information into complex but unified thought objects. PMID:11900102

  15. The intact urokinase receptor is required for efficient vitronectin binding

    DEFF Research Database (Denmark)

    Høyer-Hansen, G; Behrendt, N; Ploug, M;

    1997-01-01

    The urokinase receptor (uPAR) is a receptor for both urokinase plasminogen activator (uPA) and the adhesion protein vitronectin. There are two forms of cell surface-bound uPAR; intact uPAR and a cleaved form, uPAR(2+3), which is formed by uPA-catalyzed cleavage of uPAR. In ligand-blotting experim...

  16. Hospital Compare

    Data.gov (United States)

    U.S. Department of Health & Human Services — Hospital Compare has information about the quality of care at over 4,000 Medicare-certified hospitals across the country. You can use Hospital Compare to find...

  17. Rapid binding of plasminogen to streptokinase in a catalytic complex reveals a three-step mechanism.

    Science.gov (United States)

    Verhamme, Ingrid M; Bock, Paul E

    2014-10-01

    Rapid kinetics demonstrate a three-step pathway of streptokinase (SK) binding to plasminogen (Pg), the zymogen of plasmin (Pm). Formation of a fluorescently silent encounter complex is followed by two conformational tightening steps reported by fluorescence quenches. Forward reactions were defined by time courses of biphasic quenching during complex formation between SK or its COOH-terminal Lys(414) deletion mutant (SKΔK414) and active site-labeled [Lys]Pg ([5-(acetamido)fluorescein]-D-Phe-Phe-Arg-[Lys]Pg ([5F]FFR-[Lys]Pg)) and by the SK dependences of the quench rates. Active site-blocked Pm rapidly displaced [5F]FFR-[Lys]Pg from the complex. The encounter and final SK ·[5F]FFR-[Lys]Pg complexes were weakened similarly by SK Lys(414) deletion and blocking of lysine-binding sites (LBSs) on Pg kringles with 6-aminohexanoic acid or benzamidine. Forward and reverse rates for both tightening steps were unaffected by 6-aminohexanoic acid, whereas benzamidine released constraints on the first conformational tightening. This indicated that binding of SK Lys(414) to Pg kringle 4 plays a role in recognition of Pg by SK. The substantially lower affinity of the final SK · Pg complex compared with SK · Pm is characterized by a ∼ 25-fold weaker encounter complex and ∼ 40-fold faster off-rates for the second conformational step. The results suggest that effective Pg encounter requires SK Lys(414) engagement and significant non-LBS interactions with the protease domain, whereas Pm binding additionally requires contributions of other lysines. This difference may be responsible for the lower affinity of the SK · Pg complex and the expression of a weaker "pro"-exosite for binding of a second Pg in the substrate mode compared with SK · Pm. PMID:25138220

  18. Rapid Binding of Plasminogen to Streptokinase in a Catalytic Complex Reveals a Three-step Mechanism*

    Science.gov (United States)

    Verhamme, Ingrid M.; Bock, Paul E.

    2014-01-01

    Rapid kinetics demonstrate a three-step pathway of streptokinase (SK) binding to plasminogen (Pg), the zymogen of plasmin (Pm). Formation of a fluorescently silent encounter complex is followed by two conformational tightening steps reported by fluorescence quenches. Forward reactions were defined by time courses of biphasic quenching during complex formation between SK or its COOH-terminal Lys414 deletion mutant (SKΔK414) and active site-labeled [Lys]Pg ([5-(acetamido)fluorescein]-d-Phe-Phe-Arg-[Lys]Pg ([5F]FFR-[Lys]Pg)) and by the SK dependences of the quench rates. Active site-blocked Pm rapidly displaced [5F]FFR-[Lys]Pg from the complex. The encounter and final SK·[5F]FFR-[Lys]Pg complexes were weakened similarly by SK Lys414 deletion and blocking of lysine-binding sites (LBSs) on Pg kringles with 6-aminohexanoic acid or benzamidine. Forward and reverse rates for both tightening steps were unaffected by 6-aminohexanoic acid, whereas benzamidine released constraints on the first conformational tightening. This indicated that binding of SK Lys414 to Pg kringle 4 plays a role in recognition of Pg by SK. The substantially lower affinity of the final SK·Pg complex compared with SK·Pm is characterized by a ∼25-fold weaker encounter complex and ∼40-fold faster off-rates for the second conformational step. The results suggest that effective Pg encounter requires SK Lys414 engagement and significant non-LBS interactions with the protease domain, whereas Pm binding additionally requires contributions of other lysines. This difference may be responsible for the lower affinity of the SK·Pg complex and the expression of a weaker “pro”-exosite for binding of a second Pg in the substrate mode compared with SK·Pm. PMID:25138220

  19. Binding of Diphtheria Toxin to Phospholipids in Liposomes

    Science.gov (United States)

    Alving, Carl R.; Iglewski, Barbara H.; Urban, Katharine A.; Moss, Joel; Richards, Roberta L.; Sadoff, Jerald C.

    1980-04-01

    Diphtheria toxin bound to the phosphate portion of some, but not all, phospholipids in liposomes. Liposomes consisting of dimyristoyl phosphatidylcholine and cholesterol did not bind toxin. Addition of 20 mol% (compared to dimyristoyl phosphatidylcholine) of dipalmitoyl phosphatidic acid, dicetyl phosphate, phosphatidylinositol phosphate, cardiolipin, or phosphatidylserine in the liposomes resulted in substantial binding of toxin. Inclusion of phosphatidylinositol in dimyristol phosphatidylcholine / cholesterol liposomes did not result in toxin binding. The calcium salt of dipalmitoyl phosphatidic acid was more effective than the sodium salt, and the highest level of binding occurred with liposomes consisting only of dipalmitoyl phosphatidic acid (calcium salt) and cholesterol. Binding of toxin to liposomes was dependent on pH, and the pattern of pH dependence varied with liposomes having different compositions. Incubation of diphtheria toxin with liposomes containing dicetyl phosphate resulted in maximal binding at pH 3.6, whereas binding to liposomes containing phosphatidylinositol phosphate was maximal above pH 7. Toxin did not bind to liposomes containing 20 mol% of a free fatty acid (palmitic acid) or a sulfated lipid (3-sulfogalactosylceramide). Toxin binding to dicetyl phosphate or phosphatidylinositol phosphate was inhibited by UTP, ATP, phosphocholine, or p-nitrophenyl phosphate, but not by uracil. We conclude that (a) diphtheria toxin binds specifically to the phosphate portion of certain phospholipids, (b) binding to phospholipids in liposomes is dependent on pH, but is not due only to electrostatic interaction, and (c) binding may be strongly influenced by the composition of adjacent phospholipids that do not bind toxin. We propose that a minor membrane phospholipid (such as phosphatidylinositol phosphate or phosphatidic acid), or that some other phosphorylated membrane molecule (such as a phosphoprotein) may be important in the initial binding of

  20. Aspects of Protein, Chemistry, Part II: Oxygen-Binding Proteins

    Science.gov (United States)

    Nixon, J. E.

    1977-01-01

    Compares differences in function and behavior of two oxygen-binding proteins, myoglobin found in muscle and hemoglobin found in blood. Describes the mechanism of oxygen-binding and allosteric effect in hemoglobin; also describes the effect of pH on the affinity of hemoglobin for oxygen. (CS)

  1. Extrapolations of nuclear binding energies from new linear mass relations

    DEFF Research Database (Denmark)

    Hove, D.; Jensen, A. S.; Riisager, K.

    2013-01-01

    We present a method to extrapolate nuclear binding energies from known values for neighboring nuclei. We select four specific mass relations constructed to eliminate smooth variation of the binding energy as function nucleon numbers. The fast odd-even variations are avoided by comparing nuclei...

  2. Cooperative binding of copper(I) to the metal binding domains in Menkes disease protein

    DEFF Research Database (Denmark)

    Jensen, P Y; Bonander, N; Møller, L B;

    1999-01-01

    We have optimised the overexpression and purification of the N-terminal end of the Menkes disease protein expressed in Escherichia coli, containing one, two and six metal binding domains (MBD), respectively. The domain(s) have been characterised using circular dichroism (CD) and fluorescence...... spectroscopy, and their copper(I) binding properties have been determined. Structure prediction derived from far-UV CD indicates that the secondary structure is similar in the three proteins and dominated by beta-sheet. The tryptophan fluorescence maximum is blue-shifted in the constructs containing two...... and six MBDs relative to the monomer, suggesting more structurally buried tryptophan(s), compared to the single MBD construct. Copper(I) binding has been studied by equilibrium dialysis under anaerobic conditions. We show that the copper(I) binding to constructs containing two and six domains...

  3. Temperature dependence of estrogen binding: importance of a subzone in the ligand binding domain of a novel piscine estrogen receptor.

    Science.gov (United States)

    Tan, N S; Frecer, V; Lam, T J; Ding, J L

    1999-11-11

    The full length estrogen receptor from Oreochromis aureus (OaER) was cloned and expressed in vitro and in vivo as a functional transcription factor. Amino acid residues involved in the thermal stability of the receptor are located at/near subzones beta1 and beta3, which are highly conserved in other non-piscine species but not in OaER. Hormone binding studies, however, indicate that OaER is thermally stable but exhibited a approximately 3-fold reduced affinity for estrogen at elevated temperatures. Transfection of OaER into various cell lines cultured at different temperatures displayed a significant estrogen dose-response shift compared with that of chicken ER (cER). At 37 degrees C, OaER requires approximately 80-fold more estrogen to achieve half-maximal stimulation of CAT. Lowering of the incubation temperature from 37 degrees C to 25 degrees C or 20 degrees C resulted in a 4-fold increase in its affinity for estrogen. The thermally deficient transactivation of OaER at temperatures above 25 degrees C was fully prevented by high levels of estrogen. Thus, compared to cER, the OaER exhibits reduced affinity for estrogen at elevated temperature as reflected in its deficient transactivation capability. Amino acid replacements of OaER beta3 subzones with corresponding amino acids from cER could partially rescue this temperature sensitivity. The three-dimensional structure of the OaER ligand binding domain (LBD) was modelled based on conformational similarity and sequence homology with human RXRalpha apo, RARgamma holo and ERalpha LBDs. Unliganded and 17beta-estradiol-liganded OaER LBD retained the overall folding pattern of the nuclear receptor LBDs. The residues at/near the subzone beta3 of the LBD constitute the central core of OaER structure. Thus, amino acid alteration at this region potentially alters the structure and consequently its temperature-dependent ligand binding properties. PMID:10559464

  4. Oligosaccharide binding to barley alpha-amylase 1

    DEFF Research Database (Denmark)

    Robert, X.; Haser, R.; Mori, H.;

    2005-01-01

    Enzymatic subsite mapping earlier predicted 10 binding subsites in the active site substrate binding cleft of barley alpha-amylase isozymes. The three-dimensional structures of the oligosaccharide complexes with barley alpha-amylase isozyme 1 (AMY1) described here give for the first time a thorough...... in barley alpha-amylase isozyme 2 (AMY2), and the sugar binding modes are compared between the two isozymes. The "sugar tongs" surface binding site discovered in the AMY1-thio-DP4 complex is confirmed in the present work. A site that putatively serves as an entrance for the substrate to the active site...

  5. Megalin binds and mediates cellular internalization of folate binding protein

    DEFF Research Database (Denmark)

    Birn, Henrik; Zhai, Xiaoyue; Holm, Jan;

    2005-01-01

    Folate is an essential vitamin involved in a number of biological processes. High affinity folate binding proteins (FBPs) exist both as glycosylphosphatidylinositol-linked, membrane associated folate binding proteins and as soluble FBPs in plasma and some secretory fluids such as milk, saliva...... to bind and mediate cellular uptake of FBP. Surface plasmon resonance analysis shows binding of bovine and human milk FBP to immobilized megalin, but not to low density lipoprotein receptor related protein. Binding of (125)I-labeled folate binding protein (FBP) to sections of kidney proximal tubule, known...... to express high levels of megalin, is inhibitable by excess unlabeled FBP and by receptor associated protein, a known inhibitor of binding to megalin. Immortalized rat yolk sac cells, representing an established model for studying megalin-mediated uptake, reveal (125)I-labeled FBP uptake which is inhibited...

  6. Pretreatment of plasma samples by a novel hollow fiber centrifugal ultrafiltration technique for the determination of plasma protein binding of three coumarins using acetone as protein binding releasing agent.

    Science.gov (United States)

    Li, Junmei; Shi, Qingwen; Jiang, Ye; Liu, Yan

    2015-09-15

    A novel and practical sample pretreatment method based on hollow fiber centrifugal ultrafiltration (HFCF-UF) was developed to determine plasma protein binding by using HPLC. The samples for analyzing unbound and total concentrations could be prepared in parallel simultaneously by the same device. It only required centrifugation for a short time and the filtrate could be injected directly for HPLC analysis without further treatment. Coumarins were selected as the model drugs. Acetone was chosen as the releasing agent to free the binding drug from the drug-protein complex for the total drug concentration determination. Non-specific bindings (NSBs) between the analytes and hollow fiber membrane materials were investigated. The type and volume of protein binding releaser were optimized. Additionally, centrifugal speed and centrifugal time were considered. Under the optimized conditions, the absolute recovery rates of the unbound and total concentrations were in the range of 97.5-100.9% for the three analytes. The limits of detection were in the range of 0.0135-0.0667μgmL(-1). In vitro plasma protein binding of the three coumarins was determined at three concentrations using the validated method and the relative standard deviations (RSDs) were less than 3.4%. Compared with traditional method, the HFCF-UF method is simple to run, no specialized equipment requirement and is a more accurate plasma pretreatment procedure with almost excellent drug-protein binding equilibrium. Therefore, this method can be applied to determine the plasma protein binding in clinical practice. It also provides a reliable alternative for accurate monitoring of unbound or total drug concentration in therapeutic drug monitoring (TDM).

  7. Cortical and subcortical 5-HT2A receptor binding in neuroleptic-naive first-episode schizophrenic patients

    DEFF Research Database (Denmark)

    Erritzoe, David; Rasmussen, Hans; Kristiansen, Klaus Nyegaard;

    2008-01-01

    .5+/-5.7 years) and gender underwent a 40 min positron emission tomography (PET) study using the 5-HT(2A) antagonist, [(18)F]altanserin, as a radioligand. PET images were co-registered to 3 T magnetic resonance images (MRIs) for each individual subject, and ROIs were applied automatically onto the individual...... in the caudate nucleus was detected in the group of schizophrenic patients (0.7+/-0.1) when compared to the healthy controls (0.5+/-0.3) (p=0.02). Our results confirm other in vivo findings of no difference in cortical 5-HT(2A) receptor binding between first-episode antipsychotic-naive schizophrenic patients...... and age- and gender-matched healthy control subjects. However, a preliminary finding of increased 5-HT(2A) binding in the caudate nucleus requires further investigation to explore the relation of subcortical and cortical 5-HT(2A) receptor binding....

  8. Arsenic at very low concentrations alters glucocorticoid receptor (GR)-mediated gene activation but not GR-mediated gene repression: complex dose-response effects are closely correlated with levels of activated GR and require a functional GR DNA binding domain.

    Science.gov (United States)

    Bodwell, Jack E; Kingsley, Lauren A; Hamilton, Joshua W

    2004-08-01

    Arsenic (As) contamination of drinking water is considered a principal environmental health threat throughout the world. Chronic intake is associated with an increased risk of cancer, diabetes, and cardiovascular disease, and recent studies suggest increased health risks at levels as low as 5-10 ppb. We report here that 0.05-1 microM (6-120 ppb) As showed stimulatory effects on glucocorticoid receptor (GR)-mediated gene activation in rat EDR3 hepatoma cells of both the endogenous tyrosine aminotransferase (TAT) gene and the reporter genes containing TAT glucocorticoid response elements. At slightly higher concentrations (1-3 microM), the effects of As became inhibitory. Thus, over this narrow concentration range, the effects of As changed from a 2- to 4-fold stimulation to a greater than 2-fold suppression in activity. Interestingly, the inhibitory effect of GR on both AP1- and NF-kappa B-mediated gene activation was not affected by As. The magnitude of GR stimulation and inhibition by As was highly dependent on the cellular level of hormone-activated GR. Mutational deletion studies indicated that the central DNA binding domain (DBD) of GR is the minimal region required for the As effect and does not require free sulfhydryls. Point mutations located within the DBD that have known structural consequences significantly altered the GR response to As. In particular, point mutations in the DBD that confer a DNA-bound GR confirmation abolished the low dose As stimulatory effect but enhanced the inhibitory response, further indicating that the DBD is important for mediating these As effects. PMID:15310238

  9. Software requirements

    CERN Document Server

    Wiegers, Karl E

    2003-01-01

    Without formal, verifiable software requirements-and an effective system for managing them-the programs that developers think they've agreed to build often will not be the same products their customers are expecting. In SOFTWARE REQUIREMENTS, Second Edition, requirements engineering authority Karl Wiegers amplifies the best practices presented in his original award-winning text?now a mainstay for anyone participating in the software development process. In this book, you'll discover effective techniques for managing the requirements engineering process all the way through the development cy

  10. Molecular basis for oligomeric-DNA binding and episome maintenance by KSHV LANA.

    Directory of Open Access Journals (Sweden)

    John F Domsic

    Full Text Available LANA is the KSHV-encoded terminal repeat binding protein essential for viral replication and episome maintenance during latency. We have determined the X-ray crystal structure of LANA C-terminal DNA binding domain (LANADBD to reveal its capacity to form a decameric ring with an exterior DNA binding surface. The dimeric core is structurally similar to EBV EBNA1 with an N-terminal arm that regulates DNA binding and is required for replication function. The oligomeric interface between LANA dimers is dispensable for single site DNA binding, but is required for cooperative DNA binding, replication function, and episome maintenance. We also identify a basic patch opposite of the DNA binding surface that is responsible for the interaction with BRD proteins and contributes to episome maintenance function. The structural features of LANADBD suggest a novel mechanism of episome maintenance through DNA-binding induced oligomeric assembly.

