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Sample records for type c-repeat binding

  1. Cloning of Ammopiptanthus mongolicus C-repeat-binding factor gene and its cold-induced tolerance in transgenic tobacco

    Directory of Open Access Journals (Sweden)

    Lijiang Gu

    2013-12-01

    Full Text Available C-repeat-binding factors (CBFs are a type of important regulon in stress-related signal transduction pathways that control plant tolerance of abiotic stress. Ammopiptanthus mongolicus is the only evergreen broadleaf shrub in the northwest desert of China. The species shows strong resistance to environmental stress, especially to cold stress. An A. mongolicus CBF1 gene (AmCBF1 was cloned and transformed into tobacco. Expression of AmCBF1 could be detected in A. mongolicus shortly after exposure to low temperature of 4°C. Analysis on ratio of electrolytic leakage, soluble sugar content, free proline content, malondialdehyde (MDA content and peroxidase (POD activity before and after cold treatment (4°C for 24 h indicated AmCBF1 conferred higher cold tolerance to AmCBF1 transgenic tobacco compared with the wild type and empty vector transformed tobacco.

  2. Low-temperature-induced expression of rice ureidoglycolate amidohydrolase is mediated by a C-repeat/dehydration-responsive element that specifically interacts with rice C-repeat-binding factor 3

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    Juan eLi

    2015-11-01

    Full Text Available Nitrogen recycling and redistribution are important for the environmental stress response of plants. In non nitrogen-fixing plants, ureide metabolism is crucial to nitrogen recycling from organic sources. Various studies have suggested that the rate-limiting components of ureide metabolism respond to environmental stresses. However, the underlying regulation mechanism is not well understood. In this report, rice ureidoglycolate amidohydrolase (OsUAH, which is a recently identified enzyme catalyzing the final step of ureide degradation, was identified as low-temperature- (LT but not abscisic acid- (ABA regulated. To elucidate the LT regulatory mechanism at the transcriptional level, we isolated and characterized the promoter region of OsUAH (POsUAH. Series deletions revealed that a minimal region between -522 and -420 relative to the transcriptional start site was sufficient for the cold induction of POsUAH. Detailed analyses of this 103-bp fragment indicated that a C-repeat/dehydration-responsive (CRT/DRE element localized at position -434 was essential for LT-responsive expression. A rice C-repeat-binding factors/DRE-binding proteins 1 (CBFs/DREB1s subfamily member, OsCBF3, was screened to specifically bind to the CRT/DRE element in the minimal region both in yeast one-hybrid assays and in in vitro gel-shift analysis. Moreover, the promoter could be exclusively trans-activated by the interaction between the CRT/DRE element and OsCBF3 in vivo. These findings may help to elucidate the regulation mechanism of stress-responsive ureide metabolism genes and provide an example of the member-specific manipulation of the CBF/DREB1 subfamily.

  3. Meta-analysis of the effect of overexpression of C-repeat/dehydration-responsive element binding family genes on temperature stress tolerance and related responses

    Science.gov (United States)

    C-repeat/dehydration-responsive element binding proteins are transcription factors that play a critical role in plant response to temperature stress. Over-expression of CBF/DREB genes has been demonstrated to enhance temperature stress tolerance. A series of physiological and biochemical modificat...

  4. Transcriptome Profiling Reveals the Negative Regulation of Multiple Plant Hormone Signaling Pathways Elicited by Overexpression of C-Repeat Binding Factors

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    Aixin Li

    2017-09-01

    Full Text Available C-repeat binding factors (CBF are a subfamily of AP2 transcription factors that play critical roles in the regulation of plant cold tolerance and growth in low temperature. In the present work, we sought to perform a detailed investigation into global transcriptional regulation of plant hormone signaling associated genes in transgenic plants engineered with CBF genes. RNA samples from Arabidopsis thaliana plants overexpressing two CBF genes, CBF2 and CBF3, were subjected to Illumina HiSeq 2000 RNA sequencing (RNA-Seq. Our results showed that more than half of the hormone associated genes that were differentially expressed in CBF2 or CBF3 transgenic plants were related to auxin signal transduction and metabolism. Most of these alterations in gene expression could lead to repression of auxin signaling. Accordingly, the IAA content was significantly decreased in young tissues of plants overexpressing CBF2 and CBF3 compared with wild type. In addition, genes associated with the biosynthesis of Jasmonate (JA and Salicylic acid (SA, as well as the signal sensing of Brassinolide (BR and SA, were down-regulated, while genes associated with Gibberellin (GA deactivation were up-regulated. In general, overexpression of CBF2 and CBF3 negatively affects multiple plant hormone signaling pathways in Arabidopsis. The transcriptome analysis using CBF2 and CBF3 transgenic plants provides novel and integrated insights into the interaction between CBFs and plant hormones, particularly the modulation of auxin signaling, which may contribute to the improvement of crop yields under abiotic stress via molecular engineering using CBF genes.

  5. Cassava C-repeat binding factor 1 gene responds to low temperature and enhances cold tolerance when overexpressed in Arabidopsis and cassava.

    Science.gov (United States)

    An, Dong; Ma, Qiuxiang; Wang, Hongxia; Yang, Jun; Zhou, Wenzhi; Zhang, Peng

    2017-05-01

    Cassava MeCBF1 is a typical CBF transcription factor mediating cold responses but its low expression in apical buds along with a retarded response cause inefficient upregulation of downstream cold-related genes, rendering cassava chilling-sensitive. Low temperature is a major abiotic stress factor affecting survival, productivity and geographic distribution of important crops worldwide. The C-repeat/dehydration-responsive element binding transcription factors (CBF/DREB) are important regulators of abiotic stress response in plants. In this study, MeCBF1, a CBF-like gene, was identified in the tropical root crop cassava (Manihot esculenta Crantz). The MeCBF1 encodes a protein that shares strong homology with DREB1As/CBFs from Arabidopsis as well as other species. The MeCBF1 was localized to the nucleus and is mainly expressed in stem and mature leaves, but not in apical buds or stem cambium. MeCBF1 expression was not only highly responsive to cold, but also significantly induced by salt, PEG and ABA treatment. Several stress-associated cis-elements were found in its promoter region, e.g., ABRE-related, MYC recognition sites, and MYB responsive element. Compared with AtCBF1, the MeCBF1 expression induced by cold in cassava was retarded and upregulated only after 4 h, which was also confirmed by its promoter activity. Overexpression of MeCBF1 in transgenic Arabidopsis and cassava plants conferred enhanced crytolerance. The CBF regulon was smaller and not entirely co-regulated with MeCBF1 expression in overexpressed cassava. The retarded MeCBF1 expression in response to cold and attenuated CBF-regulon might lead cassava to chilling sensitivity.

  6. Salicylic-Acid-Induced Chilling- and Oxidative-Stress Tolerance in Relation to Gibberellin Homeostasis, C-Repeat/Dehydration-Responsive Element Binding Factor Pathway, and Antioxidant Enzyme Systems in Cold-Stored Tomato Fruit.

    Science.gov (United States)

    Ding, Yang; Zhao, Jinhong; Nie, Ying; Fan, Bei; Wu, Shujuan; Zhang, Yu; Sheng, Jiping; Shen, Lin; Zhao, Ruirui; Tang, Xuanming

    2016-11-02

    Effects of salicylic acid (SA) on gibberellin (GA) homeostasis, C-repeat/dehydration-responsive element binding factor (CBF) pathway, and antioxidant enzyme systems linked to chilling- and oxidative-stress tolerance in tomato fruit were investigated. Mature green tomatoes (Solanum lycopersicum L. cv. Moneymaker) were treated with 0, 0.5, and 1 mM SA solution for 15 min before storage at 4 °C for 28 days. In comparison to 0 or 0.5 mM SA, 1 mM SA significantly decreased the chilling injury (CI) index in tomato fruit. In the SA-treated fruit, the upregulation of GA biosynthetic gene (GA3ox1) expression was followed by gibberellic acid (GA 3 ) surge and DELLA protein degradation. CBF1 participated in the SA-modulated tolerance and stimulated the expression of GA catabolic gene (GA2ox1). Furthermore, 1 mM SA enhanced activities of antioxidant enzymes and, thus, reduced reactive oxygen species accumulation. Our findings suggest that SA might protect tomato fruit from CI and oxidative damage through regulating GA metabolism, CBF1 gene expression, and antioxidant enzyme activities.

  7. The decorin sequence SYIRIADTNIT binds collagen type I

    DEFF Research Database (Denmark)

    Kalamajski, Sebastian; Aspberg, Anders; Oldberg, Ake

    2007-01-01

    Decorin belongs to the small leucine-rich repeat proteoglycan family, interacts with fibrillar collagens, and regulates the assembly, structure, and biomechanical properties of connective tissues. The decorin-collagen type I-binding region is located in leucine-rich repeats 5-6. Site......-directed mutagenesis of this 54-residue-long collagen-binding sequence identifies Arg-207 and Asp-210 in leucine-rich repeat 6 as crucial for the binding to collagen. The synthetic peptide SYIRIADTNIT, which includes Arg-207 and Asp-210, inhibits the binding of full-length recombinant decorin to collagen in vitro....... These collagen-binding amino acids are exposed on the exterior of the beta-sheet-loop structure of the leucine-rich repeat. This resembles the location of interacting residues in other leucine-rich repeat proteins....

  8. Analysis of the ligand binding properties of recombinant bovine liver-type fatty acid binding protein

    DEFF Research Database (Denmark)

    Rolf, B; Oudenampsen-Krüger, E; Börchers, T

    1995-01-01

    The coding part of the cDNA for bovine liver-type fatty acid binding protein (L-FABP) has been amplified by RT-PCR, cloned and used for the construction of an Escherichia coli (E. coli) expression system. The recombinant protein made up to 25% of the soluble E. coli proteins and could be isolated...

  9. The Adenovirus Type 3 Dodecahedron's RGD Loop Comprises an HSPG Binding Site That Influences Integrin Binding

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    E. Gout

    2010-01-01

    Full Text Available Human type 3 adenovirus dodecahedron (a virus like particle made of twelve penton bases features the ability to enter cells through Heparan Sulphate Proteoglycans (HSPGs and integrins interaction and is used as a versatile vector to deliver DNA or proteins. Cryo-EM reconstruction of the pseudoviral particle with Heparan Sulphate (HS oligosaccharide shows an extradensity on the RGD loop. A set of mutants was designed to study the respective roles of the RGD sequence (RGE mutant and of a basic sequence located just downstream. Results showed that the RGE mutant binding to the HS deficient CHO-2241 cells was abolished and unexpectedly, mutation of the basic sequence (KQKR to AQAS dramatically decreased integrin recognition by the viral pseudoparticle. This basic sequence is thus involved in integrin docking, showing a close interplay between HSPGs and integrin receptors.

  10. Structural Analysis of Botulinum Neurotoxin Type G Receptor Binding

    Energy Technology Data Exchange (ETDEWEB)

    Schmitt, John; Karalewitz, Andrew; Benefield, Desire A.; Mushrush, Darren J.; Pruitt, Rory N.; Spiller, Benjamin W.; Barbieri, Joseph T.; Lacy, D. Borden (Vanderbilt); (MCW)

    2010-10-19

    Botulinum neurotoxin (BoNT) binds peripheral neurons at the neuromuscular junction through a dual-receptor mechanism that includes interactions with ganglioside and protein receptors. The receptor identities vary depending on BoNT serotype (A-G). BoNT/B and BoNT/G bind the luminal domains of synaptotagmin I and II, homologous synaptic vesicle proteins. We observe conditions under which BoNT/B binds both Syt isoforms, but BoNT/G binds only SytI. Both serotypes bind ganglioside G{sub T1b}. The BoNT/G receptor-binding domain crystal structure provides a context for examining these binding interactions and a platform for understanding the physiological relevance of different Syt receptor isoforms in vivo.

  11. Value of heart-type fatty acid-binding protein (H-FABP) for ...

    African Journals Online (AJOL)

    Key Words: heart-type fatty acid-binding protein, acute coronary syndrome, biomarker. ... is essential to prevent major complications and death. Routinely used biomarkers such ..... fatty acid binding proteins: their function and physiological sig-.

  12. Fungal-type carbohydrate binding modules from the coccolithophore Emiliania huxleyi show binding affinity to cellulose and chitin.

    Science.gov (United States)

    Rooijakkers, Bart J M; Ikonen, Martina S; Linder, Markus B

    2018-01-01

    Six fungal-type cellulose binding domains were found in the genome of the coccolithophore Emiliania huxleyi and cloned and expressed in Escherichia coli. Sequence comparison indicate high similarity to fungal cellulose binding domains, raising the question of why these domains exist in coccolithophores. The proteins were tested for binding with cellulose and chitin as ligands, which resulted in the identification of two functional carbohydrate binding modules: EHUX2 and EHUX4. Compared to benchmark fungal cellulose binding domain Cel7A-CBM1 from Trichoderma reesei, these proteins showed slightly lower binding to birch and bacterial cellulose, but were more efficient chitin binders. Finally, a set of cellulose binding domains was created based on the shuffling of one well-functioning and one non-functional domain. These were characterized in order to get more information of the binding domain's sequence-function relationship, indicating characteristic differences between the molecular basis of cellulose versus chitin recognition. As previous reports have showed the presence of cellulose in coccoliths and here we find functional cellulose binding modules, a possible connection is discussed.

  13. Fungal-type carbohydrate binding modules from the coccolithophore Emiliania huxleyi show binding affinity to cellulose and chitin.

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    Bart J M Rooijakkers

    Full Text Available Six fungal-type cellulose binding domains were found in the genome of the coccolithophore Emiliania huxleyi and cloned and expressed in Escherichia coli. Sequence comparison indicate high similarity to fungal cellulose binding domains, raising the question of why these domains exist in coccolithophores. The proteins were tested for binding with cellulose and chitin as ligands, which resulted in the identification of two functional carbohydrate binding modules: EHUX2 and EHUX4. Compared to benchmark fungal cellulose binding domain Cel7A-CBM1 from Trichoderma reesei, these proteins showed slightly lower binding to birch and bacterial cellulose, but were more efficient chitin binders. Finally, a set of cellulose binding domains was created based on the shuffling of one well-functioning and one non-functional domain. These were characterized in order to get more information of the binding domain's sequence-function relationship, indicating characteristic differences between the molecular basis of cellulose versus chitin recognition. As previous reports have showed the presence of cellulose in coccoliths and here we find functional cellulose binding modules, a possible connection is discussed.

  14. Crystal structure of the botulinum neurotoxin type G binding domain: insight into cell surface binding.

    Science.gov (United States)

    Stenmark, Pål; Dong, Min; Dupuy, Jérôme; Chapman, Edwin R; Stevens, Raymond C

    2010-04-16

    Botulinum neurotoxins (BoNTs) typically bind the neuronal cell surface via dual interactions with both protein receptors and gangliosides. We present here the 1.9-A 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. Copyright (c) 2010. Published by Elsevier Ltd.

  15. GABAA [gamma-aminobutyric acid] type binding sites on membranes of spermatozoa

    International Nuclear Information System (INIS)

    Erdoe, S.L.; Wekerle, L.

    1990-01-01

    The binding of [ 3 H] gamma-aminobutyric acid (GABA) to seminal membranes of swines and rams was examined. Specific, GABA binding was demonstrated in both species, which showed the features of GABA A type receptors. The affinity of binding was similar in both species, whereas the density of seminal GABA binding sites was 5 times higher in swine. Our findings suggest that GABA may have a direct effect on spermatozoa

  16. The pancreatic zymogen granule membrane protein, GP2, binds Escherichia coli type 1 Fimbriae

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    Lowe Anson W

    2009-07-01

    Full Text Available Abstract Background GP2 is the major membrane protein present in the pancreatic zymogen granule, and is cleaved and released into the pancreatic duct along with exocrine secretions. The function of GP2 is unknown. GP2's amino acid sequence is most similar to that of uromodulin, which is secreted by the kidney. Recent studies have demonstrated uromodulin binding to bacterial Type 1 fimbria. The fimbriae serve as adhesins to host receptors. The present study examines whether GP2 also shares similar binding properties to bacteria with Type 1 fimbria. Commensal and pathogenic bacteria, including E. coli and Salmonella, express type 1 fimbria. Methods An in vitro binding assay was used to assay the binding of recombinant GP2 to defined strains of E. coli that differ in their expression of Type 1 fimbria or its subunit protein, FimH. Studies were also performed to determine whether GP2 binding is dependent on the presence of mannose residues, which is a known determinant for FimH binding. Results GP2 binds E. coli that express Type 1 fimbria. Binding is dependent on GP2 glycosylation, and specifically the presence of mannose residues. Conclusion GP2 binds to Type 1 fimbria, a bacterial adhesin that is commonly expressed by members of the Enterobacteriacae family.

  17. Porcine circovirus type 2 ORF4 protein binds heavy chain ferritin

    Indian Academy of Sciences (India)

    Porcine circovirus type 2 ORF4 protein binds heavy chain ferritin. Qizhuang Lv Kangkang Guo Tao Wang ... Keywords. Cellular protein; FHC; ORF4 protein; porcine circovirus type 2 (PCV2); yeast two-hybrid ... Journal of Biosciences | News ...

  18. Urinary liver-type fatty acid-binding protein predicts progression to nephropathy in type 1 diabetic patients

    DEFF Research Database (Denmark)

    Nielsen, Stine Elkjaer; Sugaya, Takeshi; Hovind, Peter

    2010-01-01

    Urinary liver-type fatty acid-binding protein (u-LFABP) is a marker of tubulointerstitial inflammation and has been shown to be increased in patients with type 1 diabetes and is further increased in patients who progress to micro- and macroalbuminuria. Our aim was to evaluate u-LFABP as a predictor...... of progression to micro- and macroalbuminuria in type 1 diabetes....

  19. Binding of von Willebrand factor to collagen type III: role of specific amino acids in the collagen binding domain of vWF and effects of neighboring domains

    NARCIS (Netherlands)

    van der Plas, R. M.; Gomes, L.; Marquart, J. A.; Vink, T.; Meijers, J. C.; de Groot, P. G.; Sixma, J. J.; Huizinga, E. G.

    2000-01-01

    Binding of von Willebrand Factor (vWF) to sites of vascular injury is the first step of hemostasis. Collagen types I and III are important binding sites for vWF. We have previously determined the three-dimensional structure of the collagen binding A3 domain of vWF (Huizinga et al., Structure 1997;

  20. Measurement of specific [3H]-ouabain binding to different types of human leucocytes

    DEFF Research Database (Denmark)

    Boon, Arnold; Oh, V M; Taylor, John E.

    1984-01-01

    We have studied the specific binding of [3H]-ouabain to intact mononuclear leucocytes (82% lymphocytes) and polymorphonuclear leucocytes. In both types of cells [3H]-ouabain binding was saturable, confined to a single site of high affinity, slow to reach equilibrium, slow to reverse, temperature...... were expressed per square micron of cell surface area the difference between the two cell types was proportionately greater (83 and 186 sites per micron 2 respectively). We conclude that the [3H]-ouabain binding sites on mononuclear and polymorphonuclear leucocytes are similar in nature, but different...

  1. Urinary liver-type fatty acid-binding protein predicts progression to nephropathy in type 1 diabetic patients

    DEFF Research Database (Denmark)

    Nielsen, Stine Elkjaer; Sugaya, Takeshi; Hovind, Peter

    2010-01-01

    Urinary liver-type fatty acid-binding protein (u-LFABP) is a marker of tubulointerstitial inflammation and has been shown to be increased in patients with type 1 diabetes and is further increased in patients who progress to micro- and macroalbuminuria. Our aim was to evaluate u-LFABP as a predictor...

  2. Cell-type specificity of ChIP-predicted transcription factor binding sites

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    Håndstad Tony

    2012-08-01

    Full Text Available Abstract Background Context-dependent transcription factor (TF binding is one reason for differences in gene expression patterns between different cellular states. Chromatin immunoprecipitation followed by high-throughput sequencing (ChIP-seq identifies genome-wide TF binding sites for one particular context—the cells used in the experiment. But can such ChIP-seq data predict TF binding in other cellular contexts and is it possible to distinguish context-dependent from ubiquitous TF binding? Results We compared ChIP-seq data on TF binding for multiple TFs in two different cell types and found that on average only a third of ChIP-seq peak regions are common to both cell types. Expectedly, common peaks occur more frequently in certain genomic contexts, such as CpG-rich promoters, whereas chromatin differences characterize cell-type specific TF binding. We also find, however, that genotype differences between the cell types can explain differences in binding. Moreover, ChIP-seq signal intensity and peak clustering are the strongest predictors of common peaks. Compared with strong peaks located in regions containing peaks for multiple transcription factors, weak and isolated peaks are less common between the cell types and are less associated with data that indicate regulatory activity. Conclusions Together, the results suggest that experimental noise is prevalent among weak peaks, whereas strong and clustered peaks represent high-confidence binding events that often occur in other cellular contexts. Nevertheless, 30-40% of the strongest and most clustered peaks show context-dependent regulation. We show that by combining signal intensity with additional data—ranging from context independent information such as binding site conservation and position weight matrix scores to context dependent chromatin structure—we can predict whether a ChIP-seq peak is likely to be present in other cellular contexts.

  3. Tetranectin, a trimeric plasminogen-binding C-type lectin

    DEFF Research Database (Denmark)

    Holtet, T L; Graversen, Jonas Heilskov; Clemmensen, I

    1997-01-01

    -linking analysis and SDS-PAGE to be a homo-trimer in solution as are other known members of the collectin family of C-type lectins. Biochemical evidence is presented showing that an N-terminal domain encoded within exons 1 and 2 of the tetranectin gene is necessary and sufficient to govern subunit trimerization....

  4. Identification of carbohydrate-binding domains in the attachment proteins of type 1 and type 3 reoviruses.

    Science.gov (United States)

    Chappell, J D; Duong, J L; Wright, B W; Dermody, T S

    2000-09-01

    The reovirus attachment protein, sigma1, is responsible for strain-specific patterns of viral tropism in the murine central nervous system and receptor binding on cultured cells. The sigma1 protein consists of a fibrous tail domain proximal to the virion surface and a virion-distal globular head domain. To better understand mechanisms of reovirus attachment to cells, we conducted studies to identify the region of sigma1 that binds cell surface carbohydrate. Chimeric and truncated sigma1 proteins derived from prototype reovirus strains type 1 Lang (T1L) and type 3 Dearing (T3D) were expressed in insect cells by using a baculovirus vector. Assessment of expressed protein susceptibility to proteolytic cleavage, binding to anti-sigma1 antibodies, and oligomerization indicates that the chimeric and truncated sigma1 proteins are properly folded. To assess carbohydrate binding, recombinant sigma1 proteins were tested for the capacity to agglutinate mammalian erythrocytes and to bind sialic acid presented on glycophorin, the cell surface molecule bound by type 3 reovirus on human erythrocytes. Using a panel of two wild-type and ten chimeric and truncated sigma1 proteins, the sialic acid-binding domain of type 3 sigma1 was mapped to a region of sequence proposed to form the more amino terminal of two predicted beta-sheet structures in the tail. This unit corresponds to morphologic region T(iii) observed in computer-processed electron micrographs of sigma1 protein purified from virions. In contrast, the homologous region of T1L sigma1 sequence was not implicated in carbohydrate binding; rather, sequences in the distal portion of the tail known as the neck were required. Results of these studies demonstrate that a functional receptor-binding domain, which uses sialic acid as its ligand, is contained within morphologic region T(iii) of the type 3 sigma1 tail. Furthermore, our findings indicate that T1L and T3D sigma1 proteins contain different arrangements of receptor-binding

  5. The influence of cement type and temperature on chloride binding in cement paste

    DEFF Research Database (Denmark)

    Jensen, Ole Mejlhede; Korzen, Migge Sofie Hoffmann; Skibsted, Jørgen

    1998-01-01

    This paper describes effects of cement type and temperature on chloride binding in cement paste, which is an important subject in relation to life-time modelling of reinforced concrete structures. The influence of cement type on chloride binding is investigated by substituting cement with pure...... cement clinker. Both theoretical considerations and experimental data for chloride binding in cement pastes are presented. A physico-chemically based model to describe the influence of temperature on physical binding of chloride is presented. Solid-state 27Al and 29Si magic-angle spinning (MAS) nuclear...... magnetic resonance (NMR) spectroscopy has been used for quantification of the anhydrous and hydrated aluminate and silicate phases in the chloride exposed cement pastes. The 27Al isotropic chemical shift and nuclear quadrupole coupling is reported for a synthetic sample of Friedel's salt, Ca2Al(OH)6Cl×2H2O....

  6. Binding properties of Clostridium botulinum type C progenitor toxin to mucins.

    Science.gov (United States)

    Nakamura, Toshio; Takada, Noriko; Tonozuka, Takashi; Sakano, Yoshiyuki; Oguma, Keiji; Nishikawa, Atsushi

    2007-04-01

    It has been reported that Clostridium botulinum type C 16S progenitor toxin (C16S toxin) first binds to the sialic acid on the cell surface of mucin before invading cells [A. Nishikawa, N. Uotsu, H. Arimitsu, J.C. Lee, Y. Miura, Y. Fujinaga, H. Nakada, T. Watanabe, T. Ohyama, Y. Sakano, K. Oguma, The receptor and transporter for internalization of Clostridium botulinum type C progenitor toxin into HT-29 cells, Biochem. Biophys. Res. Commun. 319 (2004) 327-333]. In this study we investigated the binding properties of the C16S toxin to glycoproteins. Although the toxin bound to membrane blotted mucin derived from the bovine submaxillary gland (BSM), which contains a lot of sialyl oligosaccharides, it did not bind to neuraminidase-treated BSM. The binding of the toxin to BSM was inhibited by N-acetylneuraminic acid, N-glycolylneuraminic acid, and sialyl oligosaccharides strongly, but was not inhibited by neutral oligosaccharides. Both sialyl alpha2-3 lactose and sialyl alpha2-6 lactose prevented binding similarly. On the other hand, the toxin also bound well to porcine gastric mucin. In this case, neutral oligosaccharides might play an important role as ligand, since galactose and lactose inhibited binding. These results suggest that the toxin is capable of recognizing a wide variety of oligosaccharide structures.

  7. DNA binding properties of dioxin receptors in wild-type and mutant mouse hepatoma cells

    International Nuclear Information System (INIS)

    Cuthill, S.; Poellinger, L.

    1988-01-01

    The current model of action of 2,3,7,8-tetrachlorodibenzo-p-dioxin (dioxin) entails stimulation of target gene transcription via the formation of dioxin-receptor complexes and subsequent accumulation of the complexes within the cell nucleus. Here, the authors have analyzed the DNA binding properties of the dioxin receptor in wild-type mouse hepatoma (Hepa 1c1c7) cells and a class of nonresponsive mutant cells which fail to accumulate dioxin-receptor complexes within the nucleus in vivo. In vitro, both the wild-type and mutant [ 3 H]dioxin-receptor complexes exhibited low affinity for DNA-cellulose (5-8% and around 4% retention, respectively) in the absence of prior biochemical manipulations. However, following chromatography on heparin-Sepharose, the wild-type but not the mutant dioxin receptor was transformed to a species with an increased affinity for DNA (40-50% retention on DNA-cellulose). The gross molecular structure of the mutant, non DNA binding dioxin receptor did not appear to be altered as compared to that of the wild-type receptor. These results imply that the primary deficiency in the mutant dioxin receptor form may reside at the DNA binding level and that, in analogy to steroid hormone receptors, DNA binding of the receptor may be an essential step in the regulation of target gene transcription by dioxin

  8. Differential alterations of cortical glutamatergic binding sites in senile dementia of the Alzheimer type

    International Nuclear Information System (INIS)

    Chalmers, D.T.; Dewar, D.; Graham, D.I.; Brooks, D.N.; McCulloch, J.

    1990-01-01

    Involvement of cortical glutamatergic mechanisms in senile dementia of the Alzheimer type (SDAT) has been investigated with quantitative ligand-binding autoradiography. The distribution and density of Na(+)-dependent glutamate uptake sites and glutamate receptor subtypes--kainate, quisqualate, and N-methyl-D-aspartate--were measured in adjacent sections of frontal cortex obtained postmortem from six patients with SDAT and six age-matched controls. The number of senile plaques was determined in the same brain region. Binding of D-[3H]aspartate to Na(+)-dependent uptake sites was reduced by approximately 40% throughout SDAT frontal cortex relative to controls, indicating a general loss of glutamatergic presynaptic terminals. [3H]Kainate receptor binding was significantly increased by approximately 70% in deep layers of SDAT frontal cortex compared with controls, whereas this binding was unaltered in superficial laminae. There was a positive correlation (r = 0.914) between kainate binding and senile plaque number in deep cortical layers. Quisqualate receptors, as assessed by 2-amino-3-hydroxy-5-[3H]methylisoxazole-4-propionic acid binding, were unaltered in SDAT frontal cortex compared with controls. There was a small reduction (25%) in N-methyl-D-aspartate-sensitive [3H]glutamate binding only in superficial cortical layers of SDAT brains relative to control subjects. [3H]Glutamate binding in SDAT subjects was unrelated to senile plaque number in superficial cortical layers (r = 0.104). These results indicate that in the presence of cortical glutamatergic terminal loss in SDAT plastic alterations occur in some glutamate receptor subtypes but not in others

  9. Sex Hormone–Binding Globulin and Risk of Type 2 Diabetes in Women and Men

    Science.gov (United States)

    Ding, Eric L.; Song, Yiqing; Manson, JoAnn E.; Hunter, David J.; Lee, Cathy C.; Rifai, Nader; Buring, Julie E.; Gaziano, J. Michael; Liu, Simin

    2009-01-01

    BACKGROUND Circulating sex hormone–binding globulin levels are inversely associated with insulin resistance, but whether these levels can predict the risk of developing type 2 diabetes is uncertain. METHODS We performed a nested case–control study of postmenopausal women in the Women’s Health Study who were not using hormone therapy (359 with newly diagnosed type 2 diabetes and 359 controls). Plasma levels of sex hormone–binding globulin were measured; two polymorphisms of the gene encoding sex hormone–binding globulin, SHBG, that were robustly associated with the protein levels were genotyped and applied in mendelian randomization analyses. We then conducted a replication study in an independent cohort of men from the Physicians’ Health Study II (170 with newly diagnosed type 2 diabetes and 170 controls). RESULTS Among women, higher plasma levels of sex hormone–binding globulin were prospectively associated with a lower risk of type 2 diabetes: multivariable odds ratios were 1.00 for the first (lowest) quartile of plasma levels, 0.16 (95% confidence interval [CI], 0.08 to 0.33) for the second quartile, 0.04 (95% CI, 0.01 to 0.12) for the third quartile, and 0.09 (95% CI, 0.03 to 0.21) for the fourth (highest) quartile (P<0.001 for trend). These prospective associations were replicated among men (odds ratio for the highest quartile of plasma levels vs. the lowest quartile, 0.10; 95% CI, 0.03 to 0.36; P<0.001 for trend). As compared with homozygotes of the respective wild-type allele, carriers of a variant allele of the SHBG single-nucleotide polymorphism (SNP) rs6259 had 10% higher sex hormone–binding globulin levels (P = 0.005), and carriers of an rs6257 variant had 10% lower plasma levels (P = 0.004); variants of both SNPs were also associated with a risk of type 2 diabetes in directions corresponding to their associated sex hormone–binding globulin levels. In mendelian randomization analyses, the predicted odds ratio of type 2 diabetes per

  10. Purification and characterization of a new type lactose-binding Ulex europaeus lectin by affinity chromatography.

    Science.gov (United States)

    Konami, Y; Yamamoto, K; Osawa, T

    1991-02-01

    A new type lactose-binding lectin was purified from extracts of Ulex europaeus seeds by affinity chromatography on a column of galactose-Sepharose 4B, followed by gel filtration on Sephacryl S-300. This lectin, designated as Ulex europaeus lectin III (UEA-III), was found to be inhibited by lactose. The dimeric lectin is a glycoprotein with a molecular mass of 70,000 Da; it consists of two apparently identical subunits of a molecular mass of 34,000 Da. Compositional analysis showed that this lectin contains 30% carbohydrate and a large amount of aspartic acid, serine and valine, but no sulfur-containing amino acids. The N-terminal amino-acid sequences of L-fucose-binding Ulex europaeus lectin I (UEA-I) and di-N-acetylchitobiose-binding Ulex europaeus lectin II (UEA-II), both of which we have already purified and characterized, and that of UEA-III were determined and compared.

  11. Characterization of tissue plasminogen activator binding proteins isolated from endothelial cells and other cell types

    International Nuclear Information System (INIS)

    Beebe, D.P.; Wood, L.L.; Moos, M.

    1990-01-01

    Human tissue plasminogen activator (t-PA) was shown to bind specifically to human osteosarcoma cells (HOS), and human epidermoid carcinoma cells (A-431 cells). Crosslinking studies with DTSSP demonstrated high molecular weight complexes (130,000) between 125 I-t-PA and cell membrane protein on human umbilical vein endothelial cells (HUVEC), HOS, and A-431 cells. A 48-65,000 molecular weight complex was demonstrated after crosslinking t-PA peptide (res. 7-20) to cells. Ligand blotting of cell lysates which had been passed over a t-PA affinity column revealed binding of t-PA to 54,000 and 95,000 molecular weight proteins. Several t-PA binding proteins were identified in immunopurified cell lysates, including tubulin beta chain, plasminogen activator inhibitor type 1 and single chain urokinase

  12. A type IV pilus mediates DNA binding during natural transformation in Streptococcus pneumoniae.

    Directory of Open Access Journals (Sweden)

    Raphaël Laurenceau

    Full Text Available Natural genetic transformation is widely distributed in bacteria and generally occurs during a genetically programmed differentiated state called competence. This process promotes genome plasticity and adaptability in Gram-negative and Gram-positive bacteria. Transformation requires the binding and internalization of exogenous DNA, the mechanisms of which are unclear. Here, we report the discovery of a transformation pilus at the surface of competent Streptococcus pneumoniae cells. This Type IV-like pilus, which is primarily composed of the ComGC pilin, is required for transformation. We provide evidence that it directly binds DNA and propose that the transformation pilus is the primary DNA receptor on the bacterial cell during transformation in S. pneumoniae. Being a central component of the transformation apparatus, the transformation pilus enables S. pneumoniae, a major Gram-positive human pathogen, to acquire resistance to antibiotics and to escape vaccines through the binding and incorporation of new genetic material.