  11. Sequence similarity between the erythrocyte binding domain 1 of the Plasmodium vivax Duffy binding protein and the V3 loop of HIV-1 strain MN reveals binding residues for the Duffy Antigen Receptor for Chemokines

    Directory of Open Access Journals (Sweden)

    Garry Robert F

    2011-01-01

    Full Text Available Abstract Background The surface glycoprotein (SU, gp120 of the human immunodeficiency virus (HIV must bind to a chemokine receptor, CCR5 or CXCR4, to invade CD4+ cells. Plasmodium vivax uses the Duffy Binding Protein (DBP to bind the Duffy Antigen Receptor for Chemokines (DARC and invade reticulocytes. Results Variable loop 3 (V3 of HIV-1 SU and domain 1 of the Plasmodium vivax DBP share a sequence similarity. The site of amino acid sequence similarity was necessary, but not sufficient, for DARC binding and contained a consensus heparin binding site essential for DARC binding. Both HIV-1 and P. vivax can be blocked from binding to their chemokine receptors by the chemokine, RANTES and its analog AOP-RANTES. Site directed mutagenesis of the heparin binding motif in members of the DBP family, the P. knowlesi alpha, beta and gamma proteins abrogated their binding to erythrocytes. Positively charged residues within domain 1 are required for binding of P. vivax and P. knowlesi erythrocyte binding proteins. Conclusion A heparin binding site motif in members of the DBP family may form part of a conserved erythrocyte receptor binding pocket.

  12. Promoter-distal RNA polymerase II binding discriminates active from inactive CCAAT/ enhancer-binding protein beta binding sites

    Science.gov (United States)

    Savic, Daniel; Roberts, Brian S.; Carleton, Julia B.; Partridge, E. Christopher; White, Michael A.; Cohen, Barak A.; Cooper, Gregory M.; Gertz, Jason; Myers, Richard M.

    2015-01-01

    Transcription factors (TFs) bind to thousands of DNA sequences in mammalian genomes, but most of these binding events appear to have no direct effect on gene expression. It is unclear why only a subset of TF bound sites are actively involved in transcriptional regulation. Moreover, the key genomic features that accurately discriminate between active and inactive TF binding events remain ambiguous. Recent studies have identified promoter-distal RNA polymerase II (RNAP2) binding at enhancer elements, suggesting that these interactions may serve as a marker for active regulatory sequences. Despite these correlative analyses, a thorough functional validation of these genomic co-occupancies is still lacking. To characterize the gene regulatory activity of DNA sequences underlying promoter-distal TF binding events that co-occur with RNAP2 and TF sites devoid of RNAP2 occupancy using a functional reporter assay, we performed cis-regulatory element sequencing (CRE-seq). We tested more than 1000 promoter-distal CCAAT/enhancer-binding protein beta (CEBPB)-bound sites in HepG2 and K562 cells, and found that CEBPB-bound sites co-occurring with RNAP2 were more likely to exhibit enhancer activity. CEBPB-bound sites further maintained substantial cell-type specificity, indicating that local DNA sequence can accurately convey cell-type–specific regulatory information. By comparing our CRE-seq results to a comprehensive set of genome annotations, we identified a variety of genomic features that are strong predictors of regulatory element activity and cell-type–specific activity. Collectively, our functional assay results indicate that RNAP2 occupancy can be used as a key genomic marker that can distinguish active from inactive TF bound sites. PMID:26486725

  13. comparative Advertising

    OpenAIRE

    Anderson, Simon P.; Régis Renault

    2006-01-01

    Consumer information on products affects competition and profits. We analyze firms' decisions to impart product information through advertising: comparative advertising also allows them to impart information about rivals' products. If firms sell products of similar qualities, both want to advertise detailed product information that enables consumers to determine their matches: there is no role for comparative advertising. If qualities are sufficiently dissimilar, the high-quality one will not...

  14. Energy requirements

    NARCIS (Netherlands)

    Hulzebos, Christian V.; Sauer, Pieter J. J.

    2007-01-01

    The determination of the appropriate energy and nutritional requirements of a newborn infant requires a clear goal of the energy and other compounds to be administered, valid methods to measure energy balance and body composition, and knowledge of the neonatal metabolic capacities. Providing an appr

  15. Protein-ligand binding affinities from large-scale quantum mechanical simulations

    OpenAIRE

    Fox, Stephen J.

    2012-01-01

    The accurate prediction of protein-drug binding affinities is a major aim of computational drug optimisation and development. A quantitative measure of binding affinity is provided by the free energy of binding, and such calculations typically require extensive configurational sampling of entities such as proteins with thousands of atoms. Current binding free energy methods use force fields to perform the configurational sampling and to compute interaction energies. Due to the empirical natur...

  16. Highly selective ligand binding by Methylophilus methylotrophus cytochrome c''.

    Science.gov (United States)

    Quintas, Pedro O; Catarino, Teresa; Todorovic, Smilja; Turner, David L

    2011-06-28

    Cytochrome c'' (cyt c'') from Methylophilus methylotrophus is unusual insofar as the heme has two axial histidine ligands in the oxidized form but one is detached when the protein is reduced. Despite cyt c'' having an axial site available for binding small ligands, we show here that only NO binds readily to the ferrous cyt c''. Binding of CO, as well as CN(-), on the other hand requires considerable structural reorganization, or reduction of the disulfide bridge close to the heme. Standard free energies for the binding of NO and CO reveal high selectivity of the ferrous cyt c'' for NO, indicating its putative physiological role. In this work, we characterize in detail the kinetics of NO binding and the structural features of the Fe(2+)-NO adduct by stopped-flow and resonance Raman spectroscopy, respectively.

  17. An effective approach for identification of in vivo protein-DNA binding sites from paired-end ChIP-Seq data

    Directory of Open Access Journals (Sweden)

    Wilson Zoe A

    2010-02-01

    Full Text Available Abstract Background ChIP-Seq, which combines chromatin immunoprecipitation (ChIP with high-throughput massively parallel sequencing, is increasingly being used for identification of protein-DNA interactions in vivo in the genome. However, to maximize the effectiveness of data analysis of such sequences requires the development of new algorithms that are able to accurately predict DNA-protein binding sites. Results Here, we present SIPeS (Site Identification from Paired-end Sequencing, a novel algorithm for precise identification of binding sites from short reads generated by paired-end solexa ChIP-Seq technology. In this paper we used ChIP-Seq data from the Arabidopsis basic helix-loop-helix transcription factor ABORTED MICROSPORES (AMS, which is expressed within the anther during pollen development, the results show that SIPeS has better resolution for binding site identification compared to two existing ChIP-Seq peak detection algorithms, Cisgenome and MACS. Conclusions When compared to Cisgenome and MACS, SIPeS shows better resolution for binding site discovery. Moreover, SIPeS is designed to calculate the mappable genome length accurately with the fragment length based on the paired-end reads. Dynamic baselines are also employed to effectively discriminate closely adjacent binding sites, for effective binding sites discovery, which is of particular value when working with high-density genomes.

  18. Stepwise bending of DNA by a single TATA-box Binding Protein

    CERN Document Server

    Tolic-Norrelykke, S F; Pavone, F S; Berg-Sørensen, K; Oddershede, L B; Tolic-Norrelykke, Simon F.; Rasmusssen, Mette B.; Pavone, Francesco S.; Berg-Sorensen, Kirstine; Oddershede, Lene B.

    2006-01-01

    The TATA-box Binding Protein (TBP) is required by all three eukaryotic RNA polymerases for the initiation of transcription from most promoters. TBP recognizes, binds to, and bends promoter sequences called ``TATA-boxes'' in the DNA. We present results from the study of individual Saccharomyces cerevisia TBPs interacting with single DNA molecules containing a TATA-box. Using video microscopy, we observed the Brownian motion of beads tethered by short surface-bound DNA. When TBP binds to and bends the DNA, the conformation of the DNA changes and the amplitude of Brownian motion of the tethered bead is reduced compared to that of unbent DNA. We detected individual binding and dissociation events and derived kinetic parameters for the process. Dissociation was induced by increasing the salt concentration or by directly pulling on the tethered bead using optical tweezers. In addition to the well-defined free and bound classes of Brownian motion, we observed another two classes of motion. These extra classes were i...

  19. Hemoglobin isoform differentiation and allosteric regulation of oxygen binding in the turtle, Trachemys scripta

    DEFF Research Database (Denmark)

    Damsgaard, Christian; Storz, Jay F.; Hoffmann, Federico G.;

    2013-01-01

    When freshwater turtles acclimatize to winter hibernation, there is a gradual transition from aerobic to anaerobic metabolism, which may require adjustments of blood O2 transport before turtles become anoxic. Here, we report the effects of protons, anionic cofactors, and temperature on the O2......-binding properties of isolated hemoglobin (Hb) isoforms, HbA and HbD, in the turtle Trachemys scripta. We determined the primary structures of the constituent subunits of the two Hb isoforms, and we related the measured functional properties to differences in O2 affinity between untreated hemolysates from...... turtles that were acclimated to normoxia and anoxia. Our data show that HbD has a consistently higher O2 affinity compared with HbA, whereas Bohr and temperature effects, as well as thiol reactivity, are similar. Although sequence data show amino acid substitutions at two known β-chain ATP-binding site...

  20. Analytic QCD Binding Potentials

    CERN Document Server

    Fried, H M; Grandou, T; Sheu, Y -M

    2011-01-01

    This paper applies the analytic forms of a recent non-perturbative, manifestly gauge- and Lorentz-invariant description (of the exchange of all possible virtual gluons between quarks ($Q$) and/or anti-quarks ($\\bar{Q}$) in a quenched, eikonal approximation) to extract analytic forms for the binding potentials generating a model $Q$-$\\bar{Q}$ "pion", and a model $QQQ$ "nucleon". Other, more complicated $Q$, $\\bar{Q}$ contributions to such color-singlet states may also be identified analytically. An elementary minimization technique, relevant to the ground states of such bound systems, is adopted to approximate the solutions to a more proper, but far more complicated Schroedinger/Dirac equation; the existence of possible contributions to the pion and nucleon masses due to spin, angular momentum, and "deformation" degrees of freedom is noted but not pursued. Neglecting electromagnetic and weak interactions, this analysis illustrates how the one new parameter making its appearance in this exact, realistic formali...

  1. Diversity of Cyclic Di-GMP-Binding Proteins and Mechanisms.

    Science.gov (United States)

    Chou, Shan-Ho; Galperin, Michael Y

    2016-01-01

    Cyclic di-GMP (c-di-GMP) synthetases and hydrolases (GGDEF, EAL, and HD-GYP domains) can be readily identified in bacterial genome sequences by using standard bioinformatic tools. In contrast, identification of c-di-GMP receptors remains a difficult task, and the current list of experimentally characterized c-di-GMP-binding proteins is likely incomplete. Several classes of c-di-GMP-binding proteins have been structurally characterized; for some others, the binding sites have been identified; and for several potential c-di-GMP receptors, the binding sites remain to be determined. We present here a comparative structural analysis of c-di-GMP-protein complexes that aims to discern the common themes in the binding mechanisms that allow c-di-GMP receptors to bind it with (sub)micromolar affinities despite the 1,000-fold excess of GTP. The available structures show that most receptors use their Arg and Asp/Glu residues to bind c-di-GMP monomers, dimers, or tetramers with stacked guanine bases. The only exception is the EAL domains that bind c-di-GMP monomers in an extended conformation. We show that in c-di-GMP-binding signature motifs, Arg residues bind to the O-6 and N-7 atoms at the Hoogsteen edge of the guanine base, while Asp/Glu residues bind the N-1 and N-2 atoms at its Watson-Crick edge. In addition, Arg residues participate in stacking interactions with the guanine bases of c-di-GMP and the aromatic rings of Tyr and Phe residues. This may account for the presence of Arg residues in the active sites of every receptor protein that binds stacked c-di-GMP. We also discuss the implications of these structural data for the improved understanding of the c-di-GMP signaling mechanisms.

  2. Quarkonium Binding and Entropic Force

    CERN Document Server

    Satz, Helmut

    2015-01-01

    A Q-Qbar bound state represents a balance between repulsive kinetic and attractive potential energy. In a hot quark-gluon plasma, the interaction potential experiences medium effects. Color screening modifies the attractive binding force between the quarks, while the increase of entropy with Q-Qbar separation gives rise to a growing repulsion. We study the role of these phenomena for in-medium Q-Qbar binding and dissociation. It is found that the relevant potential for Q-Qbar binding is the free energy F; with increasing Q-Qbar separation, further binding through the internal energy U is compensated by repulsive entropic effects.

  3. Interaction of zinc and cobalt with dipeptides and their DNA binding studies

    Indian Academy of Sciences (India)

    P Rabindra Reddy; M Radhika; K Srinivas Rao

    2004-06-01

    Interactions of zinc and cobalt with peptides cysteinylglycine and histidylglycine have been studied. The binding modes were identified and geometry assigned. Stabilities of these complexes and their ability to bind DNA have been investigated. It is demonstrated that only zinc complexes bind DNA as compared to cobalt complexes.

  4. The Receptor Binding Domain of Botulinum Neurotoxin Stereotype C Binds Phosphoinositides

    Energy Technology Data Exchange (ETDEWEB)

    Zhang, Yanfeng; Varnum, Susan M.

    2012-03-01

    Botulinum neurotoxins (BoNTs) are the most toxic proteins known for humans and animals with an extremely low LD50 of {approx} 1 ng/kg. BoNTs generally require a protein and a ganglioside on the cell membrane surface for binding, which is known as a 'dual receptor' mechanism for host intoxication. Recent studies have suggested that in addition to gangliosides, other membrane lipids such as phosphoinositides may be involved in the interactions with the receptor binding domain (HCR) of BoNTs for better membrane penetration. Here, using two independent lipid-binding assays, we tested the interactions of BoNT/C-HCR with lipids in vitro. BoNT/C-HCR was found to bind negatively charged phospholipids, preferentially phosphoinositides. Additional interactions to phosphoinositides may help BoNT/C bind membrane more tightly and transduct signals for subsequent steps of intoxication. Our results provide new insights into the mechanisms of host cell membrane recognition by BoNTs.

  5. Membrane potential mediates the cellular binding of nanoparticles

    Science.gov (United States)

    Shin, Edwin H.; Li, Ye; Kumar, Umesh; Sureka, Hursh V.; Zhang, Xianren; Payne, Christine K.

    2013-06-01

    The use of nanoparticles for cellular therapeutic or sensing applications requires nanoparticles to bind, or adhere, to the cell surface. While nanoparticle parameters such as size, shape, charge, and composition are important factors in cellular binding, the cell itself must also be considered. All cells have an electrical potential across the plasma membrane driven by an ion gradient. Under standard conditions the ion gradient will result in a -10 to -100 mV potential across the membrane with a net negative charge on the cytosolic face. Using a combination of flow cytometry and fluorescence microscopy experiments and dissipative particle dynamics simulations, we have found that a decrease in membrane potential leads to decreased cellular binding of anionic nanoparticles. The decreased cellular binding of anionic nanoparticles is a general phenomenon, independent of depolarization method, nanoparticle composition, and cell type. Increased membrane potential reverses this trend resulting in increased binding of anionic nanoparticles. The cellular binding of cationic nanoparticles is minimally affected by membrane potential due to the interaction of cationic nanoparticles with cell surface proteins. The influence of membrane potential on the cellular binding of nanoparticles is especially important when considering the use of nanoparticles in the treatment or detection of diseases, such as cancer, in which the membrane potential is decreased.The use of nanoparticles for cellular therapeutic or sensing applications requires nanoparticles to bind, or adhere, to the cell surface. While nanoparticle parameters such as size, shape, charge, and composition are important factors in cellular binding, the cell itself must also be considered. All cells have an electrical potential across the plasma membrane driven by an ion gradient. Under standard conditions the ion gradient will result in a -10 to -100 mV potential across the membrane with a net negative charge on the

  6. Comparative hemorheology

    OpenAIRE

    Başkurt, Oğuz K.; Meiselman, Herbert J.

    2013-01-01

    Comparative data on blood composition and blood flow properties indicate different levels of interspecies variation for several parameters. Hematocrit and hemoglobin levels have relatively low variability among mammals, while mean cell volume and red blood cell (RBC) count are more variable. There is also a difference of variability between high and low shear rate blood viscosity in mammals, with low shear rate viscosity having a higher degree of interspecies variation. This observation paral...

  7. STARD4 Membrane Interactions and Sterol Binding.

    Science.gov (United States)

    Iaea, David B; Dikiy, Igor; Kiburu, Irene; Eliezer, David; Maxfield, Frederick R

    2015-08-01

    The steroidogenic acute regulatory protein-related lipid transfer (START) domain family is defined by a conserved 210-amino acid sequence that folds into an α/β helix-grip structure. Members of this protein family bind a variety of ligands, including cholesterol, phospholipids, sphingolipids, and bile acids, with putative roles in nonvesicular lipid transport, metabolism, and cell signaling. Among the soluble START proteins, STARD4 is expressed in most tissues and has previously been shown to transfer sterol, but the molecular mechanisms of membrane interaction and sterol binding remain unclear. In this work, we use biochemical techniques to characterize regions of STARD4 and determine their role in membrane interaction and sterol binding. Our results show that STARD4 interacts with anionic membranes through a surface-exposed basic patch and that introducing a mutation (L124D) into the Omega-1 (Ω1) loop, which covers the sterol binding pocket, attenuates sterol transfer activity. To gain insight into the attenuating mechanism of the L124D mutation, we conducted structural and biophysical studies of wild-type and L124D STARD4. These studies show that the L124D mutation reduces the conformational flexibility of the protein, resulting in a diminished level of membrane interaction and sterol transfer. These studies also reveal that the C-terminal α-helix, and not the Ω1 loop, partitions into the membrane bilayer. On the basis of these observations, we propose a model of STARD4 membrane interaction and sterol binding and release that requires dynamic movement of both the Ω1 loop and membrane insertion of the C-terminal α-helix.

  8. Alcohol Binding to the Odorant Binding Protein LUSH: Multiple Factors Affecting Binding Affinities

    OpenAIRE

    Ader, Lauren; Jones, David N. M.; Lin, Hai

    2010-01-01

    Density function theory (DFT) calculations have been carried out to investigate the binding of alcohols to the odorant binding protein LUSH from Drosophila melanogaster. LUSH is one of the few proteins known to bind to ethanol at physiologically relevant concentrations and where high-resolution structural information is available for the protein bound to alcohol at these concentrations. The structures of the LUSH–alcohol complexes identify a set of specific hydrogen-bonding interactions as cr...