  13. Exhaustive sampling of docking poses reveals binding hypotheses for propafenone type inhibitors of P-glycoprotein.

    Directory of Open Access Journals (Sweden)

    Freya Klepsch

    2011-05-01

    Full Text Available Overexpression of the xenotoxin transporter P-glycoprotein (P-gp represents one major reason for the development of multidrug resistance (MDR, leading to the failure of antibiotic and cancer therapies. Inhibitors of P-gp have thus been advocated as promising candidates for overcoming the problem of MDR. However, due to lack of a high-resolution structure the concrete mode of interaction of both substrates and inhibitors is still not known. Therefore, structure-based design studies have to rely on protein homology models. In order to identify binding hypotheses for propafenone-type P-gp inhibitors, five different propafenone derivatives with known structure-activity relationship (SAR pattern were docked into homology models of the apo and the nucleotide-bound conformation of the transporter. To circumvent the uncertainty of scoring functions, we exhaustively sampled the pose space and analyzed the poses by combining information retrieved from SAR studies with common scaffold clustering. The results suggest propafenone binding at the transmembrane helices 5, 6, 7 and 8 in both models, with the amino acid residue Y307 playing a crucial role. The identified binding site in the non-energized state is overlapping with, but not identical to, known binding areas of cyclic P-gp inhibitors and verapamil. These findings support the idea of several small binding sites forming one large binding cavity. Furthermore, the binding hypotheses for both catalytic states were analyzed and showed only small differences in their protein-ligand interaction fingerprints, which indicates only small movements of the ligand during the catalytic cycle.

  14. Thermodynamic and NMR analyses of NADPH binding to lipocalin-type prostaglandin D synthase

    International Nuclear Information System (INIS)

    Qin, Shubin; Shimamoto, Shigeru; Maruno, Takahiro; Kobayashi, Yuji; Kawahara, Kazuki; Yoshida, Takuya; Ohkubo, Tadayasu

    2015-01-01

    Lipocalin-type prostaglandin D synthase (L-PGDS) is one of the most abundant proteins in human cerebrospinal fluid (CSF) with dual functions as a prostaglandin D_2 (PGD_2) synthase and a transporter of lipophilic ligands. Recent studies revealed that L-PGDS plays important roles in protecting against various neuronal diseases induced by reactive oxygen species (ROS). However, the molecular mechanisms of such protective actions of L-PGDS remain unknown. In this study, we conducted thermodynamic and nuclear magnetic resonance (NMR) analyses, and demonstrated that L-PGDS binds to nicotinamide coenzymes, including NADPH, NADP"+, and NADH. Although a hydrophilic ligand is not common for L-PGDS, these ligands, especially NADPH showed specific interaction with L-PGDS at the upper pocket of its ligand-binding cavity with an unusually bifurcated shape. The binding affinity of L-PGDS for NADPH was comparable to that previously reported for NADPH oxidases and NADPH in vitro. These results suggested that L-PGDS potentially attenuates the activities of NADPH oxidases through interaction with NADPH. Given that NADPH is the substrate for NADPH oxidases that play key roles in neuronal cell death by generating excessive ROS, these results imply a novel linkage between L-PGDS and ROS. - Highlights: • Interactions of L-PGDS with nicotinamide coenzymes were studied by ITC and NMR. • The binding affinity of L-PGDS was strongest to NADPH among nicotinamide coenzymes. • NADPH binds to the upper part of L-PGDS ligand-binding cavity. • L-PGDS binds to both lipophilic and hydrophilic ligands. • This study implies a novel linkage between L-PGDS and reactive oxygen species.

  15. Thermodynamic and NMR analyses of NADPH binding to lipocalin-type prostaglandin D synthase

    Energy Technology Data Exchange (ETDEWEB)

    Qin, Shubin [Graduate School of Pharmaceutical Sciences, Osaka University, 1-6 Yamadaoka, Suita, Osaka 565-0871 (Japan); Shimamoto, Shigeru [Faculty of Science and Engineering, Kinki University, 3-4-1 Kowakae, Higashi-Osaka, Osaka 577-8502 (Japan); Maruno, Takahiro; Kobayashi, Yuji [Graduate School of Engineering, Osaka University, 2-1 Yamadaoka, Suita, Osaka 565-0871 (Japan); Kawahara, Kazuki; Yoshida, Takuya [Graduate School of Pharmaceutical Sciences, Osaka University, 1-6 Yamadaoka, Suita, Osaka 565-0871 (Japan); Ohkubo, Tadayasu, E-mail: ohkubo@phs.osaka-u.ac.jp [Graduate School of Pharmaceutical Sciences, Osaka University, 1-6 Yamadaoka, Suita, Osaka 565-0871 (Japan)

    2015-12-04

    Lipocalin-type prostaglandin D synthase (L-PGDS) is one of the most abundant proteins in human cerebrospinal fluid (CSF) with dual functions as a prostaglandin D{sub 2} (PGD{sub 2}) synthase and a transporter of lipophilic ligands. Recent studies revealed that L-PGDS plays important roles in protecting against various neuronal diseases induced by reactive oxygen species (ROS). However, the molecular mechanisms of such protective actions of L-PGDS remain unknown. In this study, we conducted thermodynamic and nuclear magnetic resonance (NMR) analyses, and demonstrated that L-PGDS binds to nicotinamide coenzymes, including NADPH, NADP{sup +}, and NADH. Although a hydrophilic ligand is not common for L-PGDS, these ligands, especially NADPH showed specific interaction with L-PGDS at the upper pocket of its ligand-binding cavity with an unusually bifurcated shape. The binding affinity of L-PGDS for NADPH was comparable to that previously reported for NADPH oxidases and NADPH in vitro. These results suggested that L-PGDS potentially attenuates the activities of NADPH oxidases through interaction with NADPH. Given that NADPH is the substrate for NADPH oxidases that play key roles in neuronal cell death by generating excessive ROS, these results imply a novel linkage between L-PGDS and ROS. - Highlights: • Interactions of L-PGDS with nicotinamide coenzymes were studied by ITC and NMR. • The binding affinity of L-PGDS was strongest to NADPH among nicotinamide coenzymes. • NADPH binds to the upper part of L-PGDS ligand-binding cavity. • L-PGDS binds to both lipophilic and hydrophilic ligands. • This study implies a novel linkage between L-PGDS and reactive oxygen species.

  16. Differential induction of progestin-binding sites in uterine cell types by estrogen and antiestrogen

    International Nuclear Information System (INIS)

    Ennis, B.W.; Stumpf, W.E.

    1988-01-01

    Effects of antiestrogen on progestin binding in uterine cell types were determined and compared to those of estrogen. Effects on uterine morphology were also studied. Immature rats were treated with four daily sc injections of 100 micrograms hydroxytamoxifen [TAM(OH)], 5 micrograms estradiol (E2), or oil. On day 5 the rats were injected iv with 1 microgram of the synthetic progestin [ 3 H]Org 2058, and 1 h later uteri were excised, weighed, and processed for thaw-mount autoradiography. Treatment with TAM(OH) or E2 resulted in uterine weight gain, which was greater in animals treated with E2. E2 treatment resulted in cellular hypertrophy in all tissue compartments, especially in the luminal epithelium and myometrium, but TAM(OH) treatment resulted in hypertrophy of only the luminal epithelium. Treatment with TAM(OH) or E2 changed the pattern and intensity of nuclear binding of [ 3 H]Org 2058 from that in oil-treated controls. E2 increased progestin binding in stroma and myometrium and decreased it in luminal epithelium. TAM(OH), similarly, decreased progestin binding in the luminal epithelium and increased it, albeit less than E2, in the myometrium, but left it unchanged in the stroma. The results indicate that E2 and TAM(OH) differentially effect progestin binding among the uterine tissue compartments

  17. Gentamicin binds to the megalin receptor as a competitive inhibitor using the common ligand binding motif of complement type repeats

    DEFF Research Database (Denmark)

    Dagil, Robert; O'Shea, Charlotte; Nykjær, Anders

    2013-01-01

    megalin and investigated its interaction with gentamicin. Using NMR titration data in HADDOCK, we have generated a three-dimensional model describing the complex between megalin and gentamicin. Gentamicin binds to megalin with low affinity and exploits the common ligand binding motif previously described...... to megalin is highly similar to gentamicin binding to calreticulin. We discuss the impact of this novel insight for the future structure-based design of gentamicin antagonists....

  18. Study of manganese binding to the ferroxidase centre of human H-type ferritin.

    Science.gov (United States)

    Ardini, Matteo; Howes, Barry D; Fiorillo, Annarita; Falvo, Elisabetta; Sottini, Silvia; Rovai, Donella; Lantieri, Marco; Ilari, Andrea; Gatteschi, Dante; Spina, Gabriele; Chiancone, Emilia; Stefanini, Simonetta; Fittipaldi, Maria

    2018-05-01

    Ferritins are ubiquitous and conserved proteins endowed with enzymatic ferroxidase activity, that oxidize Fe(II) ions at the dimetal ferroxidase centre to form a mineralized Fe(III) oxide core deposited within the apo-protein shell. Herein, the in vitro formation of a heterodimetal cofactor constituted by Fe and Mn ions has been investigated in human H ferritin (hHFt). Namely, Mn and Fe binding at the hHFt ferroxidase centre and its effects on Fe(II) oxidation have been investigated by UV-Vis ferroxidation kinetics, fluorimetric titrations, multifrequency EPR, and preliminary Mössbauer spectroscopy. Our results show that in hHFt, both Fe(II) and Mn(II) bind the ferroxidase centre forming a Fe-Mn cofactor. Moreover, molecular oxygen seems to favour Mn(II) binding and increases the ferroxidation activity of the Mn-loaded protein. The data suggest that Mn influences the Fe binding and the efficiency of the ferroxidation reaction. The higher efficiency of the Mn-Fe heterometallic centre may have a physiological relevance in specific cell types (i.e. glia cells), where the concentration of Mn is the same order of magnitude as iron. Copyright © 2018 Elsevier Inc. All rights reserved.

  19. The typing of Staphylococcus epidermidis by a lectin-binding assay

    DEFF Research Database (Denmark)

    Jarløv, J O; Hansen, J E; Rosdahl, V T

    1992-01-01

    A new typing method for Staphylococcus epidermidis was developed. Four biotinylated lectins--wheat germ agglutinin (WGA), soy bean agglutinin (SBA), lentil agglutinin (LCA) and Concanavalin A (ConA)--were added to immobilised whole cells of coagulase-negative staphylococci (CNS) in microtitration...... plates. The amount of bound lectin was measured by peroxidase-conjugated avidin followed by a peroxidase reaction. The method was compared to antibiotic-resistance analysis, phage typing, plasmid DNA profiles and slime production. A total of 113 isolates of CNS from 21 patients was investigated and 71...... strains of CNS, including 64 strains of S. epidermidis, were detected if all typing methods were taken into consideration. If only one typing method was used the highest discriminatory power among the S. epidermidis isolates was obtained with the lectin-binding assay which allowed 49 different strains...

  20. Sugar-binding sites of the HA1 subcomponent of Clostridium botulinum type C progenitor toxin.

    Science.gov (United States)

    Nakamura, Toshio; Tonozuka, Takashi; Ide, Azusa; Yuzawa, Takayuki; Oguma, Keiji; Nishikawa, Atsushi

    2008-02-22

    Clostridium botulinum type C 16S progenitor toxin contains a hemagglutinin (HA) subcomponent, designated HA1, which appears to play an important role in the effective internalization of the toxin in gastrointestinal epithelial cells and in creating a broad specificity for the oligosaccharide structure that corresponds to various targets. In this study, using the recombinant protein fused to glutathione S-transferase, we investigated the binding specificity of the HA1 subcomponent to sugars and estimated the binding sites of HA1 based on X-ray crystallography and soaking experiments using various sugars. N-Acetylneuraminic acid, N-acetylgalactosamine, and galactose effectively inhibited the binding that occurs between glutathione S-transferase-HA1 and mucins, whereas N-acetylglucosamine and glucose did not inhibit it. The crystal structures of HA1 complex with N-acetylneuraminic acid, N-acetylgalactosamine, and galactose were also determined. There are two sugar-binding sites, sites I and II. Site I corresponds to the electron densities noted for all sugars and is located at the C-terminal beta-trefoil domain, while site II corresponds to the electron densities noted only for galactose. An aromatic amino acid residue, Trp176, at site I has a stacking interaction with the hexose ring of the sugars. On the other hand, there is no aromatic residue at site II; thus, the interaction with galactose seems to be poor. The double mutant W176A at site I and D271F at site II has no avidity for N-acetylneuraminic acid but has avidity for galactose. In this report, the binding specificity of botulinum C16S toxin HA1 to various sugars is demonstrated based on its structural features.

  1. Reduced post-synaptic serotonin type 1A receptor binding in bipolar depression

    Science.gov (United States)

    Nugent, Allison C.; Bain, Earle E.; Carlson, Paul J.; Neumeister, Alexander; Bonne, Omer; Carson, Richard E.; Eckelman, William; Herscovitch, Peter; Zarate, Carlos A.; Charney, Dennis S.; Drevets, Wayne C.

    2013-01-01

    Multiple lines of evidence suggest that serotonin type 1A (5-HT1A) receptor dysfunction is involved in the pathophysiology of mood disorders, and that alterations in 5-HT1A receptor function play a role in the mechanisms of antidepressant and mood stabilizer treatment. The literature is in disagreement, however, as to whether 5-HT1A receptor binding abnormalities exist in bipolar disorder (BD). We acquired PET images of 5-HT1A receptor binding in 26 unmedicated BD subjects and 37 healthy controls using [18F]FCWAY, a highly selective 5-HT1A receptor radio-ligand. The mean 5-HT1A receptor binding potential (BPP) was significantly lower in BD subjects compared to controls in cortical regions where 5-HT1A receptors are expressed post-synaptically, most prominently in the mesiotemporal cortex. Post-hoc assessments involving other receptor specific binding parameters suggested that this difference particularly affected the females with BD. The mean BPP did not differ between groups in the raphe nucleus, however, where 5-HT1A receptors are predominantly expressed pre-synaptically. Across subjects the BPP in the mesiotemporal cortex was inversely correlated with trough plasma cortisol levels, consistent with preclinical literature indicating that hippocampal 5-HT1A receptor expression is inhibited by glucocorticoid receptor stimulation. These findings suggest that 5-HT1A receptor binding is abnormally reduced in BD, and this abnormality may particularly involve the postsynaptic 5-HT1A receptor system of individuals with a tendency toward cortisol hypersecretion. PMID:23434290

  2. Introduction of lysine and clot binding properties in the kringle one domain of tissue-type plasminogen activator

    NARCIS (Netherlands)

    Bakker, A.H.F.; Greef, W. van der; Rehberg, E.F.; Marotti, K.R.; Verheijen, J.H.

    1993-01-01

    Despite the high overall similarity in primary structure between kringle one (K1) and kringle two (K2) of tissue-type plasminogen activator (t-PA) there exists an enormous functional difference. It is thought that, in contrast to K1, K2 mediates lysine binding and fibrin binding and is involved in

  3. One-step purification of rat heart-type fatty acid-binding protein expressed in Escherichia coli

    NARCIS (Netherlands)

    Schaap, F. G.; Specht, B.; van der Vusse, G. J.; Börchers, T.; Glatz, J. F.

    1996-01-01

    Heart-type fatty acid-binding protein (H-FABP) is a member of a family of 14-15 kDa lipid binding proteins which are believed to enhance intracellular transport of lipids by facilitating their cytoplasmic diffusion. To obtain sufficient amounts of protein for in vitro studies, we expressed rat

  4. Null alleles and sequence variations at primer binding sites of STR loci within multiplex typing systems.

    Science.gov (United States)

    Yao, Yining; Yang, Qinrui; Shao, Chengchen; Liu, Baonian; Zhou, Yuxiang; Xu, Hongmei; Zhou, Yueqin; Tang, Qiqun; Xie, Jianhui

    2018-01-01

    Rare variants are widely observed in human genome and sequence variations at primer binding sites might impair the process of PCR amplification resulting in dropouts of alleles, named as null alleles. In this study, 5 cases from routine paternity testing using PowerPlex ® 21 System for STR genotyping were considered to harbor null alleles at TH01, FGA, D5S818, D8S1179, and D16S539, respectively. The dropout of alleles was confirmed by using alternative commercial kits AGCU Expressmarker 22 PCR amplification kit and AmpFℓSTR ® . Identifiler ® Plus Kit, and sequencing results revealed a single base variation at the primer binding site of each STR locus. Results from the collection of previous reports show that null alleles at D5S818 were frequently observed in population detected by two PowerPlex ® typing systems and null alleles at D19S433 were mostly observed in Japanese population detected by two AmpFℓSTR™ typing systems. Furthermore, the most popular mutation type appeared the transition from C to T with G to A, which might have a potential relationship with DNA methylation. Altogether, these results can provide helpful information in forensic practice to the elimination of genotyping discrepancy and the development of primer sets. Copyright © 2017 Elsevier B.V. All rights reserved.

  5. A radioisotope dilution assay for unlabelled vitamin B12-intrinsic factor complex employing the binding intrinsic factor antibody: probable evidence for two types of binding antibody

    International Nuclear Information System (INIS)

    Jacob, E.; O'Brien, H.A.W.; Mollin, D.L.

    1977-01-01

    A new radioisotope dilution assay for vitamin B 12 -intrinsic factor complex is described. The method is based on the use of the binding type intrinsic antibody (the binding reagent), which when combined with the intrinsic factor-vitamin B 12 complex (labelled ligand), is quantitatively adsorbed onto zirconium phosphate gel pH 6.25. The new assay has been shown to provide a measure of intrinsic factor comparable with other intrinsic factor assays, but it has the important advantage of being able to measure the unlabelled vitamin B 12 -intrinsic factor complex (unlabelled ligand), and will, therefore, be valuable in the study of physiological events in the gastrointestinal tract. During the study, it was found that there is some evidence for at least two types of binding intrinsic factor antibody: One which combines preferentially with the intrinsic factor-vitamin B 12 complex and one which combines equally well with this complex or with free intrinsic factor. (author)

  6. Mood stabilizer treatment increases serotonin type 1A receptor binding in bipolar depression

    Science.gov (United States)

    Nugent, Allison C; Carlson, Paul J; Bain, Earle E; Eckelman, William; Herscovitch, Peter; Manji, Husseini; Zarate, Carlos A; Drevets, Wayne C

    2013-01-01

    Abnormal serotonin type 1A (5-HT1A) receptor function and binding have been implicated in the pathophysiology of mood disorders. Preclinical studies have consistently shown that stress decreases the gene expression of 5-HT1A receptors in experimental animals, and that the associated increase in hormone secretion plays a crucial role in mediating this effect. Chronic administration of the mood stabilizers lithium and divalproex (valproate semisodium) reduces glucocorticoid signaling and function in the hippocampus. Lithium has further been shown to enhance 5-HT1A receptor function. To assess whether these effects translate to human subject with bipolar disorder (BD), positron emission tomography (PET) and [18F]trans-4-fluoro-N-(2-[4-(2-methoxyphenyl) piperazino]-ethyl)-N-(2-pyridyl) cyclohexanecarboxamide ([18F]FCWAY) were used to acquire PET images of 5-HT1A receptor binding in 10 subjects with BD, before and after treatment with lithium or divalproex. Mean 5-HT1A binding potential (BPP) significantly increased following mood stabilizer treatment, most prominently in the mesiotemporal cortex (hippocampus plus amygdala). When mood state was also controlled for, treatment was associated with increases in BPP in widespread cortical areas. These preliminary findings are consistent with the hypothesis that these mood stabilizers enhance 5-HT1A receptor expression in BD, which may underscore an important component of these agents' mechanism of action. PMID:23926239

  7. Enhanced Human-Type Receptor Binding by Ferret-Transmissible H5N1 with a K193T Mutation.

    Science.gov (United States)

    Peng, Wenjie; Bouwman, Kim M; McBride, Ryan; Grant, Oliver C; Woods, Robert J; Verheije, Monique H; Paulson, James C; de Vries, Robert P

    2018-05-15

    All human influenza pandemics have originated from avian influenza viruses. Although multiple changes are needed for an avian virus to be able to transmit between humans, binding to human-type receptors is essential. Several research groups have reported mutations in H5N1 viruses that exhibit specificity for human-type receptors and promote respiratory droplet transmission between ferrets. Upon detailed analysis, we have found that these mutants exhibit significant differences in fine receptor specificity compared to human H1N1 and H3N2 and retain avian-type receptor binding. We have recently shown that human influenza viruses preferentially bind to α2-6-sialylated branched N-linked glycans, where the sialic acids on each branch can bind to receptor sites on two protomers of the same hemagglutinin (HA) trimer. In this binding mode, the glycan projects over the 190 helix at the top of the receptor-binding pocket, which in H5N1 would create a stearic clash with lysine at position 193. Thus, we hypothesized that a K193T mutation would improve binding to branched N-linked receptors. Indeed, the addition of the K193T mutation to the H5 HA of a respiratory-droplet-transmissible virus dramatically improves both binding to human trachea epithelial cells and specificity for extended α2-6-sialylated N-linked glycans recognized by human influenza viruses. IMPORTANCE Infections by avian H5N1 viruses are associated with a high mortality rate in several species, including humans. Fortunately, H5N1 viruses do not transmit between humans because they do not bind to human-type receptors. In 2012, three seminal papers have shown how these viruses can be engineered to transmit between ferrets, the human model for influenza virus infection. Receptor binding, among others, was changed, and the viruses now bind to human-type receptors. Receptor specificity was still markedly different compared to that of human influenza viruses. Here we report an additional mutation in ferret

  8. Crystal Structure of the Receptor-Binding Domain of Botulinum Neurotoxin Type HA, Also Known as Type FA or H.

    Science.gov (United States)

    Yao, Guorui; Lam, Kwok-Ho; Perry, Kay; Weisemann, Jasmin; Rummel, Andreas; Jin, Rongsheng

    2017-03-08

    Botulinum neurotoxins (BoNTs), which have been exploited as cosmetics and muscle-disorder treatment medicines for decades, are well known for their extreme neurotoxicity to humans. They pose a potential bioterrorism threat because they cause botulism, a flaccid muscular paralysis-associated disease that requires immediate antitoxin treatment and intensive care over a long period of time. In addition to the existing seven established BoNT serotypes (BoNT/A-G), a new mosaic toxin type termed BoNT/HA (aka type FA or H) was reported recently. Sequence analyses indicate that the receptor-binding domain (H C ) of BoNT/HA is ~84% identical to that of BoNT/A1. However, BoNT/HA responds differently to some potent BoNT/A-neutralizing antibodies (e.g., CR2) that target the H C . Therefore, it raises a serious concern as to whether BoNT/HA poses a new threat to our biosecurity. In this study, we report the first high-resolution crystal structure of BoNT/HA-H C at 1.8 Å. Sequence and structure analyses reveal that BoNT/HA and BoNT/A1 are different regarding their binding to cell-surface receptors including both polysialoganglioside (PSG) and synaptic vesicle glycoprotein 2 (SV2). Furthermore, the new structure also provides explanations for the ~540-fold decreased affinity of antibody CR2 towards BoNT/HA compared to BoNT/A1. Taken together, these new findings advance our understanding of the structure and function of this newly identified toxin at the molecular level, and pave the way for the future development of more effective countermeasures.

  9. Crystal Structure of the Receptor-Binding Domain of Botulinum Neurotoxin Type HA, Also Known as Type FA or H

    Directory of Open Access Journals (Sweden)

    Guorui Yao

    2017-03-01

    Full Text Available Botulinum neurotoxins (BoNTs, which have been exploited as cosmetics and muscle-disorder treatment medicines for decades, are well known for their extreme neurotoxicity to humans. They pose a potential bioterrorism threat because they cause botulism, a flaccid muscular paralysis-associated disease that requires immediate antitoxin treatment and intensive care over a long period of time. In addition to the existing seven established BoNT serotypes (BoNT/A–G, a new mosaic toxin type termed BoNT/HA (aka type FA or H was reported recently. Sequence analyses indicate that the receptor-binding domain (HC of BoNT/HA is ~84% identical to that of BoNT/A1. However, BoNT/HA responds differently to some potent BoNT/A-neutralizing antibodies (e.g., CR2 that target the HC. Therefore, it raises a serious concern as to whether BoNT/HA poses a new threat to our biosecurity. In this study, we report the first high-resolution crystal structure of BoNT/HA-HC at 1.8 Å. Sequence and structure analyses reveal that BoNT/HA and BoNT/A1 are different regarding their binding to cell-surface receptors including both polysialoganglioside (PSG and synaptic vesicle glycoprotein 2 (SV2. Furthermore, the new structure also provides explanations for the ~540-fold decreased affinity of antibody CR2 towards BoNT/HA compared to BoNT/A1. Taken together, these new findings advance our understanding of the structure and function of this newly identified toxin at the molecular level, and pave the way for the future development of more effective countermeasures.

  10. Crystal Structure of the Receptor-Binding Domain of Botulinum Neurotoxin Type HA, Also Known as Type FA or H

    Energy Technology Data Exchange (ETDEWEB)

    Yao, Guorui; Lam, Kwok-ho; Perry, Kay; Weisemann, Jasmin; Rummel, Andreas; Jin, Rongsheng (Cornell); (Dusseldorf); (UCI)

    2017-03-01

    Botulinum neurotoxins (BoNTs), which have been exploited as cosmetics and muscle-disorder treatment medicines for decades, are well known for their extreme neurotoxicity to humans. They pose a potential bioterrorism threat because they cause botulism, a flaccid muscular paralysis-associated disease that requires immediate antitoxin treatment and intensive care over a long period of time. In addition to the existing seven established BoNT serotypes (BoNT/A–G), a new mosaic toxin type termed BoNT/HA (aka type FA or H) was reported recently. Sequence analyses indicate that the receptor-binding domain (HC) of BoNT/HA is ~84% identical to that of BoNT/A1. However, BoNT/HA responds differently to some potent BoNT/A-neutralizing antibodies (e.g., CR2) that target the HC. Therefore, it raises a serious concern as to whether BoNT/HA poses a new threat to our biosecurity. In this study, we report the first high-resolution crystal structure of BoNT/HA-HC at 1.8 Å. Sequence and structure analyses reveal that BoNT/HA and BoNT/A1 are different regarding their binding to cell-surface receptors including both polysialoganglioside (PSG) and synaptic vesicle glycoprotein 2 (SV2). Furthermore, the new structure also provides explanations for the ~540-fold decreased affinity of antibody CR2 towards BoNT/HA compared to BoNT/A1. Taken together, these new findings advance our understanding of the structure and function of this newly identified toxin at the molecular level, and pave the way for the future development of more effective countermeasures

  11. Structural and binding studies of a C-type galactose-binding lectin from Bothrops jararacussu snake venom.

    Science.gov (United States)

    Sartim, Marco A; Pinheiro, Matheus P; de Pádua, Ricardo A P; Sampaio, Suely V; Nonato, M Cristina

    2017-02-01

    BJcuL is a snake venom galactoside-binding lectin (SVgalL) isolated from Bothrops jararacussu and is involved in a wide variety of biological activities including triggering of pro-inflammatory response, disruption of microbial biofilm structure and induction of apoptosis. In the present work, we determined the crystallographic structure of BJcuL, the first holo structure of a SVgalL, and introduced the fluorescence-based thermal stability assay (Thermofluor) as a tool for screening and characterization of the binding mechanism of SVgalL ligands. BJcuL structure revealed the existence of a porous and flexible decameric arrangement composed of disulfide-linked dimers related by a five-fold symmetry. Each monomer contains the canonical carbohydrate recognition domain, a calcium ion required for BJcuL lectinic activity and a sodium ion required for protein stabilization. BJcuL thermostability was found to be induced by calcium ion and galactoside sugars which exhibit hyperbolic saturation profiles dependent on ligand concentration. Serendipitously, the gentamicin group of aminoglycoside antibiotics (gAGAs) was also identified as BJcuL ligands. On contrast, gAGAs exhibited a sigmoidal saturation profile compatible with a cooperative mechanism of binding. Thermofluor, hemagglutination inhibition assay and molecular docking strategies were used to identify a distinct binding site in BJcuL localized at the dimeric interface near the fully conserved intermolecular Cys86-Cys86 disulfide bond. The hybrid approach used in the present work provided novel insights into structural behavior and functional diversification of SVgaLs. Copyright © 2016 Elsevier Ltd. All rights reserved.

  12. In vivo labelling in several rat tissues of 'peripheral type' benzodiazepine binding sites

    Energy Technology Data Exchange (ETDEWEB)

    Benavides, J.; Guilloux, F.; Rufat, P.; Uzan, A.; Renault, C.; Dubroeucq, M.C.; Gueremy, C.; Le Fur, G. (Pharmuka Laboratoires, 92 - Gennevilliers (France))

    1984-03-16

    'Peripheral type' benzodiazepine binding sites in several rat tissues were labelled by intravenous injection of (/sup 3/H)PK 11195 and (/sup 3/H)RO5-4864. Binding was saturable in all tissues studied and regional distribution paralleled the in vitro binding. A similar potency order of displacing compounds was found in vivo and in vitro PK 11195 > PK 11211 > RO5-4864 > diazepam > dipyridamole > clonazepam. These results demonstrate the feasibility of using this technique to examine the effects of pharmacological manipulation on the binding sites in their native state. However, some properties (broader maximum during time course, higher percentage of particulate binding in the brain and independence of temperature) make (/sup 3/H)PK 11195 the most suitable ligand for this kind of studies.

  13. Evidence that kidney function but not type 2 diabetes determines retinol-binding protein 4 serum levels

    DEFF Research Database (Denmark)

    Henze, Andrea; Frey, Simone K; Raila, Jens

    2008-01-01

    It has been suggested that retinol-binding protein 4 (RBP4) links adiposity, insulin resistance, and type 2 diabetes. However, circulating RBP4 levels are also affected by kidney function. Therefore, the aim of this study was to test whether RBP4 serum levels are primarily associated with kidney...... function or type 2 diabetes....

  14. Estrogen receptor-independent catechol estrogen binding activity: protein binding studies in wild-type, Estrogen receptor-alpha KO, and aromatase KO mice tissues.

    Science.gov (United States)

    Philips, Brian J; Ansell, Pete J; Newton, Leslie G; Harada, Nobuhiro; Honda, Shin-Ichiro; Ganjam, Venkataseshu K; Rottinghaus, George E; Welshons, Wade V; Lubahn, Dennis B

    2004-06-01

    Primary evidence for novel estrogen signaling pathways is based upon well-documented estrogenic responses not inhibited by estrogen receptor antagonists. In addition to 17beta-E2, the catechol estrogen 4-hydroxyestradiol (4OHE2) has been shown to elicit biological responses independent of classical estrogen receptors in estrogen receptor-alpha knockout (ERalphaKO) mice. Consequently, our research was designed to biochemically characterize the protein(s) that could be mediating the biological effects of catechol estrogens using enzymatically synthesized, radiolabeled 4-hydroxyestrone (4OHE1) and 4OHE2. Scatchard analyses identified a single class of high-affinity (K(d) approximately 1.6 nM), saturable cytosolic binding sites in several ERalphaKO estrogen-responsive tissues. Specific catechol estrogen binding was competitively inhibited by unlabeled catechol estrogens, but not by 17beta-E2 or the estrogen receptor antagonist ICI 182,780. Tissue distribution studies indicated significant binding differences both within and among various tissues in wild-type, ERalphaKO, and aromatase knockout female mice. Ligand metabolism experiments revealed extensive metabolism of labeled catechol estrogen, suggesting that catechol estrogen metabolites were responsible for the specific binding. Collectively, our data provide compelling evidence for the interaction of catechol estrogen metabolites with a novel binding protein that exhibits high affinity, specificity, and selective tissue distribution. The extensive biochemical characterization of this binding protein indicates that this protein may be a receptor, and thus may mediate ERalpha/beta-independent effects of catechol estrogens and their metabolites.

  15. Effect of Optimization of Glycaemic Control on Mannan-Binding Lectin in Type 1 Diabetes

    Directory of Open Access Journals (Sweden)

    Gry Høst Dørflinger

    2017-01-01

    Full Text Available Objective. Mannan-binding lectin (MBL concentration in plasma is increased in subjects with type 1 diabetes and associated with increased mortality and risk of diabetic nephropathy. Recent findings show that pancreas transplantation reduces MBL concentration. Whether the increased MBL concentration is reversed by improved glycaemic control remains unknown. We investigated the effects of improved glycaemic control on MBL concentration in patients with type 1 diabetes. Methods. We measured MBL, fructosamine, and HbA1cat baseline and after 6 weeks in 52 type 1 diabetic patients following the change from conventional insulin therapy to insulin pump therapy. Results. After initiation of insulin pump therapy, the total daily insulin dose was significantly reduced (from 51 ± 18 IE/day to 39 ± 13 IE/day, P<0.0001. There was a significant decrease in HbA1c from 8.6% to 7.7% (from 70 mmol/mol to 61 mmol/mol, P<0.0001 and in fructosamine levels (from 356 μmol/L to 311 μmol/L, P<0.0001. MBL levels decreased by 10% from 2165 μg/L (IQR 919–3389 μg/L at baseline to 1928 μ/L (IQR 811–2758 μg/L at follow-up (P=0.005, but MBL change was not significantly correlated with changes in insulin dose, HbA1c, or fructosamine. Conclusions. MBL concentration decreased following the initiation of insulin pump therapy in patients with type 1 diabetes and did not correlate with changes in glycaemic control.