  9. Triplex formation by morpholino oligodeoxyribonucleotides in the HER-2/neu promoter requires the pyrimidine motif

    OpenAIRE

    Basye, Jenny; Trent, John O.; Gao, Daquan; Ebbinghaus, Scot W.

    2001-01-01

    Triplex-forming oligonucleotides (TFOs) are good candidates to be used as site-specific DNA-binding agents. Two obstacles encountered with TFOs are susceptibility to nuclease activity and a requirement for magnesium for triplex formation. Morpholino oligonucleotides were shown in one study to form triplexes in the absence of magnesium. In the current study, we have compared phosphodiester and morpholino oligonucleotides targeting a homopurine–homopyrimidine region in the human HER2/neu promot...

  10. Comparative Advantage

    DEFF Research Database (Denmark)

    Zhang, Jie; Jensen, Camilla

    2007-01-01

    The objective of this paper is to explain international tourism flows in terms of supply-side factors associated with its production in destination countries. Unlike demand-oriented analysis, the study suggests that there are parallels between tourism and international trade flows...... that are typically explained from the supply-side variables, the comparative advantage of the exporting countries. A simple model is proposed and tested. The results render strong support for the relevance of supply-side factors such as natural endowments, technology, and infrastructure in explaining international...... tourism flows....

  11. Viral Coat Protein Peptides with Limited Sequence Homology Bind Similar Domains of Alfalfa Mosaic Virus and Tobacco Streak Virus RNAs

    Science.gov (United States)

    Swanson, Maud M.; Ansel-McKinney, Patricia; Houser-Scott, Felicia; Yusibov, Vidadi; Loesch-Fries, L. Sue; Gehrke, Lee

    1998-01-01

    An unusual and distinguishing feature of alfalfa mosaic virus (AMV) and ilarviruses such as tobacco streak virus (TSV) is that the viral coat protein is required to activate the early stages of viral RNA replication, a phenomenon known as genome activation. AMV-TSV coat protein homology is limited; however, they are functionally interchangeable in activating virus replication. For example, TSV coat protein will activate AMV RNA replication and vice versa. Although AMV and TSV coat proteins have little obvious amino acid homology, we recently reported that they share an N-terminal RNA binding consensus sequence (Ansel-McKinney et al., EMBO J. 15:5077–5084, 1996). Here, we biochemically compare the binding of chemically synthesized peptides that include the consensus RNA binding sequence and lysine-rich (AMV) or arginine-rich (TSV) environment to 3′-terminal TSV and AMV RNA fragments. The arginine-rich TSV coat protein peptide binds viral RNA with lower affinity than the lysine-rich AMV coat protein peptides; however, the ribose moieties protected from hydroxyl radical attack by the two different peptides are localized in the same area of the predicted RNA structures. When included in an infectious inoculum, both AMV and TSV 3′-terminal RNA fragments inhibited AMV RNA replication, while variant RNAs unable to bind coat protein did not affect replication significantly. The data suggest that RNA binding and genome activation functions may reside in the consensus RNA binding sequence that is apparently unique to AMV and ilarvirus coat proteins. PMID:9525649

  12. Binding Energy and Enzymatic Catalysis.

    Science.gov (United States)

    Hansen, David E.; Raines, Ronald T.

    1990-01-01

    Discussed is the fundamental role that the favorable free energy of binding of the rate-determining transition state plays in catalysis. The principle that all of the catalytic factors discussed are realized by the use of this binding energy is reviewed. (CW)

  13. Tetrapyrrole binding affinity of the murine and human p22HBP heme-binding proteins.

    Science.gov (United States)

    Micaelo, Nuno M; Macedo, Anjos L; Goodfellow, Brian J; Félix, Vítor

    2010-11-01

    We present the first systematic molecular modeling study of the binding properties of murine (mHBP) and human (hHBP) p22HBP protein (heme-binding protein) with four tetrapyrrole ring systems belonging to the heme biosynthetic pathway: iron protoporphyrin IX (HEMIN), protoporphyrin IX (PPIX), coproporphyrin III (CPIII), coproporphyrin I (CPI). The relative binding affinities predicted by our computational study were found to be similar to those observed experimentally, providing a first rational structural analysis of the molecular recognition mechanism, by p22HBP, toward a number of different tetrapyrrole ligands. To probe the structure of these p22HBP protein complexes, docking, molecular dynamics and MM-PBSA methodologies supported by experimental NMR ring current shift data have been employed. The tetrapyrroles studied were found to bind murine p22HBP with the following binding affinity order: HEMIN> PPIX> CPIII> CPI, which ranged from -22.2 to -6.1 kcal/mol. In general, the protein-tetrapyrrole complexes are stabilized by non-bonded interactions between the tetrapyrrole propionate groups and basic residues of the protein, and by the preferential solvation of the complex compared to the unbound components. PMID:20800521

  14. Flavonoids with M1 Muscarinic Acetylcholine Receptor Binding Activity

    Directory of Open Access Journals (Sweden)

    Meyyammai Swaminathan

    2014-06-01

    Full Text Available Muscarinic acetylcholine receptor-active compounds have potential for the treatment of Alzheimer’s disease. In this study, a series of natural and synthetic flavones and flavonols was assayed in vitro for their ability to inhibit radioligand binding at human cloned M1 muscarinic receptors. Several compounds were found to possess competitive binding affinity (Ki = 40–110 µM, comparable to that of acetylcholine (Ki = 59 µM. Despite the fact that these compounds lack a positively-charged ammonium group under physiological conditions, molecular modelling studies suggested that they bind to the orthosteric site of the receptor, mainly through non-polar interactions.

  15. Virtual Optical Comparator

    Energy Technology Data Exchange (ETDEWEB)

    Thompson, Greg

    2008-10-20

    The Virtual Optical Comparator, VOC, was conceived as a result of the limitations of conventional optical comparators and vision systems. Piece part designs for mechanisms have started to include precision features on the face of parts that must be viewed using a reflected image rather than a profile shadow. The VOC concept uses a computer generated overlay and a digital camera to measure features on a video screen. The advantage of this system is superior edge detection compared to traditional systems. No vinyl charts are procured or inspected. The part size and expensive fixtures are no longer a concern because of the range of the X-Y table of the Virtual Optical Comparator. Product redesigns require only changes to the CAD image overlays; new vinyl charts are not required. The inspection process is more ergonomic by allowing the operator to view the part sitting at a desk rather than standing over a 30 inch screen. The procurement cost for the VOC will be less than a traditional comparator with a much smaller footprint with less maintenance and energy requirements.

  16. Comparison of Transcription Factor Binding Site Models

    KAUST Repository

    Bhuyan, Sharifulislam

    2012-05-01

    Modeling of transcription factor binding sites (TFBSs) and TFBS prediction on genomic sequences are important steps to elucidate transcription regulatory mechanism. Dependency of transcription regulation on a great number of factors such as chemical specificity, molecular structure, genomic and epigenetic characteristics, long distance interaction, makes this a challenging problem. Different experimental procedures generate evidence that DNA-binding domains of transcription factors show considerable DNA sequence specificity. Probabilistic modeling of TFBSs has been moderately successful in identifying patterns from a family of sequences. In this study, we compare performances of different probabilistic models and try to estimate their efficacy over experimental TFBSs data. We build a pipeline to calculate sensitivity and specificity from aligned TFBS sequences for several probabilistic models, such as Markov chains, hidden Markov models, Bayesian networks. Our work, containing relevant statistics and evaluation for the models, can help researchers to choose the most appropriate model for the problem at hand.

  17. Job requirements compared to dental school education: impact of a case-based learning curriculum [Integratives fallbezogenes Lernen aus Sicht zahnmedizinischer Absolventen in Bezug auf spätere Berufsanforderungen

    Directory of Open Access Journals (Sweden)

    Keeve, Philip L.

    2012-08-01

    Full Text Available [english] Introduction: Case-based learning (CBL is suggested as a key educational method of knowledge acquisition to improve dental education. The purpose of this study was to assess graduates from a patient-oriented, case-based learning (CBL-based curriculum as regards to key competencies required at their professional activity.Methods: 407 graduates from a patient-oriented, case-based learning (CBL dental curriculum who graduated between 1990 and 2006 were eligible for this study. 404 graduates were contacted between 2007 and 2008 to self-assess nine competencies as required at their day-to-day work and as taught in dental school on a 6-point Likert scale. Baseline demographics and clinical characteristics were presented as mean ± standard deviation (SD for continuous variables. To determine whether dental education sufficiently covers the job requirements of physicians, we calculated the mean difference ∆ between the ratings of competencies as required in day-to-day work and as taught in medical school by subtracting those from each other (negative mean difference ∆ indicates deficit; positive mean difference ∆ indicates surplus. Spearman’s rank correlation coefficient was calculated to reveal statistical significance (statistical significance p[german] Einführung: Das fallbezogene und patientenorientierte Studium nimmt eine Schlüsselstellung in der Ausbildung von Human- und Zahnmedizinern ein. Ziel dieser Studie war es zu prüfen, ob Studierende durch ein integratives fallbezogenes Curriculum in bestimmten Schlüsselkompetenzen den späteren Berufsanforderungen entsprechend vorbereitet werden.Methodik: Von 407 Absolventen eines integrativen Zahnmedizinstudiums mit fallbezogenen Lerninhalten, die ihre Ausbildung erfolgreich zwischen 1990 und 2006 abschlossen, wurden 404 Absolventen zwischen 2007 und 2008 anhand eines standardisierten Fragebogens zu neun Schlüsselkompetenzen befragt. Die Schlüsselkompetenzen wurden auf einer 6

  18. The function of the octamer-binding site in the DRA promoter

    Energy Technology Data Exchange (ETDEWEB)

    Voliva, C.F. [Monsanto Co., St. Louis, MO (United States); Jabrane-Ferrat, N.; Peterlin, B.M. [Univ. of California, San Francisco, CA (United States)

    1996-06-01

    The octamer binding site, which is located immediately upstream of the poorly conserved DRA TATA sequence, is important for high levels of expression of this human major histocompatibility class II gene in B cells. In this study, we demonstrate that the substitution of the DRA TATA sequence with the TATA box from the adenovirus Elb promoter removes the requirement for the octamer binding site for high levels of expression from the DRA promoter. Since only the TATA box from the Elb but not the DRA promoters binds the TATA binding protein, we conclude that the octamer binding site helps to recruit TBP to the DRA promoter. 32 refs., 7 figs.

  19. Regulation of K-Ras4B Membrane Binding by Calmodulin.

    Science.gov (United States)

    Sperlich, Benjamin; Kapoor, Shobhna; Waldmann, Herbert; Winter, Roland; Weise, Katrin

    2016-07-12

    K-Ras4B is a membrane-bound small GTPase with a prominent role in cancer development. It contains a polybasic farnesylated C-terminus that is required for the correct localization and clustering of K-Ras4B in distinct membrane domains. PDEδ and the Ca(2+)-binding protein calmodulin (CaM) are known to function as potential binding partners for farnesylated Ras proteins. However, they differ in the number of interaction sites with K-Ras4B, leading to different modes of interaction, and thus affect the subcellular distribution of K-Ras4B in different ways. Although it is clear that Ca(2+)-bound CaM can play a role in the dynamic spatial cycle of K-Ras4B in the cell, the exact molecular mechanism is only partially understood. In this biophysical study, we investigated the effect of Ca(2+)/CaM on the interaction of GDP- and GTP-loaded K-Ras4B with heterogeneous model biomembranes by using a combination of different spectroscopic and imaging techniques. The results show that Ca(2+)/CaM is able to extract K-Ras4B from negatively charged membranes in a nucleotide-independent manner. Moreover, the data demonstrate that the complex of Ca(2+)/CaM and K-Ras4B is stable in the presence of anionic membranes and shows no membrane binding. Finally, the influence of Ca(2+)/CaM on the interaction of K-Ras4B with membranes is compared with that of PDEδ, which was investigated in a previous study. Although both CaM and PDEδ exhibit a hydrophobic binding pocket for farnesyl, they have different effects on membrane binding of K-Ras4B and hence should be capable of regulating K-Ras4B plasma membrane localization in the cell. PMID:27410739

  20. Sequence specificity of formaldehyde-mediated covalent binding of anthracycline derivatives to DNA.

    Science.gov (United States)

    Szulawska, Agata; Gniazdowski, Marek; Czyz, Malgorzata

    2005-01-01

    Daunorubicin (DRB) and doxorubicin (DOX) in the presence of formaldehyde (CH2O) form covalent adducts with DNA. A G-specific adduct is formed by producing an aminal bridge between the C-3' of daunosamine and the C-2 of guanine. New derivatives of DRB, DOX and epidoxorubicin (EDOX) with an amidine group bonded to the C-3' of the daunosamine moiety, with either a morpholine or hexamethyleneimine ring attached to the amidine group, were studied in this paper. DNase I footprinting and analyses with restriction endonucleases were applied to compare the specificity of adduct formed by the amidine derivatives and their parent compounds. These approaches provide consistent results, proving that a GC pair is required for covalent binding of anthracycline derivatives to DNA and that different flanking sequences are able to modify the sequence preference of the drugs. The 5'-GC-3', 5'-CG-3' and 5'-TC-3' sequences were protected most efficiently by the parent compounds and their morpholine derivatives and some increased protection of 5'-TC-3' sequence was observed for morpholine analogues. Hexamethyleneimine derivatives bind to DNA with much lower efficiency. Finally, the sequence specificity of anthracycline derivatives was correlated with their ability to inhibit binding of transcription factors Sp1 and AP-1 to their DNA recognition sequences. The anthracycline derivatives were more potent in inhibiting Sp1 binding to its cognate GC box than in preventing AP-1 from binding to its mixed A.T and G.C site. Overall, the results indicate that the amidine derivatives of anthracyclines show similar, but not identical sequence specificity as parent compounds, though they exert their effect at a higher concentration. PMID:15588709

  1. Structural determinants within residues 180-199 of the rodent. alpha. 5 nicotinic acetylcholine receptor subunit involved in. alpha. -bungarotoxin binding

    Energy Technology Data Exchange (ETDEWEB)

    McLane, K.E.; Xiadong Wu; Conti-Tronconi, B.M. (Univ. of Minnesota, St. Paul (United States))

    1991-11-05

    Synthetic peptides corresponding to sequence segments of the nicotinic acetylcholine receptor (nAChR) {alpha} subunits have been used to identify regions that contribute to formation of the binding sites for cholinergic ligands. The authors have previously defined {alpha}-bungarotoxin ({alpha}-BTX) binding sequences between residues 180 and 199 of a putative rat neuronal nAChR {alpha} subunit, designated {alpha}5, and between residues 181 and 200 of the chick neuronal {alpha}7 and {alpha}8 subunits. These sequences are relatively divergent compared with the Torpedo and muscle nAChR {alpha}1 {alpha}-BTX binding sites, which indicates a serious limitation of predicting functional domains of proteins based on homology in general. Given the highly divergent nature of the {alpha}5 sequence, they were interested in determining the critical amino acid residues for {alpha}-BTX binding. In the present study, the effects of single amino acid substitutions of Gly or Ala for each residue of the rat {alpha}(180-199) sequence were tested, using a competition assay, in which peptides compete for {sup 125}I-{alpha}-BTX binding with native Torpedo nAChR. These results indicate that a disulfide bridge between the vicinal cysteines at positions 191 and 192 of the {alpha}5 sequence is not an absolute requirement for {alpha}-BTX binding activity.

  2. Kinetics of binding and geometry of cells on molecular biochips

    OpenAIRE

    Chechetkin, V.R.

    2011-01-01

    We examine how the shape of cells and the geometry of experiment affect the reaction-diffusion kinetics at the binding between target and probe molecules on molecular biochips. In particular, we compare the binding kinetics for the probes immobilized on surface of the semispherical and flat circular cells, the limit of thin slab of analyte solution over probe cell as well as hemispherical gel pads and cells printed in gel slab over a substrate. It is shown that hemispherical geometry provides...

  3. Cooperative binding: a multiple personality.

    Science.gov (United States)

    Martini, Johannes W R; Diambra, Luis; Habeck, Michael

    2016-06-01

    Cooperative binding has been described in many publications and has been related to or defined by several different properties of the binding behavior of the ligand to the target molecule. In addition to the commonly used Hill coefficient, other characteristics such as a sigmoidal shape of the overall titration curve in a linear plot, a change of ligand affinity of the other binding sites when a site of the target molecule becomes occupied, or complex roots of the binding polynomial have been used to define or to quantify cooperative binding. In this work, we analyze how the different properties are related in the most general model for binding curves based on the grand canonical partition function and present several examples which highlight differences between the cooperativity characterizing properties which are discussed. Our results mainly show that among the presented definitions there are not two which fully coincide. Moreover, this work poses the question whether it can make sense to distinguish between positive and negative cooperativity based on the macroscopic binding isotherm only. This article shall emphasize that scientists who investigate cooperative effects in biological systems could help avoiding misunderstandings by stating clearly which kind of cooperativity they discuss.

  4. [3]tetrahydrotrazodone binding. Association with serotonin binding sites

    International Nuclear Information System (INIS)

    High (17 nM) and low (603 nM) affinity binding sites for [3]tetrahydrotrazodone ([3] THT), a biologically active analogue of trazodone, have been identified in rat brain membranes. The substrate specificity, concentration, and subcellular and regional distributions of these sites suggest that they may represent a component of the serotonin transmitter system. Pharmacological analysis of [3]THT binding, coupled with brain lesion and drug treatment experiments, revealed that, unlike other antidepressants, [3] THT does not attach to either a biogenic amine transporter or serotonin binding sites. Rather, it would appear that [3]THT may be an antagonist ligand for the serotonin binding site. This probe may prove of value in defining the mechanism of action of trazodone and in further characterizing serotonin receptors

  5. Binding Energy and Equilibrium of Compact Objects

    Directory of Open Access Journals (Sweden)

    Germano M.