  16. Capacity for cooperative binding of thyroid hormone (T3) receptor dimers defines wild type T3 response elements.

    Science.gov (United States)

    Brent, G A; Williams, G R; Harney, J W; Forman, B M; Samuels, H H; Moore, D D; Larsen, P R

    1992-04-01

    Thyroid hormone response elements (T3REs) have been identified in a variety of promoters including those directing expression of rat GH (rGH), alpha-myosin heavy chain (rMHC), and malic enzyme (rME). A detailed biochemical and genetic analysis of the rGH element has shown that it consists of three hexamers related to the consensus [(A/G)GGT(C/A)A]. We have extended this analysis to the rMHC and rME elements. Binding of highly purified thyroid hormone receptor (T3R) to T3REs was determined using the gel shift assay, and thyroid hormone (T3) induction was measured in transient tranfections. We show that the wild type version of each of the three elements binds T3R dimers cooperatively. Mutational analysis of the rMHC and rME elements identified domains important for binding T3R dimers and allowed a direct determination of the relationship between T3R binding and function. In each element two hexamers are required for dimer binding, and mutations that interfere with dimer formation significantly reduce T3 induction. Similar to the rGH element, the rMHC T3RE contains three hexameric domains arranged as a direct repeat followed by an inverted copy, although the third domain is weaker than in rGH. All three are required for full function and T3R binding. The rME T3RE is a two-hexamer direct repeat T3RE, which also binds T3R monomer and dimer. Across a series of mutant elements, there was a strong correlation between dimer binding in vitro and function in vivo for rMHC (r = 0.99, P less than 0.01) and rME (r = 0.67, P less than 0.05) T3REs. Our results demonstrate a similar pattern of T3R dimer binding to a diverse array of hexameric sequences and arrangements in three wild type T3REs. Addition of nuclear protein enhanced T3R binding but did not alter the specificity of binding to wild type or mutant elements. Binding of purified T3R to T3REs was highly correlated with function, both with and without the addition of nuclear protein. T3R dimer formation is the common

  17. Envelope conformational changes induced by human immunodeficiency virus type 1 attachment inhibitors prevent CD4 binding and downstream entry events.

    Science.gov (United States)

    Ho, Hsu-Tso; Fan, Li; Nowicka-Sans, Beata; McAuliffe, Brian; Li, Chang-Ben; Yamanaka, Gregory; Zhou, Nannan; Fang, Hua; Dicker, Ira; Dalterio, Richard; Gong, Yi-Fei; Wang, Tao; Yin, Zhiwei; Ueda, Yasutsugu; Matiskella, John; Kadow, John; Clapham, Paul; Robinson, James; Colonno, Richard; Lin, Pin-Fang

    2006-04-01

    BMS-488043 is a small-molecule human immunodeficiency virus type 1 (HIV-1) CD4 attachment inhibitor with demonstrated clinical efficacy. The compound inhibits soluble CD4 (sCD4) binding to the 11 distinct HIV envelope gp120 proteins surveyed. Binding of BMS-488043 and that of sCD4 to gp120 are mutually exclusive, since increased concentrations of one can completely block the binding of the other without affecting the maximal gp120 binding capacity. Similarly, BMS-488043 inhibited virion envelope trimers from binding to sCD4-immunoglobulin G (IgG), with decreasing inhibition as the sCD4-IgG concentration increased, and BMS-488043 blocked the sCD4-induced exposure of the gp41 groove in virions. In both virion binding assays, BMS-488043 was active only when added prior to sCD4. Collectively, these results indicate that obstruction of gp120-sCD4 interactions is the primary inhibition mechanism of this compound and that compound interaction with envelope must precede CD4 binding. By three independent approaches, BMS-488043 was further shown to induce conformational changes within gp120 in both the CD4 and CCR5 binding regions. These changes likely prevent gp120-CD4 interactions and downstream entry events. However, BMS-488043 could only partially inhibit CD4 binding to an HIV variant containing a specific envelope truncation and altered gp120 conformation, despite effectively inhibiting the pseudotyped virus infection. Taken together, BMS-488043 inhibits viral entry primarily through altering the envelope conformation and preventing CD4 binding, and other downstream entry events could also be inhibited as a result of these induced conformational changes.

  18. C-peptide prevents SMAD3 binding to alpha promoters to inhibit collagen type IV synthesis.

    Science.gov (United States)

    Li, Yanning; Zhong, Yan; Gong, Wenjian; Gao, Xuehan; Qi, Huanli; Liu, Kun; Qi, Jinsheng

    2018-07-01

    Activation of transforming growth factor β1 (TGFB1)/SMAD3 signaling may lead to additional synthesis of collagen type IV (COL4), which is a major contributor to extracellular matrix (ECM) accumulation in diabetic nephropathy (DN). C-peptide can attenuate fibrosis to have unique beneficial effects in DN. However, whether and how C-peptide affects TGFB1/SMAD3-activated COL4 synthesis is unclear. In this study, pathological changes, expression of COL4 a1-a5 chains ( Col4a1-a5 ), COL4 distribution and protein and TGFB1 and SMAD3 protein were first assessed in a rat model of diabetes. Then, rat mesangial cells were treated with high glucose (HG) and/or C-peptide to investigate the underlying mechanism. Col4a1-a5 expression, COL4 protein and secretion, TGFB1 protein, SMAD3 nuclear translocation and binding of SMAD3 to its cognate sites in the promoters of Col4a1a2 , Col4a3a4 and Col4a5 were measured. It was found that C-peptide attenuated glomerular pathological changes and suppressed renal Col4a1 -a5 mRNA expression, COL4 protein content and TGFB1 protein content. C-peptide had a dose-dependent effect to inhibit Col4a1-a5 mRNA expression, COL4 protein content and secretion, in HG-stimulated mesangial cells. In addition, the HG-induced increase in TGFB1 protein content was significantly reduced by C-peptide. Although not apparently affecting SMAD3 nuclear translocation, C-peptide prevented SMAD3 from binding to its sites in the Col4a1a2 , Col4a3a4 and Col4a5 promoters in HG-stimulated mesangial cells. In conclusion, C-peptide could prevent SMAD3 from binding to its sites in the Col4a1a2 , Col4a3a4 and Col4a5 promoters, to inhibit COL4 generation. These results may provide a mechanism for the alleviation of fibrosis in DN by C-peptide. © 2018 Society for Endocrinology.

  19. Selective pharmacological modulation of renal peripheral-type benzodiazepine binding by treatment with diuretic drugs

    International Nuclear Information System (INIS)

    Lukeman, D.S.; Vaughn, D.A.; Fanestil, D.D.

    1988-01-01

    The authors have assessed the effects of in vivo administration of different classes of diuretic drugs on the expression of the peripheral-type benzodiazepine binding site (PBBS) in crude membranes derived from the cortex and outer medulla of rat kidney by saturation analysis with the PBBS-selective ligands [ 3 H]RO5-4864 and [ 3 H]PH 11195 in cortex and [ 3 H]RO5-4864 in outer medulla. Administration for 14-15 days of furosemide, a drug that blocks NaCl-KCl coupled transport in the thick ascending limb of the loop of Henle, produced a significant doubling in the PBBS density (B/sub max/) in outer medulla, a region of the kidney rich in thick ascending limbs, and produced a lesser but significant increase in PBBS density in the cortex. Conversely, administration for 14-15 days of the carbonic anhydrase inhibitor acetazolamide, which acts predominantly in the proximal tubule, and hydrochlorothiazide, which acts predominantly in the early distal tubule, elicited statistically significant increases in PBBS density in renal cortex but not in renal outer medulla. Furthermore, all drug treatments were without effect on the equilibrium dissociation constants (K/sub d/s) of [ 3 H]RO5-4864 and [ 3 H]PK 11195 binding to cortical and outer medullary membrane preparations. These findings demonstrate that the PBBS can be selectively up-regulated in different regions of the kidney by diuretic drugs with different modes/sites of action. 50 references, 1 table

  20. The association between sex hormone-binding globulin and type 2 diabetes in Nigerian men

    Directory of Open Access Journals (Sweden)

    Fayefori M. Abbiyesuku

    2013-07-01

    Full Text Available Background: Epidemiological studies have shown that sex hormone-binding globulin (SHBG has a role in glucose homeostasis in both men and women. However, a prospective study on Japanese-American subjects concluded that SHBG was not a significant risk factor in either men or women, suggesting ethnic differences. We were not aware of any evaluation of SHBG in subjects of African ancestry. Objectives: We investigated the association between SHBG and insulin resistance in type 2 diabetic diabetic men in a hospital in Nigeria. Method: Forty-eight male subjects with type 2 diabetes and 20 non-diabetic male subjects were recruited in this cross-sectional hospital-based study by the convenient sampling method.Height and circumferences around the waist and hip were measured to the nearest 0.5 cm and the waist–hip ratio was calculated from this measurement. Weight was measured and body mass index was calculated. Fasting plasma glucose concentration was measured by the glucose oxidase method with a between-run coefficient of variation of 3%. Insulin and SHBG were measured by means of enzyme-linked immunosorbent assay (ELISA. Results: There was a statistically-significant difference between test results for the diabetic and non-diabetic patients. The mean SHBG concentration was higher in the non-diabetic group (42.2 nmol/L than the diabetic group (30.5 nmol/L. A significant inverse association between insulin resistance and SHBG was observed (r = 0.353, p < 0.015. Conclusion: This study supported earlier observations that a significant inverse correlation exists between SHBG and insulin resistance and provides evidence that the relationship may extend to type 2 diabetic men of African ancestry in Nigeria.

  1. Contribution of cellular retinol-binding protein type 1 to retinol metabolism during mouse development.

    Science.gov (United States)

    Matt, Nicolas; Schmidt, Carsten K; Dupé, Valérie; Dennefeld, Christine; Nau, Heinz; Chambon, Pierre; Mark, Manuel; Ghyselinck, Norbert B

    2005-05-01

    Within cells, retinol (ROL) is bound to cytoplasmic proteins (cellular retinol-binding proteins [CRBPs]), whose proposed function is to protect it from unspecific enzymes through channeling to retinoid-metabolizing pathways. We show that, during development, ROL and retinyl ester levels are decreased in CRBP type 1 (CRBP1) -deficient embryos and fetuses by 50% and 80%, respectively. The steady state level of retinoic acid (RA) is also decreased but to a lesser extent. However, CRBP1-null fetuses do not exhibit the abnormalities characteristic of a vitamin A-deficiency syndrome. Neither CRBP1 deficiency alters the expression patterns of RA-responding genes during development, nor does CRBP1 availability modify the expression of an RA-dependent gene in primary embryonic fibroblasts treated with ROL. Therefore, CRBP1 is required in prenatal life to maintain normal amounts of ROL and to ensure its efficient storage but seems of secondary importance for RA synthesis, at least under conditions of maternal vitamin A sufficiency. Copyright 2005 Wiley-Liss, Inc.

  2. Mannose-Binding Lectin Levels and Carotid Intima-Media Thickness in Type 2 Diabetic Patients

    Directory of Open Access Journals (Sweden)

    Miklós Káplár

    2016-01-01

    Full Text Available Introduction. Mannose-binding lectin (MBL activates complement system and has been suggested to play a role in vascular complications in diabetics. Carotid intima-media thickness (cIMT detects subclinical atherosclerosis. We evaluated the association of MBL and IMT in type 2 diabetic (T2DM patients. Methods. Serum MBL levels and cIMT were measured in a total of 103 diabetics and in 98 age-matched healthy controls. Results. There was no significant difference in MBL level in T2DM versus controls. As expected, IMT was significantly higher in T2DM patients than in controls (P=0.001. In T2DM, the lowest cIMT was seen in patients with normal MBL level (500–1000 while cIMT continuously increased with both high MBL and absolute MBL deficiency states. This was especially significant in high MBL versus normal MBL T2DM patients (P=0.002. According to multiple regression analysis the main predictors of IMT in T2DM are age (P<0.003, ApoA level (P=0.023, and the MBL (P=0.036. Conclusions. Our results suggest a dual role of MBL as a risk factor for cIMT in T2DM. MBL may also be used as a marker of macrovascular disease, as both low and high levels indicate the susceptibility for atherosclerosis in T2DM.

  3. Serum heart type fatty acid binding protein levels are not changed in hyperthyroidism.

    Science.gov (United States)

    Ozbek, Mustafa; Gungunes, Askin; Sahin, Mustafa; Ginis, Zeynep; Ucan, Bekir; Sayki, Muyesser; Tutal, Esra; Cakal, Erman; Kuşkonmaz, Serife M; Öztürk, Mehmet A; Delibasi, Tuncay

    2016-09-01

    Heart type fatty acid binding protein (H-FABP) is a small protein and released into the circulation when myocardial damage has occurred. Previous studies have demonstrated that H-FABP is closely associated with cardiac and some endocrinologic disorders including prediabetes, metabolic syndrome, and acromegaly. Hyperthyroism is a well-known disorder associated with cardiovascular diseases. We aimed to investigate the effect of hyperthyrodism on H-FABP levels. Forty six patients with hyperthyroidism with no known history of coronary artery disease and 40 healthy controls are involved in the study. Serum H-FABP levels are measured using sandwich enzyme-linked immunosorbent assay. There was no significant difference between serum H-FABP levels of patients with hyperthyroidism and controls (871±66 pg/mL, and 816±66 pg/mL, respectively P=0.56). There was no significant correlation between H-FABP, free triiodothyronine (fT3), free thyroxine (fT4), and thyroid stimulating hormone (TSH) levels in patients and controls. Serum H-FABP levels are not altered in patients with hyperthyroidism.

  4. Alternate thermoregulation and functional binding of Escherichia coli type 1 fimbriae in environmental and animal isolates.

    Science.gov (United States)

    Marshall, Jacqueline; Rossez, Yannick; Mainda, Geoffrey; Gally, David L; Daniell, Tim J; Holden, Nicola J

    2016-11-01

    Type 1 fimbriae (T1F) are well characterised cell surface organelles expressed by Escherichia coli and required for adherence to mannosylated host tissue. They satisfy molecular Koch's postulates as a virulence determinant and a host-adapted role has been reinforced by reports that T1F expression is repressed at submammalian temperatures. Analysis of a group of 136 environmental and animal E. coli isolates that express T1F at 37°C showed that 28% are also capable of expression at 20°C, in a phase variable manner. The heterogeneous proportions varied widely, and although growth temperature impacted the total proportion expressing T1F, there was no direct correlation between growth at 37°C and 20°C, indicative of differences in thermoregulation of the genetic switch (fimS) that controls phase variation. Specificities of the adhesin (FimH) also varied between the isolates: most bound to α-(1-3) mannan and yeast extracts as expected, but some recognised β-(1-4)-mannans and N-linked glycoproteins from plants, and T1F from two of the isolates mediated binding to plant roots. The results expand our view of a well-described adherence factor to show alternative expression profiles and adhesin specificities, which in turn may confer an advantage for certain isolates in alternative hosts and habitats. © FEMS 2016. All rights reserved. For permissions, please e-mail: journals.permissions@oup.com.

  5. Clinical significance of urinary liver-type fatty acid binding protein at various stages of nephropathy

    Directory of Open Access Journals (Sweden)

    V Viswanathan

    2015-01-01

    Full Text Available This cross-sectional study was to evaluate the levels of urinary liver-type fatty acid binding protein (u-LFABP pg/mg urine creatinine ratio at different stages of diabetic nephropathy and to see its correlation with other clinical parameters in South Indian patients with type 2 diabetes mellitus (T2DM. A total of 65 (M: F; 42:23 T2DM subjects were divided into three groups, and were compared with 13 (M: F; 3:10 nondiabetic controls. The study groups were as follows: normoalbuminuric (n = 22, microalbuminuric (n = 22 and macroalbuminuric (n = 21. Estimated glomerular filtration rate (eGFR was calculated using Cockcroft and Gault formula. u-LFABP levels in spot urine samples were measured with a solid phase enzyme linked immunosorbent assay. This study showed that u-LFABP levels were undetectable in healthy controls and was very low in the normoalbuminuric subjects. Elevated levels of u-LFABP are evident from the microalbuminuric stage indicating tubular damage. The levels of u-LFABP increased gradually with declining renal function. Geometric mean (95% confidence interval for normoalbuminuria was 0.65 (0.47-0.97, microalbuminuria was 0.99 (0.55-1.97 and macroalbuminuria was 5.16 (1.8-14.5, (P = 0.005. In conclusion, u-LFABP levels were elevated in patients with reduced eGFR and showed a positive correlation with systolic blood pressure and protein to creatinine ratio in the total study subjects.

  6. Water binding of proteins in the processing frankfurter-type sausages. Part. 1. Water-binding ability of freeze-dried meat fractions containing myofibrillar and stromal proteins.

    Science.gov (United States)

    Heinevetter, L; Gassmann, B; Kroll, J

    1987-01-01

    As soon as possible and 48 h after slaughter respectively, from both blade-bone muscle groups of cattle and pig carcasses the "thick pieces" were excised, extracted, and fractionated. Residues and precipitates from water and salt extracts resulted were freeze-dried, and an improved Baumann capillary suction apparatus was used to measure their water binding capacity (WBC) with and without addition of 2% sodium chloride and/or heating to 80 degrees C. With one exception the WBC results followed a relative pattern demonstrating the final residues (stromal proteins and leavings of myofibrillar proteins) binding the highest amount of added water, precipitates of dialysis (mainly containing myofibrillar proteins) a remarkable amount and powdered meats the least. As scanning electron micrographs confirmed, there were no fibrous structures in the precipitates resulted from dialysis of salt solutions (1.0 mol/1). Heating decreased the spontaneous water uptake of all fractions. Addition of sodium chloride had only a noticeable capillary-suction and swelling effect on unheated samples. Hence swelling of undissolved protein structures (extraction of myosin and possibly of actomyosin) is therefore not the only way for water binding in frankfurter-type sausages.

  7. A single mutation in Taiwanese H6N1 influenza hemagglutinin switches binding to human-type receptors

    Energy Technology Data Exchange (ETDEWEB)

    de Vries, Robert P.; Tzarum, Netanel; Peng, Wenjie; Thompson, Andrew J.; Ambepitiya Wickramasinghe, Iresha N.; de la Pena, Alba T. Torrents; van Breemen, Marielle J.; Bouwman, Kim M.; Zhu, Xueyong; McBride, Ryan; Yu, Wenli; Sanders, Rogier W.; Verheije, Monique H.; Wilson, Ian A.; Paulson, James C.

    2017-07-10

    In June 2013, the first case of human infection with an avian H6N1 virus was reported in a Taiwanese woman. Although this was a single non-fatal case, the virus continues to circulate in Taiwanese poultry. As with any emerging avian virus that infects humans, there is concern that acquisition of human-type receptor specificity could enable transmission in the human population. Despite mutations in the receptor-binding pocket of the human H6N1 isolate, it has retained avian-type (NeuAcα2-3Gal) receptor specificity. However, we show here that a single nucleotide substitution, resulting in a change from Gly to Asp at position 225 (G225D), completely switches specificity to human-type (NeuAcα2-6Gal) receptors. Significantly, G225D H6 loses binding to chicken trachea epithelium and is now able to bind to human tracheal tissue. Structural analysis reveals that Asp225 directly interacts with the penultimate Gal of the human-type receptor, stabilizing human receptor binding.

  8. Bedside Heart Type Fatty Acid Binding Protein (H-FABP): Is an Early Predictive Marker of Cardiac Syncope

    International Nuclear Information System (INIS)

    Sonmez, B. M.; Yilmaz, F.; Durdu, T.; Hakbilir, O.; Ongar, M.; Ozturk, D.; Altinbilek, E.; Kavalci, C.; Turhan, T.

    2015-01-01

    Objective: To determine the value of bedside heart-type fatty acid binding protein in diagnosis of cardiac syncope in patients presenting with syncope or presyncope. Methods: The prospective study was conducted at Ankara Numune Training and Research Hospital, Ankara, Turkey, between September 1, 2010, and January 1, 2011, and comprised patients aged over 18 years who presented with syncope or presyncope. Patients presenting to emergency department within 4 hours of syncope or presyncope underwent a bedside heart-type fatty acid binding protein test measurement. SPSS 16 was used for statistical analysis, Results: Of the 100 patients evaluated, 22(22 percent) were diagnosed with cardiac syncope. Of them, 13(59.1 percent) patients had a positive and 9(40.9 percent) had a negative heart-type fatty acid binding protein result. Consequently, the test result was 12.64 times more positive in patients with cardiac syncope compared to those without. Conclusions: Bedside heart-type fatty acid binding protein, particularly at early phase of myocardial injury, reduces diagnostic and therapeutic uncertainity of cardiac origin in syncope patients. (author)

  9. Histamine type I (H1) receptor radioligand binding studies on normal T cell subsets, B cells, and monocytes

    International Nuclear Information System (INIS)

    Cameron, W.; Doyle, K.; Rocklin, R.E.

    1986-01-01

    A single, specific binding site for [ 3 H]pyrilamine on normal human T helper, T suppressor, B cells, and monocytes was documented. The binding of the radioligand to its receptor is reversible with cold H 1 antagonist, saturates at 40 to 60 nM, and binding equilibrium is achieved in 2 to 4 min. Using a computer program (Ligand), the authors calculated the dissociation constants, binding capacities, and numbers of receptors per cell for each of the different cell types. Monocytes were found to have the highest affinity for [ 3 H]pyrilamine, followed by T helper cells, B cells and T suppressor cells (K/sub D/ = 44.6 +/- 49.4 nM). T suppressor cells were found to express the higher number of H 1 receptors per cell followed by B cells, T helper cells, and monocytes. The binding affinity for [ 3 H]pyrilamine increased over a 48-hr period, whereas the number of receptors per T cell was essentially unchanged. In contrast, T cells stimulated with Con A or PHA were shown to have a greater than fourfold increase in the number of receptors per cell, whereas the binding affinity for [ 3 H]pyrilamine decreased over the 48-hr period. Although the function of H 1 receptors on T cells, B cells, and monocytes has not been completely defined, this receptor has the potential of playing an important role in the modulating the immune response

  10. Different types of working memory binding in epilepsy patients with unilateral anterior temporal lobectomy.

    Science.gov (United States)

    van Geldorp, Bonnie; Bouman, Zita; Hendriks, Marc P H; Kessels, Roy P C

    2014-03-01

    The medial temporal lobe is an important structure for long-term memory formation, but its role in working memory is less clear. Recent studies have shown hippocampal involvement during working memory tasks requiring binding of information. It is yet unclear whether this is limited to tasks containing spatial features. The present study contrasted three binding conditions and one single-item condition in patients with unilateral anterior temporal lobectomy. A group of 43 patients with temporal lobectomy (23 left; 20 right) and 20 matched controls were examined with a working memory task assessing spatial relational binding (object-location), non-spatial relational binding (object-object), conjunctive binding (object-colour) and working memory for single items. We varied the delay period (3 or 6s), as there is evidence showing that delay length may modulate working memory performance. The results indicate that performance was worse for patients than for controls in both relational binding conditions, whereas patients were unimpaired in conjunctive binding. Single-item memory was found to be marginally impaired, due to a deficit on long-delay trials only. In conclusion, working memory binding deficits are found in patients with unilateral anterior temporal lobectomy. The role of the medial temporal lobe in working memory is not limited to tasks containing spatial features. Rather, it seems to be involved in relational binding, but not in conjunctive binding. The medial temporal lobe gets involved when working memory capacity does not suffice, for example when relations have to be maintained or when the delay period is long. Copyright © 2014 Elsevier Inc. All rights reserved.

  11. dsRNA binding characterization of full length recombinant wild type and mutants Zaire ebolavirus VP35.

    Science.gov (United States)

    Zinzula, Luca; Esposito, Francesca; Pala, Daniela; Tramontano, Enzo

    2012-03-01

    The Ebola viruses (EBOVs) VP35 protein is a multifunctional major virulence factor involved in EBOVs replication and evasion of the host immune system. EBOV VP35 is an essential component of the viral RNA polymerase, it is a key participant of the nucleocapsid assembly and it inhibits the innate immune response by antagonizing RIG-I like receptors through its dsRNA binding function and, hence, by suppressing the host type I interferon (IFN) production. Insights into the VP35 dsRNA recognition have been recently revealed by structural and functional analysis performed on its C-terminus protein. We report the biochemical characterization of the Zaire ebolavirus (ZEBOV) full-length recombinant VP35 (rVP35)-dsRNA binding function. We established a novel in vitro magnetic dsRNA binding pull down assay, determined the rVP35 optimal dsRNA binding parameters, measured the rVP35 equilibrium dissociation constant for heterologous in vitro transcribed dsRNA of different length and short synthetic dsRNA of 8bp, and validated the assay for compound screening by assessing the inhibitory ability of auryntricarboxylic acid (IC(50) value of 50μg/mL). Furthermore, we compared the dsRNA binding properties of full length wt rVP35 with those of R305A, K309A and R312A rVP35 mutants, which were previously reported to be defective in dsRNA binding-mediated IFN inhibition, showing that the latter have measurably increased K(d) values for dsRNA binding and modified migration patterns in mobility shift assays with respect to wt rVP35. Overall, these results provide the first characterization of the full-length wt and mutants VP35-dsRNA binding functions. Copyright © 2012 Elsevier B.V. All rights reserved.

  12. The role of the lysyl binding site of tissue-type plasminogen activator in the interaction with a forming fibrin clot

    NARCIS (Netherlands)

    Bakker, A.H.F.; Weening-Verhoeff, E.J.D.; Verheijen, J.H.

    1995-01-01

    To describe the role of the lysyl binding site in the interaction of tissue-type plasminogen activator (t-PA, FGK1K2P) with a forming fibrin clot, we performed binding experiments with domain deletion mutants GK1K2P, K2P, and the corresponding point mutants lacking the lysyl binding site in the

  13. Nitrate binding to Limulus polyphemus subunit type II hemocyanin and its functional implications

    NARCIS (Netherlands)

    Hazes, B; Magnus, KA; Kalk, KH; Bonaventura, C; Hol, WGJ

    1996-01-01

    The horseshoe crab, Limulus polyphemus, employs hemocyanin as an oxygen carrier in its hemolymph. This hemocyanin displays cooperative oxygen binding and heterotropic allosteric regulation by protons, chloride ions and divalent cations. Here, we report the crystal structure of Limulus polyphemus

  14. Synthesis of Heparan Sulfate with Cyclophilin B-binding Properties Is Determined by Cell Type-specific Expression of Sulfotransferases*

    Science.gov (United States)

    Deligny, Audrey; Denys, Agnès; Marcant, Adeline; Melchior, Aurélie; Mazurier, Joël; van Kuppevelt, Toin H.; Allain, Fabrice

    2010-01-01

    Cyclophilin B (CyPB) induces migration and adhesion of T lymphocytes via a mechanism that requires interaction with 3-O-sulfated heparan sulfate (HS). HS biosynthesis is a complex process with many sulfotransferases involved. N-Deacetylases/N-sulfotransferases are responsible for N-sulfation, which is essential for subsequent modification steps, whereas 3-O-sulfotransferases (3-OSTs) catalyze the least abundant modification. These enzymes are represented by several isoforms, which differ in term of distribution pattern, suggesting their involvement in making tissue-specific HS. To elucidate how the specificity of CyPB binding is determined, we explored the relationships between the expression of these sulfotransferases and the generation of HS motifs with CyPB-binding properties. We demonstrated that high N-sulfate density and the presence of 2-O- and 3-O-sulfates determine binding of CyPB, as evidenced by competitive experiments with heparin derivatives, soluble HS, and anti-HS antibodies. We then showed that target cells, i.e. CD4+ lymphocyte subsets, monocytes/macrophages, and related cell lines, specifically expressed high levels of NDST2 and 3-OST3 isoforms. Silencing the expression of NDST1, NDST2, 2-OST, and 3-OST3 by RNA interference efficiently decreased binding and activity of CyPB, thus confirming their involvement in the biosynthesis of binding sequences for CyPB. Moreover, we demonstrated that NDST1 was able to partially sulfate exogenous substrate in the absence of NDST2 but not vice versa, suggesting that both isoenzymes do not have redundant activities but do have rather complementary activities in making N-sulfated sequences with CyPB-binding properties. Altogether, these results suggest a regulatory mechanism in which cell type-specific expression of certain HS sulfotransferases determines the specific binding of CyPB to target cells. PMID:19940140

  15. Synthesis of heparan sulfate with cyclophilin B-binding properties is determined by cell type-specific expression of sulfotransferases.

    Science.gov (United States)

    Deligny, Audrey; Denys, Agnès; Marcant, Adeline; Melchior, Aurélie; Mazurier, Joël; van Kuppevelt, Toin H; Allain, Fabrice

    2010-01-15

    Cyclophilin B (CyPB) induces migration and adhesion of T lymphocytes via a mechanism that requires interaction with 3-O-sulfated heparan sulfate (HS). HS biosynthesis is a complex process with many sulfotransferases involved. N-Deacetylases/N-sulfotransferases are responsible for N-sulfation, which is essential for subsequent modification steps, whereas 3-O-sulfotransferases (3-OSTs) catalyze the least abundant modification. These enzymes are represented by several isoforms, which differ in term of distribution pattern, suggesting their involvement in making tissue-specific HS. To elucidate how the specificity of CyPB binding is determined, we explored the relationships between the expression of these sulfotransferases and the generation of HS motifs with CyPB-binding properties. We demonstrated that high N-sulfate density and the presence of 2-O- and 3-O-sulfates determine binding of CyPB, as evidenced by competitive experiments with heparin derivatives, soluble HS, and anti-HS antibodies. We then showed that target cells, i.e. CD4+ lymphocyte subsets, monocytes/macrophages, and related cell lines, specifically expressed high levels of NDST2 and 3-OST3 isoforms. Silencing the expression of NDST1, NDST2, 2-OST, and 3-OST3 by RNA interference efficiently decreased binding and activity of CyPB, thus confirming their involvement in the biosynthesis of binding sequences for CyPB. Moreover, we demonstrated that NDST1 was able to partially sulfate exogenous substrate in the absence of NDST2 but not vice versa, suggesting that both isoenzymes do not have redundant activities but do have rather complementary activities in making N-sulfated sequences with CyPB-binding properties. Altogether, these results suggest a regulatory mechanism in which cell type-specific expression of certain HS sulfotransferases determines the specific binding of CyPB to target cells.

  16. Identification of binding domains in the herpes simplex virus type 1 small capsid protein pUL35 (VP26).

    Science.gov (United States)

    Apcarian, Arin; Cunningham, Anthony L; Diefenbach, Russell J

    2010-11-01

    In this study, fragments of the small capsid protein pUL35 (VP26) from herpes simplex virus type 1 (HSV-1) were generated to identify binding domains for a number of known ligands. Analysis of the binding of dynein light chain subunits, DYNLT1 and DYNLT3, as well the HSV-1 structural proteins pUL19 (VP5) and pUL37 was then undertaken using the LexA yeast two-hybrid assay. The N-terminal half of pUL35, in particular residues 30-43, was identified as a common region for the binding of DYNLT1 and DYNLT3. Additional distinct regions in the C terminus of pUL35 also contribute to the binding of DYNLT1 and DYNLT3. In contrast, only the C-terminal half of pUL35 was found to mediate the binding of pUL19 and pUL37 through distinct regions. The relevance of this information to the role of pUL35 in viral transport and assembly is discussed.

  17. In vivo autoradiographic demonstration of β-adrenergic binding sites in adult rat type II alveolar epithelial cells

    International Nuclear Information System (INIS)

    Smith, D.M.; Sidhu, M.K.

    1984-01-01

    Adult male rats were injected intravenously with the muscarinic binding probe 3 H-Quinuclidinyl benzilate (QNB) or the β-adrenergic probe 3 H-dihydroalprenolol (DHA). Other rats were pre-treated with an intraperitoneal injection of a 500-fold excess of L-isoproterenol prior to the DHA. Light microscopic autoradiography of 0.5 μm sections of lung from the QNB group demonstrated very little labelling even after 6 months of exposure. In constrast, trachealis smooth muscle from these animals contained substantial labelling. Autoradiographs of lung from rats injected with DHA demonstrated labelling which was well localized over alveolar septa and concentrated over the cytoplasm of type II cells. Quantitative analysis of labelling in the DHA groups indicated a significant reduction of labelling in animals treated with L-isoproterenol prior to DHA, in both the alveolar parenchyma in general and over type II cells. The results of this study provide morphologic evidence for the uptake and specific binding of β-adrenergic antagonists by the adult lung in vivo, while failing to demonstrate similar binding of a muscarinic probe. In addition, the results demonstrate specific β-adrenergic receptors on type II cells in vivo and substantiate the view of a direct effect of β-adrenergic agonists on alveolar type II cells

  18. Heart-type fatty-acid-binding protein (FABP3 is a lysophosphatidic acid-binding protein in human coronary artery endothelial cells

    Directory of Open Access Journals (Sweden)

    Ryoko Tsukahara

    2014-01-01

    Full Text Available Fatty-acid-binding protein 3, muscle and heart (FABP3, also known as heart-type FABP, is a member of the family of intracellular lipid-binding proteins. It is a small cytoplasmic protein with a molecular mass of about 15 kDa. FABPs are known to be carrier proteins for transporting fatty acids and other lipophilic substances from the cytoplasm to the nucleus, where these lipids are released to a group of nuclear receptors such as peroxisome proliferator-activated receptors (PPARs. In this study, using lysophosphatidic acid (LPA-coated agarose beads, we have identified FABP3 as an LPA carrier protein in human coronary artery endothelial cells (HCAECs. Administration of LPA to HCAECs resulted in a dose-dependent increase in PPARγ activation. Furthermore, the LPA-induced PPARγ activation was abolished when the FABP3 expression was reduced using small interfering RNA (siRNA. We further show that the nuclear fraction of control HCAECs contained a significant amount of exogenously added LPA, whereas FABP3 siRNA-transfected HCAECs had a decreased level of LPA in the nucleus. Taken together, these results suggest that FABP3 governs the transcriptional activities of LPA by targeting them to cognate PPARγ in the nucleus.

  19. Binding characteristics of 9-fluoropropyl-(+)-dihydrotetrabenzazine (AV-133) to the vesicular monoamine transporter type 2 in rats

    Energy Technology Data Exchange (ETDEWEB)

    Tsao, H.-H. [Department of Medical Imaging and Radiological Sciences, Chang Gung University, Taoyuan, Taiwan (China); Lin, K.-J. [Department of Medical Imaging and Radiological Sciences, Chang Gung University, Taoyuan, Taiwan (China); Department of Nuclear Medicine, Chang Gung University and Memorial Hospital, Taoyuan, Taiwan (China); Juang, J.-H. [Division of Endocrinology and Metabolism, Chung Gung University and Chung Gung Memorial Hospital, Taoyuan, Taiwan (China); Skovronsky, Daniel M. [Avid Radiopharmaceuticals, Philadelphia, PA (United States); Department of Radiology, University of Pennsylvania, Philadelphia, PA 19104 (United States); Yen, T.-C. [Department of Nuclear Medicine, Chang Gung University and Memorial Hospital, Taoyuan, Taiwan (China); Wey, S.-P. [Department of Medical Imaging and Radiological Sciences, Chang Gung University, Taoyuan, Taiwan (China); Kung, M.-P. [Department of Medical Imaging and Radiological Sciences, Chang Gung University, Taoyuan, Taiwan (China); Department of Radiology, University of Pennsylvania, Philadelphia, PA 19104 (United States)], E-mail: kungmp@sunmac.spect.upenn.edu

    2010-05-15

    The vesicular monoamine transporter type 2 (VMAT2) is highly expressed in pancreatic {beta}-cells and thus has been proposed to be a potential target for measuring {beta}-cell mass (BCM) by molecular imaging. C-11- and F-18-labeled tetrabenazine derivatives targeting VMAT2 have shown some promising results as potential biomarkers for BCM. In the present study, we examined the binding characteristics of 9-fluoropropyl-(+)-dihydrotetrabenzazine ([{sup 18}F]AV-133), a potential PET tracer for BCM imaging, in rat pancreas and rat brain. Methods: Pancreatic exocrine cells and pancreatic islet cells were isolated and purified from Sprague-Dawley rats. Membrane homogenates, prepared from both pancreatic exocrine and islet cells as well as from brain striatum and hypothalamus regions, were used for in vitro binding studies. In vitro and ex vivo autoradiography studies with [{sup 18}F]AV-133 were performed on rat brain and rat pancreas sections. Immunohistochemistry studies were performed to confirm the distribution of VMAT2 on islet {beta}-cells. Results: Excellent binding affinities of [{sup 18}F]AV-133 were observed in rat striatum and hypothalamus homogenates with K{sub d} values of 0.19 and 0.25 nM, respectively. In contrast to single-site binding observed in rat striatum homogenates, rat islet cell homogenates showed two saturable binding sites (site A: K{sub d}=6.76 nM, B{sub max}=60 fmol/mg protein; site B: K{sub d}=241 nM, B{sub max}=1500 fmol/mg protein). Rat exocrine pancreas homogenates showed only a single low-affinity binding site (K{sub d}=209 nM), which was similar to site B in islet cells. In vitro autoradiography of [{sup 18}F]AV-133 using frozen sections of rat pancreas showed specific labeling of islets, as evidenced by co-localization with anti-insulin antibody. Ex vivo VMAT2 pancreatic autoradiography in the rat, however, was not successful, in contrast to the excellent ex vivo autoradiography of VMAT2 binding sites in the brain. In vivo/ex vivo islet

  20. Association of ATP-Binding Cassette Transporter A1 Gene Polymorphisms in Type 2 Diabetes Mellitus among Malaysians

    OpenAIRE

    Haghvirdizadeh, Polin; Ramachandran, Vasudevan; Etemad, Ali; Heidari, Farzad; Ghodsian, Nooshin; Bin Ismail, Norzian; Ismail, Patimah

    2015-01-01

    Background. Type 2 diabetes mellitus (T2DM) is a complex polygenic disorder characterized by impaired insulin resistance, insulin secretion, and dysregulation of lipid and protein metabolism with environmental and genetic factors. ATP-binding cassette transporter A1 (ABCA1) gene polymorphisms are reported as the one of the genetic risk factors for T2DM in various populations with conflicting results. This study was conducted based on PCR-HRM to determine the frequency of ABCA1 gene by rs22308...

  1. Heart type fatty acid binding protein response and subsequent development of atherosclerosis in insulin resistant polycystic ovary syndrome patients

    OpenAIRE

    Cakir Evrim; Ozbek Mustafa; Sahin Mustafa; Cakal Erman; Gungunes Askin; Ginis Zeynep; Demirci Taner; Delibasi Tuncay

    2012-01-01

    Abstract Background Women with polycystic ovary syndrome (PCOS) have higher risk for cardiovascular disease (CVD). Heart type fatty acid binding protein (HFABP) has been found to be predictive for myocardial ischemia.Wet ested whether HFABP is the predictor for CVD in PCOS patients, who have an increased risk of cardiovascular disease. Methods This was a prospective, cross sectional controlled study conducted in a training and research hospital.The study population consisted of 46 reproductiv...

  2. Genetic evidence that raised sex hormone binding globulin (SHBG) levels reduce the risk of type 2 diabetes

    DEFF Research Database (Denmark)

    Perry, John R B; Weedon, Michael N; Langenberg, Claudia

    2009-01-01

    Epidemiological studies consistently show that circulating sex hormone binding globulin (SHBG) levels are lower in type 2 diabetes patients than non-diabetic individuals, but the causal nature of this association is controversial. Genetic studies can help dissect causal directions...... used data from this SNP, or closely correlated SNPs, in 27 657 type 2 diabetes patients and 58 481 controls from 15 studies. We then used data from additional studies to estimate the difference in SHBG levels between type 2 diabetes patients and controls. The SHBG SNP rs1799941 was associated with type...... 2 diabetes [odds ratio (OR) 0.94, 95% CI: 0.91, 0.97; P = 2 x 10(-5)], with the SHBG raising allele associated with reduced risk of type 2 diabetes. This effect was very similar to that expected (OR 0.92, 95% CI: 0.88, 0.96), given the SHBG-SNP versus SHBG levels association (SHBG levels are 0.2...

  3. The ligand-binding domain of the cell surface receptor for urokinase-type plasminogen activator

    DEFF Research Database (Denmark)

    Behrendt, N; Ploug, M; Patthy, L

    1991-01-01

    with the internal repeats of u-PAR constitute the extracellular part of Ly-6 antigens and of the squid glycoprotein Sgp-2. Like u-PAR, these proteins are attached to the membrane by a glycosyl-phosphatidylinositol anchor. The hydrophilic, ligand-binding u-PAR domain identified in the present study has potential...

  4. New insights into structure and function of the different types of fatty acid-binding protein

    NARCIS (Netherlands)

    Zimmerman, Augusta Wilhelmina

    2002-01-01

    Fatty acid binding proteins (FABPs) are small cytosolic proteins with virtually identical backbone structures that facilitate the solubility and intracellular transport of fatty acids. They may also modulate the effect of fatty acids on various metabolic enzymes and receptors and on cellular

  5. Binding and functional pharmacological characteristics of gepant-type antagonists in rat brain and mesenteric arteries

    DEFF Research Database (Denmark)

    Sheykhzade, Majid; Amandi, Nilofar; Pla, Monica Vidal

    2017-01-01

    AIM: The neuropeptide calcitonin gene-related peptide (CGRP) is found in afferent sensory nerve fibers innervating the resistance arteries and plays a pivotal role in a number of neurovascular diseases such as migraine and subarachnoid bleedings. The present study investigates the binding and ant...

  6. Different types of working memory binding in epilepsy patients with unilateral anterior temporal lobectomy

    NARCIS (Netherlands)

    Geldorp, B. van; Bouman, Z.; Hendriks, M.P.H.; Kessels, R.P.C.

    2014-01-01

    The medial temporal lobe is an important structure for long-term memory formation, but its role in working memory is less clear. Recent studies have shown hippocampal involvement during working memory tasks requiring binding of information. It is yet unclear whether this is limited to tasks

  7. The human 64-kDa polyadenylylation factor contains a ribonucleoprotein-type RNA binding domain and unusual auxiliary motifs

    International Nuclear Information System (INIS)

    Takagaki, Yoshio; Manley, J.L.; MacDonald, C.C.; Shenk, T.

    1992-01-01

    Cleavage stimulation factor is one of the multiple factors required for 3'-end cleavage of mammalian pre-mRNAs. The authors have shown previously that this factor is composed of three subunits with estimated molecular masses of 77, 64, and 50 kDa and that the 64-kDa subunit can be UV-cross linked to RNA in a polyadenylylation signal (AAUAAA)-dependent manner. They have now isolated cDNAs encoding the 64-kDa subunit of human cleavage stimulation factor. The 64-kDa subunit contains a ribonucleoprotein-type RNA binding domain in the N-terminal region and a repeat structure in the C-terminal region in which a pentapeptide sequence (consensus MEARA/G) is repeated 12 times and the formation of a long α-helix stabilized by salt bridges is predicted. An ∼270-amino acid segment surrounding this repeat structure is highly enriched in proline and glycine residues (∼20% for each). When cloned 64-kDa subunit was expressed in Escherichia coli, an N-terminal fragment containing the RNA binding domain bound to RNAs in a polyadenylylation-signal-independent manner, suggesting that the RNA binding domain is directly involved in the binding of the 64-kDa subunit to pre-mRNAs

  8. 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. Copyright © 2010 Elsevier Ltd. All rights reserved.

  9. Binding of mutant and wild type p53 proteins to supercoiled DNA

    Czech Academy of Sciences Publication Activity Database

    Brázdová, Marie; Němcová, Kateřina; Pivoňková, Hana; Walter, K.; Warnecke, Gabriele; Živanovic, Marko; Krstic, Dušan; Paleček, Emil; Deppert, W.; Fojta, Miroslav

    2005-01-01

    Roč. 3, č. 1 (2005), S4-S5 ISSN 1214-021X. [Cells VI - Biological Days /18./. 24.10.2005-26.10.2005, České Budějovice] R&D Projects: GA MŠk(CZ) 1K04119; GA ČR(CZ) GA301/05/0416 Institutional research plan: CEZ:AV0Z50040507 Keywords : wtp53 * hot spot mutp53 * SCS binding Subject RIV: BO - Biophysics

  10. Exploring the water-binding pocket of the type II dehydroquinase enzyme in the structure-based design of inhibitors.

    Science.gov (United States)

    Blanco, Beatriz; Sedes, Antía; Peón, Antonio; Otero, José M; van Raaij, Mark J; Thompson, Paul; Hawkins, Alastair R; González-Bello, Concepción

    2014-04-24

    Structural and computational studies to explore the WAT1 binding pocket in the structure-based design of inhibitors against the type II dehydroquinase (DHQ2) enzyme are reported. The crystal structures of DHQ2 from M. tuberculosis in complex with four of the reported compounds are described. The electrostatic interaction observed between the guanidinium group of the essential arginine and the carboxylate group of one of the inhibitors in the reported crystal structures supports the recently suggested role of this arginine as the residue that triggers the release of the product from the active site. The results of the structural and molecular dynamics simulation studies revealed that the inhibitory potency is favored by promoting interactions with WAT1 and the residues located within this pocket and, more importantly, by avoiding situations where the ligands occupy the WAT1 binding pocket. The new insights can be used to advantage in the structure-based design of inhibitors.

  11. Genetic and epigenetic mutations affect the DNA binding capability of human ZFP57 in transient neonatal diabetes type 1.

    Science.gov (United States)

    Baglivo, Ilaria; Esposito, Sabrina; De Cesare, Lucia; Sparago, Angela; Anvar, Zahra; Riso, Vincenzo; Cammisa, Marco; Fattorusso, Roberto; Grimaldi, Giovanna; Riccio, Andrea; Pedone, Paolo V

    2013-05-21

    In the mouse, ZFP57 contains three classical Cys2His2 zinc finger domains (ZF) and recognizes the methylated TGC(met)CGC target sequence using the first and the second ZFs. In this study, we demonstrate that the human ZFP57 (hZFP57) containing six Cys2His2 ZFs, binds the same methylated sequence through the third and the fourth ZFs, and identify the aminoacids critical for DNA interaction. In addition, we present evidences indicating that hZFP57 mutations and hypomethylation of the TNDM1 ICR both associated with Transient Neonatal Diabetes Mellitus type 1 result in loss of hZFP57 binding to the TNDM1 locus, likely causing PLAGL1 activation. Copyright © 2013 Federation of European Biochemical Societies. Published by Elsevier B.V. All rights reserved.

  12. IGF2 mRNA-binding protein 2: biological function and putative role in type 2 diabetes

    DEFF Research Database (Denmark)

    Christiansen, J.; Kolte, A.M.; Hansen, T.O.

    2009-01-01

    Recent genome-wide association (GWA) studies of type 2 diabetes (T2D) have implicated IGF2 mRNA-binding protein 2 (IMP2/IGF2BP2) as one of the several factors in the etiology of late onset diabetes. IMP2 belongs to a family of oncofetal mRNA-binding proteins implicated in RNA localization...... and T2D Udgivelsesdato: 2009/11......, stability, and translation that are essential for normal embryonic growth and development. This review provides a background to the IMP protein family with an emphasis on human IMP2, followed by a closer look at the GWA studies to evaluate the significance, if any, of the proposed correlation between IMP2...

  13. Analysis of chitin-binding proteins from Manduca sexta provides new insights into evolution of peritrophin A-type chitin-binding domains in insects.

    Science.gov (United States)

    Tetreau, Guillaume; Dittmer, Neal T; Cao, Xiaolong; Agrawal, Sinu; Chen, Yun-Ru; Muthukrishnan, Subbaratnam; Haobo, Jiang; Blissard, Gary W; Kanost, Michael R; Wang, Ping

    2015-07-01

    In insects, chitin is a major structural component of the cuticle and the peritrophic membrane (PM). In nature, chitin is always associated with proteins among which chitin-binding proteins (CBPs) are the most important for forming, maintaining and regulating the functions of these extracellular structures. In this study, a genome-wide search for genes encoding proteins with ChtBD2-type (peritrophin A-type) chitin-binding domains (CBDs) was conducted. A total of 53 genes encoding 56 CBPs were identified, including 15 CPAP1s (cuticular proteins analogous to peritrophins with 1 CBD), 11 CPAP3s (CPAPs with 3 CBDs) and 17 PMPs (PM proteins) with a variable number of CBDs, which are structural components of cuticle or of the PM. CBDs were also identified in enzymes of chitin metabolism including 6 chitinases and 7 chitin deacetylases encoded by 6 and 5 genes, respectively. RNA-seq analysis confirmed that PMP and CPAP genes have differential spatial expression patterns. The expression of PMP genes is midgut-specific, while CPAP genes are widely expressed in different cuticle forming tissues. Phylogenetic analysis of CBDs of proteins in insects belonging to different orders revealed that CPAP1s from different species constitute a separate family with 16 different groups, including 6 new groups identified in this study. The CPAP3s are clustered into a separate family of 7 groups present in all insect orders. Altogether, they reveal that duplication events of CBDs in CPAP1s and CPAP3s occurred prior to the evolutionary radiation of insect species. In contrast to the CPAPs, all CBDs from individual PMPs are generally clustered and distinct from other PMPs in the same species in phylogenetic analyses, indicating that the duplication of CBDs in each of these PMPs occurred after divergence of insect species. Phylogenetic analysis of these three CBP families showed that the CBDs in CPAP1s form a clearly separate family, while those found in PMPs and CPAP3s were clustered

  14. Tight-binding calculation of Ti-Rh--type phase diagram

    International Nuclear Information System (INIS)

    Sluiter, M.; Turchi, P.; Fu Zezhong; de Fontaine, D.

    1988-01-01

    Tight-binding electronic band-structure calculations were combined with a free-energy expression from a statistical mechanical method called the cluster-variation method. The effective pair interactions used in the cluster-variation calculation were evaluated by the generalized perturbation method. Only d orbitals were included and the numbers of d electrons per atom were taken to be three for the pure A element and eight for the pure B. A phase diagram was constructed incorporating, for the first time, both fcc and bcc lattices and their simple-ordered superstructures. The calculated diagram agreed reasonably well with those determined empirically for Ti-Rh or Ti-Ir

  15. Carrageenan activates monocytes via type-specific binding with interleukin-8: an implication for design of immuno-active biomaterials.

    Science.gov (United States)

    Chan, Weng-I; Zhang, Guangpan; Li, Xin; Leung, Chung-Hang; Ma, Dik-Lung; Dong, Lei; Wang, Chunming

    2017-02-28

    Polymers that can activate the immune system may become useful biomaterials tools, given that the mechanisms underlying their actions are well understood. Herein, we report a novel type of interaction between polymers and immune cells - in studying the influence of the three major types of carrageenan (CGN) polysaccharides on monocyte behaviour in vitro, we found only the λ-type induced monocyte adhesion and this action requires the presence of an adequate amount of serum. Further analyses indicated λ-CGN bound interleukin-8 (IL-8) in the serum and activated the cultured monocytes through an IL-8-dependent pathway. This is the first demonstration that a polymer, with a renowned immunostimulatory effect, activates the immune system via binding and harnessing the function of a specific cytokine in the microenvironment. This is a new mechanism underlying polymer-immunity interactions that may shed light on future design and application of biomaterials tools targeting the immune system for a wide variety of therapeutic applications.

  16. Expression Profiles of Cellular Retinol-binding Protein, Type II (CRBP II in Erlang Mountainous Chickens

    Directory of Open Access Journals (Sweden)

    H. D. Yin

    2014-03-01

    Full Text Available Cellular retinol-binding protein II (CRBP II belongs to the family of cellular retinol-binding proteins and plays a major role in absorption, transport, and metabolism of vitamin A. In addition, because vitamin A is correlated with reproductive performance, we measured CRBP II mRNA abundance in erlang mountainous chickens by real-time PCR using the relative quantification method. The expression of CRBP II showed a tissue-specific pattern and egg production rate-dependent changes. The expression was very high (p<0.05 in jejunum and liver, intermediate in kidney, ovary, and oviduct, and lowest (p<0.05 in heart, hypothalamus, and pituitary. In the hypothalamus, oviduct, ovary, and pituitary, CRBP II mRNA abundance were correlated to egg production rate, which increased from 12 wk to 32 wk, peaked at 32 wk relative to the other time points, and then decreased from 32 wk to 45 wk. In contrast, the expression of CRBP II mRNA in heart, jejunum, kidney, and liver was not different at any of the ages evaluated in this study. These data may help to understand the genetic basis of vitamin A metabolism, and suggest that CRBP II may be a candidate gene to affect egg production traits in chickens.

  17. The N-terminus of porcine circovirus type 2 replication protein is required for nuclear localization and ori binding activities

    International Nuclear Information System (INIS)

    Lin, W.-L.; Chien, M.-S.; Du, Y.-W.; Wu, P.-C.; Huang Chienjin

    2009-01-01

    Porcine circovirus type 2 possesses a circular, single-stranded DNA genome that requires the replication protein (Rep) for virus replication. To characterize the DNA binding potential and the significant region that confers the nuclear localization of the Rep protein, the defined coding regions of rep gene were cloned and expressed. All of the recombinant proteins except for the N-terminal 110 residues deletion mutant could bind to the double-stranded minimal binding site of replication origin (ori). In addition, the N-terminal deletion mutant lacking 110 residues exhibited mainly cytoplasmic staining in the transfected cells in contrast to the others, which localized dominantly in the nucleus, suggesting that this N-terminal domain is essential for nuclear localization. Furthermore, a series of green fluorescence proteins (GFP) containing potential nuclear localization signal (NLS) sequences were tested for their cellular distribution. The ability of the utmost 20 residues of the N-terminal region to target the GFP to the nucleus confirmed its role as a functional NLS.

  18. Characterization of Small Molecule Scaffolds that Bind to the Shigella Type III Secretion System Protein IpaD

    Science.gov (United States)

    Dey, Supratim; Anbanandam, Asokan; Mumford, Ben E.; De Guzman, Roberto N.

    2017-01-01

    Many pathogens such as Shigella and other bacteria assemble the type III secretion system (T3SS) nanoinjector to inject virulence proteins into their target cells to cause infectious diseases in humans. The rise of drug resistance among pathogens that rely on the T3SS for infectivity, plus the dearth of new antibiotics require alternative strategies in developing new antibiotics. The Shigella T3SS tip protein IpaD is an attractive target for developing anti-infectives because of its essential role in virulence and its exposure on the bacterial surface. Currently, the only known small molecules that bind to IpaD are bile salts sterols. Here, we identified four new small molecule scaffolds that bind to IpaD based on the methylquinoline, pyrrolidin-aniline, hydroxyindole, and morpholinoaniline scaffolds. NMR mapping revealed potential hotspots in IpaD for binding small molecules. These scaffolds can be used as building blocks in developing small molecule inhibitors of IpaD that could lead to new anti-infectives. PMID:28750143

  19. Apo calmodulin binding to the L-type voltage-gated calcium channel Cav1.2 IQ peptide

    International Nuclear Information System (INIS)

    Lian Luyun; Myatt, Daniel; Kitmitto, Ashraf

    2007-01-01

    The influx of calcium through the L-type voltage-gated calcium channels (LTCCs) is the trigger for the process of calcium-induced calcium release (CICR) from the sarcoplasmic recticulum, an essential step for cardiac contraction. There are two feedback mechanisms that regulate LTCC activity: calcium-dependent inactivation (CDI) and calcium-dependent facilitation (CDF), both of which are mediated by calmodulin (CaM) binding. The IQ domain (aa 1645-1668) housed within the cytoplasmic domain of the LTCC Ca v 1.2 subunit has been shown to bind both calcium-loaded (Ca 2+ CaM ) and calcium-free CaM (apoCaM). Here, we provide new data for the structural basis for the interaction of apoCaM with the IQ peptide using NMR, revealing that the apoCaM C-lobe residues are most significantly perturbed upon complex formation. In addition, we have employed transmission electron microscopy of purified LTCC complexes which shows that both apoCaM and Ca 2+ CaM can bind to the intact channel

  20. Crystal Structure of the Receptor-Binding Domain of Botulinum Neurotoxin Type HA, Also Known as Type FA or H

    OpenAIRE

    Yao, Guorui; Lam, Kwok-ho; Perry, Kay; Weisemann, Jasmin; Rummel, Andreas; Jin, Rongsheng

    2017-01-01

    Botulinum neurotoxins (BoNTs), which have been exploited as cosmetics and muscle-disorder treatment medicines for decades, are well known for their extreme neurotoxicity to humans. They pose a potential bioterrorism threat because they cause botulism, a flaccid muscular paralysis-associated disease that requires immediate antitoxin treatment and intensive care over a long period of time. In addition to the existing seven established BoNT serotypes (BoNT/A–G), a new mosaic toxin type termed Bo...

  1. Differential effect of detergents on [3H]Ro 5-4864 and [3H]PK 11195 binding to peripheral-type benzodiazepine-binding sites

    International Nuclear Information System (INIS)

    Awad, M.; Gavish, M.

    1988-01-01

    The present study demonstrates a differential effect of various detergent treatments on [ 3 H]Ro 5-4864 and [ 3 H]PK 11195 binding to peripheral benzodiazepine binding sites (PBS). Triton X-100 caused a decrease of about 70% in [ 3 H]Ro 5-4864 binding to membranes from various peripheral tissues of rat, but had only a negligible effect on [ 3 H]PK 11195 binding. A similar effect of Triton X-100 was observed on guinea pig and rabbit kidney membranes. The decrease in [ 3 H]Ro 5-4864 binding after treatment with Triton X-100 was apparently due to a decrease in the density of PBS, since the affinity remained unaltered. The detergents 3-[(3-cholamidopropyl)-dimethylammonio]-1-propane sulfonate (CHAPS), Tween 20, deoxycholic acid, or digitonin (0.0125%) caused only a minor change in [ 3 H]Ro 5-4864 and [ 3 H]PK 11195 binding to rat kidney membranes; but when concentrations were substantially increased (0.1%), all detergents caused a decrease of at least 50% in [ 3 H]Ro 5-4864 binding, while [ 3 H]PK 11195 binding to rat kidney membranes remained unaffected by the first three detergents, with only a minor decrease (15%) after treatment with digitonin

  2. Characterization of the SUMO-binding activity of the myeloproliferative and mental retardation (MYM-type zinc fingers in ZNF261 and ZNF198.

    Directory of Open Access Journals (Sweden)

    Catherine M Guzzo

    Full Text Available SUMO-binding proteins interact with SUMO modified proteins to mediate a wide range of functional consequences. Here, we report the identification of a new SUMO-binding protein, ZNF261. Four human proteins including ZNF261, ZNF198, ZNF262, and ZNF258 contain a stretch of tandem zinc fingers called myeloproliferative and mental retardation (MYM-type zinc fingers. We demonstrated that MYM-type zinc fingers from ZNF261 and ZNF198 are necessary and sufficient for SUMO-binding and that individual MYM-type zinc fingers function as SUMO-interacting motifs (SIMs. Our binding studies revealed that the MYM-type zinc fingers from ZNF261 and ZNF198 interact with the same surface on SUMO-2 recognized by the archetypal consensus SIM. We also present evidence that MYM-type zinc fingers in ZNF261 contain zinc, but that zinc is not required for SUMO-binding. Immunofluorescence microscopy studies using truncated fragments of ZNF198 revealed that MYM-type zinc fingers of ZNF198 are necessary for localization to PML-nuclear bodies (PML-NBs. In summary, our studies have identified and characterized the SUMO-binding activity of the MYM-type zinc fingers in ZNF261 and ZNF198.

  3. Enhanced binding affinity, remarkable selectivity, and high capacity of CO 2 by dual functionalization of a rht-type metal-organic framework

    KAUST Repository

    Li, Baiyan; Zhang, Zhijuan; Li, Yi; Yao, Kexin; Zhu, Yihan; Deng, Zhiyong; Yang, Fen; Zhou, Xiaojing; Li, Guanghua; Wu, Haohan; Nijem, Nour; Chabal, Yves Jean; Lai, Zhiping; Han, Yu; Shi, Zhan; Feng, Shouhua; Li, Jing

    2011-01-01

    Open and friendly: The smallest member of the rht-type metal-organic frameworks (MOFs, see picture) constructed by a hexacarboxylate ligand with a nitrogen-rich imino triazine backbone shows a significantly enhanced gas binding affinity relative

  4. Photoconversion and fluorescence properties of a red/green-type cyanobacteriochrome AM1_C0023g2 that binds not only phycocyanobilin but also biliverdin.

    Directory of Open Access Journals (Sweden)

    Keiji eFushimi

    2016-04-01

    Full Text Available Cyanobacteriochromes (CBCRs are distantly related to the red/far-red responsive phytochromes. Red/green-type CBCRs are widely distributed among various cyanobacteria. The red/green-type CBCRs covalently bind phycocyanobilin (PCB and show red/green reversible photoconversion. Recent studies revealed that some red/green-type CBCRs from chlorophyll d-bearing cyanobacterium Acaryochloris marina covalently bind not only PCB but also biliverdin (BV. The BV-binding CBCRs show far-red/orange reversible photoconversion. Here, we identified another CBCR (AM1_C0023g2 from A. marina that also covalently binds not only PCB but also BV with high binding efficiencies, although BV chromophore is unstable in the presence of urea. Replacement of Ser334 with Gly resulted in significant improvement in the yield of the BV-binding holoprotein, thereby ensuring that the mutant protein is a fine platform for future development of optogenetic switches. We also succeeded in detecting near-infrared fluorescence from mammalian cells harboring PCB-binding AM1_C0023g2 whose fluorescence quantum yield is 3.0%. Here the PCB-binding holoprotein is shown as a platform for future development of fluorescent probes.

  5. Heart type fatty acid binding protein response and subsequent development of atherosclerosis in insulin resistant polycystic ovary syndrome patients.

    Science.gov (United States)

    Cakir, Evrim; Ozbek, Mustafa; Sahin, Mustafa; Cakal, Erman; Gungunes, Askin; Ginis, Zeynep; Demirci, Taner; Delibasi, Tuncay

    2012-12-18

    Women with polycystic ovary syndrome (PCOS) have higher risk for cardiovascular disease (CVD). Heart type fatty acid binding protein (HFABP) has been found to be predictive for myocardial ischemia.Wet ested whether HFABP is the predictor for CVD in PCOS patients, who have an increased risk of cardiovascular disease. This was a prospective, cross sectional controlled study conducted in a training and research hospital.The study population consisted of 46 reproductive-age PCOS women and 28 control subjects. We evaluated anthropometric and metabolic parameters, carotid intima media thickness and HFABP levels in both PCOS patients and control group. Mean fasting insulin, homeostasis model assessment insulin resistance index (HOMA-IR), triglyceride, total cholesterol, low density lipoprotein cholesterol, free testosterone, total testosterone, carotid intima media thickness (CIMT) levels were significantly higher in PCOS patients. Although HFABP levels were higher in PCOS patients, the difference did not reach statistically significant in early age groups. After adjustment for age and body mass index, HFABP level was positive correlated with hsCRP, free testosterone levels, CIMT and HOMA-IR. Heart type free fatty acid binding protein appeared to have an important role in metabolic response and subsequent development of atherosclerosis in insulin resistant, hyperandrogenemic PCOS patients.

  6. Heart type fatty acid binding protein response and subsequent development of atherosclerosis in insulin resistant polycystic ovary syndrome patients

    Directory of Open Access Journals (Sweden)

    Cakir Evrim

    2012-12-01

    Full Text Available Abstract Background Women with polycystic ovary syndrome (PCOS have higher risk for cardiovascular disease (CVD. Heart type fatty acid binding protein (HFABP has been found to be predictive for myocardial ischemia.Wet ested whether HFABP is the predictor for CVD in PCOS patients, who have an increased risk of cardiovascular disease. Methods This was a prospective, cross sectional controlled study conducted in a training and research hospital.The study population consisted of 46 reproductive-age PCOS women and 28 control subjects. We evaluated anthropometric and metabolic parameters, carotid intima media thickness and HFABP levels in both PCOS patients and control group. Results Mean fasting insulin, homeostasis model assessment insulin resistance index (HOMA-IR, triglyceride, total cholesterol, low density lipoprotein cholesterol, free testosterone, total testosterone, carotid intima media thickness (CIMT levels were significantly higher in PCOS patients. Although HFABP levels were higher in PCOS patients, the difference did not reach statistically significant in early age groups. After adjustment for age and body mass index, HFABP level was positive correlated with hsCRP, free testosterone levels, CIMT and HOMA-IR. Conclusions Heart type free fatty acid binding protein appeared to have an important role in metabolic response and subsequent development of atherosclerosis in insulin resistant, hyperandrogenemic PCOS patients.

  7. Molecular characterization of a novel human hybrid-type receptor that binds the alpha2-macroglobulin receptor-associated protein

    DEFF Research Database (Denmark)

    Jacobsen, Linda; Madsen, P; Moestrup, S K

    1996-01-01

    the corresponding cDNA. The gene, designated SORL1, maps to chromosome 11q 23/24 and encodes a 2214-residue type 1 receptor containing a furin cleavage site immediately preceding the N terminus determined in the purified protein. The receptor, designated sorLA-1, has a short cytoplasmic tail containing a tyrosine...... density lipoprotein receptor gene family receptors, and 3) six tandemly arranged fibronectin type III repeats also found in certain neural adhesion proteins. sorLA-1 may therefore be classified as a hybrid receptor. Northern blotting revealed specific mRNA transcripts in brain, spinal cord, and testis......The 39-40-kDa receptor-associated protein (RAP) binds to the members of the low density lipoprotein receptor gene family and functions as a specialized endoplasmic reticulum/Golgi chaperone. Using RAP affinity chromatography, we have purified a novel approximately 250-kDa brain protein and isolated...

  8. Memory Effects of Benzodiazepines: Memory Stages and Types Versus Binding-Site Subtypes

    Directory of Open Access Journals (Sweden)

    Miroslav M. Savic

    2005-01-01

    Full Text Available Benzodiazepines are well established as inhibitory modulators of memory processing. This effect is especially prominent when applied before the acquisition phase of a memory task. This minireview concentrates on the putative subtype selectivity of the acquisition-impairing action of benzodiazepines. Namely, recent genetic studies and standard behavioral tests employing subtype-selective ligands pointed to the predominant involvement of two subtypes of benzodiazepine binding sites in memory modulation. Explicit memory learning seems to be affected through the GABAA receptors containing the α1 and α5 subunits, whereas the effects on procedural memory can be mainly mediated by the α1 subunit. The pervading involvement of the α1 subunit in memory modulation is not at all unexpected because this subunit is the major subtype, present in 60% of all GABAA receptors. On the other hand, the role of α5 subunits, mainly expressed in the hippocampus, in modulating distinct forms of memory gives promise of selective pharmacological coping with certain memory deficit states.