    2014-04-01

    Full Text Available The theoretical analysis of the existence of a limit mass for compact astronomic ob- jects requires the solution of the Einstein’s equations of g eneral relativity together with an appropriate equation of state. Analytical solutions exi st in some special cases like the spherically symmetric static object without energy sou rces that is here considered. Solutions, i.e. the spacetime metrics, can have a singular m athematical form (the so called Schwarzschild metric due to Hilbert or a nonsingula r form (original work of Schwarzschild. The former predicts a limit mass and, conse quently, the existence of black holes above this limit. Here it is shown that, the origi nal Schwarzschild met- ric permits compact objects, without mass limit, having rea sonable values for central density and pressure. The lack of a limit mass is also demonst rated analytically just imposing reasonable conditions on the energy-matter densi ty, of positivity and decreas- ing with radius. Finally the ratio between proper mass and to tal mass tends to 2 for high values of mass so that the binding energy reaches the lim it m (total mass seen by a distant observer. As it is known the negative binding energ y reduces the gravitational mass of the object; the limit of m for the binding energy provides a mechanism for stable equilibrium of any amount of mass to contrast the gravitatio nal collapse.

  6. LEGACY MANAGEMENT REQUIRES INFORMATION

    Energy Technology Data Exchange (ETDEWEB)

    CONNELL, C.W.; HILDEBRAND, R.D.

    2006-12-14

    ''Legacy Management Requires Information'' describes the goal(s) of the US Department of Energy's Office of Legacy Management (LM) relative to maintaining critical records and the way those goals are being addressed at Hanford. The paper discusses the current practices for document control, as well as the use of modern databases for both storing and accessing the data to support cleanup decisions. In addition to the information goals of LM, the Hanford Federal Facility Agreement and Consent Order, known as the ''Tri-Party Agreement'' (TPA) is one of the main drivers in documentation and data management. The TPA, which specifies discrete milestones for cleaning up the Hanford Site, is a legally binding agreement among the US Department of Energy (DOE), the Washington State Department of Ecology (Ecology), and the US Environmental Protection Agency (EPA). The TPA requires that DOE provide the lead regulatory agency with the results of analytical laboratory and non-laboratory tests/readings to help guide them in making decisions. The Agreement also calls for each signatory to preserve--for at least ten years after the Agreement has ended--all of the records in its or its contractors, possession related to sampling, analysis, investigations, and monitoring conducted. The tools used at Hanford to meet TPA requirements are also the tools that can satisfy the needs of LM.

  7. Defining NELF-E RNA binding in HIV-1 and promoter-proximal pause regions.

    Directory of Open Access Journals (Sweden)

    John M Pagano

    2014-01-01

    Full Text Available The four-subunit Negative Elongation Factor (NELF is a major regulator of RNA Polymerase II (Pol II pausing. The subunit NELF-E contains a conserved RNA Recognition Motif (RRM and is proposed to facilitate Poll II pausing through its association with nascent transcribed RNA. However, conflicting ideas have emerged for the function of its RNA binding activity. Here, we use in vitro selection strategies and quantitative biochemistry to identify and characterize the consensus NELF-E binding element (NBE that is required for sequence specific RNA recognition (NBE: CUGAGGA(U for Drosophila. An NBE-like element is present within the loop region of the transactivation-response element (TAR of HIV-1 RNA, a known regulatory target of human NELF-E. The NBE is required for high affinity binding, as opposed to the lower stem of TAR, as previously claimed. We also identify a non-conserved region within the RRM that contributes to the RNA recognition of Drosophila NELF-E. To understand the broader functional relevance of NBEs, we analyzed promoter-proximal regions genome-wide in Drosophila and show that the NBE is enriched +20 to +30 nucleotides downstream of the transcription start site. Consistent with the role of NELF in pausing, we observe a significant increase in NBEs among paused genes compared to non-paused genes. In addition to these observations, SELEX with nuclear run-on RNA enrich for NBE-like sequences. Together, these results describe the RNA binding behavior of NELF-E and supports a biological role for NELF-E in promoter-proximal pausing of both HIV-1 and cellular genes.

  8. Computational design of a PAK1 binding protein.

    Science.gov (United States)

    Jha, Ramesh K; Leaver-Fay, Andrew; Yin, Shuangye; Wu, Yibing; Butterfoss, Glenn L; Szyperski, Thomas; Dokholyan, Nikolay V; Kuhlman, Brian

    2010-07-01

    We describe a computational protocol, called DDMI, for redesigning scaffold proteins to bind to a specified region on a target protein. The DDMI protocol is implemented within the Rosetta molecular modeling program and uses rigid-body docking, sequence design, and gradient-based minimization of backbone and side-chain torsion angles to design low-energy interfaces between the scaffold and target protein. Iterative rounds of sequence design and conformational optimization were needed to produce models that have calculated binding energies that are similar to binding energies calculated for native complexes. We also show that additional conformation sampling with molecular dynamics can be iterated with sequence design to further lower the computed energy of the designed complexes. To experimentally test the DDMI protocol, we redesigned the human hyperplastic discs protein to bind to the kinase domain of p21-activated kinase 1 (PAK1). Six designs were experimentally characterized. Two of the designs aggregated and were not characterized further. Of the remaining four designs, three bound to the PAK1 with affinities tighter than 350 muM. The tightest binding design, named Spider Roll, bound with an affinity of 100 muM. NMR-based structure prediction of Spider Roll based on backbone and (13)C(beta) chemical shifts using the program CS-ROSETTA indicated that the architecture of human hyperplastic discs protein is preserved. Mutagenesis studies confirmed that Spider Roll binds the target patch on PAK1. Additionally, Spider Roll binds to full-length PAK1 in its activated state but does not bind PAK1 when it forms an auto-inhibited conformation that blocks the Spider Roll target site. Subsequent NMR characterization of the binding of Spider Roll to PAK1 revealed a comparably small binding 'on-rate' constant (design the site of novel protein-protein interactions is an important step towards creating new proteins that are useful as therapeutics or molecular probes.

  9. Methodological issues in the preparation and assay of platelet 3H-imipramine binding.

    Science.gov (United States)

    Severson, J A; Schneider, L S; Fredrickson, E R

    1990-07-01

    Several methodological factors in the preparation of platelets and the determination of platelet 3H-imipramine (3H-IMI) binding were examined. The ionic composition of the assay significantly affected platelet 3H-IMI binding. Approximately 25% of the specific binding of 3H-IMI to intact platelet preparations was retained in the absence of sodium and chloride ions. The addition of sodium ions enhanced the specific binding of 3H-IMI, but the addition of chloride in the presence of sodium had a more pronounced effect, enhancing binding approximately five-fold over that observed with the addition of sodium. Sodium was the only cation tested that enhanced binding. Only halides enhanced binding in the presence of sodium with the following order of potency: Cl- greater than Br- greater than I- = F-. Ions increased the density of binding sites (Bmax) and did not affect the affinity of the binding sites for 3H-IMI. In the presence of sodium and chloride, the use of serotonin (5HT) to define nonspecific binding in saturation experiments resulted in lower binding densities (Bmax) than when desipramine was used to define nonspecific binding. The component of binding that was insensitive to 5HT was roughly equal to the Bmax of 3H-IMI binding obtained in the absence of sodium and chloride using desipramine to define nonspecific binding. Overall, these data suggest that not all 3H-IMI binding that is displaced by desipramine is related to serotonergic mechanisms, and suggest that 5HT is a better choice than desipramine for the determination of the nonspecific binding of 3H-IMI. In addition, the binding of 3H-IMI to different platelet preparations was compared. The binding of 3H-IMI to intact platelets was less than that obtained using lysed platelet membranes when data were expressed per mg protein. The Coomassie Blue dye-binding method to determine platelet protein resulted in greater Bmax values than were obtained with the Folin phenol reagent method. The method of platelet

  10. Dissecting electrostatic screening, specific ion binding, and ligand binding in an energetic model for glycine riboswitch folding

    Energy Technology Data Exchange (ETDEWEB)

    Lipfert, Jan; Sim, Adelene Y.L.; Herschlag, Daniel; Doniach, Sebastian (Stanford)

    2010-09-17

    Riboswitches are gene-regulating RNAs that are usually found in the 5{prime}-untranslated regions of messenger RNA. As the sugar-phosphate backbone of RNA is highly negatively charged, the folding and ligand-binding interactions of riboswitches are strongly dependent on the presence of cations. Using small angle X-ray scattering (SAXS) and hydroxyl radical footprinting, we examined the cation dependence of the different folding stages of the glycine-binding riboswitch from Vibrio cholerae. We found that the partial folding of the tandem aptamer of this riboswitch in the absence of glycine is supported by all tested mono- and divalent ions, suggesting that this transition is mediated by nonspecific electrostatic screening. Poisson-Boltzmann calculations using SAXS-derived low-resolution structural models allowed us to perform an energetic dissection of this process. The results showed that a model with a constant favorable contribution to folding that is opposed by an unfavorable electrostatic term that varies with ion concentration and valency provides a reasonable quantitative description of the observed folding behavior. Glycine binding, on the other hand, requires specific divalent ions binding based on the observation that Mg{sup 2+}, Ca{sup 2+}, and Mn{sup 2+} facilitated glycine binding, whereas other divalent cations did not. The results provide a case study of how ion-dependent electrostatic relaxation, specific ion binding, and ligand binding can be coupled to shape the energetic landscape of a riboswitch and can begin to be quantitatively dissected.

  11. Liver fatty acid-binding protein binds monoacylglycerol in vitro and in mouse liver cytosol.

    Science.gov (United States)

    Lagakos, William S; Guan, Xudong; Ho, Shiu-Ying; Sawicki, Luciana Rodriguez; Corsico, Betina; Kodukula, Sarala; Murota, Kaeko; Stark, Ruth E; Storch, Judith

    2013-07-01

    Liver fatty acid-binding protein (LFABP; FABP1) is expressed both in liver and intestinal mucosa. Mice null for LFABP were recently shown to have altered metabolism of not only fatty acids but also monoacylglycerol, the two major products of dietary triacylglycerol hydrolysis (Lagakos, W. S., Gajda, A. M., Agellon, L., Binas, B., Choi, V., Mandap, B., Russnak, T., Zhou, Y. X., and Storch, J. (2011) Am. J. Physiol. Gastrointest. Liver Physiol. 300, G803-G814). Nevertheless, the binding and transport of monoacylglycerol (MG) by LFABP are uncertain, with conflicting reports in the literature as to whether this single chain amphiphile is in fact bound by LFABP. In the present studies, gel filtration chromatography of liver cytosol from LFABP(-/-) mice shows the absence of the low molecular weight peak of radiolabeled monoolein present in the fractions that contain LFABP in cytosol from wild type mice, indicating that LFABP binds sn-2 MG in vivo. Furthermore, solution-state NMR spectroscopy demonstrates two molecules of sn-2 monoolein bound in the LFABP binding pocket in positions similar to those found for oleate binding. Equilibrium binding affinities are ∼2-fold lower for MG compared with fatty acid. Finally, kinetic studies examining the transfer of a fluorescent MG analog show that the rate of transfer of MG is 7-fold faster from LFABP to phospholipid membranes than from membranes to membranes and occurs by an aqueous diffusion mechanism. These results provide strong support for monoacylglycerol as a physiological ligand for LFABP and further suggest that LFABP functions in the efficient intracellular transport of MG. PMID:23658011

  12. Liver Fatty Acid-binding Protein Binds Monoacylglycerol in Vitro and in Mouse Liver Cytosol*

    Science.gov (United States)

    Lagakos, William S.; Guan, Xudong; Ho, Shiu-Ying; Sawicki, Luciana Rodriguez; Corsico, Betina; Kodukula, Sarala; Murota, Kaeko; Stark, Ruth E.; Storch, Judith

    2013-01-01

    Liver fatty acid-binding protein (LFABP; FABP1) is expressed both in liver and intestinal mucosa. Mice null for LFABP were recently shown to have altered metabolism of not only fatty acids but also monoacylglycerol, the two major products of dietary triacylglycerol hydrolysis (Lagakos, W. S., Gajda, A. M., Agellon, L., Binas, B., Choi, V., Mandap, B., Russnak, T., Zhou, Y. X., and Storch, J. (2011) Am. J. Physiol. Gastrointest. Liver Physiol. 300, G803–G814). Nevertheless, the binding and transport of monoacylglycerol (MG) by LFABP are uncertain, with conflicting reports in the literature as to whether this single chain amphiphile is in fact bound by LFABP. In the present studies, gel filtration chromatography of liver cytosol from LFABP−/− mice shows the absence of the low molecular weight peak of radiolabeled monoolein present in the fractions that contain LFABP in cytosol from wild type mice, indicating that LFABP binds sn-2 MG in vivo. Furthermore, solution-state NMR spectroscopy demonstrates two molecules of sn-2 monoolein bound in the LFABP binding pocket in positions similar to those found for oleate binding. Equilibrium binding affinities are ∼2-fold lower for MG compared with fatty acid. Finally, kinetic studies examining the transfer of a fluorescent MG analog show that the rate of transfer of MG is 7-fold faster from LFABP to phospholipid membranes than from membranes to membranes and occurs by an aqueous diffusion mechanism. These results provide strong support for monoacylglycerol as a physiological ligand for LFABP and further suggest that LFABP functions in the efficient intracellular transport of MG. PMID:23658011

  13. Thyroxine binding to serum thyronine-binding globulin in thyroidectomized adult and normal neonatal rats

    International Nuclear Information System (INIS)

    The amount of tracer [125I]T4 bound to serum thyronine-binding globulin (TBG) was measured by polyacrylamide gel electrophoresis in adult thyroidectomized (TX) rats and normal 1-day to 4-week-old rat puts. Thyroidectomy was associated with the appearance of significant amounts of [125I]T4 binding to serum TBG in lean rats, but not in obese Zucker rats. Treatment of the TX rats in vivo with replacement doses of T4 prevented this increase in TBG binding, but enrichment of serum from TX rats with T4 did not. Significant amounts of tracer [125I]T4 binding to TBG was present in serum from 1- to 3-week-old normal rat pups, but not in 1-day- or 4-week-old pups. There were significantly higher levels of TBG binding of [125I]T4 in serum from 2-week-old rat pups raised in litters of 16 pups compared to those raised in litters of 4 pups. All manipulations that result in the appearance of TBG in rat serum also result in either weight loss or a slowing in the rate of growth, suggesting that the appearance of TBG in rat serum has a nutritional component. This possibility is further supported by the observations that increases in TBG binding of [125I]T4 are not found in obese Zucker rats fed a low protein-high carbohydrate diet for 14 days or fasted for 7 days, or after thyroidectomy, perhaps owing to the large stores of fuel in the obese rat

  14. Oligomerization of Mannan-binding Lectin Dictates Binding Properties and Complement Activation.

    Science.gov (United States)

    Kjaer, T R; Jensen, L; Hansen, A; Dani, R; Jensenius, J C; Dobó, J; Gál, P; Thiel, S

    2016-07-01

    The complement system is a part of the innate immune system and is involved in recognition and clearance of pathogens and altered-self structures. The lectin pathway of the complement system is initiated when soluble pattern recognition molecules (PRMs) with collagen-like regions bind to foreign or altered self-surfaces. Associated with the collagen-like stems of these PRMs are three mannan-binding lectin (MBL)-associated serine proteases (MASPs) and two MBL-associated proteins (MAps). The most studied of the PRMs, MBL, is present in serum mainly as trimeric and tetrameric oligomers of the structural subunit. We hypothesized that oligomerization of MBL may influence both the potential to bind to micro organisms and the interaction with the MASPs and MAps, thus influencing the ability to initiate complement activation. When testing binding at 37 °C, we found higher binding of tetrameric MBL to Staphylococcus aureus (S. aureus) than trimeric and dimeric MBL. In serum, we found that tetrameric MBL was the main oligomeric form present in complexes with the MASPs and MAp44. Such preference was confirmed using purified forms of recombinant MBL (rMBL) oligomers, where tetrameric rMBL interacted stronger with all of the MASPs and MAp44, compared to trimeric MBL. As a direct consequence of the weaker interaction with the MASPs, we found that trimeric rMBL was inferior to tetrameric rMBL in activating the complement system. Our data suggest that the oligomeric state of MBL is crucial both for the binding properties and the effector function of MBL.

  15. An Investigation of Online Homework: Required or Not Required?

    Science.gov (United States)

    Wooten, Tommy; Dillard-Eggers, Jane

    2013-01-01

    In our research we investigate the use of online homework in principles of accounting classes where some classes required online homework while other classes did not. Users of online homework, compared to nonusers, had a higher grade point average and earned a higher grade in class. On average, both required and not-required users rated the online…

  16. Structural studies of sugar binding proteins

    OpenAIRE

    Sooriyaarachchi, Sanjeewani

    2010-01-01

    Binding proteins, which are themselves non-enzymatic, play an important role in enzymatic reactions as well as non-enzymatic processes by providing a binding platform for the specific recognition of particular molecules. For example, periplasmic binding proteins play a vital role in nutrient uptake in Gram-negative bacteria. In the present study, three sugar binding proteins, including two periplasmic binding proteins and a β-glucan binding protein, are described. The glucose/galactose bindin...

  17. Alternative polyadenylation and RNA-binding proteins.

    Science.gov (United States)

    Erson-Bensan, Ayse Elif

    2016-08-01

    Our understanding of the extent of microRNA-based gene regulation has expanded in an impressive pace over the past decade. Now, we are beginning to better appreciate the role of 3'-UTR (untranslated region) cis-elements which harbor not only microRNA but also RNA-binding protein (RBP) binding sites that have significant effect on the stability and translational rate of mRNAs. To add further complexity, alternative polyadenylation (APA) emerges as a widespread mechanism to regulate gene expression by producing shorter or longer mRNA isoforms that differ in the length of their 3'-UTRs or even coding sequences. Resulting shorter mRNA isoforms generally lack cis-elements where trans-acting factors bind, and hence are differentially regulated compared with the longer isoforms. This review focuses on the RBPs involved in APA regulation and their action mechanisms on APA-generated isoforms. A better understanding of the complex interactions between APA and RBPs is promising for mechanistic and clinical implications including biomarker discovery and new therapeutic approaches. PMID:27208003

  18. Characterization of soluble fibronectin binding to Bacille Calmette-Guérin.

    Science.gov (United States)

    Aslanzadeh, J; Brown, E J; Quillin, S P; Ritchey, J K; Ratliff, T L

    1989-10-01

    Fibronectin (FN), a 420 kDa glycoprotein, consists of two similar subunits linked by a disulphide bond near the C-terminal end. FN is present in soluble and matrix forms in various body fluids and tissues and has been shown to bind to variety of organisms. We characterized the conditions required for 125I-FN binding to Bacille Calmette-Guérin (BCG). The binding was dose-dependent, reached saturation within 3 min, and was essentially irreversible for at least 24 h under optimal binding conditions at pH 6.0. In contrast, the binding was reversible (greater than 90% in 24 h) when the pH was increased to 10.0. Scatchard analysis of the dose-response experiments produced a straight line, suggesting the presence of a single class of FN receptor on BCG. 125I-FN binding was trypsin-sensitive, suggesting that the BCG-binding molecule is a protein. The number of FN receptors was determined to be 8000-15,000 per bacterium. 125I-FN binding was pH dependent, with maximal binding at acidic pH. 125I-FN binding was sensitive to the presence of NaCl, with 0.08 M-NaCl inhibiting binding by 85%. These data demonstrate that soluble FN binds to a trypsin-sensitive cell-surface component of BCG in an essentially irreversible manner.