  9. Comparative studies of vertebrate scavenger receptor class B type 1: a high-density lipoprotein binding protein

    Directory of Open Access Journals (Sweden)

    Holmes RS

    2012-06-01

    Full Text Available Roger S Holmes,1,2 Laura A Cox11Department of Genetics and Southwest National Primate Research Center, Texas Biomedical Research Institute, San Antonio, TX, USA; 2School of Biomolecular and Physical Sciences, Griffith University, Nathan, Queensland, AustraliaAbstract: Scavenger receptor class B type 1 protein (SCARB1 plays an essential role in cholesterol homeostasis and functions in binding high density lipoprotein cholesterol (HDL in liver and other tissues of the body. SCARB1 also functions in lymphocyte homeostasis and in the uptake of hepatitis C virus (HCV by the liver. A genetic deficiency of this protein results in autoimmune disorders and significant changes in blood cholesterol phenotype. Comparative SCARB1 amino acid sequences and structures and SCARB1 gene locations were examined using data from several vertebrate genome projects. Vertebrate SCARB1 sequences shared 50%–99% identity as compared with 28%–31% sequence identities with other CD36-like superfamily members, ie, SCARB2 and SCARB3 (also called CD36. At least eight N-glycosylation sites were conserved among most of the vertebrate SCARB1 proteins examined. Sequence alignments, key amino acid residues, and conserved predicted secondary structures were also studied, including: cytoplasmic, transmembrane, and exoplasmic sequences; conserved N-terminal and C-terminal transmembrane glycines which participate in oligomer formation; conserved cystine disulfides and a free SH residue which participates in lipid transport; carboxyl terminal PDZ-binding domain sequences (Ala507-Arg/Lys508-Leu509; and 30 conserved proline and 18 conserved glycine residues, which may contribute to short loop formation within the exoplasmic HDL-binding sequence. Vertebrate SCARB1 genes usually contained 12 coding exons. The human SCARB1 gene contained CpG islands, micro RNA binding sites, and several transcription factor binding sites (including PPARG which may contribute to the high level (13.7 times

  10. The angiotensin II type 1 receptor antagonist Losartan binds and activates bradykinin B2 receptor signaling

    DEFF Research Database (Denmark)

    Bonde, Marie Mi; Olsen, Kristine Boisen; Erikstrup, Niels

    2011-01-01

    The angiotensin II type 1 receptor (AT1R) blocker (ARB) Losartan has cardioprotective effects during ischemia-reperfusion injury and inhibits reperfusion arrhythmias -effects that go beyond the benefits of lowering blood pressure. The renin-angiotensin and kallikrein-kinin systems are intricately...

  11. Allosteric ligands and their binding sites define γ-aminobutyric acid (GABA) type A receptor subtypes.

    Science.gov (United States)

    Olsen, Richard W

    2015-01-01

    GABAA receptors (GABA(A)Rs) mediate rapid inhibitory transmission in the brain. GABA(A)Rs are ligand-gated chloride ion channel proteins and exist in about a dozen or more heteropentameric subtypes exhibiting variable age and brain regional localization and thus participation in differing brain functions and diseases. GABA(A)Rs are also subject to modulation by several chemotypes of allosteric ligands that help define structure and function, including subtype definition. The channel blocker picrotoxin identified a noncompetitive channel blocker site in GABA(A)Rs. This ligand site is located in the transmembrane channel pore, whereas the GABA agonist site is in the extracellular domain at subunit interfaces, a site useful for low energy coupled conformational changes of the functional channel domain. Two classes of pharmacologically important allosteric modulatory ligand binding sites reside in the extracellular domain at modified agonist sites at other subunit interfaces: the benzodiazepine site and the high-affinity, relevant to intoxication, ethanol site. The benzodiazepine site is specific for certain GABA(A)R subtypes, mainly synaptic, while the ethanol site is found at a modified benzodiazepine site on different, extrasynaptic, subtypes. In the transmembrane domain are allosteric modulatory ligand sites for diverse chemotypes of general anesthetics: the volatile and intravenous agents, barbiturates, etomidate, propofol, long-chain alcohols, and neurosteroids. The last are endogenous positive allosteric modulators. X-ray crystal structures of prokaryotic and invertebrate pentameric ligand-gated ion channels, and the mammalian GABA(A)R protein, allow homology modeling of GABA(A)R subtypes with the various ligand sites located to suggest the structure and function of these proteins and their pharmacological modulation. © 2015 Elsevier Inc. All rights reserved.

  12. Estimation of the Binding Free Energy of AC1NX476 to HIV-1 Protease Wild Type and Mutations Using Free Energy Perturbation Method.

    Science.gov (United States)

    Ngo, Son Tung; Mai, Binh Khanh; Hiep, Dinh Minh; Li, Mai Suan

    2015-10-01

    The binding mechanism of AC1NX476 to HIV-1 protease wild type and mutations was studied by the docking and molecular dynamics simulations. The binding free energy was calculated using the double-annihilation binding free energy method. It is shown that the binding affinity of AC1NX476 to wild type is higher than not only ritonavir but also darunavir, making AC1NX476 become attractive candidate for HIV treatment. Our theoretical results are in excellent agreement with the experimental data as the correlation coefficient between calculated and experimentally measured binding free energies R = 0.993. Residues Asp25-A, Asp29-A, Asp30-A, Ile47-A, Gly48-A, and Val50-A from chain A, and Asp25-B from chain B play a crucial role in the ligand binding. The mutations were found to reduce the receptor-ligand interaction by widening the binding cavity, and the binding propensity is mainly driven by the van der Waals interaction. Our finding may be useful for designing potential drugs to combat with HIV. © 2015 John Wiley & Sons A/S.

  13. SECRET domain of variola virus CrmB protein can be a member of poxviral type II chemokine-binding proteins family

    Directory of Open Access Journals (Sweden)

    Shchelkunov Sergei N

    2010-10-01

    Full Text Available Abstract Background Variola virus (VARV the causative agent of smallpox, eradicated in 1980, have wide spectrum of immunomodulatory proteins to evade host immunity. Recently additional biological activity was discovered for VARV CrmB protein, known to bind and inhibit tumour necrosis factor (TNF through its N-terminal domain homologous to cellular TNF receptors. Besides binding TNF, this protein was also shown to bind with high affinity several chemokines which recruit B- and T-lymphocytes and dendritic cells to sites of viral entry and replication. Ability to bind chemokines was shown to be associated with unique C-terminal domain of CrmB protein. This domain named SECRET (Smallpox virus-Encoded Chemokine Receptor is unrelated to the host proteins and lacks significant homology with other known viral chemokine-binding proteins or any other known protein. Findings De novo modelling of VARV-CrmB SECRET domain spatial structure revealed its apparent structural homology with cowpox virus CC-chemokine binding protein (vCCI and vaccinia virus A41 protein, despite low sequence identity between these three proteins. Potential ligand-binding surface of modelled VARV-CrmB SECRET domain was also predicted to bear prominent electronegative charge which is characteristic to known orthopoxviral chemokine-binding proteins. Conclusions Our results suggest that SECRET should be included into the family of poxviral type II chemokine-binding proteins and that it might have been evolved from the vCCI-like predecessor protein.

  14. SECRET domain of variola virus CrmB protein can be a member of poxviral type II chemokine-binding proteins family.

    Science.gov (United States)

    Antonets, Denis V; Nepomnyashchikh, Tatyana S; Shchelkunov, Sergei N

    2010-10-27

    Variola virus (VARV) the causative agent of smallpox, eradicated in 1980, have wide spectrum of immunomodulatory proteins to evade host immunity. Recently additional biological activity was discovered for VARV CrmB protein, known to bind and inhibit tumour necrosis factor (TNF) through its N-terminal domain homologous to cellular TNF receptors. Besides binding TNF, this protein was also shown to bind with high affinity several chemokines which recruit B- and T-lymphocytes and dendritic cells to sites of viral entry and replication. Ability to bind chemokines was shown to be associated with unique C-terminal domain of CrmB protein. This domain named SECRET (Smallpox virus-Encoded Chemokine Receptor) is unrelated to the host proteins and lacks significant homology with other known viral chemokine-binding proteins or any other known protein. De novo modelling of VARV-CrmB SECRET domain spatial structure revealed its apparent structural homology with cowpox virus CC-chemokine binding protein (vCCI) and vaccinia virus A41 protein, despite low sequence identity between these three proteins. Potential ligand-binding surface of modelled VARV-CrmB SECRET domain was also predicted to bear prominent electronegative charge which is characteristic to known orthopoxviral chemokine-binding proteins. Our results suggest that SECRET should be included into the family of poxviral type II chemokine-binding proteins and that it might have been evolved from the vCCI-like predecessor protein.

  15. Atuoradiographic detection of multiple sclerosis plaques with an ω3 (peripheral type benzodiazepine) binding site radiogland

    International Nuclear Information System (INIS)

    HAUW, J.J.; Sazdovitch, V.; Cornu, P.; Dubois, A.; Benavides, J.; Mac Kenzie, E.; Scatton, B.

    1989-01-01

    In Multiple Sclerosis (MS), the presence of monocyte-macrophages and microglial cells in active plaques is a constant feature. They are numerous at the border of active lesions where they constiture, with other cell types such as lymphocytes and oligodendrocytes the so-called flial wall. Monocyte-macrophages and microglial cells are thought to be directly involved in the process of demyelination. Some macrophages laden with meylin degradation products are also seen in the center of the plaqie, often located in the perivascular cuffs. In addition, astrocytic gliosis occurs in the center of the plaques. As all these cell types are richly endowed with ω3 sites, the feasibility of using ω3 site autoradiagraphy to detect the demyelination plaques in the brain of post-mortem cases of Multiple Sclerosis has been investigated. (Author). 8 refs

  16. The Inner Nuclear Membrane Protein Nemp1 Is a New Type of RanGTP-Binding Protein in Eukaryotes.

    Directory of Open Access Journals (Sweden)

    Takashi Shibano

    Full Text Available The inner nuclear membrane (INM protein Nemp1/TMEM194A has previously been suggested to be involved in eye development in Xenopus, and contains two evolutionarily conserved sequences in the transmembrane domains (TMs and the C-terminal region, named region A and region B, respectively. To elucidate the molecular nature of Nemp1, we analyzed its interacting proteins through those conserved regions. First, we found that Nemp1 interacts with itself and lamin through the TMs and region A, respectively. Colocalization of Nemp1 and lamin at the INM suggests that the interaction with lamin participates in the INM localization of Nemp1. Secondly, through yeast two-hybrid screening using region B as bait, we identified the small GTPase Ran as a probable Nemp1-binding partner. GST pulldown and co-immunoprecipitation assays using region B and Ran mutants revealed that region B binds directly to the GTP-bound Ran through its effector domain. Immunostaining experiments using transfected COS-7 cells revealed that full-length Nemp1 recruits Ran near the nuclear envelope, suggesting a role for Nemp1 in the accumulation of RanGTP at the nuclear periphery. At the neurula-to-tailbud stages of Xenopus embryos, nemp1 expression overlapped with ran in several regions including the eye vesicles. Co-knockdown using antisense morpholino oligos for nemp1 and ran caused reduction of cell densities and severe eye defects more strongly than either single knockdown alone, suggesting their functional interaction. Finally we show that Arabidopsis thaliana Nemp1-orthologous proteins interact with A. thaliana Ran, suggesting their evolutionally conserved physical and functional interactions possibly in basic cellular functions including nuclear transportation. Taken together, we conclude that Nemp1 represents a new type of RanGTP-binding protein.

  17. High Serum Adipocyte Fatty Acid Binding Protein Is Associated with Metabolic Syndrome in Patients with Type 2 Diabetes

    Directory of Open Access Journals (Sweden)

    Jer-Chuan Li

    2016-01-01

    Full Text Available Adipocyte fatty acid binding protein (A-FABP is a key mediator of obesity-related metabolic syndrome (MetS. The aim of this study was to evaluate the relationship between A-FABP concentration and MetS in type 2 diabetes mellitus (DM patients. Fasting blood samples were obtained from 165 type 2 DM volunteers. MetS and its components were defined using diagnostic criteria from the International Diabetes Federation. Among 165 DM patients, 113 patients (68.5% had MetS. Diabetic persons who had MetS had significantly higher A-FABP levels (P<0.001 than those without MetS. Female DM persons had higher A-FABP level than man (P<0.001. No statistically significant differences in A-FABP levels were found in use of statin, fibrate, or antidiabetic drugs. Multivariate forward stepwise linear regression analysis revealed that body fat mass (P<0.001, logarithmically transformed creatinine (log-creatinine; P<0.001, female DM patients (P<0.001, and logarithmically transformed high sensitive C-reactive protein (log-hs-CRP; P=0.013 were positively correlated, while albumin (P=0.004 and glomerular filtration rate (GFR; P=0.043 were negatively correlated with serum A-FABP levels in type 2 DM patients. In this study, higher serum A-FABP level was positively associated with MetS in type 2 DM patients.

  18. Receptor type I and type II binding regions and the peptidyl-prolyl isomerase site of cyclophilin B are required for enhancement of T-lymphocyte adhesion to fibronectin.

    Science.gov (United States)

    Carpentier, Mathieu; Allain, Fabrice; Slomianny, Marie-Christine; Durieux, Sandrine; Vanpouille, Christophe; Haendler, Bernard; Spik, Geneviève

    2002-04-23

    Cyclophilin B (CyPB), a cyclosporin A (CsA) binding protein, interacts with two types of binding sites at the surface of T-lymphocytes. The type I sites correspond to functional receptors involved in endocytosis and the type II sites to sulfated glycosaminoglycans (GAGs). Mutational analysis of CyPB has revealed that W128, which is part of the CsA-binding pocket, is implicated in the binding to the functional type I receptors and that two amino acid clusters located in the N-terminus ensure the binding to GAGs. The peptidyl-prolyl isomerase activity of CyPB is not required for receptor binding. We have recently demonstrated that CyPB enhances adhesion of peripheral blood T-lymphocytes to fibronectin, a component of the extracellular matrix. We intended to identify additional amino acids involved in the binding of CyPB to its functional type I receptor and to determine regions responsible for the stimulation of peripheral blood T-lymphocyte adhesion. We determined that residues R76, G77, K132, D155, and D158 of the calcineurin (CN) interacting region were implicated in the recognition of type I receptor but not of GAGs. We also found that two different changes in the N-terminal extension that abated binding to GAGs prevented adhesion of peripheral blood T-lymphocytes to coated CyPB, whereas abbrogation of the PPIase activity had no effect. On the other hand, the adhesion of peripheral blood T-lymphocytes to coated fibronectin was not stimulated by CyPB mutants devoid of either type I receptor or GAGs binding activity or by mutants of the PPIase site. Altogether, the results demonstrate that different regions of CyPB are involved in peripheral blood T-lymphocyte activation and imply a novel important physiological function for peptidyl-prolyl isomerase activity.

  19. Contribution of electrostatics to the binding of pancreatic-type ribonucleases to membranes.

    Science.gov (United States)

    Sundlass, Nadia K; Eller, Chelcie H; Cui, Qiang; Raines, Ronald T

    2013-09-17

    Pancreatic-type ribonucleases show clinical promise as chemotherapeutic agents but are limited in efficacy by the inefficiency of their uptake by human cells. Cellular uptake can be increased by the addition of positive charges to the surface of ribonucleases, either by site-directed mutagenesis or by chemical modification. This observation has led to the hypothesis that ribonuclease uptake by cells depends on electrostatics. Here, we use a combination of experimental and computational methods to ascertain the contribution of electrostatics to the cellular uptake of ribonucleases. We focus on three homologous ribonucleases: Onconase (frog), ribonuclease A (cow), and ribonuclease 1 (human). Our results support the hypothesis that electrostatics are necessary for the cellular uptake of Onconase. In contrast, specific interactions with cell-surface components likely contribute more to the cellular uptake of ribonuclease A and ribonuclease 1 than do electrostatics. These findings provide insight for the design of new cytotoxic ribonucleases.

  20. Structural analogs of human insulin-like growth factor I with reduced affinity for serum binding proteins and the type 2 insulin-like growth factor receptor

    International Nuclear Information System (INIS)

    Bayne, M.L.; Applebaum, J.; Chicchi, G.G.; Hayes, N.S.; Green, B.G.; Cascieri, M.A.

    1988-01-01

    Four structural analogs of human insulin-like growth factor I (hIGF-I) have been prepared by site-directed mutagenesis of a synthetic IGF-I gene and subsequent expression and purification of the mutant protein from the conditioned media of transformed yeast. [Phe -1 , Val 1 , Asn 2 , Gln 3 , His 4 , Ser 8 , His 9 , Glu 12 , Tyr 15 , Leu 16 ]IGF-I (B-chain mutant), in which the first 16 amino acids of hIGF-I were replaced with the first 17 amino acids of the B-chain of insulin, has >1000-, 100-, and 2-fold reduced potency for human serum binding proteins, the rat liver type 2 IGF receptor, and the human placental type 1 IGF receptor, respectively. The B-chain mutant also has 4-fold increased affinity for the human placental insulin receptor. [Gln 3 , Ala 4 ] IGF-I has 4-fold reduced affinity for human serum binding proteins, but is equipotent to hIGF-I at the types 1 and 2 IGF and insulin receptors. [Tyr 15 , Leu 16 ] IGH-I has 4-fold reduced affinity for human serum binding proteins and 10-fold increased affinity for the insulin receptor. The peptide in which these four-point mutations are combined, [Gln 3 , Ala 4 , Tyr 15 ,Leu 16 ]IGF-I, has 600-fold reduced affinity for the serum binding proteins. All four of these mutants stimulate DNA synthesis in the rat vascular smooth muscle cell line A10 with potencies reflecting their potency at the type 1 IGF receptor. These studies identify some of the domains of hIGF-I which are responsible for maintaining high affinity binding with the serum binding protein and the type 2 IGF receptor. In addition, These peptides will be useful in defining the role of the type 2 IGF receptor and serum binding proteins in the physiological actions of hIGF-I

  1. Relation of Absolute or Relative Adiposity to Insulin Resistance, Retinol Binding Protein-4, Leptin, and Adiponectin in Type 2 Diabetes

    Directory of Open Access Journals (Sweden)

    You Lim Kim

    2012-12-01

    Full Text Available BackgroundCentral fat mass (CFM correlates with insulin resistance and increases the risk of type 2 diabetes and cardiovascular complications; however, peripheral fat mass (PFM is associated with insulin sensitivity. The aim of this study was to investigate the relation of absolute and relative regional adiposity to insulin resistance index and adipokines in type 2 diabetes.MethodsTotal of 83 overweighted-Korean women with type 2 diabetes were enrolled, and rate constants for plasma glucose disappearance (KITT and serum adipokines, such as retinol binding protein-4 (RBP4, leptin, and adiponectin, were measured. Using dual X-ray absorptiometry, trunk fat mass (in kilograms was defined as CFM, sum of fat mass on the lower extremities (in kilograms as PFM, and sum of CFM and PFM as total fat mass (TFM. PFM/TFM ratio, CFM/TFM ratio, and PFM/CFM ratio were defined as relative adiposity.ResultsMedian age was 55.9 years, mean body mass index 27.2 kg/m2, and mean HbA1c level 7.12±0.84%. KITT was positively associated with PMF/TFM ratio, PMF/CFM ratio, and negatively with CFM/TFM ratio, but was not associated with TFM, PFM, or CFM. RBP4 levels also had a significant relationship with PMF/TFM ratio and PMF/CFM ratio. Adiponectin, leptin, and apolipoprotein A levels were related to absolute adiposity, while only adiponectin to relative adiposity. In correlation analysis, KITT in type 2 diabetes was positively related with HbA1c, fasting glucose, RBP4, and free fatty acid.ConclusionThese results suggest that increased relative amount of peripheral fat mass may aggravate insulin resistance in type 2 diabetes.

  2. Patterns of binding of aluminum-containing adjuvants to Haemophilus influenzae type b and meningococcal group C conjugate vaccines and components.

    Science.gov (United States)

    Otto, Robert B D; Burkin, Karena; Amir, Saba Erum; Crane, Dennis T; Bolgiano, Barbara

    2015-09-01

    The basis of Haemophilus influenzae type b (Hib) and Neisseria meningitidis serogroup C (MenC) glycoconjugates binding to aluminum-containing adjuvants was studied. By measuring the amount of polysaccharide and protein in the non-adsorbed supernatant, the adjuvant, aluminum phosphate, AlPO4, was found to be less efficient than aluminum hydroxide, Al(OH)3 at binding to the conjugates, at concentrations relevant to licensed vaccine formulations and when equimolar. At neutral pH, binding of TT conjugates to AlPO4 was facilitated through the carrier protein, with only weak binding of AlPO4 to CRM197 being observed. There was slightly higher binding of either adjuvant to tetanus toxoid conjugates, than to CRM197 conjugates. This was verified in AlPO4 formulations containing DTwP-Hib, where the adsorption of TT-conjugated Hib was higher than CRM197-conjugated Hib. At neutral pH, the anionic Hib and MenC polysaccharides did not appreciably bind to AlPO4, but did bind to Al(OH)3, due to electrostatic interactions. Phosphate ions reduced the binding of the conjugates to the adjuvants. These patterns of adjuvant adsorption can form the basis for future formulation studies with individual and combination vaccines containing saccharide-protein conjugates. Crown Copyright © 2015. Published by Elsevier Ltd. All rights reserved.

  3. Patterns of binding of aluminum-containing adjuvants to Haemophilus influenzae type b and meningococcal group C conjugate vaccines and components

    Science.gov (United States)

    Otto, Robert B.D.; Burkin, Karena; Amir, Saba Erum; Crane, Dennis T.; Bolgiano, Barbara

    2015-01-01

    The basis of Haemophilus influenzae type b (Hib) and Neisseria meningitidis serogroup C (MenC) glycoconjugates binding to aluminum-containing adjuvants was studied. By measuring the amount of polysaccharide and protein in the non-adsorbed supernatant, the adjuvant, aluminum phosphate, AlPO4, was found to be less efficient than aluminum hydroxide, Al(OH)3 at binding to the conjugates, at concentrations relevant to licensed vaccine formulations and when equimolar. At neutral pH, binding of TT conjugates to AlPO4 was facilitated through the carrier protein, with only weak binding of AlPO4 to CRM197 being observed. There was slightly higher binding of either adjuvant to tetanus toxoid conjugates, than to CRM197 conjugates. This was verified in AlPO4 formulations containing DTwP–Hib, where the adsorption of TT-conjugated Hib was higher than CRM197-conjugated Hib. At neutral pH, the anionic Hib and MenC polysaccharides did not appreciably bind to AlPO4, but did bind to Al(OH)3, due to electrostatic interactions. Phosphate ions reduced the binding of the conjugates to the adjuvants. These patterns of adjuvant adsorption can form the basis for future formulation studies with individual and combination vaccines containing saccharide-protein conjugates. PMID:26194164

  4. The role of solvation in the binding selectivity of the L-type calcium channel.

    Science.gov (United States)

    Boda, Dezső; Henderson, Douglas; Gillespie, Dirk

    2013-08-07

    We present grand canonical Monte Carlo simulation results for a reduced model of the L-type calcium channel. While charged residues of the protein amino acids in the selectivity filter are treated explicitly, most of the degrees of freedom (including the rest of the protein and the solvent) are represented by their dielectric response, i.e., dielectric continua. The new aspect of this paper is that the dielectric coefficient in the channel is different from that in the baths. The ions entering the channel, thus, cross a dielectric boundary at the entrance of the channel. Simulating this case has been made possible by our recent methodological development [D. Boda, D. Henderson, B. Eisenberg, and D. Gillespie, J. Chem. Phys. 135, 064105 (2011)]. Our main focus is on the effect of solvation energy (represented by the Born energy) on monovalent vs. divalent ion selectivity in the channel. We find no significant change in selectivity by changing the dielectric coefficient in the channel because the larger solvation penalty is counterbalanced by the enhanced Coulomb attraction inside the channel as soon as we use the Born radii (fitted to experimental hydration energies) to compute the solvation penalty from the Born equation.

  5. Mutations in type 3 reovirus that determine binding to sialic acid are contained in the fibrous tail domain of viral attachment protein sigma1.

    Science.gov (United States)

    Chappell, J D; Gunn, V L; Wetzel, J D; Baer, G S; Dermody, T S

    1997-03-01

    The reovirus attachment protein, sigma1, determines numerous aspects of reovirus-induced disease, including viral virulence, pathways of spread, and tropism for certain types of cells in the central nervous system. The sigma1 protein projects from the virion surface and consists of two distinct morphologic domains, a virion-distal globular domain known as the head and an elongated fibrous domain, termed the tail, which is anchored into the virion capsid. To better understand structure-function relationships of sigma1 protein, we conducted experiments to identify sequences in sigma1 important for viral binding to sialic acid, a component of the receptor for type 3 reovirus. Three serotype 3 reovirus strains incapable of binding sialylated receptors were adapted to growth in murine erythroleukemia (MEL) cells, in which sialic acid is essential for reovirus infectivity. MEL-adapted (MA) mutant viruses isolated by serial passage in MEL cells acquired the capacity to bind sialic acid-containing receptors and demonstrated a dependence on sialic acid for infection of MEL cells. Analysis of reassortant viruses isolated from crosses of an MA mutant virus and a reovirus strain that does not bind sialic acid indicated that the sigma1 protein is solely responsible for efficient growth of MA mutant viruses in MEL cells. The deduced sigma1 amino acid sequences of the MA mutant viruses revealed that each strain contains a substitution within a short region of sequence in the sigma1 tail predicted to form beta-sheet. These studies identify specific sequences that determine the capacity of reovirus to bind sialylated receptors and suggest a location for a sialic acid-binding domain. Furthermore, the results support a model in which type 3 sigma1 protein contains discrete receptor binding domains, one in the head and another in the tail that binds sialic acid.

  6. Intestinal fatty acid binding protein Ala54Thr polymorphism is associated with peripheral atherosclerosis combined with type 2 diabetes mellitus.

    Science.gov (United States)

    Khattab, Salma A; Abo-Elmatty, Dina M; Ghattas, Maivel H; Mesbah, Noha M; Mehanna, Eman T

    2017-09-01

    Intestinal fatty acid-binding protein 2 (FABP2) is expressed in enterocytes and binds saturated and unsaturated long-chain fatty acids. The FABP2 Ala54Thr polymorphism has been reported to effect lipid metabolism. The aim of the present study was to assess the relationship between this polymorphism and peripheral atherosclerosis combined with type 2 diabetes mellitus (T2DM) in an Egyptian population. The study was performed on 100 T2DM patients with peripheral atherosclerosis and 100 control subjects. The Ala54Thr polymorphism was analyzed by polymerase chain reaction-restriction fragment length polymorphism, whereas serum FABP2 levels were determined using ELISA. Fasting blood glucose, fasting serum insulin concentrations, HbA1c, lipid profile, body mass index (BMI) and systolic and diastolic blood pressure (SBP and DBP, respectively) were determined. There was a higher frequency of the Thr54 allele among the patient group (P = 0.002). In Ala54/Thr54 heterozygotes and carriers of the rare Thr54/Thr54 genotype, there were significant increases in BMI and FABP2. Those with the Thr54/Thr54 genotype had significantly decreased high-density lipoprotein cholesterol (HDL-C) concentrations; in addition, those with the Thr54/Thr54 genotype had significantly higher SBP and DBP than subjects with the Ala54/Ala54 and Ala54/Thr54 genotypes. There was a positive correlation between FABP2 levels and BMI, SBP and DBP, and a negative correlation with HDL-C. The Thr54 allele of the FABP2 Ala54Thr polymorphism was associated with an increased incidence of peripheral atherosclerosis combined with T2DM in the population studied. © 2016 Ruijin Hospital, Shanghai Jiaotong University School of Medicine and John Wiley & Sons Australia, Ltd.

  7. Glycosaminoglycans mediate retention of the poxvirus type I interferon binding protein at the cell surface to locally block interferon antiviral responses

    Science.gov (United States)

    Montanuy, Imma; Alejo, Ali; Alcami, Antonio

    2011-01-01

    Eradication of smallpox was accomplished 30 yr ago, but poxviral infections still represent a public health concern due to the potential release of variola virus or the emergence of zoonotic poxviruses, such as monkeypox virus. A critical determinant of poxvirus virulence is the inhibition of interferons (IFNs) by the virus-encoded type I IFN-binding protein (IFNα/βBP). This immunomodulatory protein is secreted and has the unique property of interacting with the cell surface in order to prevent IFN-mediated antiviral responses. However, the mechanism of its attachment to the cell surface remains unknown. Using surface plasmon resonance and cell-binding assays, we report that the IFNα/βBP from vaccinia virus, the smallpox vaccine, interacts with cell surface glycosaminoglycans (GAGs). Analysis of the contribution of different regions of the protein to cell surface binding demonstrated that clusters of basic residues in the first immunoglobulin domain mediate GAG interactions. Furthermore, mutation of the GAG-interaction motifs does not affect its IFN-binding and -blocking capacity. Functional conservation of GAG-binding sites is demonstrated for the IFNα/βBP from variola and monkeypox viruses, extending our understanding of immune modulation by the most virulent human poxviruses. These results are relevant for the design of improved vaccines and intervention strategies.—Montanuy, I., Alejo, A., Alcami, A. Glycosaminoglycans mediate retention of the poxvirus type I interferon binding protein at the cell surface to locally block interferon antiviral responses. PMID:21372110

  8. Elevation of urinary liver-type fatty acid binding protein after cardiac catheterization related to cardiovascular events

    Directory of Open Access Journals (Sweden)

    Kamijo-Ikemori A

    2015-08-01

    Full Text Available Atsuko Kamijo-Ikemori,1,3 Nobuyuki Hashimoto,2 Takeshi Sugaya,1 Katsuomi Matsui,1 Mikako Hisamichi,1 Yugo Shibagaki,1 Fumihiko Miyake,2 Kenjiro Kimura1 1Department of Nephrology and Hypertension, 2Department of Cardiology, 3Department of Anatomy, St Marianna University School of Medicine, Kawasaki, Kanagawa, Japan Purpose: Contrast medium (CM induces tubular hypoxia via endothelial damage due to direct cytotoxicity or viscosity. Urinary liver-type fatty acid binding protein (L-FABP increases along with tubular hypoxia and may be a detector of systemic circulation injury. The aim of this study was to evaluate the clinical usefulness of detecting increases in urinary L-FABP levels due to administration of CM, as a prognostic biomarker for cardiovascular disease in patients without occurrence of CM-induced nephropathy undergoing cardiac catheterization procedure (CCP. Methods: Retrospective longitudinal analyses of the relationship between urinary L-FABP levels and occurrence of cardiovascular events were performed (n=29. Urinary L-FABP was measured by ELISA before CCP, and at 6, 12, 24, and 48 hours after CCP. Results: Urinary L-FABP levels were significantly higher at 12 hours (P<0.05 and 24 hours (P<0.005 after CCP compared with before CCP, only in the patients with occurrence of cardiovascular events (n=17, but not in those without cardiovascular events (n=12. The parameter with the largest area under the curve (0.816 for predicting the occurrence of cardiovascular events was the change in urinary L-FABP at 24 hours after CCP. The difference in urinary L-FABP levels (ΔL-FABP ≥11.0 µg/g creatinine between before CCP and at 24 hours after CCP was a risk factor for the occurrence of cardiovascular events (hazard ratio, 4.93; 95% confidence interval, 1.27–19.13; P=0.021. Conclusion: Measurement of urinary L-FABP before CCP and at 24 hours after CCP in patients with mild to moderate renal dysfunction may be an important indicator for risk

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

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    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.

  10. Characterisation of the binding of [{sup 3}H]methyllycaconitine: a new radioligand for labelling {alpha}7-type neuronal nicotinic acetylcholine receptors

    Energy Technology Data Exchange (ETDEWEB)

    Davies, A.R.L.; Wolstenholme, A.J.; Wonnacott, S. [Department of Biology and Biochemistry, University of Bath, Bath BA2 7AY (United Kingdom); Hardick, D.J.; Blagbrough, I.S.; Potter, B.V.L. [Department of Pharmacy and Pharmacology, University of Bath, Bath BA2 7AY (United Kingdom)

    1999-05-15

    Methyllycaconitine (MLA), a norditerpenoid alkaloid isolated from Delphinium seeds, is one of the most potent non-proteinacious ligands that is selective for {alpha}bungarotoxin-sensitive neuronal nicotinic acetylcholine receptors (nAChR). [{sup 3}H]MLA bound to rat brain membranes with high affinity (K{sub d}=1.86{+-}0.31 nM) with a good ratio of specific to non-specific binding. The binding of [{sup 3}H]MLA was characterised by rapid association (t{sub 1/2}=2.3 min) and dissociation (t{sub 1/2}=12.6 min) kinetics. The radioligand binding displayed nicotinic pharmacology, consistent with an interaction with {alpha}bungarotoxin-sensitive nAChR. The snake {alpha}-toxins, {alpha}bungarotoxin and {alpha}cobratoxin, displaced [{sup 3}H]MLA with high affinity (K{sub i}=1.8{+-}0.5 and 5.5{+-}0.9 nM, respectively), whereas nicotine was less potent (K{sub i}=6.1{+-}1.1 {mu}M). The distribution of [{sup 3}H]MLA binding sites in crudely dissected rat brain regions was identical to that of [{sup 125}I]{alpha}bungarotoxin binding sites, with a high binding site density in hippocampus and hypothalamus, but low density in striatum and cerebellum. [{sup 3}H]MLA also labelled a sub-population of binding sites which are not sensitive to the snake {alpha}toxins, but which did not differ significantly from the major population with respect to their other pharmacological properties or regional distribution. [{sup 3}H]MLA, therefore, is a novel radiolabel for characterising {alpha}7-type nAChR. A good signal to noise ratio and rapid binding kinetics provide advantages over the use of radiolabelled {alpha}bungarotoxin for rapid and accurate equilibrium binding assays. (Copyright (c) 1999 Elsevier Science B.V., Amsterdam. All rights reserved.)

  11. Characterisation of the binding of [3H]methyllycaconitine: a new radioligand for labelling α7-type neuronal nicotinic acetylcholine receptors

    International Nuclear Information System (INIS)

    Davies, A.R.L.; Wolstenholme, A.J.; Wonnacott, S.; Hardick, D.J.; Blagbrough, I.S.; Potter, B.V.L.

    1999-01-01

    Methyllycaconitine (MLA), a norditerpenoid alkaloid isolated from Delphinium seeds, is one of the most potent non-proteinacious ligands that is selective for αbungarotoxin-sensitive neuronal nicotinic acetylcholine receptors (nAChR). [ 3 H]MLA bound to rat brain membranes with high affinity (K d =1.86±0.31 nM) with a good ratio of specific to non-specific binding. The binding of [ 3 H]MLA was characterised by rapid association (t 1/2 =2.3 min) and dissociation (t 1/2 =12.6 min) kinetics. The radioligand binding displayed nicotinic pharmacology, consistent with an interaction with αbungarotoxin-sensitive nAChR. The snake α-toxins, αbungarotoxin and αcobratoxin, displaced [ 3 H]MLA with high affinity (K i =1.8±0.5 and 5.5±0.9 nM, respectively), whereas nicotine was less potent (K i =6.1±1.1 μM). The distribution of [ 3 H]MLA binding sites in crudely dissected rat brain regions was identical to that of [ 125 I]αbungarotoxin binding sites, with a high binding site density in hippocampus and hypothalamus, but low density in striatum and cerebellum. [ 3 H]MLA also labelled a sub-population of binding sites which are not sensitive to the snake αtoxins, but which did not differ significantly from the major population with respect to their other pharmacological properties or regional distribution. [ 3 H]MLA, therefore, is a novel radiolabel for characterising α7-type nAChR. A good signal to noise ratio and rapid binding kinetics provide advantages over the use of radiolabelled αbungarotoxin for rapid and accurate equilibrium binding assays. (Copyright (c) 1999 Elsevier Science B.V., Amsterdam. All rights reserved.)

  12. Association of ATP-Binding Cassette Transporter A1 Gene Polymorphisms in Type 2 Diabetes Mellitus among Malaysians

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    Polin Haghvirdizadeh

    2015-01-01

    Full Text Available Background. Type 2 diabetes mellitus (T2DM is a complex polygenic disorder characterized by impaired insulin resistance, insulin secretion, and dysregulation of lipid and protein metabolism with environmental and genetic factors. ATP-binding cassette transporter A1 (ABCA1 gene polymorphisms are reported as the one of the genetic risk factors for T2DM in various populations with conflicting results. This study was conducted based on PCR-HRM to determine the frequency of ABCA1 gene by rs2230806 (R219K, rs1800977 (C69T, and rs9282541 (R230C polymorphisms Malaysian subjects. Methods. A total of 164 T2DM and 165 controls were recruited and their genotypes for ABCA1 gene polymorphisms were determined based on the real time high resolution melting analysis. Results. There was a significant difference between the subjects in terms of age, BMI, FPG, HbA1c, HDL, LDL, and TG P<0.05. There was a significant association between HOM of R219K P=0.005, among Malaysian subjects; moreover, allele frequency revealed the significant difference in A allele of R219K P=0.003. But, there was no significant difference in genotypic and allelic frequencies of C69T and R230C polymorphism. Conclusion. R219K polymorphism of ABCA1 gene can be considered as a genetic risk factor for T2DM subjects among Malaysians.

  13. Identification of the gene encoding the 65-kilodalton DNA-binding protein of herpes simplex virus type 1

    International Nuclear Information System (INIS)

    Parris, D.S.; Cross, A.; Orr, A.; Frame, M.C.; Murphy, M.; McGeoch, D.J.; Marsden, H.S.; Haarr, L.

    1988-01-01

    Hybrid arrest of in vitro translation was used to localize the region of the herpes simplex virus type 1 genome encoding the 65-kilodalton DNA-binding protein (65K DBP ) to between genome coordinates 0.592 and 0.649. Knowledge of the DNA sequence of this region allowed us to identify three open reading frames as likely candidates for the gene encoding 65K DBP . Two independent approaches were used to determine which of these three open reading frames encoded the protein. For the first approach a monoclonal antibody, MAb 6898, which reacted specifically with 65K DBP , was isolated. This antibody was used, with the techniques of hybrid arrest of in vitro translation and in vitro translation of selected mRNA, to identify the gene encoding 65K DBP . The second approach involved preparation of antisera directed against oligopeptides corresponding to regions of the predicted amino acid sequence of this gene. These antisera reacted specifically with 65K DBP , thus confirming the gene assignment

  14. Prion Fragment Peptides Are Digested with Membrane Type Matrix Metalloproteinases and Acquire Enzyme Resistance through Cu2+-Binding

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    Aya Kojima

    2014-05-01

    Full Text Available Prions are the cause of neurodegenerative disease in humans and other mammals. The structural conversion of the prion protein (PrP from a normal cellular protein (PrPC to a protease-resistant isoform (PrPSc is thought to relate to Cu2+ binding to histidine residues. In this study, we focused on the membrane-type matrix metalloproteinases (MT-MMPs such as MT1-MMP and MT3-MMP, which are expressed in the brain as PrPC-degrading proteases. We synthesized 21 prion fragment peptides. Each purified peptide was individually incubated with recombinant MT1-MMP or MT3-MMP in the presence or absence of Cu2+ and the cleavage sites determined by LC-ESI-MS analysis. Recombinant MMP-7 and human serum (HS were also tested as control. hPrP61-90, from the octapeptide-repeat region, was cleaved by HS but not by the MMPs tested here. On the other hand, hPrP92-168 from the central region was cleaved by MT1-MMP and MT3-MMP at various sites. These cleavages were inhibited by treatment with Cu2+. The C-terminal peptides had higher resistance than the central region. The data obtained from this study suggest that MT-MMPs expressed in the brain might possess PrPC-degrading activity.

  15. Heart-type fatty acid binding protein is an early marker of myocardial damage after radiofrequency catheter ablation.

    Science.gov (United States)

    Giannessi, Daniela; Piacenti, Marcello; Maltinti, Maristella; Rossi, Andrea; Di Cecco, Pietro; Startari, Umberto; Cabiati, Manuela; Panchetti, Luca; Del Ry, Silvia; Morales, Maria-Aurora

    2010-10-01

    Radiofrequency (RF) ablation of arrhythmias induces myocardial damage and release of biomarkers. This study aimed to assess the kinetics of heart-type fatty acid-binding protein (h-FABP), a cytosolic protein released after myocardial injury incurred by both atrial and ventricular RF ablation, compared to other markers of myocardial injury. h-FABP, cTnI, CK-MB(mass) and myoglobin were evaluated in 30 patients with atrial or ventricular tachyarrhythmias before, immediately after and at 3, 6 and 24h after the procedure. h-FABP increased immediately after the procedure in all subjects (6.6 ± 1.2 μg/L vs 2.7 ± 0.3, pvalues of time for mean power of RF application in both the entire patient cohort and in ventricular ablations. h-FABP may be an early parameter for monitoring RF-induced lesions and the site of ablation was relevant for biomarker increase. Copyright © 2010 Elsevier Inc. All rights reserved.

  16. Tissue-type plasminogen activator-binding RNA aptamers inhibiting low-density lipoprotein receptor family-mediated internalisation.

    Science.gov (United States)

    Bjerregaard, Nils; Bøtkjær, Kenneth A; Helsen, Nicky; Andreasen, Peter A; Dupont, Daniel M

    2015-07-01

    Recombinant tissue-type plasminogen activator (tPA, trade name Alteplase), currently the only drug approved by the US Food and Drug Administration and the European Medicines Agency for the treatment of cerebral ischaemic stroke, has been implicated in a number of adverse effects reportedly mediated by interactions with the low-density lipoprotein (LDL) family receptors, including neuronal cell death and an increased risk of cerebral haemorrhage. The tissue-type plasminogen activator is the principal initiator of thrombolysis in human physiology, an effect that is mediated directly via localised activation of the plasmin zymogen plasminogen at the surface of fibrin clots in the vascular lumen. Here, we sought to identify a ligand to tPA capable of inhibiting the relevant LDL family receptors without interfering with the fibrinolytic activity of tPA. Systematic evolution of ligands by exponential enrichment (SELEX) was employed to isolate tPA-binding RNA aptamers, which were characterised in biochemical assays of tPA association to low density lipoprotein receptor-related protein-1 (LRP-1, an LDL receptor family member); tPA-mediated in vitro and ex vivo clot lysis; and tPA-mediated plasminogen activation in the absence and presence of a stimulating soluble fibrin fragment. Two aptamers, K18 and K32, had minimal effects on clot lysis, but were able to efficiently inhibit tPA-LRP-1 association and LDL receptor family-mediated endocytosis in human vascular endothelial cells and astrocytes. These observations suggest that coadministration alongside tPA may be a viable strategy to improve the safety of thrombolytic treatment of cerebral ischaemic stroke by restricting tPA activity to the vascular lumen.

  17. An Aromatic Cap Seals the Substrate Binding Site in an ECF-Type S Subunit for Riboflavin

    Energy Technology Data Exchange (ETDEWEB)

    Karpowich, Nathan K.; Song, Jinmei; Wang, Da-Neng

    2016-06-13

    ECF transporters are a family of active membrane transporters for essential micronutrients, such as vitamins and trace metals. Found exclusively in archaea and bacteria, these transporters are composed of four subunits: an integral membrane substrate-binding subunit (EcfS), a transmembrane coupling subunit (EcfT), and two ATP-binding cassette ATPases (EcfA and EcfA'). We have characterized the structural basis of substrate binding by the EcfS subunit for riboflavin from Thermotoga maritima, TmRibU. TmRibU binds riboflavin with high affinity, and the protein–substrate complex is exceptionally stable in solution. The crystal structure of riboflavin-bound TmRibU reveals an electronegative binding pocket at the extracellular surface in which the substrate is completely buried. Analysis of the intermolecular contacts indicates that nearly every available substrate hydrogen bond is satisfied. A conserved aromatic residue at the extracellular end of TM5, Tyr130, caps the binding site to generate a substrate-bound, occluded state, and non-conservative mutation of Tyr130 reduces the stability of this conformation. Using a novel fluorescence binding assay, we find that an aromatic residue at this position is essential for high-affinity substrate binding. Comparison with other S subunit structures suggests that TM5 and Loop5-6 contain a dynamic, conserved motif that plays a key role in gating substrate entry and release by S subunits of ECF transporters.

  18. Improved methods for binding acma-type protein anchor fusions yo cell-wall material of micro-organisms

    NARCIS (Netherlands)

    Leenhouts, Cornelis; Ramasamy, R.; Steen, Anton; Kok, Jan; Buist, Girbe; Kuipers, Oscar

    2002-01-01

    The invention provides a method for improving binding of a proteinaceous substance to cell-wall material of a Gram-positive bacterium, said substance comprising an AcmA cell wall binding domain or homolog or functional derivative thereof, said method comprising treating said cell-wall material with

  19. Expression of peanut agglutinin-binding mucin-type glycoprotein in human esophageal squamous cell carcinoma as a marker

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    Balakrishnan Ramathilakam

    2003-11-01

    Full Text Available Abstract Background The TF (Thomson – Friedenreich blood group antigen behaves as an onco-foetal carcinoma-associated antigen, showing increased expression in malignancies and its detection and quantification can be used in serologic diagnosis mainly in adenocarcinomas. This study was undertaken to analyze the sera and tissue level detectable mucin-type glycoprotein (TF-antigen by Peanut agglutinin (PNA and its diagnostic index in serum as well tissues of human esophageal squamous cell carcinoma as marker. Results We examined 100 patients for serological analysis by Enzyme Linked Lectin Assay (ELISA and demonstrated a sensitivity of 87.5%, specificity of 90% and a positive predictive value of 95%. The immuno-histochemical localization of TF antigen by Fluorescence Antigen Technique (FAT in 25 specimens of normal esophageal squamous epithelium specimens and 92 specimens with different grades of, allowed a quicker and more precise identification of its increased expression and this did not correlate with gender and tumor size. There was a positive correlation between membrane bound TF antigen expression with different histological progression, from well differentiated to poorly differentiated, determined by PNA binding. Specimens showed morphological changes and a pronounced increase in PNA binding in Golgi apparatus, secretory granules of the cytosol of well differentiated and an increased cell membrane labeling in moderately and poorly differentiated, when compared with ESCC and normal tissues. Conclusion The authors propose that the expression of TF-antigen in human may play an important role during tumorigenesis establishing it as a chemically well-defined carcinoma-associated antigen. Identification of the circulating TF-antigen as a reactive form and as a cryptic form in the healthy individuals, using PNA-ELLA and Immunohistochemical analysis of TF antigen by FAT is positively correlated with the different histological grades as a simple

  20. Zinc finger arrays binding human papillomavirus types 16 and 18 genomic DNA: precursors of gene-therapeutics for in-situ reversal of associated cervical neoplasia

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    Wayengera Misaki

    2012-07-01

    Full Text Available Abstract Background Human papillomavirus (HPV types 16 and 18 are the high-risk, sexually transmitted infectious causes of most cervical intraepithelial neoplasias (CIN or cancers. While efficacious vaccines to reduce the sexual acquisition of these high-risk HPVs have recently been introduced, no virus-targeted therapies exist for those already exposed and infected. Considering the oncogenic role of the transforming (E6 and E7 genes of high-risk HPVs in the slow pathogenesis of cervical cancer, we hypothesize that timely disruption or abolition of HPV genome expression within pre-cancerous lesions identified at screening may reverse neoplasia. We aimed to derive model zinc finger nucleases (ZFNs for mutagenesis of the genomes of two high-risk HPV (types 16 & 18. Methods and results Using ZiFiT software and the complete genomes of HPV types16 and 18, we computationally generated the consensus amino acid sequences of the DNA-binding domains (F1, F2, & F3 of (i 296 & 327 contextually unpaired (or single three zinc-finger arrays (sZFAs and (ii 9 & 13 contextually paired (left and right three- zinc-finger arrays (pZFAs that bind genomic DNA of HPV-types 16 and 18 respectively, inclusive of the E7 gene (s/pZFAHpV/E7. In the absence of contextually paired three-zinc-finger arrays (pZFAs that bind DNA corresponding to the genomic context of the E6 gene of either HPV type, we derived the DNA binding domains of another set of 9 & 14 contextually unpaired E6 gene-binding ZFAs (sZFAE6 to aid the future quest for paired ZFAs to target E6 gene sequences in both HPV types studied (pZFAE6. This paper presents models for (i synthesis of hybrid ZFNs that cleave within the genomic DNA of either HPV type, by linking the gene sequences of the DNA-cleavage domain of the FokI endonuclease FN to the gene sequences of a member of the paired-HPV-binding ZFAs (pZFAHpV/E7 + FN, and (ii delivery of the same into precancerous lesions using HPV-derived viral plasmids or

  1. Endogenous Androgens and Sex Hormone-Binding Globulin in Women and Risk of Metabolic Syndrome and Type 2 Diabetes.

    Science.gov (United States)

    Fenske, Benjamin; Kische, Hanna; Gross, Stefan; Wallaschofski, Henri; Völzke, Henry; Dörr, Marcus; Nauck, Matthias; Keevil, Brian G; Brabant, Georg; Haring, Robin

    2015-12-01

    The association of endogenous androgens and sex hormone-binding globulin (SHBG) with metabolic syndrome (MetS) and type 2 diabetes mellitus (T2DM) mostly 23562 refers to small and selected study samples with immunoassay-based measurements. Thus, we investigated the association of hormone levels with MetS and T2DM in women from a large population-based sample. A total of 2077 women from the Study of Health in Pomerania were assessed at baseline (N = 3160, 1997-2001) and 5-year follow-up (N = 1711, 2002-2006). We investigated associations of total testosterone (T) and androstenedione measured by liquid chromatography-tandem mass spectrometry, SHBG by immunoassay, and free T and free androgen index with MetS and T2DM. Baseline prevalence of MetS and T2DM was 23.1% (N = 365) and 9.5% (N = 196), with an incidence of 17.7 and 7.0 per 1.000 person-years, respectively. Cross-sectional analyses yielded inverse associations of SHBG with MetS (relative risk [RR], 0.67; 95% confidence interval [CI], 0.60-0.74) and T2DM (RR, 0.61; 95% CI, 0.50-0.74) after multivariable adjustment. In longitudinal analyses, only age-adjusted models showed an inverse association of baseline SHBG with incident MetS (RR, 0.61; 95% CI, 0.51-0.73) and T2DM (RR, 0.58; 95% CI, 0.43-0.78). Multivariable-adjusted models stratified by menopausal status revealed an inverse association between SHBG and incident MetS risk in postmenopausal women (RR, 0.65; 95% CI, 0.51-0.81). This longitudinal population-based study revealed independent inverse associations of SHBG with MetS and T2DM, suggesting low SHBG as a potential risk marker for cardiometabolic morbidity, especially among postmenopausal women.

  2. Human heart-type fatty acid-binding protein as an early diagnostic marker of doxorubicin cardiac toxicity

    Directory of Open Access Journals (Sweden)

    Ashraf H. ElGhandour

    2009-04-01

    Full Text Available Progressive cardiotoxicity following treatment with doxorubicin-based chemotherapy in patients with non-Hodgkin’s lymphoma (NHL may lead to late onset cardiomyopathy. So, early prediction of toxicity can lead to prevention of heart failure in these patients. The aim of this work was to investigate the role of H-FABP as an early diagnostic marker of anthracycline-induced cardiac toxicity together with brain natriuretic peptide (BNP as an indication of ventricular dysfunction in such patients. Our study was conducted on 40 NHL patients who received 6 cycles of a doxorubicin containing chemotherapy protocol (CHOP, not exceeding the total allowed dose of doxorubicin (500 mg/m2. Ten healthy controls were included in our study. Human heart-type fatty acid binding protein (H-FABP was assessed 24 hours after the first cycle of CHOP. Plasma levels of BNP were estimated both before starting chemotherapy and after the last cycle of CHOP. Resting echocardiography was also performed before and at the end of chemotherapy cycles. The ejection fraction (EF of 8 of our patients decreased below 50% at the end of the sixth cycle. Elevated levels of both H-FABP and BNP were found in all patients wth EF below 50% and both markers showed a positive correlation with each other. We concluded that H-FABP may serve as a reliable early marker for prediction of cardiomyopathy induced by doxorubicin. Thus, in patients with elevated H-FABP, alternative treatment modalities with no cardiac toxicity may be considered in order to prevent subsequent heart failure in these patients.

  3. The UL5 and UL52 subunits of the herpes simplex virus type 1 helicase-primase subcomplex exhibit a complex interdependence for DNA binding.

    Science.gov (United States)

    Biswas, N; Weller, S K

    2001-05-18

    Herpes simplex virus type 1 encodes a heterotrimeric helicase-primase complex composed of the products of the UL5, UL52, and UL8 genes. The UL5 protein contains seven motifs found in all members of helicase Superfamily 1 (SF1), and the UL52 protein contains several conserved motifs found in primases; however, the contributions of each subunit to the biochemical activities of the subcomplex are not clear. In this work, the DNA binding properties of wild type and mutant subcomplexes were examined using single-stranded, duplex, and forked substrates. A gel mobility shift assay indicated that the UL5-UL52 subcomplex binds more efficiently to the forked substrate than to either single strand or duplex DNA. Although nucleotides are not absolutely required for DNA binding, ADP stimulated the binding of UL5-UL52 to single strand DNA whereas ATP, ADP, and adenosine 5'-O-(thiotriphosphate) stimulated the binding to a forked substrate. We have previously shown that both subunits contact single-stranded DNA in a photocross-linking assay (Biswas, N., and Weller, S. K. (1999) J. Biol. Chem. 274, 8068-8076). In this study, photocross-linking assays with forked substrates indicate that the UL5 and UL52 subunits contact the forked substrates at different positions, UL52 at the single-stranded DNA tail and UL5 near the junction between single-stranded and double-stranded DNA. Neither subunit was able to cross-link a forked substrate when 5-iododeoxyuridine was located within the duplex portion. Photocross-linking experiments with subcomplexes containing mutant versions of UL5 and wild type UL52 indicated that the integrity of the ATP binding region is important for DNA binding of both subunits. These results support our previous proposal that UL5 and UL52 exhibit a complex interdependence for DNA binding (Biswas, N., and Weller, S. K. (1999) J. Biol. Chem. 274, 8068-8076) and indicate that the UL52 subunit may play a more active role in helicase activity than had previously been

  4. Conformational changes in IpaD from Shigella flexneri upon binding bile salts provide insight into the second step of type III secretion.

    Science.gov (United States)

    Dickenson, Nicholas E; Zhang, Lingling; Epler, Chelsea R; Adam, Philip R; Picking, Wendy L; Picking, William D

    2011-01-18

    Shigella flexneri uses its type III secretion apparatus (TTSA) to inject host-altering proteins into targeted eukaryotic cells. The TTSA is composed of a basal body and an exposed needle with invasion plasmid antigen D (IpaD) forming a tip complex that controls secretion. The bile salt deoxycholate (DOC) stimulates recruitment of the translocator protein IpaB into the maturing TTSA needle tip complex. This process appears to be triggered by a direct interaction between DOC and IpaD. Fluorescence spectroscopy and NMR spectroscopy are used here to confirm the DOC-IpaD interaction and to reveal that IpaD conformational changes upon DOC binding trigger the appearance of IpaB at the needle tip. Förster resonance energy transfer between specific sites on IpaD was used here to identify changes in distances between IpaD domains as a result of DOC binding. To further explore the effects of DOC binding on IpaD structure, NMR chemical shift mapping was employed. The environments of residues within the proposed DOC binding site and additional residues within the "distal" globular domain were perturbed upon DOC binding, further indicating that conformational changes occur within IpaD upon DOC binding. These events are proposed to be responsible for the recruitment of IpaB at the TTSA needle tip. Mutation analyses combined with additional spectroscopic analyses confirm that conformational changes in IpaD induced by DOC binding contribute to the recruitment of IpaB to the S. flexneri TTSA needle tip. These findings lay the foundation for determining how environmental factors promote TTSA needle tip maturation prior to host cell contact.

  5. Conformational Changes in IpaD from Shigella flexneri Upon Binding Bile Salts Provide Insight into the Second Step of Type III Secretion†

    Science.gov (United States)

    Dickenson, Nicholas E.; Zhang, Lingling; Epler, Chelsea R.; Adam, Philip R.; Picking, Wendy L.; Picking, William D.

    2011-01-01

    Shigella flexneri uses its type III secretion apparatus (TTSA) to inject host-altering proteins into targeted eukaryotic cells. The TTSA is composed of a basal body and an exposed needle with invasion plasmid antigen D (IpaD) forming a tip complex that controls secretion. The bile salt deoxycholate (DOC) stimulates recruitment of the translocator protein IpaB into the maturing TTSA needle tip complex. This process appears to be triggered by a direct interaction between DOC and IpaD. Fluorescence spectroscopy and NMR spectroscopy are used here to confirm the DOC-IpaD interaction and to reveal that IpaD conformational changes upon DOC binding trigger the appearance of IpaB at the needle tip. Förster resonance energy transfer between specific sites on IpaD was used here to identify changes in distances between IpaD domains as a result of DOC binding. To further explore the effects of DOC binding on IpaD structure, NMR chemical shift mapping was employed. The environments of residues within the proposed DOC binding site and additional residues within the “distal” globular domain were perturbed upon DOC binding, further indicating that conformational changes occur within IpaD upon DOC binding. These events are proposed to be responsible for the recruitment of IpaB at the TTSA needle tip. Mutation analyses combined with additional spectroscopic analyses confirms that conformational changes in IpaD induced by DOC binding contribute to the recruitment of IpaB to the S. flexneri TTSA needle tip. These findings lay the foundation for determining how environmental factors promote TTSA needle tip maturation prior to host cell contact. PMID:21126091

  6. Analysis of Metal-Binding Features of the Wild Type and Two Domain-Truncated Mutant Variants of Littorina littorea Metallothionein Reveals Its Cd-Specific Character

    Directory of Open Access Journals (Sweden)

    Òscar Palacios

    2017-07-01

    Full Text Available After the resolution of the 3D structure of the Cd9-aggregate of the Littorina littorea metallothionein (MT, we report here a detailed analysis of the metal binding capabilities of the wild type MT, LlwtMT, and of two truncated mutants lacking either the N-terminal domain, Lltr2MT, or both the N-terminal domain, plus four extra flanking residues (SSVF, Lltr1MT. The recombinant synthesis and in vitro studies of these three proteins revealed that LlwtMT forms unique M9-LlwtMT complexes with Zn(II and Cd(II, while yielding a complex mixture of heteronuclear Zn,Cu-LlwtMT species with Cu(I. As expected, the truncated mutants gave rise to unique M6-LltrMT complexes and Zn,Cu-LltrMT mixtures of lower stoichiometry with respect to LlwtMT, with the SSVF fragment having an influence on their metal binding performance. Our results also revealed a major specificity, and therefore a better metal-coordinating performance of the three proteins for Cd(II than for Zn(II, although the analysis of the Zn(II/Cd(II displacement reaction clearly demonstrates a lack of any type of cooperativity in Cd(II binding. Contrarily, the analysis of their Cu(I binding abilities revealed that every LlMT domain is prone to build Cu4-aggregates, the whole MT working by modules analogously to, as previously described, certain fungal MTs, like those of C. neoformans and T. mesenterica. It is concluded that the Littorina littorea MT is a Cd-specific protein that (beyond its extended binding capacity through an additional Cd-binding domain confers to Littorina littorea a particular adaptive advantage in its changeable marine habitat.

  7. Human papillomavirus type 16 E2 and E6 are RNA-binding proteins and inhibit in vitro splicing of pre-mRNAs with suboptimal splice sites

    International Nuclear Information System (INIS)

    Bodaghi, Sohrab; Jia Rong; Zheng Zhiming

    2009-01-01

    Human papillomavirus type 16 (HPV16) genome expresses six regulatory proteins (E1, E2, E4, E5, E6, and E7) which regulate viral DNA replication, gene expression, and cell function. We expressed HPV16 E2, E4, E6, and E7 from bacteria as GST fusion proteins and examined their possible functions in RNA splicing. Both HPV16 E2, a viral transactivator protein, and E6, a viral oncoprotein, inhibited splicing of pre-mRNAs containing an intron with suboptimal splice sites, whereas HPV5 E2 did not. The N-terminal half and the hinge region of HPV16 E2 as well as the N-terminal and central portions of HPV16 E6 are responsible for the suppression. HPV16 E2 interacts with pre-mRNAs through its C-terminal DNA-binding domain. HPV16 E6 binds pre-mRNAs via nuclear localization signal (NLS3) in its C-terminal half. Low-risk HPV6 E6, a cytoplasmic protein, does not bind RNA. Notably, both HPV16 E2 and E6 selectively bind to the intron region of pre-mRNAs and interact with a subset of cellular SR proteins. Together, these findings suggest that HPV16 E2 and E6 are RNA binding proteins and might play roles in posttranscriptional regulation during virus infection

  8. Importance of chemical binding type between As and iron-oxide on bioaccessibility in soil: Test with synthesized two line ferrihydrite

    Energy Technology Data Exchange (ETDEWEB)

    Jeong, Seulki [Seoul Center, Korea Basic Science Institute, 6-7, Inchon-ro 22-gil, Seongbuk-gu, Seoul 02855 (Korea, Republic of); Yang, Kyung [Division of Public Infrastructure Assessment, Environmental Assessment Group, Korea Environmental Institute, Sejong 30147 (Korea, Republic of); Jho, Eun Hea [Department of Environmental Science, Hankuk University of Foreign Studies, 81 Oedae-ro, Mohyeonmyeon, Cheoin-gu, Yongin-si, Gyeonggi-do 17035 (Korea, Republic of); Nam, Kyoungphile, E-mail: kpnam@snu.ac.kr [Department of Civil and Environmental Engineering, Seoul National University, Gwanak-ro 1, Gwanak-gu, Seoul 08826 (Korea, Republic of)

    2017-05-15

    Highlights: • Arsenic (As)-adsorbed and As-coprecipiated two-line ferrihydrites were synthesized. • Bioaccessibility was closely related to chemical binding type of As in Fe oxide. • Chemical binding type needs to be considered to characterize the risk of As in soil. - Abstract: Bioaccessible concentrations of As associated with Fe oxide as different chemical binding types were determined in soils using the in vitro Physiologically Based Extraction Test (PBET). When compared to the five-step sequential extraction data, most of the As extracted by in vitro PBET originated from the amorphous Fe oxide-bound fraction, and more importantly, the bioaccessibility of As ranged from 0 to 58.8% in 24 soil samples. Two batches of ferrihydrite were synthesized separately. For one batch, As was adsorbed onto the ferrihydrite after synthesis; for the other one, As was added while synthesizing ferrihydrite to co-precipitate. The bioaccessible concentration of As determined by in vitro PBET of the former was 415 mg of As/kg of ferrihydrite and that of the latter was 67 mg of As/kg of ferrihydrite. X-ray photoelectron spectra (XPS) analysis indicated that As–O–Fe bonds were evident in As-associated ferrihydrite sample and especially, As was found within the Fe oxide lattice in the co-precipitated sample. Our data suggest that binding type between As and Fe oxide should be considered when determining the bioaccessibility of As in soil, which, in turn, greatly influences the realistic risk of As present in soil.

  9. Effects of anesthetics on vesicular monoamine transporter type 2 binding to 18F-FP-(+)-DTBZ: a biodistribution study in rat brain

    International Nuclear Information System (INIS)

    Chen, Zhengping; Tang, Jie; Liu, Chunyi; Li, Xiaomin; Huang, Hongbo; Xu, Xijie; Yu, Huixin

    2016-01-01

    Objectives: The in vivo binding analysis of vesicular monoamine transporter type 2 (VMAT2) to radioligand has provided a means of investigating related disorders. Anesthesia is often inevitable when the investigations are performed in animals. In the present study, we tested effects of four commonly-used anesthetics: isoflurane, pentobarbital, chloral hydrate and ketamine, on in vivo VMAT2 binding to 18 F-FP-(+)-DTBZ, a specific VMAT2 radioligand, in rat brain. Methods: The transient equilibrium time window for in vivo binding of 18 F-FP-(+)-DTBZ after a bolus injection was firstly determined. The brain biodistribution studies under anesthetized and awake rats were then performed at the equilibrium time. Standard uptake values (SUVs) of the interest brain regions: the striatum (ST), hippocampus (HP), cortex (CX) and cerebellum (CB) were obtained; and ratios of tissue to cerebellum were calculated. Results: Isoflurane and pentobarbital did not alter distribution of 18 F-FP-(+)-DTBZ in the brain relative to the awake group; neither SUVs nor ratios of ST/CB and HP/CB were altered significantly. Chloral hydrate significantly increased SUVs of all the brain regions, but did not significantly alter ratios of ST/CB and HP/CB. Ketamine significantly increased SUVs of the striatum, hippocampus and cortex, and insignificantly increased the SUV of the cerebellum; consequently, ketamine significantly increased ratios of ST/CB and HP/CB. Conclusions: It is concluded that in vivo VMAT2 binding to 18 F-FP-(+)-DTBZ are not altered by isoflurane and pentobarbital, but altered by chloral hydrate and ketamine. Isoflurane and pentobarbital may be promising anesthetic compounds for investigating in vivo VMAT2 binding. Further studies are warranted to investigate the interactions of anesthetics with VMAT2 binding potential with in vivo PET studies.

  10. NC1 domain of type VII collagen binds to the beta3 chain of laminin 5 via a unique subdomain within the fibronectin-like repeats.

    Science.gov (United States)

    Chen, M; Marinkovich, M P; Jones, J C; O'Toole, E A; Li, Y Y; Woodley, D T

    1999-02-01

    Type VII collagen, the major component of anchoring fibrils, consists of a central collagenous triple-helical domain flanked by two noncollagenous, globular domains, NC1 and NC2. Approximately 50% of the molecular mass of the molecule is consumed by the NC1 domain. We previously demonstrated that NC1 binds to various extracellular matrix components including a complex of laminin 5 and laminin 6 (Chen et al, 1997a). In this study, we examined the interaction of NC1 with laminin 5 (a component of anchoring filaments). Both authentic and purified recombinant NC1 bound to human and rat laminin 5 as measured by enzyme-linked immunosorbant assay and by binding of 125I-radiolabeled NC1 to laminin 5-coated wells, but not to laminin 1 or albumin. NC1 bound predominantly to the beta3 chain of laminin 5, but also to the gamma2 chain when examined by a protein overlay assay. The binding of 125I-NC1 to laminin 5 was inhibited by a 50-fold excess of unlabeled NC1 or de-glycosylated NC1, as well as a polyclonal antibody to laminin 5 or a monoclonal antibody to the beta3 chain. In contrast, the NC1-laminin 5 interaction was not affected by a monoclonal antibody to the alpha3 chain. Using NC1 deletion mutant recombinant proteins, a 285 AA (residues 760-1045) subdomain of NC1 was identified as the binding site for laminin 5. IgG from an epidermolysis bullosa acquisita serum containing autoantibodies to epitopes within NC1 that colocalized with the laminin 5 binding site inhibited the binding of NC1 to laminin 5. Thus, perturbation of the NC1-laminin 5 interaction may contribute to the pathogenesis of epidermolysis bullosa acquisita.

  11. Magnesium-adenosine diphosphate binding sites in wild-type creatine kinase and in mutants: role of aromatic residues probed by Raman and infrared spectroscopies.