  19. Critical role of the H6-H7 loop in the conformational adaptation of all-trans retinoic acid and synthetic retinoids within the ligand-binding site of RARalpha.

    Science.gov (United States)

    Mailfait, S; Thoreau, E; Belaiche, D; Formstecher And B Sablonniè, P

    2000-06-01

    The pleiotropic effects of the natural and synthetic retinoids are mediated by the activation of the two subfamilies of nuclear receptors, the retinoic acid receptors (RARs) and the retinoic X receptors (RXRs). At the molecular level, these events begin with the specific ligand recognition by a nuclear receptor subtype. The adaptation of ligands to the receptor binding site leads to an optimal number of interactions for binding and selectivity which justifies elucidation of the structural requirements of the ligand binding pocket. To explore the contribution of H6-H7 loop folding in the ligand-induced conformational changes explained by the mouse-trap model, four RARalpha mutants were constructed. Ligand binding and transactivation studies revealed that three residues from the H6-H7 loop (Gly(301), Phe(302) and Gly(303)) are critical for the conformational adaptation of both synthetic agonists and antagonists. Model building and analysis of both RARalpha-ATRA and RARalpha-CD367 complexes demonstrate that accommodation of CD367 results in a less tight contact of the saturated ring of this ligand with the amino acid side chains of the receptor ligand-binding pocket compared with that of ATRA. According to the flexibility of the agonists tested (ATRA>TTNPB=Am580> CD367), we observed a decrease in binding that was dependent on ligand structure rigidity. In contrast, the binding and transactivating activities of the L266A mutant confirmed the structural constraints imposed by synthetic ligands on binding affinity for the receptor and revealed that subtle local rearrangements induced by specific conformational adaptation changes result in different binding affinities. Our results illustrate the dynamic nature of the interaction between RARalpha and its ligands and demonstrate the critical role of the H6-H7 loop in the binding of both synthetic retinoid agonists and antagonists.

  20. Brain hyaluronan binding protein inhibits tumor growth

    Institute of Scientific and Technical Information of China (English)

    高锋; 曹曼林; 王蕾

    2004-01-01

    Background Great efforts have been made to search for the angiogenic inhibitors in avascular tissues. Several proteins isolated from cartilage have been proved to have anti-angiogenic or anti-tumour effects. Because cartilage contains a great amount of hyaluronic acid (HA) oligosaccharides and abundant HA binding proteins (HABP), therefore, we speculated that HABP might be one of the factors regulating vascularization in cartilage or anti-angiogenesis in tumours. The purpose of this research was to evaluale the effects of hyaluronan binding protein on inhibiting tumour growth both in vivo and vitro. Methods A unique protein termed human brain hyaluronan (HA) binding protein (b-HABP) was cloned from human brain cDNA library. MDA-435 human breast cancer cell line was chosen as a transfectant. The in vitro underlying mechanisms were investigated by determining the possibilities of MDA-435/b-HABP colony formation on soft agar, the effects of the transfectant on the proliferation of endothelial cells and the expression levels of caspase 3 and FasL from MDA-435/b-HABP. The in vivo study included tumour growth on the chorioallantoic membrane (CAM) of chicken embryos and nude mice. Results Colony formation assay revealed that the colonies formed by MDA-435/b-HABP were greatly reduced compared to mock transfectants. The conditioned media from MDA-435/b-HABP inhibited the growth of endothelial cells in culture. Caspase 3 and FasL expressions were induced by MDA-435/b-HABP. The size of tumours of MDA-435/b-HABP in both CAM and nude mice was much smaller than that of MDA-435 alone. Conclusions Human brain hyaluronan binding protein (b-HABP) may represent a new kind of naturally existing anti-tumour substance. This brain-derived glycoprotein may block tumour growth by inducing apoptosis of cancer cells or by decreasing angiogenesis in tumour tissue via inhibiting proliferation of endothelial cells.

  1. HTLV-1 Tax Protein Stimulation of DNA Binding of bZIP Proteins by Enhancing Dimerization

    Science.gov (United States)

    Wagner, Susanne; Green, Michael R.

    1993-10-01

    The Tax protein of human T cell leukemia virus type-1 (HTLV-I) transcriptionally activates the HTLV-I promoter. This activation requires binding sites for activating transcription factor (ATF) proteins, a family of cellular proteins that contain basic region-leucine zipper (bZIP) DNA binding domains. Data are presented showing that Tax increases the in vitro DNA binding activity of multiple ATF proteins. Tax also stimulated DNA binding by other bZIP proteins, but did not affect DNA binding proteins that lack a bZIP domain. The increase in DNA binding occurred because Tax promotes dimerization of the bZIP domain in the absence of DNA, and the elevated concentration of the bZIP homodimer then facilitates the DNA binding reaction. These results help explain how Tax activates viral transcription and transforms cells.

  2. Inhibition of RNA Polymerase II Transcription in Human Cells by Synthetic DNA-Binding Ligands

    Science.gov (United States)

    Dickinson, Liliane A.; Gulizia, Richard J.; Trauger, John W.; Baird, Eldon E.; Mosier, Donald E.; Gottesfeld, Joel M.; Dervan, Peter B.

    1998-10-01

    Sequence-specific DNA-binding small molecules that can permeate human cells potentially could regulate transcription of specific genes. Multiple cellular DNA-binding transcription factors are required by HIV type 1 for RNA synthesis. Two pyrrole--imidazole polyamides were designed to bind DNA sequences immediately adjacent to binding sites for the transcription factors Ets-1, lymphoid-enhancer binding factor 1, and TATA-box binding protein. These synthetic ligands specifically inhibit DNA-binding of each transcription factor and HIV type 1 transcription in cell-free assays. When used in combination, the polyamides inhibit virus replication by >99% in isolated human peripheral blood lymphocytes, with no detectable cell toxicity. The ability of small molecules to target predetermined DNA sequences located with RNA polymerase II promoters suggests a general approach for regulation of gene expression, as well as a mechanism for the inhibition of viral replication.

  3. Influence of target concentration and background binding on in vitro selection of affinity reagents.

    Directory of Open Access Journals (Sweden)

    Jinpeng Wang

    Full Text Available Nucleic acid-based aptamers possess many useful features that make them a promising alternative to antibodies and other affinity reagents, including well-established chemical synthesis, reversible folding, thermal stability and low cost. However, the selection process typically used to generate aptamers (SELEX often requires significant resources and can fail to yield aptamers with sufficient affinity and specificity. A number of seminal theoretical models and numerical simulations have been reported in the literature offering insights into experimental factors that govern the effectiveness of the selection process. Though useful, these previous models have not considered the full spectrum of experimental factors or the potential impact of tuning these parameters at each round over the course of a multi-round selection process. We have developed an improved mathematical model to address this important question, and report that both target concentration and the degree of non-specific background binding are critical determinants of SELEX efficiency. Although smaller target concentrations should theoretically offer superior selection outcome, we show that the level of background binding dramatically affect the target concentration that will yield maximum enrichment at each round of selection. Thus, our model enables experimentalists to determine appropriate target concentrations as a means for protocol optimization. Finally, we perform a comparative analysis of two different selection methods over multiple rounds of selection, and show that methods with inherently lower background binding offer dramatic advantages in selection efficiency.

  4. Assessment of Density Functional Methods for Exciton Binding Energies and Related Optoelectronic Properties

    CERN Document Server

    Lee, Jui-Che; Lin, Shiang-Tai

    2015-01-01

    The exciton binding energy, the energy required to dissociate an excited electron-hole pair into free charge carriers, is one of the key factors to the optoelectronic performance of organic materials. However, it remains unclear whether modern quantum-mechanical calculations, mostly based on Kohn-Sham density functional theory (KS-DFT) and time-dependent density functional theory (TDDFT), are reliably accurate for exciton binding energies. In this study, the exciton binding energies and related optoelectronic properties (e.g., the ionization potentials, electron affinities, fundamental gaps, and optical gaps) of 121 small- to medium-sized molecules are calculated using KS-DFT and TDDFT with various density functionals. Our KS-DFT and TDDFT results are compared with those calculated using highly accurate CCSD and EOM-CCSD methods, respectively. The omegaB97, omegaB97X, and omegaB97X-D functionals are shown to generally outperform (with a mean absolute error of 0.36 eV) other functionals for the properties inve...

  5. Sulfated hyaluronan improves bone regeneration of diabetic rats by binding sclerostin and enhancing osteoblast function.

    Science.gov (United States)

    Picke, Ann-Kristin; Salbach-Hirsch, Juliane; Hintze, Vera; Rother, Sandra; Rauner, Martina; Kascholke, Christian; Möller, Stephanie; Bernhardt, Ricardo; Rammelt, Stefan; Pisabarro, M Teresa; Ruiz-Gómez, Gloria; Schnabelrauch, Matthias; Schulz-Siegmund, Michaela; Hacker, Michael C; Scharnweber, Dieter; Hofbauer, Christine; Hofbauer, Lorenz C

    2016-07-01

    Bone fractures in patients with diabetes mellitus heal poorly and require innovative therapies to support bone regeneration. Here, we assessed whether sulfated hyaluronan included in collagen-based scaffold coatings can improve fracture healing in diabetic rats. Macroporous thermopolymerized lactide-based scaffolds were coated with collagen including non-sulfated or sulfated hyaluronan (HA/sHA3) and inserted into 3 mm femoral defects of non-diabetic and diabetic ZDF rats. After 12 weeks, scaffolds coated with collagen/HA or collagen/sHA3 accelerated bone defect regeneration in diabetic, but not in non-diabetic rats as compared to their non-coated controls. At the tissue level, collagen/sHA3 promoted bone mineralization and decreased the amount of non-mineralized bone matrix. Moreover, collagen/sHA3-coated scaffolds from diabetic rats bound more sclerostin in vivo than the respective controls. Binding assays confirmed a high binding affinity of sHA3 to sclerostin. In vitro, sHA3 induced BMP-2 and lowered the RANKL/OPG expression ratio, regardless of the glucose concentration in osteoblastic cells. Both sHA3 and high glucose concentrations decreased the differentiation of osteoclastic cells. In summary, scaffolds coated with collagen/sHA3 represent a potentially suitable biomaterial to improve bone defect regeneration in diabetic conditions. The underlying mechanism involves improved osteoblast function and binding sclerostin, a potent inhibitor of Wnt signaling and osteoblast function. PMID:27131598

  6. Predicting the Effect of Mutations on Protein-Protein Binding Interactions through Structure-Based Interface Profiles.

    Science.gov (United States)

    Brender, Jeffrey R; Zhang, Yang

    2015-10-01

    The formation of protein-protein complexes is essential for proteins to perform their physiological functions in the cell. Mutations that prevent the proper formation of the correct complexes can have serious consequences for the associated cellular processes. Since experimental determination of protein-protein binding affinity remains difficult when performed on a large scale, computational methods for predicting the consequences of mutations on binding affinity are highly desirable. We show that a scoring function based on interface structure profiles collected from analogous protein-protein interactions in the PDB is a powerful predictor of protein binding affinity changes upon mutation. As a standalone feature, the differences between the interface profile score of the mutant and wild-type proteins has an accuracy equivalent to the best all-atom potentials, despite being two orders of magnitude faster once the profile has been constructed. Due to its unique sensitivity in collecting the evolutionary profiles of analogous binding interactions and the high speed of calculation, the interface profile score has additional advantages as a complementary feature to combine with physics-based potentials for improving the accuracy of composite scoring approaches. By incorporating the sequence-derived and residue-level coarse-grained potentials with the interface structure profile score, a composite model was constructed through the random forest training, which generates a Pearson correlation coefficient >0.8 between the predicted and observed binding free-energy changes upon mutation. This accuracy is comparable to, or outperforms in most cases, the current best methods, but does not require high-resolution full-atomic models of the mutant structures. The binding interface profiling approach should find useful application in human-disease mutation recognition and protein interface design studies.

  7. β-Subunit Binding Is Sufficient for Ligands to Open the Integrin αIIbβ3 Headpiece.

    Science.gov (United States)

    Lin, Fu-Yang; Zhu, Jianghai; Eng, Edward T; Hudson, Nathan E; Springer, Timothy A

    2016-02-26

    The platelet integrin αIIbβ3 binds to a KQAGDV motif at the fibrinogen γ-chain C terminus and to RGD motifs present in loops in many extracellular matrix proteins. These ligands bind in a groove between the integrin α and β-subunits; the basic Lys or Arg side chain hydrogen bonds to the αIIb-subunit, and the acidic Asp side chain coordinates to a metal ion held by the β3-subunit. Ligand binding induces headpiece opening, with conformational change in the β-subunit. During this opening, RGD slides in the ligand-binding pocket toward αIIb, with movement of the βI-domain β1-α1 loop toward αIIb, enabling formation of direct, charged hydrogen bonds between the Arg side chain and αIIb. Here we test whether ligand interactions with β3 suffice for stable ligand binding and headpiece opening. We find that the AGDV tetrapeptide from KQAGDV binds to the αIIbβ3 headpiece with affinity comparable with the RGDSP peptide from fibronectin. AGDV induced complete headpiece opening in solution as shown by increase in hydrodynamic radius. Soaking of AGDV into closed αIIbβ3 headpiece crystals induced intermediate states similarly to RGDSP. AGDV has very little contact with the α-subunit. Furthermore, as measured by epitope exposure, AGDV, like the fibrinogen γ C-terminal peptide and RGD, caused integrin extension on the cell surface. Thus, pushing by the β3-subunit on Asp is sufficient for headpiece opening and ligand sliding, and no pulling by the αIIb subunit on Arg is required.

  8. Plasmonic-Based Electrochemical Impedance Spectroscopy: Application to Molecular Binding

    Science.gov (United States)

    Lu, Jin; Wang, Wei; Wang, Shaopeng; Shan, Xiaonan; Li, Jinghong; Tao, Nongjian

    2012-01-01

    Plasmonic-based electrochemical impedance spectroscopy (P-EIS) is developed to investigate molecular binding on surfaces. Its basic principle relies on the sensitive dependence of surface plasmon resonance (SPR) signal on surface charge density, which is modulated by applying an AC potential to a SPR chip surface. The AC component of the SPR response gives the electrochemical impedance, and the DC component provides the conventional SPR detection. The plasmonic-based impedance measured over a range of frequency is in quantitative agreement with the conventional electrochemical impedance. Compared to the conventional SPR detection, P-EIS is sensitive to molecular binding taking place on the chip surface, and less sensitive to bulk refractive index changes or non-specific binding. Moreover, this new approach allows for simultaneous SPR and surface impedance analysis of molecular binding processes. PMID:22122514

  9. Kinetics characterization of c-Src binding to lipid membranes: Switching from labile to persistent binding.

    Science.gov (United States)

    Le Roux, Anabel-Lise; Busquets, Maria Antònia; Sagués, Francesc; Pons, Miquel

    2016-02-01

    Cell signaling by the c-Src proto-oncogen requires the attachment of the protein to the inner side of the plasma membrane through the myristoylated N-terminal region, known as the SH4 domain. Additional binding regions of lower affinity are located in the neighbor intrinsically disordered Unique domain and the structured SH3 domain. Here we present a surface plasmon resonance study of the binding of a myristoylated protein including the SH4, Unique and SH3 domains of c-Src to immobilized liposomes. Two distinct binding processes were observed: a fast and a slow one. The second process lead to a persistently bound form (PB) with a slower binding and a much slower dissociation rate than the first one. The association and dissociation of the PB form could be detected using an anti-SH4 antibody. The kinetic analysis revealed that binding of the PB form follows a second order rate law suggesting that it involves the formation of c-Src dimers on the membrane surface. A kinetically equivalent PB form is observed in a myristoylated peptide containing only the SH4 domain but not in a construct including the three domains but with a 12-carbon lauroyl substituent instead of the 14-carbon myristoyl group. The PB form is observed with neutral lipids but its population increases when the immobilized liposomes contain negatively charged lipids. We suggest that the PB form may represent the active signaling form of c-Src while the labile form provides the capacity for fast 2D search of the target signaling site on the membrane surface.

  10. Chemokine cooperativity is caused by competitive glycosaminoglycan binding

    NARCIS (Netherlands)

    Verkaar, F.; Offenbeek, J. van; Lee, M. van der; Lith, L.H. van; Watts, A.O.; Rops, A.L.; Aguilar, D.C.; Ziarek, J.J.; Vlag, J. van der; Handel, T.M.; Volkman, B.F.; Proudfoot, A.E.; Vischer, H.F.; Zaman, G.J.; Smit, M.J.

    2014-01-01

    Chemokines comprise a family of secreted proteins that activate G protein-coupled chemokine receptors and thereby control the migration of leukocytes during inflammation or immune surveillance. The positional information required for such migratory behavior is governed by the binding of chemokines t

  11. A 3D-QSAR-driven approach to binding mode and affinity prediction

    DEFF Research Database (Denmark)

    Tosco, Paolo; Balle, Thomas

    2012-01-01

    A method for predicting the binding mode of a series of ligands is proposed. The procedure relies on three-dimensional quantitative structure-activity relationships (3D-QSAR) and does not require structural knowledge of the binding site. Candidate alignments are automatically built and ranked...... according to a consensus scoring function. 3D-QSAR analysis based on the selected binding mode enables affinity prediction of new drug candidates having less than 10 rotatable bonds....