    Science.gov (United States)

    Hagemann, H; Marcillat, O; Buchet, R; Vial, C

    2000-08-08

    Two distinct methods were used to investigate the role of Trp residues during Mg-ADP binding to cytosolic creatine kinase (CK) from rabbit muscle: (1) Raman spectroscopy, which is very sensitive to the environment of aromatic side-chain residues, and (2) reaction-induced infrared difference spectroscopy (RIDS) and photolabile substrate (ADP[Et(PhNO(2))]), combined with site-directed mutagenesis on the four Trp residues of CK. Our Raman results indicated that the environment of Trp and of Tyr were not affected during Mg-ADP binding to CK. Analysis of RIDS of wild-type CK, inactive W227Y, and active W210,217,272Y mutants suggested that Trp227 was not involved in the stacking interactions. Results are consistent with Trp227 being essential to prevent water molecules from entering in the active site [as suggested by Gross, M., Furter-Graves, E. M., Wallimann, T., Eppenberger, H. M., and Furter, R. (1994) Protein Sci. 3, 1058-1068] and that another Trp could in addition help to steer the nucleotide in the binding site, although it is not essential for the activity of CK. Raman and infrared spectra indicated that Mg-ADP binding does not involve large secondary structure changes. Only 3-4 residues absorbing in the amide I region are directly implicated in the Mg-ADP binding (corresponding to secondary structure changes less than 1%), suggesting that movement of protein domains due to Mg-nucleotide binding do not promote large secondary structure changes.

  12. Nucleoprotein of influenza B virus binds to its type A counterpart and disrupts influenza A viral polymerase complex formation

    International Nuclear Information System (INIS)

    Jaru-ampornpan, Peera; Narkpuk, Jaraspim; Wanitchang, Asawin; Jongkaewwattana, Anan

    2014-01-01

    Highlights: •FluB nucleoprotein (BNP) can bind to FluA nucleoprotein (ANP). •BNP–ANP interaction inhibits FluA polymerase activity. •BNP binding prevents ANP from forming a functional FluA polymerase complex. •Nuclear localization of BNP is necessary for FluA polymerase inhibition. •Viral RNA is not required for the BNP–ANP interaction. -- Abstract: Upon co-infection with influenza B virus (FluB), influenza A virus (FluA) replication is substantially impaired. Previously, we have shown that the nucleoprotein of FluB (BNP) can inhibit FluA polymerase machinery, retarding the growth of FluA. However, the molecular mechanism underlying this inhibitory action awaited further investigation. Here, we provide evidence that BNP hinders the proper formation of FluA polymerase complex by competitively binding to the nucleoprotein of FluA. To exert this inhibitory effect, BNP must be localized in the nucleus. The interaction does not require the presence of the viral RNA but needs an intact BNP RNA-binding motif. The results highlight the novel role of BNP as an anti-influenza A viral agent and provide insights into the mechanism of intertypic interference

  13. Direct binding of retromer to human papillomavirus type 16 minor capsid protein L2 mediates endosome exit during viral infection.

    Directory of Open Access Journals (Sweden)

    Andreea Popa

    2015-02-01

    Full Text Available Trafficking of human papillomaviruses to the Golgi apparatus during virus entry requires retromer, an endosomal coat protein complex that mediates the vesicular transport of cellular transmembrane proteins from the endosome to the Golgi apparatus or the plasma membrane. Here we show that the HPV16 L2 minor capsid protein is a retromer cargo, even though L2 is not a transmembrane protein. We show that direct binding of retromer to a conserved sequence in the carboxy-terminus of L2 is required for exit of L2 from the early endosome and delivery to the trans-Golgi network during virus entry. This binding site is different from known retromer binding motifs and can be replaced by a sorting signal from a cellular retromer cargo. Thus, HPV16 is an unconventional particulate retromer cargo, and retromer binding initiates retrograde transport of viral components from the endosome to the trans-Golgi network during virus entry. We propose that the carboxy-terminal segment of L2 protein protrudes through the endosomal membrane and is accessed by retromer in the cytoplasm.

  14. Nucleoprotein of influenza B virus binds to its type A counterpart and disrupts influenza A viral polymerase complex formation

    Energy Technology Data Exchange (ETDEWEB)

    Jaru-ampornpan, Peera, E-mail: peera.jar@biotec.or.th; Narkpuk, Jaraspim; Wanitchang, Asawin; Jongkaewwattana, Anan, E-mail: anan.jon@biotec.or.th

    2014-01-03

    Highlights: •FluB nucleoprotein (BNP) can bind to FluA nucleoprotein (ANP). •BNP–ANP interaction inhibits FluA polymerase activity. •BNP binding prevents ANP from forming a functional FluA polymerase complex. •Nuclear localization of BNP is necessary for FluA polymerase inhibition. •Viral RNA is not required for the BNP–ANP interaction. -- Abstract: Upon co-infection with influenza B virus (FluB), influenza A virus (FluA) replication is substantially impaired. Previously, we have shown that the nucleoprotein of FluB (BNP) can inhibit FluA polymerase machinery, retarding the growth of FluA. However, the molecular mechanism underlying this inhibitory action awaited further investigation. Here, we provide evidence that BNP hinders the proper formation of FluA polymerase complex by competitively binding to the nucleoprotein of FluA. To exert this inhibitory effect, BNP must be localized in the nucleus. The interaction does not require the presence of the viral RNA but needs an intact BNP RNA-binding motif. The results highlight the novel role of BNP as an anti-influenza A viral agent and provide insights into the mechanism of intertypic interference.

  15. Exploring variation in binding of Protein A and Protein G to immunoglobulin type G by isothermal titration calorimetry

    DEFF Research Database (Denmark)

    Lund, L. N.; Christensen, T.; Toone, E.

    2011-01-01

    ligands and for the establishment of generic processes for the purification of various antibodies. In this paper, the interactions between the two IgG-binding proteins and IgG of two different subclasses, IgG1 and IgG4, as well as their analogous Fc fragments have been studied by isothermal titration...

  16. Surface-bound capsular polysaccharide of type Ia group B Streptococcus mediates C1 binding and activation of the classic complement pathway

    International Nuclear Information System (INIS)

    Levy, N.J.; Kasper, D.L.

    1986-01-01

    The role of surface-bound type Ia group B Streptococcus (GBS) capsular polysaccharide in anti-body-independent binding of C1 and activation of the classic component pathway was investigated. In a radiolabeled bacterial-polymorphonuclear leukocyte (PMN) association assay, a measure of bacterial opsonization, preincubation of 3 H-type Ia GBS with purified F(ab') 2 to the organism blocked the association of the bacteria with PMN', and the inhibitory effect was dose dependent. The specificity of F(ab') 2 blocking was shown after adsorption of F(ab') 2 with type Ia polysaccharide-sensitized erythrocytes. Polysaccharide-adsorbed F(ab') 2 had a 70% decrease in ability to block the association of bacteria with PMN. Neuraminidase digestion removed 80% of the terminal sialic acid residues from the native polysaccharide. These neuraminidase-digested organisms had a 72% decrease in binding and transfer of purified C1 compared with non-enzyme-treated organisms. Type Ia capsular polysaccharide bound to sheep erythrocytes promoted classic complement pathway-mediated hemolysis of the cells. The role of C1 inhibitor (INH) in modulation of C1 activation by the organisms was investigated. The possibility existed that the C1 INH could be bound by the bacteria, allowing C1 activation to occur in the fluid phase. The inhibitor was purified from human serum, and its activity was measured before and after incubation with type Ia GBS. The organisms had no effect on C1 INH activity. Thus surface-bound capsular polysacchardie of type Ia GBS mediates C1 binding and classic pathway activation, and this does not involve the C1 INH

  17. Enhanced binding affinity, remarkable selectivity, and high capacity of CO 2 by dual functionalization of a rht-type metal-organic framework

    KAUST Repository

    Li, Baiyan

    2011-12-23

    Open and friendly: The smallest member of the rht-type metal-organic frameworks (MOFs, see picture) constructed by a hexacarboxylate ligand with a nitrogen-rich imino triazine backbone shows a significantly enhanced gas binding affinity relative to all other isoreticular rht-type MOFs. The high adsorption capacity and remarkable selectivity of CO 2 are attributed to the high density of open metal and Lewis basic sites in the framework. Copyright © 2012 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

  18. AGO2 Negatively Regulates Type I Interferon Signaling Pathway by Competition Binding IRF3 with CBP/p300

    Directory of Open Access Journals (Sweden)

    Shengyu Wang

    2017-05-01

    Full Text Available Viral infection triggers a series of signaling cascades and host innate immune responses, including interferon (IFN production, which depends on coordinated activity of multiple transcription factors. IFN regulatory factor 3 (IRF3 and transcriptional coactivator CREB binding protein (CBP and/or p300 are core factors that participate in transcriptional complex formation in the nucleus. In general, cells balance the production of IFNs through suppressive and stimulative mechanisms, but viral infections can disrupt such equilibrium. This study determined that H5N1 viral infection reduced the distribution of human argonaute 2 (AGO2 in A549 cell nucleus. AGO2 did not block phosphorylation, nuclear translocation, and DNA binding ability of IRF3 but inhibited its association with CBP. Therefore, this newly revealed mechanism shows that cellular response leads to transfer of AGO2 from cell nucleus and promotes IFN-β expression to increase host survival during viral infection.

  19. Binding of the urokinase-type plasminogen activator to its cell surface receptor is inhibited by low doses of suramin

    DEFF Research Database (Denmark)

    Behrendt, N; Rønne, E; Danø, K

    1993-01-01

    micrograms/ml when using U937 cells and a ligand concentration of 0.3 nM. This concentration of the drug is well below the serum levels found in suramin-treated patients. Inhibition of binding was also demonstrated at the molecular level, using chemical cross-linking or an enzyme-linked immunosorbent assay...... to the anti-invasive properties of suramin by destroying the cellular potential for localized plasminogen activation and proteolytic matrix degradation....

  20. Overlapping binding sites for trypsin and papain on a Kunitz-type proteinase inhibitor from Prosopis juliflora.

    Science.gov (United States)

    Franco, Octávio L; Grossi de Sá, Maria F; Sales, Maurício P; Mello, Luciane V; Oliveira, Adeliana S; Rigden, Daniel J

    2002-11-15

    Proteinase inhibitors are among the most promising candidates for expression by transgenic plants and consequent protection against insect predation. However, some insects can respond to the threat of the proteinase inhibitor by the production of enzymes insensitive to inhibition. Inhibitors combining more than one favorable activity are therefore strongly favored. Recently, a known small Kunitz trypsin inhibitor from Prosopis juliflora (PTPKI) has been shown to possess unexpected potent cysteine proteinase inhibitory activity. Here we show, by enzyme assay and gel filtration, that, unlike other Kunitz inhibitors with dual activities, this inhibitor is incapable of simultaneous inhibition of trypsin and papain. These data are most readily interpreted by proposing overlapping binding sites for the two enzymes. Molecular modeling and docking experiments favor an interaction mode in which the same inhibitor loop that interacts in a canonical fashion with trypsin can also bind into the papain catalytic site cleft. Unusual residue substitutions at the proposed interface can explain the relative rarity of twin trypsin/papain inhibition. Other changes seem responsible for the relative low affinity of PTPKI for trypsin. The predicted coincidence of trypsin and papain binding sites, once confirmed, would facilitate the search, by phage display for example, for mutants highly active against both proteinases. Copyright 2002 Wiley-Liss, Inc.

  1. Determining the Specificity of Cascade Binding, Interference, and Primed Adaptation In Vivo in the Escherichia coli Type I-E CRISPR-Cas System

    Directory of Open Access Journals (Sweden)

    Lauren A. Cooper

    2018-04-01

    Full Text Available In clustered regularly interspaced short palindromic repeat (CRISPR-Cas (CRISPR-associated immunity systems, short CRISPR RNAs (crRNAs are bound by Cas proteins, and these complexes target invading nucleic acid molecules for degradation in a process known as interference. In type I CRISPR-Cas systems, the Cas protein complex that binds DNA is known as Cascade. Association of Cascade with target DNA can also lead to acquisition of new immunity elements in a process known as primed adaptation. Here, we assess the specificity determinants for Cascade-DNA interaction, interference, and primed adaptation in vivo, for the type I-E system of Escherichia coli. Remarkably, as few as 5 bp of crRNA-DNA are sufficient for association of Cascade with a DNA target. Consequently, a single crRNA promotes Cascade association with numerous off-target sites, and the endogenous E. coli crRNAs direct Cascade binding to >100 chromosomal sites. In contrast to the low specificity of Cascade-DNA interactions, >18 bp are required for both interference and primed adaptation. Hence, Cascade binding to suboptimal, off-target sites is inert. Our data support a model in which the initial Cascade association with DNA targets requires only limited sequence complementarity at the crRNA 5′ end whereas recruitment and/or activation of the Cas3 nuclease, a prerequisite for interference and primed adaptation, requires extensive base pairing.

  2. Characterisation of the selective binding of antibiotics vancomycin and teicoplanin by the VanS receptor regulating type A vancomycin resistance in the enterococci.

    Science.gov (United States)

    Hughes, C S; Longo, E; Phillips-Jones, M K; Hussain, R

    2017-08-01

    A-type resistance towards "last-line" glycopeptide antibiotic vancomycin in the leading hospital acquired infectious agent, the enterococci, is the most common in the UK. Resistance is regulated by the VanR A S A two-component system, comprising the histidine sensor kinase VanS A and the partner response regulator VanR A . The nature of the activating ligand for VanS A has not been identified, therefore this work sought to identify and characterise ligand(s) for VanS A . In vitro approaches were used to screen the structural and activity effects of a range of potential ligands with purified VanS A protein. Of the screened ligands (glycopeptide antibiotics vancomycin and teicoplanin, and peptidoglycan components N-acetylmuramic acid, D-Ala-D-Ala and Ala-D-y-Glu-Lys-D-Ala-D-Ala) only glycopeptide antibiotics vancomycin and teicoplanin were found to bind VanS A with different affinities (vancomycin 70μM; teicoplanin 30 and 170μM), and were proposed to bind via exposed aromatic residues tryptophan and tyrosine. Furthermore, binding of the antibiotics induced quicker, longer-lived phosphorylation states for VanS A , proposing them as activators of type A vancomycin resistance in the enterococci. Copyright © 2017 Diamond Light Source Ltd. Published by Elsevier B.V. All rights reserved.

  3. Insights into the molecular basis for substrate binding and specificity of the wild-type L-arginine/agmatine antiporter AdiC.

    Science.gov (United States)

    Ilgü, Hüseyin; Jeckelmann, Jean-Marc; Gapsys, Vytautas; Ucurum, Zöhre; de Groot, Bert L; Fotiadis, Dimitrios

    2016-09-13

    Pathogenic enterobacteria need to survive the extreme acidity of the stomach to successfully colonize the human gut. Enteric bacteria circumvent the gastric acid barrier by activating extreme acid-resistance responses, such as the arginine-dependent acid resistance system. In this response, l-arginine is decarboxylated to agmatine, thereby consuming one proton from the cytoplasm. In Escherichia coli, the l-arginine/agmatine antiporter AdiC facilitates the export of agmatine in exchange of l-arginine, thus providing substrates for further removal of protons from the cytoplasm and balancing the intracellular pH. We have solved the crystal structures of wild-type AdiC in the presence and absence of the substrate agmatine at 2.6-Å and 2.2-Å resolution, respectively. The high-resolution structures made possible the identification of crucial water molecules in the substrate-binding sites, unveiling their functional roles for agmatine release and structure stabilization, which was further corroborated by molecular dynamics simulations. Structural analysis combined with site-directed mutagenesis and the scintillation proximity radioligand binding assay improved our understanding of substrate binding and specificity of the wild-type l-arginine/agmatine antiporter AdiC. Finally, we present a potential mechanism for conformational changes of the AdiC transport cycle involved in the release of agmatine into the periplasmic space of E. coli.

  4. Genome-Wide Progesterone Receptor Binding: Cell Type-Specific and Shared Mechanisms in T47D Breast Cancer Cells and Primary Leiomyoma Cells

    Science.gov (United States)

    Huang, Lei; Owen, Jonas K.; Xie, Anna; Navarro, Antonia; Monsivais, Diana; Coon V, John S.; Kim, J. Julie; Dai, Yang; Bulun, Serdar E.

    2012-01-01

    Background Progesterone, via its nuclear receptor (PR), exerts an overall tumorigenic effect on both uterine fibroid (leiomyoma) and breast cancer tissues, whereas the antiprogestin RU486 inhibits growth of these tissues through an unknown mechanism. Here, we determined the interaction between common or cell-specific genome-wide binding sites of PR and mRNA expression in RU486-treated uterine leiomyoma and breast cancer cells. Principal Findings ChIP-sequencing revealed 31,457 and 7,034 PR-binding sites in breast cancer and uterine leiomyoma cells, respectively; 1,035 sites overlapped in both cell types. Based on the chromatin-PR interaction in both cell types, we statistically refined the consensus progesterone response element to G•ACA• • •TGT•C. We identified two striking differences between uterine leiomyoma and breast cancer cells. First, the cis-regulatory elements for HSF, TEF-1, and C/EBPα and β were statistically enriched at genomic RU486/PR-targets in uterine leiomyoma, whereas E2F, FOXO1, FOXA1, and FOXF sites were preferentially enriched in breast cancer cells. Second, 51.5% of RU486-regulated genes in breast cancer cells but only 6.6% of RU486-regulated genes in uterine leiomyoma cells contained a PR-binding site within 5 kb from their transcription start sites (TSSs), whereas 75.4% of RU486-regulated genes contained a PR-binding site farther than 50 kb from their TSSs in uterine leiomyoma cells. RU486 regulated only seven mRNAs in both cell types. Among these, adipophilin (PLIN2), a pro-differentiation gene, was induced via RU486 and PR via the same regulatory region in both cell types. Conclusions Our studies have identified molecular components in a RU486/PR-controlled gene network involved in the regulation of cell growth, cell migration, and extracellular matrix function. Tissue-specific and common patterns of genome-wide PR binding and gene regulation may determine the therapeutic effects of antiprogestins in uterine fibroids and

  5. Heparin (GAG-hed) inhibits LCR activity of Human Papillomavirus type 18 by decreasing AP1 binding

    International Nuclear Information System (INIS)

    Villanueva, Rita; Morales-Peza, Néstor; Castelán-Sánchez, Irma; García-Villa, Enrique; Tapia, Rocio; Cid-Arregui, Ángel; García-Carrancá, Alejandro; López-Bayghen, Esther; Gariglio, Patricio

    2006-01-01

    High risk HPVs are causative agents of anogenital cancers. Viral E6 and E7 genes are continuously expressed and are largely responsible for the oncogenic activity of these viruses. Transcription of the E6 and E7 genes is controlled by the viral Long Control Region (LCR), plus several cellular transcription factors including AP1 and the viral protein E2. Within the LCR, the binding and activity of the transcription factor AP1 represents a key regulatory event in maintaining E6/E7 gene expression and uncontrolled cell proliferation. Glycosaminoglycans (GAGs), such as heparin, can inhibit tumour growth; they have also shown antiviral effects and inhibition of AP1 transcriptional activity. The purpose of this study was to test the heparinoid GAG-hed, as a possible antiviral and antitumoral agent in an HPV18 positive HeLa cell line. Using in vivo and in vitro approaches we tested GAG-hed effects on HeLa tumour cell growth, cell proliferation and on the expression of HPV18 E6/E7 oncogenes. GAG-hed effects on AP1 binding to HPV18-LCR-DNA were tested by EMSA. We were able to record the antitumoral effect of GAG-hed in vivo by using as a model tumours induced by injection of HeLa cells into athymic female mice. The antiviral effect of GAG-hed resulted in the inhibition of LCR activity and, consequently, the inhibition of E6 and E7 transcription. A specific diminishing of cell proliferation rates was observed in HeLa but not in HPV-free colorectal adenocarcinoma cells. Treated HeLa cells did not undergo apoptosis but the percentage of cells in G 2 /M phase of the cell cycle was increased. We also detected that GAG-hed prevents the binding of the transcription factor AP1 to the LCR. Direct interaction of GAG-hed with the components of the AP1 complex and subsequent interference with its ability to correctly bind specific sites within the viral LCR may contribute to the inhibition of E6/E7 transcription and cell proliferation. Our data suggest that GAG-hed could have

  6. The Fifth Transmembrane Domain of Angiotensin II Type 1 Receptor Participates in the Formation of the Ligand-binding Pocket and Undergoes a Counterclockwise Rotation upon Receptor Activation*

    Science.gov (United States)

    Domazet, Ivana; Martin, Stéphane S.; Holleran, Brian J.; Morin, Marie-Ève; Lacasse, Patrick; Lavigne, Pierre; Escher, Emanuel; Leduc, Richard; Guillemette, Gaétan

    2009-01-01

    The octapeptide hormone angiotensin II exerts a wide variety of cardiovascular effects through the activation of the angiotensin II Type 1 (AT1) receptor, which belongs to the G protein-coupled receptor superfamily. Like other G protein- coupled receptors, the AT1 receptor possesses seven transmembrane domains that provide structural support for the formation of the ligand-binding pocket. The role of the fifth transmembrane domain (TMD5) was investigated using the substituted cysteine accessibility method. All of the residues within Thr-190 to Leu-217 region were mutated one at a time to cysteine, and after expression in COS-7 cells, the mutant receptors were treated with the sulfhydryl-specific alkylating agent methanethiosulfonate-ethylammonium (MTSEA). MTSEA reacts selectively with water-accessible, free sulfhydryl groups of endogenous or introduced point mutation cysteines. If a cysteine is found in the binding pocket, the covalent modification will affect the binding kinetics of the ligand. MTSEA substantially decreased the binding affinity of L197C-AT1, N200C-AT1, I201C-AT1, G203C-AT1, and F204C-AT1 mutant receptors, which suggests that these residues orient themselves within the water-accessible binding pocket of the AT1 receptor. Interestingly, this pattern of acquired MTSEA sensitivity was altered for TMD5 reporter cysteines engineered in a constitutively active N111G-AT1 receptor background. Indeed, mutant I201C-N111G-AT1 became more sensitive to MTSEA, whereas mutant G203C-N111G-AT1 lost some sensitivity. Our results suggest that constitutive activation of AT1 receptor causes an apparent counterclockwise rotation of TMD5 as viewed from the extracellular side. PMID:19773549

  7. The fifth transmembrane domain of angiotensin II Type 1 receptor participates in the formation of the ligand-binding pocket and undergoes a counterclockwise rotation upon receptor activation.

    Science.gov (United States)

    Domazet, Ivana; Martin, Stéphane S; Holleran, Brian J; Morin, Marie-Eve; Lacasse, Patrick; Lavigne, Pierre; Escher, Emanuel; Leduc, Richard; Guillemette, Gaétan

    2009-11-13

    The octapeptide hormone angiotensin II exerts a wide variety of cardiovascular effects through the activation of the angiotensin II Type 1 (AT(1)) receptor, which belongs to the G protein-coupled receptor superfamily. Like other G protein- coupled receptors, the AT(1) receptor possesses seven transmembrane domains that provide structural support for the formation of the ligand-binding pocket. The role of the fifth transmembrane domain (TMD5) was investigated using the substituted cysteine accessibility method. All of the residues within Thr-190 to Leu-217 region were mutated one at a time to cysteine, and after expression in COS-7 cells, the mutant receptors were treated with the sulfhydryl-specific alkylating agent methanethiosulfonate-ethylammonium (MTSEA). MTSEA reacts selectively with water-accessible, free sulfhydryl groups of endogenous or introduced point mutation cysteines. If a cysteine is found in the binding pocket, the covalent modification will affect the binding kinetics of the ligand. MTSEA substantially decreased the binding affinity of L197C-AT(1), N200C-AT(1), I201C-AT(1), G203C-AT(1), and F204C-AT(1) mutant receptors, which suggests that these residues orient themselves within the water-accessible binding pocket of the AT(1) receptor. Interestingly, this pattern of acquired MTSEA sensitivity was altered for TMD5 reporter cysteines engineered in a constitutively active N111G-AT(1) receptor background. Indeed, mutant I201C-N111G-AT(1) became more sensitive to MTSEA, whereas mutant G203C-N111G-AT(1) lost some sensitivity. Our results suggest that constitutive activation of AT(1) receptor causes an apparent counterclockwise rotation of TMD5 as viewed from the extracellular side.

  8. Computational Studies of a Mechanism for Binding and Drug Resistance in the Wild Type and Four Mutations of HIV-1 Protease with a GRL-0519 Inhibitor

    Directory of Open Access Journals (Sweden)

    Guodong Hu

    2016-05-01

    Full Text Available Drug resistance of mutations in HIV-1 protease (PR is the most severe challenge to the long-term efficacy of HIV-1 PR inhibitor in highly active antiretroviral therapy. To elucidate the molecular mechanism of drug resistance associated with mutations (D30N, I50V, I54M, and V82A and inhibitor (GRL-0519 complexes, we have performed five molecular dynamics (MD simulations and calculated the binding free energies using the molecular mechanics Poisson–Boltzmann surface area (MM-PBSA method. The ranking of calculated binding free energies is in accordance with the experimental data. The free energy spectra of each residue and inhibitor interaction for all complexes show a similar binding model. Analysis based on the MD trajectories and contribution of each residues show that groups R2 and R3 mainly contribute van der Waals energies, while groups R1 and R4 contribute electrostatic interaction by hydrogen bonds. The drug resistance of D30N can be attributed to the decline in binding affinity of residues 28 and 29. The size of Val50 is smaller than Ile50 causes the residue to move, especially in chain A. The stable hydrophobic core, including the side chain of Ile54 in the wild type (WT complex, became unstable in I54M because the side chain of Met54 is flexible with two alternative conformations. The binding affinity of Ala82 in V82A decreases relative to Val82 in WT. The present study could provide important guidance for the design of a potent new drug resisting the mutation inhibitors.

  9. 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.

  10. Corticotropin-Releasing Hormone Receptor Type 1 (CRHR1 Clustering with MAGUKs Is Mediated via Its C-Terminal PDZ Binding Motif.

    Directory of Open Access Journals (Sweden)

    Julia Bender

    Full Text Available The corticotropin-releasing hormone receptor type 1 (CRHR1 plays an important role in orchestrating neuroendocrine, behavioral, and autonomic responses to stress. To identify molecules capable of directly modulating CRHR1 signaling, we performed a yeast-two-hybrid screen using the C-terminal intracellular tail of the receptor as bait. We identified several members of the membrane-associated guanylate kinase (MAGUK family: postsynaptic density protein 95 (PSD95, synapse-associated protein 97 (SAP97, SAP102 and membrane associated guanylate kinase, WW and PDZ domain containing 2 (MAGI2. CRHR1 is co-expressed with the identified MAGUKs and with the additionally investigated PSD93 in neurons of the adult mouse brain and in primary hippocampal neurons, supporting the probability of a physiological interaction in vivo. The C-terminal PDZ (PSD-95, discs large, zona occludens 1 binding motif of CRHR1 is essential for its physical interaction with MAGUKs, as revealed by the CRHR1-STAVA mutant, which harbors a functionally impaired PDZ binding motif. The imitation of a phosphorylation at Thr413 within the PDZ binding motif also disrupted the interaction with MAGUKs. In contrast, distinct PDZ domains within the identified MAGUKs are involved in the interactions. Expression of CRHR1 in primary neurons demonstrated its localization throughout the neuronal plasma membrane, including the excitatory post synapse, where the receptor co-localized with PSD95 and SAP97. The co-expression of CRHR1 and respective interacting MAGUKs in HEK293 cells resulted in a clustered subcellular co-localization which required an intact PDZ binding motif. In conclusion, our study characterized the PDZ binding motif-mediated interaction of CRHR1 with multiple MAGUKs, which directly affects receptor function.

  11. Reformulated tight binding calculation for band discontinuity at CdTe/Hg xCd1-xTe heterointerfaces and their type I-type III transitions

    International Nuclear Information System (INIS)

    Ekpunobi, A.J.

    2005-01-01

    A recently reformulated tight binding method is used to calculate the valence band discontinuity at the CdTe/Hg x Cd 1-x Te interface in the s 2 p 2 configuration. The calculated valence band discontinuity of 0.31 eV at CdTe/HgTe interface is in good agreement with self-consistent calculation and accepted experimental value. Calculations were extended to alloy interfaces, which enabled the investigation of the band-offset problem at the transition point. Both valence band discontinuity ratio and conduction band discontinuity ratio show inflexions at the transition point

  12. Plasma levels of galectin-3-binding protein reflect type I interferon activity and are increased in patients with systemic lupus erythematosus

    DEFF Research Database (Denmark)

    Nielsen, Christoffer T; Lood, Christian; Østergaard, Ole

    2014-01-01

    OBJECTIVE: Simple measures of type I interferon (IFN) activity constitute highly attractive biomarkers in systemic lupus erythematosus (SLE). We explore galectin-3-binding protein (G3BP) as a novel measure of type I IFN activity and serum/plasma biomarker in large independent cohorts of patients...... parameters including disease activity in the four SLE cohorts was performed. RESULTS: G3BP concentrations correlated significantly with the IFN-α reporter gene assay (r=0.56, p=0.0005) and with IFN-α gene expression scores (r=0.54, p=0.0002). Plasma concentrations were significantly increased in the SLE......BP levels in the consecutive SLE-samples and was significantly associated with changes in disease activity (r=0.44, p=0.014). CONCLUSIONS: G3BP plasma levels reflect type I IFN activity and are increased in SLE. Associations with disease activity or clinical manifestations are uncertain. This study...

  13. Regulation of steroid 5-{alpha} reductase type 2 (Srd5a2) by sterol regulatory element binding proteins and statin

    Energy Technology Data Exchange (ETDEWEB)

    Seo, Young-Kyo [Department of Molecular Biology and Biochemistry, 3244 McGaugh Hall, University of California, UC Irvine, Irvine, CA 92697-3900 (United States); Zhu, Bing [Department of Pathology, University of Texas Medical Branch, Galveston, TX 77555-0144 (United States); Jeon, Tae-Il [Department of Molecular Biology and Biochemistry, 3244 McGaugh Hall, University of California, UC Irvine, Irvine, CA 92697-3900 (United States); Osborne, Timothy F., E-mail: tfosborn@uci.edu [Department of Molecular Biology and Biochemistry, 3244 McGaugh Hall, University of California, UC Irvine, Irvine, CA 92697-3900 (United States)

    2009-11-01

    In this study, we show that sterol regulatory element binding proteins (SREBPs) regulate expression of Srd5a2, an enzyme that catalyzes the irreversible conversion of testosterone to dihydroxytestosterone in the male reproductive tract and is highly expressed in androgen-sensitive tissues such as the prostate and skin. We show that Srd5a2 is induced in livers and prostate from mice fed a chow diet supplemented with lovastatin plus ezitimibe (L/E), which increases the activity of nuclear SREBP-2. The three fold increase in Srd5a2 mRNA mediated by L/E treatment was accompanied by the induction of SREBP-2 binding to the Srd5a2 promoter detected by a ChIP-chip assay in liver. We identified a SREBP-2 responsive region within the first 300 upstream bases of the mouse Srd5a2 promoter by co-transfection assays which contain a site that bound SREBP-2 in vitro by an EMSA. Srd5a2 protein was also induced in cells over-expressing SREBP-2 in culture. The induction of Srd5a2 through SREBP-2 provides a mechanistic explanation for why even though statin therapy is effective in reducing cholesterol levels in treating hypercholesterolemia it does not compromise androgen production in clinical studies.

  14. Regulation of steroid 5-α reductase type 2 (Srd5a2) by sterol regulatory element binding proteins and statin

    International Nuclear Information System (INIS)

    Seo, Young-Kyo; Zhu, Bing; Jeon, Tae-Il; Osborne, Timothy F.

    2009-01-01

    In this study, we show that sterol regulatory element binding proteins (SREBPs) regulate expression of Srd5a2, an enzyme that catalyzes the irreversible conversion of testosterone to dihydroxytestosterone in the male reproductive tract and is highly expressed in androgen-sensitive tissues such as the prostate and skin. We show that Srd5a2 is induced in livers and prostate from mice fed a chow diet supplemented with lovastatin plus ezitimibe (L/E), which increases the activity of nuclear SREBP-2. The three fold increase in Srd5a2 mRNA mediated by L/E treatment was accompanied by the induction of SREBP-2 binding to the Srd5a2 promoter detected by a ChIP-chip assay in liver. We identified a SREBP-2 responsive region within the first 300 upstream bases of the mouse Srd5a2 promoter by co-transfection assays which contain a site that bound SREBP-2 in vitro by an EMSA. Srd5a2 protein was also induced in cells over-expressing SREBP-2 in culture. The induction of Srd5a2 through SREBP-2 provides a mechanistic explanation for why even though statin therapy is effective in reducing cholesterol levels in treating hypercholesterolemia it does not compromise androgen production in clinical studies.

  15. Increased Autoreactivity of the Complement-Activating Molecule Mannan-Binding Lectin in a Type 1 Diabetes Model

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    Jakob Appel Østergaard

    2016-01-01

    Full Text Available Background. Diabetic kidney disease is the leading cause of end-stage renal failure despite intensive treatment of modifiable risk factors. Identification of new drug targets is therefore of paramount importance. The complement system is emerging as a potential new target. The lectin pathway of the complement system, initiated by the carbohydrate-recognition molecule mannan-binding lectin (MBL, is linked to poor kidney prognosis in diabetes. We hypothesized that MBL activates complement upon binding within the diabetic glomerulus. Methods. We investigated this by comparing complement deposition and activation in kidneys from streptozotocin-induced diabetic mice and healthy control mice. Results. After 20 weeks of diabetes, glomerular deposition of MBL was significantly increased. Diabetic animals had 2.0-fold higher (95% CI 1.6–2.5 immunofluorescence intensity from anti-MBL antibodies compared with controls (P<0.001. Diabetes and control groups did not differ in glomerular immunofluorescence intensity obtained by antibodies against complement factors C4, C3, and C9. However, the circulating complement activation product C3a was increased in diabetes as compared to control mice (P=0.04. Conclusion. 20 weeks of diabetes increased MBL autoreactivity in the kidney and circulating C3a concentration. Together with previous findings, these results indicate direct effects of MBL within the kidney in diabetes.