  12. Degradation of the starch components amylopectin and amylose by barley α-amylase 1: Role of surface binding site 2

    DEFF Research Database (Denmark)

    Nielsen, Jonas Willum; Kramhøft, Birte; Bozonnet, Sophie;

    2012-01-01

    is required for binding of the amylose helix mimic, β-cyclodextrin. Also, mutant enzymes altered at position 380 displayed reduced binding to starch granules. Similarly, binding of wild type AMY1 to starch granules was suppressed in the presence of β-cyclodextrin. We investigated the role of SBS2 by comparing......Barley α-amylase isozyme 1 (AMY1, EC 3.2.1.1) contains two surface binding sites, SBS1 and SBS2, involved in the degradation of starch granules. The distinct role of SBS1 and SBS2 remains to be fully understood. Mutational analysis of Tyr-380 situated at SBS2 previously revealed that Tyr-380...... kinetic properties of the wild type AMY1 and the Y380A mutant enzyme in hydrolysis of amylopectin, amylose and β-limit dextrin, and the inhibition by β-cyclodextrin. Progress curves of the release of reducing ends revealed a bi-exponential hydrolysis of amylopectin and β-limit dextrin, whereas hydrolysis...

  13. Molecular Weight, Protein Binding Affinity and Methane Mitigation of Condensed Tannins from Mangosteen-peel (Garcinia mangostana L).

    Science.gov (United States)

    Paengkoum, P; Phonmun, T; Liang, J B; Huang, X D; Tan, H Y; Jahromi, M F

    2015-10-01

    The objectives of this study were to determine the molecular weight of condensed tannins (CT) extracted from mangosteen (Garcinia mangostana L) peel, its protein binding affinity and effects on fermentation parameters including total gas, methane (CH4) and volatile fatty acids (VFA) production. The average molecular weight (Mw) of the purified CT was 2,081 Da with a protein binding affinity of 0.69 (the amount needed to bind half the maximum bovine serum albumin). In vitro gas production declined by 0.409, 0.121, and 0.311, respectively, while CH4 production decreased by 0.211, 0.353, and 0.549, respectively, with addition of 10, 20, and 30 mg CT/500 mg dry matter (DM) compared to the control (p<0.05). The effects of CT from mangosteen-peel on in vitro DM degradability (IVDMD) and in vitro N degradability was negative and linear (p<0.01). Total VFA, concentrations of acetic, propionic, butyric and isovaleric acids decreased linearly with increasing amount of CT. The aforementioned results show that protein binding affinity of CT from mangosteen-peel is lower than those reported for Leucaena forages, however, the former has stronger negative effect on IVDMD. Therefore, the use of mangosteen-peel as protein source and CH4 mitigating agent in ruminant feed requires further investigations. PMID:26323400

  14. Architecture and RNA binding of the human negative elongation factor

    Science.gov (United States)

    Vos, Seychelle M; Pöllmann, David; Caizzi, Livia; Hofmann, Katharina B; Rombaut, Pascaline; Zimniak, Tomasz; Herzog, Franz; Cramer, Patrick

    2016-01-01

    Transcription regulation in metazoans often involves promoter-proximal pausing of RNA polymerase (Pol) II, which requires the 4-subunit negative elongation factor (NELF). Here we discern the functional architecture of human NELF through X-ray crystallography, protein crosslinking, biochemical assays, and RNA crosslinking in cells. We identify a NELF core subcomplex formed by conserved regions in subunits NELF-A and NELF-C, and resolve its crystal structure. The NELF-AC subcomplex binds single-stranded nucleic acids in vitro, and NELF-C associates with RNA in vivo. A positively charged face of NELF-AC is involved in RNA binding, whereas the opposite face of the NELF-AC subcomplex binds NELF-B. NELF-B is predicted to form a HEAT repeat fold, also binds RNA in vivo, and anchors the subunit NELF-E, which is confirmed to bind RNA in vivo. These results reveal the three-dimensional architecture and three RNA-binding faces of NELF. DOI: http://dx.doi.org/10.7554/eLife.14981.001 PMID:27282391

  15. Acid Gas Capture Using CO2-Binding Organic Liquids

    Energy Technology Data Exchange (ETDEWEB)

    Heldebrant, David J.; Koech, Phillip K.; Rainbolt, James E.; Zheng, Feng

    2010-11-10

    Current chemical CO2 scrubbing technology is primarily aqueous alkanolamine based. These systems rapidly bind CO2 (forming water-soluble carbamate and bicarbonate salts) however, the process has serious disadvantages. The concentration of monoethanolamine rarely exceeds 30 wt % due to the corrosive nature of the solution, and this reduces the maximum CO2 volumetric (≤108 g/L) and gravimetric capacity (≤7 wt%) of the CO2 scrubber. The ≤30 wt % loading of ethanolamine also means that a large excess of water must be pumped and heated during CO2 capture and release, and this greatly increases the energy requirements especially considering the high specific heat of water (4 j/g-1K-1). Our approach is to switch to organic systems that chemically bind CO2 as liquid alkylcarbonate salts. Our CO2-binding organic liquids have higher CO2 solubility, lower specific heats, potential for less corrosion and lower binding energies for CO2 than aqueous systems. CO2BOLs also reversibly bind and release mixed sulfur oxides. Furthermore the CO2BOL system can be direct solvent replacements for any solvent based CO2 capture systems because they are commercially available reagents and because they are fluids they would not require extensive process re-engineering.

  16. Insulin requirements in type 1 diabetic pregnancy

    DEFF Research Database (Denmark)

    Callesen, Nicoline; Ringholm, Lene; Stage, Edna;

    2012-01-01

    To evaluate the insulin requirements in women with type 1 diabetes during twin pregnancy compared with singleton pregnancy.......To evaluate the insulin requirements in women with type 1 diabetes during twin pregnancy compared with singleton pregnancy....

  17. Probing protein phosphatase substrate binding

    DEFF Research Database (Denmark)

    Højlys-Larsen, Kim B.; Sørensen, Kasper Kildegaard; Jensen, Knud Jørgen;

    2012-01-01

    profile of the integrin-linked kinase associated phosphatase (ILKAP), a member of the protein phosphatase 2C (PP2C) family. Phosphatases can potentially dephosphorylate these phosphopeptide substrates but, interestingly, performing the binding studies at 4 °C allowed efficient binding to phosphopeptides...... around the phosphorylated residue are important for the binding affinity of ILKAP. We conclude that solid-phase affinity pull-down of proteins from complex mixtures can be applied in phosphoproteomics and systems biology.......Proteomics and high throughput analysis for systems biology can benefit significantly from solid-phase chemical tools for affinity pull-down of proteins from complex mixtures. Here we report the application of solid-phase synthesis of phosphopeptides for pull-down and analysis of the affinity...

  18. Water binding in legume seeds

    Science.gov (United States)

    Vertucci, C. W.; Leopold, A. C.

    1987-01-01

    The physical status of water in seeds has a pivotal role in determining the physiological reactions that can take place in the dry state. Using water sorption isotherms from cotyledon and axis tissue of five leguminous seeds, the strength of water binding and the numbers of binding sites have been estimated using van't Hoff analyses and the D'Arcy/Watt equation. These parameters of water sorption are calculated for each of the three regions of water binding and for a range of temperatures. Water sorption characteristics are reflective of the chemical composition of the biological materials as well as the temperature at which hydration takes place. Changes in the sorption characteristics with temperature and hydration level may suggest hydration-induced structural changes in cellular components.

  19. Comparative study on implementation of management requires from ABNT NBR ISO/IEC 17025; Estudo comparativo de implementacao de requisitos gerenciais da ABNT NBR ISO/TEC 17025 e boas praticas de gestao em laboratorios de calibracao da area de radiacoes ionizantes

    Energy Technology Data Exchange (ETDEWEB)

    Suplino Filho, Carlos Alberto Lucas; Souza, Luciane de Rezende; Oliveira, Estela Maria de, E-mail: clucas@ird.gov.br, E-mail: rluciane@ird.gov.br, E-mail: estela@ird.gov.br [Instituto de Radioprotecao e Dosimetria, (IRD/CNEN-RJ), Rio de Janeiro, RJ (Brazil)

    2014-07-01

    This work was developed in order to emphasize the importance of laboratory management system in the direction and control of the company or institute with regard to the quality of delivery of ionizing radiation service to society. It was developed a comparative study of managerial points of Deming with the managerial requirements of ISO / IEC 17025, which found that the difficulties of the laboratories indicated by nonconformities tracked during audits, are related to specific points cited by Deming. (author)

  20. Membrane binding of Neuronal Calcium Sensor-1 (NCS1).

    Science.gov (United States)

    Lemire, Samuel; Jeromin, Andreas; Boisselier, Élodie

    2016-03-01

    Neuronal Calcium Sensor-1 (NCS1) belongs to the family of Neuronal Calcium Sensor (NCS) proteins. NCS1 is composed of four EF-hand motifs and an N-terminal myristoylation. However, the presence of a calcium-myristoyl switch in NCS1 and its role in the membrane binding are controversial. The model of Langmuir lipid monolayers is thus used to mimic the cell membrane in order to characterize the membrane interactions of NCS1. Two binding parameters are calculated from monolayer measurements: the maximum insertion pressure, up to which protein binding is energetically favorable, and the synergy, reporting attractive or repulsive interactions with the lipid monolayers. Binding membrane measurements performed in the presence of myristoylated NCS1 reveal better binding interactions for phospholipids composed of phosphoethanolamine polar head groups and unsaturated fatty acyl chains. In the absence of calcium, the membrane binding measurements are drastically modified and suggest that the protein is more strongly bound to the membrane. Indeed, the binding of calcium by three EF-hand motifs of NCS1 leads to a conformation change. NCS1 arrangement at the membrane could thus be reshuffled for better interactions with its substrates. The N-terminal peptide of NCS1 is composed of two amphiphilic helices involved in the membrane interactions of NCS1. Moreover, the presence of the myristoyl group has a weak influence on the membrane binding of NCS1 suggesting the absence of a calcium-myristoyl switch mechanism in this protein. The myristoylation could thus have a structural role required in the folding/unfolding of NCS1 which is essential to its multiple biological functions.

  1. Characterization of cap binding proteins associated with the nucleus

    International Nuclear Information System (INIS)

    Eucaryotic mRNAs a carry 7-methylguanosine triphosphate residue (called cap structure) at their 5' terminus. The cap plays an important role in RNA recognition. Cap binding proteins (CBP) of HeLa cells were identified by photoaffinity labelling using the cap analogue γ-(32P)-(4-(benzoyl-phenyl)methylamido)-7-methylguanosine-5'-triphosphate (BP-m7GTP). Photoreaction of this cap analogue with HeLa cell initiation factors resulted in specific labelling of two polypeptides of Msub(r) 37000 and 26000. The latter was also labelled in crude initiation factors prepared from reticulocytes and is identical to the cap binding protein CBP I previously identified. These cap binding proteins were also affinity labelled in poliovirus infected cell extracts. Photoaffinity reaction with BP-m7GTP of whole HeLa cell homogenate showed three additional polypeptides with Msub(r) 120000, 89000 and 80000. These cap binding proteins were found to be associated with the nucleus and are therefore referred to as nuclear cap binding proteins, i.e. NCBP 1, NCBP 2 and NCBP 3. They were also present in splicing extracts. Photoaffinity labelling in these nuclear extracts was differentially inhibited by various cap analogues and capped mRNAs. Affinity chromatography on immobilized globin mRNA led to a partial separation of the three nuclear cap binding proteins. Chromatography on m7GTP-Sepharose resulted in a specific binding of NCBP 3. The different behaviour of the cap binding proteins suggests that they are functionally distinct and that they might be involved in different processes requiring cap recognition. (Author)

  2. Computational Prediction of RNA-Binding Proteins and Binding Sites.

    Science.gov (United States)

    Si, Jingna; Cui, Jing; Cheng, Jin; Wu, Rongling

    2015-01-01

    Proteins and RNA interaction have vital roles in many cellular processes such as protein synthesis, sequence encoding, RNA transfer, and gene regulation at the transcriptional and post-transcriptional levels. Approximately 6%-8% of all proteins are RNA-binding proteins (RBPs). Distinguishing these RBPs or their binding residues is a major aim of structural biology. Previously, a number of experimental methods were developed for the determination of protein-RNA interactions. However, these experimental methods are expensive, time-consuming, and labor-intensive. Alternatively, researchers have developed many computational approaches to predict RBPs and protein-RNA binding sites, by combining various machine learning methods and abundant sequence and/or structural features. There are three kinds of computational approaches, which are prediction from protein sequence, prediction from protein structure, and protein-RNA docking. In this paper, we review all existing studies of predictions of RNA-binding sites and RBPs and complexes, including data sets used in different approaches, sequence and structural features used in several predictors, prediction method classifications, performance comparisons, evaluation methods, and future directions.

  3. Computational Prediction of RNA-Binding Proteins and Binding Sites

    Directory of Open Access Journals (Sweden)

    Jingna Si

    2015-11-01

    Full Text Available Proteins and RNA interaction have vital roles in many cellular processes such as protein synthesis, sequence encoding, RNA transfer, and gene regulation at the transcriptional and post-transcriptional levels. Approximately 6%–8% of all proteins are RNA-binding proteins (RBPs. Distinguishing these RBPs or their binding residues is a major aim of structural biology. Previously, a number of experimental methods were developed for the determination of protein–RNA interactions. However, these experimental methods are expensive, time-consuming, and labor-intensive. Alternatively, researchers have developed many computational approaches to predict RBPs and protein–RNA binding sites, by combining various machine learning methods and abundant sequence and/or structural features. There are three kinds of computational approaches, which are prediction from protein sequence, prediction from protein structure, and protein-RNA docking. In this paper, we review all existing studies of predictions of RNA-binding sites and RBPs and complexes, including data sets used in different approaches, sequence and structural features used in several predictors, prediction method classifications, performance comparisons, evaluation methods, and future directions.

  4. Yeast Golgi-localized, γ-Ear–containing, ADP-Ribosylation Factor-binding Proteins Are but Adaptor Protein-1 Is Not Required for Cell-free Transport of Membrane Proteins from the Trans-Golgi Network to the Prevacuolar Compartment

    OpenAIRE

    Abazeed, Mohamed E.; Fuller, Robert S.

    2008-01-01

    Golgi-localized, γ-Ear–containing, ADP-ribosylation factor-binding proteins (GGAs) and adaptor protein-1 (AP-1) mediate clathrin-dependent trafficking of transmembrane proteins between the trans-Golgi network (TGN) and endosomes. In yeast, the vacuolar sorting receptor Vps10p follows a direct pathway from the TGN to the late endosome/prevacuolar compartment (PVC), whereas, the processing protease Kex2p partitions between the direct pathway and an indirect pathway through the early endosome. T...

  5. Skyrmions with low binding energies

    Directory of Open Access Journals (Sweden)

    Mike Gillard

    2015-06-01

    Full Text Available Nuclear binding energies are investigated in two variants of the Skyrme model: the first replaces the usual Skyrme term with a term that is sixth order in derivatives, and the second includes a potential that is quartic in the pion fields. Solitons in the first model are shown to deviate significantly from ansätze previously assumed in the literature. The binding energies obtained in both models are lower than those obtained from the standard Skyrme model, and those obtained in the second model are close to the experimental values.

  6. Skyrmions with low binding energies

    Energy Technology Data Exchange (ETDEWEB)

    Gillard, Mike, E-mail: m.n.gillard@leeds.ac.uk; Harland, Derek, E-mail: d.g.harland@leeds.ac.uk; Speight, Martin, E-mail: speight@maths.leeds.ac.uk

    2015-06-15

    Nuclear binding energies are investigated in two variants of the Skyrme model: the first replaces the usual Skyrme term with a term that is sixth order in derivatives, and the second includes a potential that is quartic in the pion fields. Solitons in the first model are shown to deviate significantly from ansätze previously assumed in the literature. The binding energies obtained in both models are lower than those obtained from the standard Skyrme model, and those obtained in the second model are close to the experimental values.

  7. Cell-Binding Assays for Determining the Affinity of Protein-Protein Interactions: Technologies and Considerations.

    Science.gov (United States)

    Hunter, S A; Cochran, J R

    2016-01-01

    Determining the equilibrium-binding affinity (Kd) of two interacting proteins is essential not only for the biochemical study of protein signaling and function but also for the engineering of improved protein and enzyme variants. One common technique for measuring protein-binding affinities uses flow cytometry to analyze ligand binding to proteins presented on the surface of a cell. However, cell-binding assays require specific considerations to accurately quantify the binding affinity of a protein-protein interaction. Here we will cover the basic assumptions in designing a cell-based binding assay, including the relevant equations and theory behind determining binding affinities. Further, two major considerations in measuring binding affinities-time to equilibrium and ligand depletion-will be discussed. As these conditions have the potential to greatly alter the Kd, methods through which to avoid or minimize them will be provided. We then outline detailed protocols for performing direct- and competitive-binding assays against proteins displayed on the surface of yeast or mammalian cells that can be used to derive accurate Kd values. Finally, a comparison of cell-based binding assays to other types of binding assays will be presented. PMID:27586327

  8. Multiple sup 3 H-oxytocin binding sites in rat myometrial plasma membranes

    Energy Technology Data Exchange (ETDEWEB)

    Crankshaw, D.; Gaspar, V.; Pliska, V. (McMaster Univ., Hamilton, Ontario, (Canada))

    1990-01-01

    The affinity spectrum method has been used to analyse binding isotherms for {sup 3}H-oxytocin to rat myometrial plasma membranes. Three populations of binding sites with dissociation constants (Kd) of 0.6-1.5 x 10(-9), 0.4-1.0 x 10(-7) and 7 x 10(-6) mol/l were identified and their existence verified by cluster analysis based on similarities between Kd, binding capacity and Hill coefficient. When experimental values were compared to theoretical curves constructed using the estimated binding parameters, good fits were obtained. Binding parameters obtained by this method were not influenced by the presence of GTP gamma S (guanosine-5'-O-3-thiotriphosphate) in the incubation medium. The binding parameters agree reasonably well with those found in uterine cells, they support the existence of a medium affinity site and may allow for an explanation of some of the discrepancies between binding and response in this system.

  9. Synthetic heparin-binding growth factor analogs

    Science.gov (United States)

    Pena, Louis A.; Zamora, Paul; Lin, Xinhua; Glass, John D.

    2007-01-23

    The invention provides synthetic heparin-binding growth factor analogs having at least one peptide chain that binds a heparin-binding growth factor receptor, covalently bound to a hydrophobic linker, which is in turn covalently bound to a non-signaling peptide that includes a heparin-binding domain. The synthetic heparin-binding growth factor analogs are useful as soluble biologics or as surface coatings for medical devices.

  10. Ice-binding proteins that accumulate on different ice crystal planes produce distinct thermal hysteresis dynamics.

    Science.gov (United States)

    Drori, Ran; Celik, Yeliz; Davies, Peter L; Braslavsky, Ido

    2014-09-01

    Ice-binding proteins that aid the survival of freeze-avoiding, cold-adapted organisms by inhibiting the growth of endogenous ice crystals are called antifreeze proteins (AFPs). The binding of AFPs to ice causes a separation between the melting point and the freezing point of the ice crystal (thermal hysteresis, TH). TH produced by hyperactive AFPs is an order of magnitude higher than that produced by a typical fish AFP. The basis for this difference in activity remains unclear. Here, we have compared the time dependence of TH activity for both hyperactive and moderately active AFPs using a custom-made nanolitre osmometer and a novel microfluidics system. We found that the TH activities of hyperactive AFPs were time-dependent, and that the TH activity of a moderate AFP was almost insensitive to time. Fluorescence microscopy measurement revealed that despite their higher TH activity, hyperactive AFPs from two insects (moth and beetle) took far longer to accumulate on the ice surface than did a moderately active fish AFP. An ice-binding protein from a bacterium that functions as an ice adhesin rather than as an antifreeze had intermediate TH properties. Nevertheless, the accumulation of this ice adhesion protein and the two hyperactive AFPs on the basal plane of ice is distinct and extensive, but not detectable for moderately active AFPs. Basal ice plane binding is the distinguishing feature of antifreeze hyperactivity, which is not strictly needed in fish that require only approximately 1°C of TH. Here, we found a correlation between the accumulation kinetics of the hyperactive AFP at the basal plane and the time sensitivity of the measured TH.

  11. Using engineered single-chain antibodies to correlate molecular binding properties and nanoparticle adhesion dynamics.

    Science.gov (United States)

    Haun, Jered B; Pepper, Lauren R; Boder, Eric T; Hammer, Daniel A

    2011-11-15

    Elucidation of the relationship between targeting molecule binding properties and the adhesive behavior of therapeutic or diagnostic nanocarriers would aid in the design of optimized vectors and lead to improved efficacy. We measured the adhesion of 200-nm-diameter particles under fluid flow that was mediated by a diverse array of molecular interactions, including recombinant single-chain antibodies (scFvs), full antibodies, and the avidin/biotin interaction. Within the panel of scFvs, we used a family of mutants that display a spectrum of binding kinetics, allowing us to compare nanoparticle adhesion to bond chemistry. In addition, we explored the effect of molecular size by inserting a protein linker into the scFv fusion construct and by employing scFvs that are specific for targets with vastly different sizes. Using computational models, we extracted multivalent kinetic rate constants for particle attachment and detachment from the adhesion data and correlated the results to molecular binding properties. Our results indicate that the factors that increase encounter probability, such as adhesion molecule valency and size, directly enhance the rate of nanoparticle attachment. Bond kinetics had no influence on scFv-mediated nanoparticle attachment within the kinetic range tested, however, but did appear to affect antibody/antigen and avidin/biotin mediated adhesion. We attribute this finding to a combination of multivalent binding and differences in bond mechanical strength between recombinant scFvs and the other adhesion molecules. Nanoparticle detachment probability correlated directly with adhesion molecule valency and size, as well as the logarithm of the affinity for all molecules tested. On the basis of this work, scFvs can serve as viable targeting receptors for nanoparticles, but improvements to their bond mechanical strength would likely be required to fully exploit their tunable kinetic properties and maximize the adhesion efficiency of nanoparticles that

  12. Is Fortran Still Relevant? Comparing Fortran with Java and C++

    Directory of Open Access Journals (Sweden)

    Shahid Alam

    2014-06-01

    Full Text Available This paper presents a comparative study to evaluate and compare Fortran with the two most popular programming languages Java and C++. Fortran has gone through major and minor extensions in the years 2003 and 2008. (1 How much have these extensions made Fortran comparable to Java and ++? (2 What are the differences and similarities, in supporting features like: Templates, object constructors and destructors, abstract data types and dynamic binding? These are the main questions we are trying to answer in this study. An object-oriented ray tracing application is implemented in these three languages to compare them. By using only one program we ensured there was only one set of requirements thus making the comparison homogeneous. Based on our literature survey this is the first study carried out to compare these languages by applying software metrics to the ray tracing application and comparing these results with the similarities and differences found in practice. We motivate the language implementers and compiler developers, by providing binary analysis and profiling of the application, to improve Fortran object handling and processing, and hence making it more prolific and general. This study facilitates and encourages the reader to further explore, study and use these languages more effectively and productively, especially Fortran.

  13. Tropomyosin-binding properties modulate competition between tropomodulin isoforms.

    Science.gov (United States)

    Colpan, Mert; Moroz, Natalia A; Gray, Kevin T; Cooper, Dillon A; Diaz, Christian A; Kostyukova, Alla S

    2016-06-15

    The formation and fine-tuning of cytoskeleton in cells are governed by proteins that influence actin filament dynamics. Tropomodulin (Tmod) regulates the length of actin filaments by capping the pointed ends in a tropomyosin (TM)-dependent manner. Tmod1, Tmod2 and Tmod3 are associated with the cytoskeleton of non-muscle cells and their expression has distinct consequences on cell morphology. To understand the molecular basis of differences in the function and localization of Tmod isoforms in a cell, we compared the actin filament-binding abilities of Tmod1, Tmod2 and Tmod3 in the presence of Tpm3.1, a non-muscle TM isoform. Tmod3 displayed preferential binding to actin filaments when competing with other isoforms. Mutating the second or both TM-binding sites of Tmod3 destroyed its preferential binding. Our findings clarify how Tmod1, Tmod2 and Tmod3 compete for binding actin filaments. Different binding mechanisms and strengths of Tmod isoforms for Tpm3.1 contribute to their divergent functional capabilities.

  14. Tropomyosin-binding properties modulate competition between tropomodulin isoforms.

    Science.gov (United States)

    Colpan, Mert; Moroz, Natalia A; Gray, Kevin T; Cooper, Dillon A; Diaz, Christian A; Kostyukova, Alla S

    2016-06-15

    The formation and fine-tuning of cytoskeleton in cells are governed by proteins that influence actin filament dynamics. Tropomodulin (Tmod) regulates the length of actin filaments by capping the pointed ends in a tropomyosin (TM)-dependent manner. Tmod1, Tmod2 and Tmod3 are associated with the cytoskeleton of non-muscle cells and their expression has distinct consequences on cell morphology. To understand the molecular basis of differences in the function and localization of Tmod isoforms in a cell, we compared the actin filament-binding abilities of Tmod1, Tmod2 and Tmod3 in the presence of Tpm3.1, a non-muscle TM isoform. Tmod3 displayed preferential binding to actin filaments when competing with other isoforms. Mutating the second or both TM-binding sites of Tmod3 destroyed its preferential binding. Our findings clarify how Tmod1, Tmod2 and Tmod3 compete for binding actin filaments. Different binding mechanisms and strengths of Tmod isoforms for Tpm3.1 contribute to their divergent functional capabilities. PMID:27091317

  15. Sex hormone binding globulin phenotypes

    DEFF Research Database (Denmark)

    Cornelisse, M M; Bennett, Patrick; Christiansen, M;

    1994-01-01

    Human sex hormone binding globulin (SHBG) is encoded by a normal and a variant allele. The resulting SHBG phenotypes (the homozygous normal SHBG, the heterozygous SHBG and the homozygous variant SHBG phenotype) can be distinguished by their electrophoretic patterns. We developed a novel detection...

  16. BINDING ISOTHERMS SURFACTANT-PROTEINS

    OpenAIRE

    Elena Irina Moater; Cristiana Radulescu; Ionica Ionita

    2011-01-01

    The interactions between surfactants and proteins shows some similarities with interactions between surfactants and polymers, but the hydrophobic amphoteric nature of proteins and their secondary and tertiary structure components make them different from conventional polymer systems. Many studies from the past about surfactant - proteins bonding used the dialysis techniques. Other techniques used to determine the binding isotherm, included ultrafiltration, ultracentrifugation, potentiometry, ...

  17. Cellulose binding domain fusion proteins

    Energy Technology Data Exchange (ETDEWEB)

    Shoseyov, Oded (Karmey Yosef, IL); Shpiegl, Itai (Rehovot, IL); Goldstein, Marc A. (Davis, CA); Doi, Roy H. (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.

  18. Reflection-Based Python-C++ Bindings

    International Nuclear Information System (INIS)

    Python is a flexible, powerful, high-level language with excellent interactive and introspective capabilities and a very clean syntax. As such, it can be a very effective tool for driving physics analysis. Python is designed to be extensible in low-level C-like languages, and its use as a scientific steering language has become quite widespread. To this end, existing and custom-written C or C++ libraries are bound to the Python environment as so-called extension modules. A number of tools for easing the process of creating such bindings exist, such as SWIG and Boost. Python. Yet, the process still requires a considerable amount of effort and expertise. The C++ language has few built-in introspective capabilities, but tools such as LCGDict and CINT add this by providing so-called dictionaries: libraries that contain information about the names, entry points, argument types, etc. of other libraries. The reflection information from these dictionaries can be used for the creation of bindings and so the process can be fully automated, as dictionaries are already provided for many end-user libraries for other purposes, such as object persistency. PyLCGDict is a Python extension module that uses LCG dictionaries, as PyROOT uses CINT reflection information, to allow /cwPython users to access C++ libraries with essentially no preparation on the users' behalf. In addition, and in a similar way, PyROOT gives ROOT users access to Python libraries

  19. The PUF binding landscape in metazoan germ cells

    Science.gov (United States)

    Prasad, Aman; Porter, Douglas F.; Kroll-Conner, Peggy L.; Mohanty, Ipsita; Ryan, Anne R.; Crittenden, Sarah L.; Wickens, Marvin; Kimble, Judith

    2016-01-01

    PUF (Pumilio/FBF) proteins are RNA-binding proteins and conserved stem cell regulators. The Caenorhabditis elegans PUF proteins FBF-1 and FBF-2 (collectively FBF) regulate mRNAs in germ cells. Without FBF, adult germlines lose all stem cells. A major gap in our understanding of PUF proteins, including FBF, is a global view of their binding sites in their native context (i.e., their “binding landscape”). To understand the interactions underlying FBF function, we used iCLIP (individual-nucleotide resolution UV crosslinking and immunoprecipitation) to determine binding landscapes of C. elegans FBF-1 and FBF-2 in the germline tissue of intact animals. Multiple iCLIP peak-calling methods were compared to maximize identification of both established FBF binding sites and positive control target mRNAs in our iCLIP data. We discovered that FBF-1 and FBF-2 bind to RNAs through canonical as well as alternate motifs. We also analyzed crosslinking-induced mutations to map binding sites precisely and to identify key nucleotides that may be critical for FBF–RNA interactions. FBF-1 and FBF-2 can bind sites in the 5′UTR, coding region, or 3′UTR, but have a strong bias for the 3′ end of transcripts. FBF-1 and FBF-2 have strongly overlapping target profiles, including mRNAs and noncoding RNAs. From a statistically robust list of 1404 common FBF targets, 847 were previously unknown, 154 were related to cell cycle regulation, three were lincRNAs, and 335 were shared with the human PUF protein PUM2. PMID:27165521

  20. Melittin binding to mixed phosphatidylglycerol/phosphatidylcholine membranes

    Energy Technology Data Exchange (ETDEWEB)

    Beschiaschvili, G.; Seelig, J. (Univ. of Basel (Switzerland))

    1990-01-09

    The binding of bee venom melittin to negatively charged unilamellar vesicles and planar lipid bilayers composed of 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine (POPC) and 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphoglycerol (POPG) was studied with circular dichroism and deuterium NMR spectroscopy. The melittin binding isotherm was measured for small unilamellar vesicles containing 10 or 20 mol % POPG. Due to electrostatic attraction, binding of the positively charged melittin was much enhanced as compared to the binding to neutral lipid vesicles. However, after correction for electrostatic effects by means of the Gouy-Chapman theory, all melittin binding isotherms could be described by a partition Kp = (4.5 +/- 0.6) x 10(4) M-1. It was estimated that about 50% of the total melittin surface was embedded in a hydrophobic environment. The melittin partition constant for small unilamellar vesicles was by a factor of 20 larger than that of planar bilayers and attests to the tighter lipid packing in the nonsonicated bilayers. Deuterium NMR studies were performed with coarse lipid dispersions. Binding of melittin to POPC/POPG (80/20 mol/mol) membranes caused systematic changes in the conformation of the phosphocholine and phosphoglycerol head groups which were ascribed to the influence of electrostatic charge on the choline dipole. While the negative charge of phosphatidylglycerol moved the N+ end of the choline -P-N+ dipole toward the bilayer interior, the binding of melittin reversed this effect and rotated the N+ end toward the aqueous phase. No specific melittin-POPG complexes could be detected. The phosphoglycerol head group was less affected by melittin binding than its choline counterpart.

  1. Probing heterobivalent binding to the endocytic AP-2 adaptor complex by DNA-based spatial screening.

    Science.gov (United States)

    Diezmann, F; von Kleist, L; Haucke, V; Seitz, O

    2015-08-01

    The double helical DNA scaffold offers a unique set of properties, which are particularly useful for studies of multivalency in biomolecular interactions: (i) multivalent ligand displays can be formed upon nucleic acid hybridization in a self-assembly process, which facilitates spatial screening (ii) valency and spatial arrangement of the ligand display can be precisely controlled and (iii) the flexibility of the ligand display can be adjusted by integrating nick sites and unpaired template regions. Herein we describe the use of DNA-based spatial screening for the characterization of the adaptor complex 2 (AP-2), a central interaction hub within the endocytic protein network in clathrin-mediated endocytosis. AP-2 is comprised of a core domain and two, so-called appendage domains, the α- and the β2-ear, which associate with cytoplasmatic proteins required for the formation or maturation of clathrin/AP-2 coated pits. Each appendage domain has two binding grooves which recognize distinct peptide motives with micromolar affinity. This provides opportunities for enhanced interactions with protein molecules that contain two (or more) different peptide motives. To determine whether a particular, spatial arrangement of binding motifs is required for high affinity binding we probed the distance-affinity relationships by means of DNA-programmed spatial screening with self-assembled peptide-DNA complexes. By using trimolecular and tetramolecular assemblies two different peptides were positioned in 2-22 nucleotide distance. The binding data obtained with both recombinant protein in well-defined buffer systems and native AP-2 in brain extract suggests that the two binding sites of the AP-2 α-appendage can cooperate to provide up to 40-fold enhancement of affinity compared to the monovalent interaction. The distance between the two recognized peptide motives was less important provided that the DNA duplex segments were connected by flexible, single strand segments. By

  2. The Cobalamin-binding Protein in Zebrafish is an Intermediate Between the Three Cobalamin-binding Proteins in Human

    DEFF Research Database (Denmark)

    Greibe, Eva Holm; Fedosov, Sergey; Nexø, Ebba

    2012-01-01

    In humans, three soluble extracellular cobalamin-binding proteins; transcobalamin (TC), intrinsic factor (IF), and haptocorrin (HC), are involved in the uptake and transport of cobalamin. In this study, we investigate a cobalamin-binding protein from zebrafish (Danio rerio) and summarize current...... knowledge concerning the phylogenetic evolution of kindred proteins. We identified a cobalamin binding capacity in zebrafish protein extracts (8.2 pmol/fish) and ambient water (13.5 pmol/fish) associated with a single protein. The protein showed resistance toward degradation by trypsin and chymotrypsin...... (like human IF, but unlike human HC and TC). The cobalamin analogue, cobinamide, bound weaker to the zebrafish cobalamin binder than to human HC, but stronger than to human TC and IF. Affinity for another analogue, adenosyl-pseudo-cobalamin was low compared with human HC and TC, but high compared...

  3. Comparative integromics on Eph family.

    Science.gov (United States)

    Katoh, Masuko; Katoh, Masaru

    2006-05-01

    EPHA1, EPHA2, EPHA3, EPHA4, EPHA5, EPHA6, EPHA7, EPHA8, EPHA10, EPHB1, EPHB2, EPHB3, EPHB4 and EPHB6 are EPH family receptors for Ephrin family ligands. Ephrin/EPH signaling pathway networks with the WNT signaling pathway during embryogenesis, tissue regeneration, and carcinogenesis. TCF/LEF-binding sites within the promoter region of human EPH family members were searched for by using bioinformatics and human intelligence. Because five TCF/LEF-binding sites were identified within the 5'-promoter region of the EPHA7 gene, comparative genomics analyses on EPHA7 orthologs were further performed. EPHA7-MANEA-FHL5 locus at human chromosome 6q16.1 and EPHA10-MANEAL-FHL3 locus at human chromosome 1p34.3 were paralogous regions within the human genome. Human EPHA7 mRNA was expressed in embryonic stem (ES) cells, neural tissues, duodenal cancer and parathyroid tumors, while mouse Epha7 mRNA was expressed in fertilized egg, Rathke's pouche, visual cortex, pituitary gland, other neural tissues, pancreas, lung tumors and mammary tumors. The chimpanzee EPHA7 gene and cow Epha7 gene were identified within NW_107969.1 and AC155055.2 genome sequences, respectively. Five TCF/LEF-binding sites within human EPHA7 promoter were conserved in the chimpanzee EPHA7 promoter, and three TCF/LEF-binding sites in the cow Epha7 promoter, but none in the mouse Epha7 promoter. Primates and cow EPHA7 orthologs were identified as evolutionarily conserved targets of the WNT/beta-catenin signaling pathway. D6S1056 microsatellite marker within EPHA7 gene is deleted in prostate cancer. Deletion and/or promoter CpG hypermethylation could explain the EPHA7 down-regulation in human tumors. EPHA7 is a target of systems medicine, especially in the fields of regenerative medicine and oncology. PMID:16596241

  4. Role of insulin secretagogues in the regulation of somatostatin binding by isolated rat islets.

    OpenAIRE

    Mehler, P S; Sussman, A L; Maman, A; Leitner, J W; Sussman, K E

    1980-01-01

    To study the possible role of the secretion vesicle inligant-receptor interaction, somatostatin binding was measured in islets in the presence of various substances known to promote secretion vesicle migration and fusion with the plasma membrane and insulin release. Rat islets were incubated with glucose, 30 and 300 mg/dl, for 60 min. After inculation, somatostatin binding was measured. In islets preincubated with glucose, 300 mg/dl, somatostatin binding was increased 250% when compared with ...

  5. Assessment of algorithms for inferring positional weight matrix motifs of transcription factor binding sites using protein binding microarray data.

    Directory of Open Access Journals (Sweden)

    Yaron Orenstein

    Full Text Available The new technology of protein binding microarrays (PBMs allows simultaneous measurement of the binding intensities of a transcription factor to tens of thousands of synthetic double-stranded DNA probes, covering all possible 10-mers. A key computational challenge is inferring the binding motif from these data. We present a systematic comparison of four methods developed specifically for reconstructing a binding site motif represented as a positional weight matrix from PBM data. The reconstructed motifs were evaluated in terms of three criteria: concordance with reference motifs from the literature and ability to predict in vivo and in vitro bindings. The evaluation encompassed over 200 transcription factors and some 300 assays. The results show a tradeoff between how the methods perform according to the different criteria, and a dichotomy of method types. Algorithms that construct motifs with low information content predict PBM probe ranking more faithfully, while methods that produce highly informative motifs match reference motifs better. Interestingly, in predicting high-affinity binding, all methods give far poorer results for in vivo assays compared to in vitro assays.

  6. Binding energy and fine structure of the He- ion

    Institute of Scientific and Technical Information of China (English)

    ZHUO; Lin; ZHU; Jing-jing; GOU; Bing-cong

    2007-01-01

    The variational method using a multiconfiguration wavefunction is carried out on the core-excited state 1s2s2p 4P0 for helium negative ion, including mass polarization and relativistic corrections. Binding energy and fine structure are reported. The results are compared with other theoretical and experimental date in the literature.

  7. D-2 receptor binding in dopa-responsive dystonia

    NARCIS (Netherlands)

    Kunig, G; Leenders, KL; Antonini, A; Vontobel, P; Weindl, A; Meinck, HM

    1998-01-01

    We have studied dopamine D-2 receptor binding by [C-11]raclopride positron emission tomography in 14 patients with dopa-responsive dystonia (DRD). Data were compared with 16 levodopa-treated patients with Parkinson's disease (PD) and 26 healthy controls. The results revealed an elevated [C-11]raclop

  8. Voluntary Binding Arbitration as an Alternative to Tax Court Litigation

    OpenAIRE

    Sansing, Richard

    1997-01-01

    Develops and analyzes a model of Tax Court litigation, in which each side possesses private information regarding the facts under dispute. Characterizes equilibrium behavior for both conventional and final-offer arbitration. Compares the results of the two arbitration methods, both in terms of the probability that the parties will agree to binding arbitration and the outcomes of the two arbitration methods.

  9. Comprehensive human transcription factor binding site map for combinatory binding motifs discovery.

    Directory of Open Access Journals (Sweden)

    Arnoldo J Müller-Molina

    Full Text Available To know the map between transcription factors (TFs and their binding sites is essential to reverse engineer the regulation process. Only about 10%-20% of the transcription factor binding motifs (TFBMs have been reported. This lack of data hinders understanding gene regulation. To address this drawback, we propose a computational method that exploits never used TF properties to discover the missing TFBMs and their sites in all human gene promoters. The method starts by predicting a dictionary of regulatory "DNA words." From this dictionary, it distills 4098 novel predictions. To disclose the crosstalk between motifs, an additional algorithm extracts TF combinatorial binding patterns creating a collection of TF regulatory syntactic rules. Using these rules, we narrowed down a list of 504 novel motifs that appear frequently in syntax patterns. We tested the predictions against 509 known motifs confirming that our system can reliably predict ab initio motifs with an accuracy of 81%-far higher than previous approaches. We found that on average, 90% of the discovered combinatorial binding patterns target at least 10 genes, suggesting that to control in an independent manner smaller gene sets, supplementary regulatory mechanisms are required. Additionally, we discovered that the new TFBMs and their combinatorial patterns convey biological meaning, targeting TFs and genes related to developmental functions. Thus, among all the possible available targets in the genome, the TFs tend to regulate other TFs and genes involved in developmental functions. We provide a comprehensive resource for regulation analysis that includes a dictionary of "DNA words," newly predicted motifs and their corresponding combinatorial patterns. Combinatorial patterns are a useful filter to discover TFBMs that play a major role in orchestrating other factors and thus, are likely to lock/unlock cellular functional clusters.

  10. A novel p53-binding domain in CUL7.

    Science.gov (United States)

    Kasper, Jocelyn S; Arai, Takehiro; DeCaprio, James A

    2006-09-15

    CUL7 is a member of the cullin RING ligase family and forms an SCF-like complex with SKP1 and FBXW8. CUL7 is required for normal mouse embryonic development and cellular proliferation, and is highly homologous to PARC, a p53-associated, parkin-like cytoplasmic protein. We determined that CUL7, in a manner similar to PARC, can bind directly to p53 but does not affect p53 expression. We identified a discrete, co-linear domain in CUL7 that is conserved in PARC and HERC2, and is necessary and sufficient for p53-binding. The presence of p53 stabilized expression of this domain and we demonstrate that this p53-binding domain of CUL7 contributes to the cytoplasmic localization of CUL7. The results support the model that p53 plays a role in regulation of CUL7 activity. PMID:16875676

  11. Isothermal Titration Calorimetry Measurements of Metal Ions Binding to Proteins.

    Science.gov (United States)

    Quinn, Colette F; Carpenter, Margaret C; Croteau, Molly L; Wilcox, Dean E

    2016-01-01

    ITC measurements involving metal ions are susceptible to a number of competing reactions (oxidation, precipitation, and hydrolysis) and coupled reactions involving the buffer and protons. Stabilization and delivery of the metal ion as a well-defined and well-characterized complex with the buffer, or a specific ligand, can suppress undesired solution chemistry and, depending on the stability of the metal complex, allow accurate measurements of higher affinity protein-binding sites. This requires, however, knowledge of the thermodynamics of formation of the metal complex and accounting for its contribution to the experimentally measured values (KITC and ΔHITC) through a post hoc analysis that provides the condition-independent binding thermodynamics (K, ΔG(o), ΔH, ΔS, and ΔCP). This analysis also quantifies the number of protons that are displaced when the metal ion binds to the protein.

  12. Altering Antibody-Drug Conjugate Binding to the Neonatal Fc Receptor Impacts Efficacy and Tolerability.

    Science.gov (United States)

    Hamblett, Kevin J; Le, Tiep; Rock, Brooke M; Rock, Dan A; Siu, Sophia; Huard, Justin N; Conner, Kip P; Milburn, Robert R; O'Neill, Jason W; Tometsko, Mark E; Fanslow, William C

    2016-07-01

    Antibody-drug conjugates (ADC) rely on the target-binding specificity of an antibody to selectively deliver potent drugs to cancer cells. IgG antibody half-life is regulated by neonatal Fc receptor (FcRn) binding. Histidine 435 of human IgG was mutated to alanine (H435A) to explore the effect of FcRn binding on the pharmacokinetics, efficacy, and tolerability of two separate maytansine-based ADC pairs with noncleavable linkers, (c-DM1 and c-H435A-DM1) and (7v-Cys-may and 7v-H435A-Cys-may). The in vitro cell-killing potency of each pair of ADCs was similar, demonstrating that H435A showed no measurable impact on ADC bioactivity. The H435A mutant antibodies showed no detectable binding to human or mouse FcRn in vitro, whereas their counterpart wild-type IgG ADCs were found to bind to FcRn at pH = 6.0. In xenograft bearing SCID mice expressing mouse FcRn, the AUC of 7v-Cys-may was 1.6-fold higher than that of 7v-H435A-may, yet the observed efficacy was similar. More severe thrombocytopenia was observed with 7v-H435A-Cys-may as compared to 7v-Cys-may at multiple dose levels. The AUC of c-DM1 was approximately 3-fold higher than that of c-H435A-DM1 in 786-0 xenograft bearing SCID mice, which led to a 3-fold difference in efficacy by dose. Murine FcRn knockout, human FcRn transgenic line 32 SCID animals bearing 786-0 xenografts showed an amplified exposure difference between c-DM1 and c-H435A-DM1 as compared to murine FcRn expressing SCID mice, leading to a 10-fold higher dose required for efficacy despite a 6-fold higher AUC of the c-H435A-DM1. The accelerated clearance observed for the noncleavable maytansine ADCs with the H435A FcRn mutation led to reduced efficacy at equivalent doses and exacerbation of clinical pathology parameters (decreased tolerability) at equivalent doses. The results show that reduced ADC clearance mediated by FcRn modulation can improve therapeutic index. PMID:27248573

  13. Using Carbohydrate Interaction Assays to Reveal Novel Binding Sites in Carbohydrate Active Enzymes

    DEFF Research Database (Denmark)

    Cockburn, Darrell; Wilkens, Casper; Dilokpimol, Adiphol;

    2016-01-01

    Carbohydrate active enzymes often contain auxiliary binding sites located either on independent domains termed carbohydrate binding modules (CBMs) or as so-called surface binding sites (SBSs) on the catalytic module at a certain distance from the active site. The SBSs are usually critical...... for the activity of their cognate enzyme, though they are not readily detected in the sequence of a protein, but normally require a crystal structure of a complex for their identification. A variety of methods, including affinity electrophoresis (AE), insoluble polysaccharide pulldown (IPP) and surface plasmon...... resonance (SPR) have been used to study auxiliary binding sites. These techniques are complementary as AE allows monitoring of binding to soluble polysaccharides, IPP to insoluble polysaccharides and SPR to oligosaccharides. Here we show that these methods are useful not only for analyzing known binding...

  14. Characteristics of human erythrocyte insulin binding sites.

    OpenAIRE

    Okada, Yoshio

    1981-01-01

    Insulin and human erythrocyte cell membrane interactions were studied with respect to binding and dissociation. The per cent of specific binding of 125I-labeled insulin to erythrocytes was directly proportional to the cell concentration. The optimum pH for binding was 8.1. The initial binding rate was directly proportional to, and the steady state insulin binding was reversely proportional to, the incubation temperature. The per cent of specific binding of 125I-labeled insulin was 12.10 +/- 1...

  15. Natural history of S-adenosylmethionine-binding proteins

    Directory of Open Access Journals (Sweden)

    Mushegian Arcady R

    2005-10-01

    Full Text Available Abstract Background S-adenosylmethionine is a source of diverse chemical groups used in biosynthesis and modification of virtually every class of biomolecules. The most notable reaction requiring S-adenosylmethionine, transfer of methyl group, is performed by a large class of enzymes, S-adenosylmethionine-dependent methyltransferases, which have been the focus of considerable structure-function studies. Evolutionary trajectories of these enzymes, and especially of other classes of S-adenosylmethionine-binding proteins, nevertheless, remain poorly understood. We addressed this issue by computational comparison of sequences and structures of various S-adenosylmethionine-binding proteins. Results Two widespread folds, Rossmann fold and TIM barrel, have been repeatedly used in evolution for diverse types of S-adenosylmethionine conversion. There were also cases of recruitment of other relatively common folds for S-adenosylmethionine binding. Several classes of proteins have unique unrelated folds, specialized for just one type of chemistry and unified by the theme of internal domain duplications. In several cases, functional divergence is evident, when evolutionarily related enzymes have changed the mode of binding and the type of chemical transformation of S-adenosylmethionine. There are also instances of functional convergence, when biochemically similar processes are performed by drastically different classes of S-adenosylmethionine-binding proteins. Comparison of remote sequence similarities and analysis of phyletic patterns suggests that the last universal common ancestor of cellular life had between 10 and 20 S-adenosylmethionine-binding proteins from at least 5 fold classes, providing for S-adenosylmethionine formation, polyamine biosynthesis, and methylation of several substrates, including nucleic acids and peptide chain release factor. Conclusion We have observed several novel relationships between families that were not known to be

  16. Modification of the loops in the ligand-binding site turns avidin into a steroid-binding protein

    Directory of Open Access Journals (Sweden)

    Kulomaa Markku S

    2011-06-01

    Full Text Available Abstract Background Engineered proteins, with non-immunoglobulin scaffolds, have become an important alternative to antibodies in many biotechnical and therapeutic applications. When compared to antibodies, tailored proteins may provide advantageous properties such as a smaller size or a more stable structure. Results Avidin is a widely used protein in biomedicine and biotechnology. To tailor the binding properties of avidin, we have designed a sequence-randomized avidin library with mutagenesis focused at the loop area of the binding site. Selection from the generated library led to the isolation of a steroid-binding avidin mutant (sbAvd-1 showing micromolar affinity towards testosterone (Kd ~ 9 μM. Furthermore, a gene library based on the sbAvd-1 gene was created by randomizing the loop area between β-strands 3 and 4. Phage display selection from this library led to the isolation of a steroid-binding protein with significantly decreased biotin binding affinity compared to sbAvd-1. Importantly, differential scanning calorimetry and analytical gel-filtration revealed that the high stability and the tetrameric structure were preserved in these engineered avidins. Conclusions The high stability and structural properties of avidin make it an attractive molecule for the engineering of novel receptors. This methodology may allow the use of avidin as a universal scaffold in the development of novel receptors for small molecules.

  17. Dissection of the Critical Binding Determinants of Cellular Retinoic Acid Binding Protein II by Mutagenesis and Fluorescence Binding Assay

    OpenAIRE

    Vasileiou, Chrysoula; Lee, Kin Sing Stephen; Crist, Rachael M.; Vaezeslami, Soheila; Goins, Sarah M.; Geiger, James H.; Borhan, Babak

    2009-01-01

    The binding of retinoic acid to mutants of Cellular Retinoic Acid Binding Protein II (CRABPII) was evaluated to better understand the importance of the direct protein/ligand interactions. The important role of Arg111 for the correct structure and function of the protein was verified and other residues that directly affect retinoic acid binding have been identified. Furthermore, retinoic acid binding to CRABPII mutants that lack all previously identified interacting amino acids was rescued by ...

  18. Study on Synthesis and Binding Ability of a New Anion Receptor Containing NH Binding Sites

    Institute of Scientific and Technical Information of China (English)

    QIAO,Yan-Hong; LIN,Hai; LIN,Hua-Kuan

    2007-01-01

    A new colorimetric recognition receptor 1 based on the dual capability containing NH binding sites of selectively sensing anionic guest species has been synthesized. Compared with other halide anions, its UV/Vis absorption spectrum in dimethyl sulfoxide showed the response toward the presence of fluoride anion with high selectivity,and also displayed dramatic color changes from colorless to yellow in the presence of TBAF (5 × 10-5 mol/L). The similar UV/Vis absorption spectrum change also occurred when 1 was treated with AcO- while a little change with H2PO-4 and OH-. Receptor 1 has almost not affinity abilities to Cl-, Br- and I-. The binding ability of receptor 1to fluoride with high selectivity over other halides contributes to the anion size and the ability of forming hydrogen bonding. While the different ability of binding with geometrically triangular (AcO-), tetrahedral (H2PO-4 ) and linear (OH-) anions maybe result from their geometry configuration.

  19. Characterization of Heparin-binding Site of Tissue Transglutaminase

    Science.gov (United States)

    Wang, Zhuo; Collighan, Russell J.; Pytel, Kamila; Rathbone, Daniel L.; Li, Xiaoling; Griffin, Martin

    2012-01-01

    Tissue transglutaminase (TG2) is a multifunctional Ca2+-activated protein cross-linking enzyme secreted into the extracellular matrix (ECM), where it is involved in wound healing and scarring, tissue fibrosis, celiac disease, and metastatic cancer. Extracellular TG2 can also facilitate cell adhesion important in wound healing through a nontransamidating mechanism via its association with fibronectin, heparan sulfates (HS), and integrins. Regulating the mechanism how TG2 is translocated into the ECM therefore provides a strategy for modulating these physiological and pathological functions of the enzyme. Here, through molecular modeling and mutagenesis, we have identified the HS-binding site of TG2 202KFLKNAGRDCSRRSSPVYVGR222. We demonstrate the requirement of this binding site for translocation of TG2 into the ECM through a mechanism involving cell surface shedding of HS. By synthesizing a peptide NPKFLKNAGRDCSRRSS corresponding to the HS-binding site within TG2, we also demonstrate how this mimicking peptide can in isolation compensate for the RGD-induced loss of cell adhesion on fibronectin via binding to syndecan-4, leading to activation of PKCα, pFAK-397, and ERK1/2 and the subsequent formation of focal adhesions and actin cytoskeleton organization. A novel regulatory mechanism for TG2 translocation into the extracellular compartment that depends upon TG2 conformation and the binding of HS is proposed. PMID:22298777

  20. Human plasminogen binding protein tetranectin

    DEFF Research Database (Denmark)

    Kastrup, J S; Rasmussen, H; Nielsen, B B;

    1997-01-01

    The recombinant human plasminogen binding protein tetranectin (TN) and the C-type lectin CRD of this protein (TN3) have been crystallized. TN3 crystallizes in the tetragonal space group P4(2)2(1)2 with cell dimensions a = b = 64.0, c = 75.7 A and with one molecule per asymmetric unit. The crystals...... to at least 2.5 A. A full data set has been collected to 3.0 A. The asymmetric unit contains one monomer of TN. Molecular replacement solutions for TN3 and TN have been obtained using the structure of the C-type lectin CRD of rat mannose-binding protein as search model. The rhombohedral space group indicates...