  16. Identification of a regulatory variant that binds FOXA1 and FOXA2 at the CDC123/CAMK1D type 2 diabetes GWAS locus.

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    Marie P Fogarty

    2014-09-01

    Full Text Available Many of the type 2 diabetes loci identified through genome-wide association studies localize to non-protein-coding intronic and intergenic regions and likely contain variants that regulate gene transcription. The CDC123/CAMK1D type 2 diabetes association signal on chromosome 10 spans an intergenic region between CDC123 and CAMK1D and also overlaps the CDC123 3'UTR. To gain insight into the molecular mechanisms underlying the association signal, we used open chromatin, histone modifications and transcription factor ChIP-seq data sets from type 2 diabetes-relevant cell types to identify SNPs overlapping predicted regulatory regions. Two regions containing type 2 diabetes-associated variants were tested for enhancer activity using luciferase reporter assays. One SNP, rs11257655, displayed allelic differences in transcriptional enhancer activity in 832/13 and MIN6 insulinoma cells as well as in human HepG2 hepatocellular carcinoma cells. The rs11257655 risk allele T showed greater transcriptional activity than the non-risk allele C in all cell types tested. Using electromobility shift and supershift assays we demonstrated that the rs11257655 risk allele showed allele-specific binding to FOXA1 and FOXA2. We validated FOXA1 and FOXA2 enrichment at the rs11257655 risk allele using allele-specific ChIP in human islets. These results suggest that rs11257655 affects transcriptional activity through altered binding of a protein complex that includes FOXA1 and FOXA2, providing a potential molecular mechanism at this GWAS locus.

  17. Hepatic 123I-insulin binding kinetics in non-insulin-dependent (Type 2) diabetic patients after i.v. bolus administration

    International Nuclear Information System (INIS)

    Oolbekkink, M.; Veen, E.A. van der; Heine, R.J.; Hollander, W. den; Nauta, J.J.P.

    1989-01-01

    Insulin binding kinetics in the liver were studied in non insulin dependent (Type 2) diabetic patients, by i.v. bolus administration of 123 I-insulin. Eight Type 2 diabetic patients were compared with six male volunteers. Uptake of 123 I-insulin by liver and kidneys was measured by dynamic scintigraphy with a gamma camera during 30 min. Images of liver and kidneys appeared within 2-3 min after administration of 123 I-insulin at a dose of 1 mCi (37 MBq). Peak radioactivity for the liver was found 7.5±0.2 and 6.9±0.3 min after injection for the healthy and the diabetic subjects, respectively (N.S.). The percentage 123 I-insulin hepatic uptake was not significantly different for the diabetic and the healthy subjects. Although a large variation exists for maximal uptake of radioactivity within both groups, the data suggest that binding differences in the liver in Type 2 diabetic patients, as compared to healthy subjects, may not account for hepatic insulin resistance. (orig.)

  18. Membrane fusion proteins of type I secretion system and tripartite efflux pumps share a binding motif for TolC in gram-negative bacteria.

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    Minho Lee

    Full Text Available The Hly translocator complex of Escherichia coli catalyzes type I secretion of the toxin hemolysin A (HlyA. In this complex, HlyB is an inner membrane ABC (ATP Binding Cassette-type transporter, TolC is an outer membrane channel protein, and HlyD is a periplasmic adaptor anchored in the inner membrane that bridges HlyB to TolC. This tripartite organization is reminiscent of that of drug efflux systems such as AcrA-AcrB-TolC and MacA-MacB-TolC of E. coli. We have previously shown the crucial role of conserved residues located at the hairpin tip region of AcrA and MacA adaptors during assembly of their cognate systems. In this study, we investigated the role of the putative tip region of HlyD using HlyD mutants with single amino acid substitutions at the conserved positions. In vivo and in vitro data show that all mutations abolished HlyD binding to TolC and resulted in the absence of HlyA secretion. Together, our results suggest that, similarly to AcrA and MacA, HlyD interacts with TolC in a tip-to-tip manner. A general model in which these conserved interactions induce opening of TolC during drug efflux and type I secretion is discussed.

  19. Structure of thrombospondin type 3 repeats in bacterial outer membrane protein A reveals its intra-repeat disulfide bond-dependent calcium-binding capability

    Energy Technology Data Exchange (ETDEWEB)

    Dai, Shuyan; Sun, Cancan; Tan, Kemin; Ye, Sheng; Zhang, Rongguang

    2017-09-01

    Eukaryotic thrombospondin type 3 repeat (TT3R) is an efficient calcium ion (Ca2+) binding motif only found in mammalian thrombospondin family. TT3R has also been found in prokaryotic cellulase Cel5G, which was thought to forfeit the Ca2+-binding capability due to the formation of intra-repeat disulfide bonds, instead of the inter-repeat ones possessed by eukaryotic TT3Rs. In this study, we have identified an enormous number of prokaryotic TT3R-containing proteins belonging to several different protein families, including outer membrane protein A (OmpA), an important structural protein connecting the outer membrane and the periplasmic peptidoglycan layer in gram-negative bacteria. Here, we report the crystal structure of the periplasmic region of OmpA from Capnocytophaga gingivalis, which contains a linker region comprising five consecutive TT3Rs. The structure of OmpA-TT3R exhibits a well-ordered architecture organized around two tightly-coordinated Ca2+ and confirms the presence of abnormal intra-repeat disulfide bonds. Further mutagenesis studies showed that the Ca2+-binding capability of OmpA-TT3R is indeed dependent on the proper formation of intra-repeat disulfide bonds, which help to fix a conserved glycine residue at its proper position for Ca2+ coordination. Additionally, despite lacking inter repeat disulfide bonds, the interfaces between adjacent OmpA-TT3Rs are enhanced by both hydrophobic and conserved aromatic-proline interactions.

  20. Identification of a single‐copy gene encoding a Type I chlorophyll a/b‐binding polypeptide of photosystem I in Arabidopsis thaliana

    DEFF Research Database (Denmark)

    Jensen, Poul E; Kristensen, Michael; Hoff, Tine

    1992-01-01

    We have isolated and sequenced cDNA and genomic clones from Arabidopsis thaliana which specify a 241 residue protein with 84% sequence identity to a photosystem I Type I chlorophyll a/b-binding (CAB) protein from tomato. The open reading frame is interrupted by three introns which are found...... at equivalent positions as the corresponding introns in the tomato gene. Comparison to the amino acid sequence of other CAB proteins confirms that all CAB proteins share two regions of very high similarity. However, near the N-terminus and between the conserved regions this light-harvesting complex I (LHCI...

  1. The Breast Cancer-Associated Glycoforms of MUC1, MUC1-Tn and sialyl-Tn, Are Expressed in COSMC Wild-Type Cells and Bind the C-Type Lectin MGL.

    Directory of Open Access Journals (Sweden)

    Richard Beatson

    Full Text Available Aberrant glycosylation occurs in the majority of human cancers and changes in mucin-type O-glycosylation are key events that play a role in the induction of invasion and metastases. These changes generate novel cancer-specific glyco-antigens that can interact with cells of the immune system through carbohydrate binding lectins. Two glyco-epitopes that are found expressed by many carcinomas are Tn (GalNAc-Ser/Thr and STn (NeuAcα2,6GalNAc-Ser/Thr. These glycans can be carried on many mucin-type glycoproteins including MUC1. We show that the majority of breast cancers carry Tn within the same cell and in close proximity to extended glycan T (Galβ1,3GalNAc the addition of Gal to the GalNAc being catalysed by the T synthase. The presence of active T synthase suggests that loss of the private chaperone for T synthase, COSMC, does not explain the expression of Tn and STn in breast cancer cells. We show that MUC1 carrying both Tn or STn can bind to the C-type lectin MGL and using atomic force microscopy show that they bind to MGL with a similar dead adhesion force. Tumour associated STn is associated with poor prognosis and resistance to chemotherapy in breast carcinomas, inhibition of DC maturation, DC apoptosis and inhibition of NK activity. As engagement of MGL in the absence of TLR triggering may lead to anergy, the binding of MUC1-STn to MGL may be in part responsible for some of the characteristics of STn expressing tumours.

  2. Human T-cell leukemia virus type 1 Tax requires direct access to DNA for recruitment of CREB binding protein to the viral promoter.

    Science.gov (United States)

    Lenzmeier, B A; Giebler, H A; Nyborg, J K

    1998-02-01

    Efficient human T-cell leukemia virus type 1 (HTLV-1) replication and viral gene expression are dependent upon the virally encoded oncoprotein Tax. To activate HTLV-1 transcription, Tax interacts with the cellular DNA binding protein cyclic AMP-responsive element binding protein (CREB) and recruits the coactivator CREB binding protein (CBP), forming a nucleoprotein complex on the three viral cyclic AMP-responsive elements (CREs) in the HTLV-1 promoter. Short stretches of dG-dC-rich (GC-rich) DNA, immediately flanking each of the viral CREs, are essential for Tax recruitment of CBP in vitro and Tax transactivation in vivo. Although the importance of the viral CRE-flanking sequences is well established, several studies have failed to identify an interaction between Tax and the DNA. The mechanistic role of the viral CRE-flanking sequences has therefore remained enigmatic. In this study, we used high resolution methidiumpropyl-EDTA iron(II) footprinting to show that Tax extended the CREB footprint into the GC-rich DNA flanking sequences of the viral CRE. The Tax-CREB footprint was enhanced but not extended by the KIX domain of CBP, suggesting that the coactivator increased the stability of the nucleoprotein complex. Conversely, the footprint pattern of CREB on a cellular CRE lacking GC-rich flanking sequences did not change in the presence of Tax or Tax plus KIX. The minor-groove DNA binding drug chromomycin A3 bound to the GC-rich flanking sequences and inhibited the association of Tax and the Tax-CBP complex without affecting CREB binding. Tax specifically cross-linked to the viral CRE in the 5'-flanking sequence, and this cross-link was blocked by chromomycin A3. Together, these data support a model where Tax interacts directly with both CREB and the minor-groove viral CRE-flanking sequences to form a high-affinity binding site for the recruitment of CBP to the HTLV-1 promoter.

  3. Characterization of two novel bacterial type A exo-chitobiose hydrolases having C-terminal 5/12-type carbohydrate-binding modules

    DEFF Research Database (Denmark)

    Binti Jamek, Shariza; Nyffenegger, Christian; Muschiol, Jan

    2017-01-01

    "exo-chitobiose hydrolases." In this study, the chitinase type A from Serratia marcescens (SmaChiA) was used as a template for identifying two novel exo-chitobiose hydrolase type A enzymes, FbalChi18A and MvarChi18A, originating from the marine organisms Ferrimonas balearica and Microbulbifer...

  4. Micro-ribonucleic acid-binding site variants of type 2 diabetes candidate loci predispose to gestational diabetes mellitus in Chinese Han women.

    Science.gov (United States)

    Wang, Xiaojing; Li, Wei; Ma, Liangkun; Ping, Fan; Liu, Juntao; Wu, Xueyan; Mao, Jiangfeng; Wang, Xi; Nie, Min

    2018-01-20

    Emerging evidence has suggested that the genetic background of gestational diabetes mellitus (GDM) was analogous to type 2 diabetes mellitus. In contrast to type 2 diabetes mellitus, the genetic studies for GDM were limited. Accordingly, the aim of the present study was to extensively explore the influence of micro-ribonucleic acid-binding single-nucleotide polymorphisms (SNPs) in type 2 diabetes mellitus candidate loci on GDM susceptibility in Chinese. A total of 839 GDM patients and 900 controls were enrolled. Six micro-ribonucleic acid-binding SNPs were selected from 30 type 2 diabetes mellitus susceptibility loci and genotyped using TaqMan allelic discrimination assays. The minor allele of three SNPs, PAX4 rs712699 (OR 1.366, 95% confidence interval 1.021-1.828, P = 0.036), KCNB1 rs1051295 (OR 1.579, 95% confidence interval 1.172-2.128, P = 0.003) and MFN2 rs1042842 (OR 1.398, 95% confidence interval 1.050-1.862, P = 0.022) were identified to significantly confer higher a risk of GDM in the additive model. The association between rs1051295 and increased fasting plasma glucose (b = 0.006, P = 0.008), 3-h oral glucose tolerance test plasma glucose (b = 0.058, P = 0.025) and homeostatic model assessment of insulin resistance (b = 0.065, P = 0.017) was also shown. Rs1042842 was correlated with higher 3-h oral glucose tolerance test plasma glucose (b = 0.056, P = 0.028). However, no significant correlation between the other included SNPs (LPIN1 rs1050800, VPS26A rs1802295 and NLRP3 rs10802502) and GDM susceptibility were observed. The present findings showed that micro-ribonucleic acid-binding SNPs in type 2 diabetes mellitus candidate loci were also associated with GDM susceptibility, which further highlighted the similar genetic basis underlying GDM and type 2 diabetes mellitus. © 2018 The Authors. Journal of Diabetes Investigation published by Asian Association for the Study of Diabetes (AASD) and John Wiley & Sons Australia, Ltd.

  5. Exercise increases hyper-acetylation of histones on the Cis-element of NRF-1 binding to the Mef2a promoter: Implications on type 2 diabetes.

    Science.gov (United States)

    Joseph, Jitcy S; Ayeleso, Ademola O; Mukwevho, Emmanuel

    2017-04-22

    Exercise brings changes on the chromatin ensuing the upregulation of many genes that confer protection from type 2 diabetes. In type-2 diabetes, critical genes are down-regulated such as those involved in glucose transport (GLUT4, MEF2A) and also oxidative phosphorylation (NRF-1 and its target genes). Recent reports have shown that NRF-1 not only regulate mitochondrial oxidative genes but also controls MEF2A, the main transcription factor for glucose transporter, GLUT4. Such dual control of the two pathways by NRF-1 place it as critical gene in the design of therapeutic modalities much needed to cure or better manage type 2 diabetes. Although it is known that NRF-1 controls these dual pathways (glucose transport and oxidative phosphorylation), the actual molecular mechanisms involved surrounding this regulation remains elusive. NRF-1 itself is regulated through posttranslational modifications (acetylation, methylation and phosphorylation) resulting in enhanced binding to its target genes. This study is therefore aimed at assessing whether CaMKII, a kinase activated by exercise brings about hyper-acetylation of histones in the vicinity of NRF-1 target gene, Mef2a. Five to six weeks old male Wistar rats were used in this study. Chromatin immunoprecipitation (ChIP) assay was used to investigate the extent through which NRF-1 is bound to the Mef2a gene and if this was associated with hyper-acetylation of histones in the region of NRF-1 binding site of the Mef2a gene. Quantitative real time PCR (qPCR) was used to determine the gene expression of MEF2A and NRF-1. Results from this study indicated that exercise-induced CaMKII activation increased hyper-acetylation of histones in the region of NRF-1 binding site on vicinity of Mef2a gene and this was associated with the increased binding of NRF-1 to Mef2a gene. Exercise also increased the expression of NRF-1 and MEF2A genes. Administration of CaMKII inhibitor (KN93) prior to exercise attenuated the observed exercise

  6. Altered 5-HT2A Receptor Binding after Recovery from Bulimia-Type Anorexia Nervosa: Relationships to Harm Avoidance and Drive for Thinness

    Science.gov (United States)

    Bailer, Ursula F; Price, Julie C; Meltzer, Carolyn C; Mathis, Chester A; Frank, Guido K; Weissfeld, Lisa; McConaha, Claire W; Henry, Shannan E; Brooks-Achenbach, Sarah; Barbarich, Nicole C; Kaye, Walter H

    2015-01-01

    Several lines of evidence suggest that a disturbance of serotonin neuronal pathways may contribute to the pathogenesis of anorexia nervosa (AN) and bulimia nervosa (BN). This study applied positron emission tomography (PET) to investigate the brain serotonin 2A (5-HT2A) receptor, which could contribute to disturbances of appetite and behavior in AN and BN. To avoid the confounding effects of malnutrition, we studied 10 women recovered from bulimia-type AN (REC AN–BN, >1 year normal weight, regular menstrual cycles, no binging, or purging) compared with 16 healthy control women (CW) using PET imaging and a specific 5-HT2A receptor antagonist, [18F]altanserin. REC AN–BN women had significantly reduced [18F]altanserin binding potential relative to CW in the left subgenual cingulate, the left parietal cortex, and the right occipital cortex. [18F]altanserin binding potential was positively related to harm avoidance and negatively related to novelty seeking in cingulate and temporal regions only in REC AN–BN subjects. In addition, REC AN–BN had negative relationships between [18F]altanserin binding potential and drive for thinness in several cortical regions. In conclusion, this study extends research suggesting that altered 5-HT neuronal system activity persists after recovery from bulimia-type AN, particularly in subgenual cingulate regions. Altered 5-HT neurotransmission after recovery also supports the possibility that this may be a trait-related disturbance that contributes to the pathophysiology of eating disorders. It is possible that subgenual cingulate findings are not specific for AN–BN, but may be related to the high incidence of lifetime major depressive disorder diagnosis in these subjects. PMID:15054474

  7. Identification of the gene encoding a type 1 RNase H with an N-terminal double-stranded RNA binding domain from a psychrotrophic bacterium.

    Science.gov (United States)

    Tadokoro, Takashi; Chon, Hyongi; Koga, Yuichi; Takano, Kazufumi; Kanaya, Shigenori

    2007-07-01

    The gene encoding a bacterial type 1 RNase H, termed RBD-RNase HI, was cloned from the psychrotrophic bacterium Shewanella sp. SIB1, overproduced in Escherichia coli, and the recombinant protein was purified and biochemically characterized. SIB1 RBD-RNase HI consists of 262 amino acid residues and shows amino acid sequence identities of 26% to SIB1 RNase HI, 17% to E. coli RNase HI, and 32% to human RNase H1. SIB1 RBD-RNase HI has a double-stranded RNA binding domain (RBD) at the N-terminus, which is commonly present at the N-termini of eukaryotic type 1 RNases H. Gel mobility shift assay indicated that this domain binds to an RNA/DNA hybrid in an isolated form, suggesting that this domain is involved in substrate binding. SIB1 RBD-RNase HI exhibited the enzymatic activity both in vitro and in vivo. Its optimum pH and metal ion requirement were similar to those of SIB1 RNase HI, E. coli RNase HI, and human RNase H1. The specific activity of SIB1 RBD-RNase HI was comparable to that of E. coli RNase HI and was much higher than those of SIB1 RNase HI and human RNase H1. SIB1 RBD-RNase HI showed poor cleavage-site specificity for oligomeric substrates. SIB1 RBD-RNase HI was less stable than E. coli RNase HI but was as stable as human RNase H1. Database searches indicate that several bacteria and archaea contain an RBD-RNase HI. This is the first report on the biochemical characterization of RBD-RNase HI.

  8. The Scl1 protein of M6-type group A Streptococcus binds the human complement regulatory protein, factor H, and inhibits the alternative pathway of complement.

    Science.gov (United States)

    Caswell, Clayton C; Han, Runlin; Hovis, Kelley M; Ciborowski, Pawel; Keene, Douglas R; Marconi, Richard T; Lukomski, Slawomir

    2008-02-01

    Non-specific activation of the complement system is regulated by the plasma glycoprotein factor H (FH). Bacteria can avoid complement-mediated opsonization and phagocytosis through acquiring FH to the cell surface. Here, we characterize an interaction between the streptococcal collagen-like protein Scl1.6 of M6-type group A Streptococcus (GAS) and FH. Using affinity chromatography with immobilized recombinant Scl1.6 protein, we co-eluted human plasma proteins with molecular weight of 155 kDa, 43 kDa and 38 kDa. Mass spectrometry identified the 155 kDa band as FH and two other bands as isoforms of the FH-related protein-1. The identities of all three bands were confirmed by Western immunoblotting with specific antibodies. Structure-function relation studies determined that the globular domain of the Scl1.6 variant specifically binds FH while fused to collagenous tails of various lengths. This binding is not restricted to Scl1.6 as the phylogenetically linked Scl1.55 variant also binds FH. Functional analyses demonstrated the cofactor activity of the rScl1.6-bound FH for factor I-mediated cleavage of C3b. Finally, purified FH bound to the Scl1.6 protein present in the cell wall material obtained from M6-type GAS. In conclusion, we have identified a functional interaction between Scl1 and plasma FH, which may contribute to GAS evasion of complement-mediated opsonization and phagocytosis.

  9. Residues 28 to 39 of the Extracellular Loop 1 of Chicken Na+/H+ Exchanger Type I Mediate Cell Binding and Entry of Subgroup J Avian Leukosis Virus.

    Science.gov (United States)

    Guan, Xiaolu; Zhang, Yao; Yu, Mengmeng; Ren, Chaoqi; Gao, Yanni; Yun, Bingling; Liu, Yongzhen; Wang, Yongqiang; Qi, Xiaole; Liu, Changjun; Cui, Hongyu; Zhang, Yanping; Gao, Li; Li, Kai; Pan, Qing; Zhang, Baoshan; Wang, Xiaomei; Gao, Yulong

    2018-01-01

    Chicken Na + /H + exchanger type I (chNHE1), a multispan transmembrane protein, is a cellular receptor of the subgroup J avian leukosis virus (ALV-J). To identify the functional determinants of chNHE1 responsible for the ALV-J receptor activity, a series of chimeric receptors was created by exchanging the extracellular loops (ECL) of human NHE1 (huNHE1) and chNHE1 and by ECL replacement with a hemagglutinin (HA) tag. These chimeric receptors then were used in binding and entry assays to map the minimal ALV-J gp85-binding domain of chNHE1. We show that ECL1 of chNHE1 (chECL1) is the critical functional ECL that interacts directly with ALV-J gp85; ECL3 is also involved in ALV-J gp85 binding. Amino acid residues 28 to 39 of the N-terminal membrane-proximal region of chECL1 constitute the minimal domain required for chNHE1 binding of ALV-J gp85. These residues are sufficient to mediate viral entry into ALV-J nonpermissive cells. Point mutation analysis revealed that A30, V33, W38, and E39 of chECL1 are the key residues mediating the binding between chNHE1 and ALV-J gp85. Further, the replacement of residues 28 to 39 of huNHE1 with the corresponding chNHE1 residues converted the nonfunctional ALV-J receptor huNHE1 to a functional one. Importantly, soluble chECL1 and huECL1 harboring chNHE1 residues 28 to 39 both could effectively block ALV-J infection. Collectively, our findings indicate that residues 28 to 39 of chNHE1 constitute a domain that is critical for receptor function and mediate ALV-J entry. IMPORTANCE chNHE1 is a cellular receptor of ALV-J, a retrovirus that causes infections in chickens and serious economic losses in the poultry industry. Until now, the domains determining the chNHE1 receptor function remained unknown. We demonstrate that chECL1 is critical for receptor function, with residues 28 to 39 constituting the minimal functional domain responsible for chNHE1 binding of ALV-J gp85 and efficiently mediating ALV-J cell entry. These residues are

  10. A 48 kDa collagen-binding phosphoprotein isolated from bovine aortic endothelial cells interacts with the collagenous domain, but not the globular domain, of collagen type IV.

    OpenAIRE

    Yannariello-Brown, J; Madri, J A

    1990-01-01

    We have identified collagen-binding proteins in detergent extracts of metabolically labelled bovine aortic endothelial cells (BAEC) by collagen type IV-Sepharose affinity chromatography. The major collagen type IV-binding protein identified by SDS/PAGE had a molecular mass of 48 kDa, which we term the 'collagen-binding 48 kDa protein' (CB48). The pI of CB48 was 8.0-8.3 in a two-dimensional gel system, running non-equilibrium pH gel electrophoresis in the first dimension and SDS/PAGE in the se...

  11. Purification and crystallization of the ABC-type transport substrate-binding protein OppA from Thermoanaerobacter tengcongensis

    Energy Technology Data Exchange (ETDEWEB)

    Gao, Jinlan; Li, Xiaolu [State Key Laboratory of Medical Molecular Biology, Institute of Basic Medical Sciences, Chinese Academy of Medical Sciences, Peking Union Medical College, Tsinghua University, Beijing 100005, People' s Republic of China (China); Tsinghua-Peking Joint Center for Life Sciences, Center for Structural Biology, School of Life Sciences, Tsinghua University, Beijing 100084, People' s Republic of China (China); Feng, Yue; Zhang, Bo [Tsinghua-Peking Joint Center for Life Sciences, Center for Structural Biology, School of Life Sciences, Tsinghua University, Beijing 100084, People' s Republic of China (China); Miao, Shiying [State Key Laboratory of Medical Molecular Biology, Institute of Basic Medical Sciences, Chinese Academy of Medical Sciences, Peking Union Medical College, Tsinghua University, Beijing 100005, People' s Republic of China (China); Wang, Linfang, E-mail: lfwangz@yahoo.com [State Key Laboratory of Medical Molecular Biology, Institute of Basic Medical Sciences, Chinese Academy of Medical Sciences, Peking Union Medical College, Tsinghua University, Beijing 100005, People' s Republic of China (China); Wang, Na, E-mail: nawang@tsinghua.edu.cn [Tsinghua-Peking Joint Center for Life Sciences, Center for Structural Biology, School of Life Sciences, Tsinghua University, Beijing 100084, People' s Republic of China (China)

    2012-06-22

    Highlights: Black-Right-Pointing-Pointer We truncated the signal peptide of OppA{sub TTE0054} to make it express in Escherichia coli as a soluble protein. Black-Right-Pointing-Pointer Crystals of OppA{sub TTE0054} were grown by sitting-drop vapor diffusion method. Black-Right-Pointing-Pointer The crystal of OppA{sub TTE0054} diffracted to 2.25 A. -- Abstract: Di- and oligopeptide- binding protein OppAs play important roles in solute and nutrient uptake, sporulation, biofilm formation, cell wall muropeptides recycling, peptide-dependent quorum-sensing responses, adherence to host cells, and a variety of other biological processes. Soluble OppA from Thermoanaerobacter tengcongensis was expressed in Escherichia coli. The protein was found to be >95% pure with SDS-PAGE after a series of purification steps and the purity was further verified by mass spectrometry. The protein was crystallized using the sitting-drop vapour-diffusion method with PEG 400 as the precipitant. Crystal diffraction extended to 2.25 A. The crystal belonged to space group C222{sub 1}, with unit-cell parameters of a = 69.395, b = 199.572, c = 131.673 A, and {alpha} = {beta} = {gamma} = 90 Degree-Sign .

  12. A novel type of DNA-binding protein interacts with a conserved sequence in an early nodulin ENOD12 promoter

    DEFF Research Database (Denmark)

    Christiansen, H; Hansen, A C; Vijn, I

    1996-01-01

    The pea genes PsENOD12A and PsENOD12B are expressed in the root hairs shortly after infection with the nitrogen-fixing bacterium Rhizobium leguminosarum bv. viciae or after application of purified Nod factors. A 199 bp promoter fragment of the PsENOD12B gene contains sufficient information for Nod...... factor-induced tissue-specific expression. We have isolated a Vicia sativa cDNA encoding a 1641 amino acid protein, ENBP1, that interacts with the 199 bp ENOD12 promoter. Two different DNA-binding domains were identified in ENBP1. A domain containing six AT-hooks interacts specifically with an AT...... of the ENBP1 transcript in cells expressing ENOD12 strongly suggest that ENBP1 is a transcription factor involved in the regulation of ENOD12. Finally, the C-terminal region of ENBP1 shows strong homology to a protein from rat that is specifically expressed in testis tissue. Udgivelsesdato: 1996-Dec...

  13. Global Autorecognition and Activation of Complement by Mannan-Binding Lectin in a Mouse Model of Type 1 Diabetes

    Directory of Open Access Journals (Sweden)

    Esben Axelgaard

    2017-01-01

    Full Text Available Increasing evidence links mannan-binding lectin (MBL to late vascular complications of diabetes. MBL is a complement-activating pattern recognition molecule of the innate immune system that can mediate an inflammation response through activation of the lectin pathway. In two recent animal studies, we have shown that autoreactivity of MBL is increased in the kidney in diabetic nephropathy. We hypothesize that long-term exposure to uncontrolled high blood glucose in diabetes may mediate formation of neoepitopes in several tissues and that MBL is able to recognize these structures and thus activate the lectin pathway. To test this hypothesis, we induced diabetes by injection of low-dose streptozotocin in MBL double-knockout (MBL/DKO mice. Development of diabetes was followed by measurements of blood glucose and urine albumin-to-creatinine ratio. Fluorophore-labelled recombinant MBL was injected intravenously in diabetic and nondiabetic mice followed by ex vivo imaging of several organs. We observed that MBL accumulated in the heart, liver, brain, lung, pancreas, and intestines of diabetic mice. We furthermore detected increased systemic complement activation after administration of MBL, thus indicating MBL-mediated systemic complement activation in these animals. These new findings indicate a global role of MBL during late diabetes-mediated vascular complications in various tissues.

  14. The association of metabotropic glutamate receptor type 5 with the neuronal Ca2+-binding protein 2 modulates receptor function.

    Science.gov (United States)

    Canela, Laia; Fernández-Dueñas, Víctor; Albergaria, Catarina; Watanabe, Masahiko; Lluís, Carme; Mallol, Josefa; Canela, Enric I; Franco, Rafael; Luján, Rafael; Ciruela, Francisco

    2009-10-01

    Metabotropic glutamate (mGlu) receptors mediate in part the CNS effects of glutamate. These receptors interact with a large array of intracellular proteins in which the final role is to regulate receptor function. Here, using co-immunoprecipitation and pull-down experiments we showed a close and specific interaction between mGlu(5) receptor and NECAB2 in both transfected human embryonic kidney cells and rat hippocampus. Interestingly, in pull-down experiments increasing concentrations of calcium drastically reduced the ability of these two proteins to interact, suggesting that NECAB2 binds to mGlu(5) receptor in a calcium-regulated manner. Immunoelectron microscopy detection of NECAB2 and mGlu(5) receptor in the rat hippocampal formation indicated that both proteins are codistributed in the same subcellular compartment of pyramidal cells. In addition, the NECAB2/mGlu(5) receptor interaction regulated mGlu(5b)-mediated activation of both inositol phosphate accumulation and the extracellular signal-regulated kinase/mitogen-activated protein kinase pathway. Overall, these findings indicate that NECAB2 by its physical interaction with mGlu(5b) receptor modulates receptor function.

  15. 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.

  16. Human T-lymphotropic virus type 1 Tax protein complexes with P-TEFb and competes for Brd4 and 7SK snRNP/HEXIM1 binding.

    Science.gov (United States)

    Cho, Won-Kyung; Jang, Moon Kyoo; Huang, Keven; Pise-Masison, Cynthia A; Brady, John N

    2010-12-01

    Positive transcription elongation factor b (P-TEFb) plays an important role in stimulating RNA polymerase II elongation for viral and cellular gene expression. P-TEFb is found in cells in either an active, low-molecular-weight (LMW) form or an inactive, high-molecular-weight (HMW) form. We report here that human T-lymphotropic virus type 1 (HTLV-1) Tax interacts with the cyclin T1 subunit of P-TEFb, forming a distinct Tax/P-TEFb LMW complex. We demonstrate that Tax can play a role in regulating the amount of HMW complex present in the cell by decreasing the binding of 7SK snRNP/HEXIM1 to P-TEFb. This is seen both in vitro using purified Tax protein and in vivo in cells transduced with Tax expression constructs. Further, we find that a peptide of cyclin T1 spanning the Tax binding domain inhibits the ability of Tax to disrupt HMW P-TEFb complexes. These results suggest that the direct interaction of Tax with cyclin T1 can dissociate P-TEFb from the P-TEFb/7SK snRNP/HEXIM1 complex for activation of the viral long terminal repeat (LTR). We also show that Tax competes with Brd4 for P-TEFb binding. Chromatin immunoprecipitation (ChIP) assays demonstrated that Brd4 and P-TEFb are associated with the basal HTLV-1 LTR, while Tax and P-TEFb are associated with the activated template. Furthermore, the knockdown of Brd4 by small interfering RNA (siRNA) activates the HTLV-1 LTR promoter, which results in an increase in viral expression and production. Our studies have identified Tax as a regulator of P-TEFb that is capable of affecting the balance between its association with the large inactive complex and the small active complex.

  17. A novel C-type lectin from the sea cucumber Apostichopus japonicus (AjCTL-2) with preferential binding of d-galactose.

    Science.gov (United States)

    Wang, Hui; Xue, Zhuang; Liu, Zhaoqun; Wang, Weilin; Wang, Feifei; Wang, Ying; Wang, Lingling; Song, Linsheng

    2018-05-15

    C-type lectins (CTLs) are Ca 2+ dependent carbohydrate-binding proteins that share structural homology in their carbohydrate-recognition domains (CRDs). In the present study, a novel CTL was identified from sea cucumber Apostichopus japonicus (named as AjCTL-2). The deduced amino acid sequence of AjCTL-2 was homologous to CTLs from other animals with the identities ranging from 33% to 40%. It contained a canonical signal peptide at the N-terminus, a low density lipoprotein receptor class A (LDLa), a C1r/C1s/Uegf/bone morphogenetic protein 1 (CUB), and a CRD with two motifs Glu-Pro-Asn (EPN) and Trp-Asn-Asp (WND) in Ca 2+ binding site 2. The mRNA transcripts of AjCTL-2 were extensively expressed in all the tested tissues including respiratory tree, muscle, gut, coelomocyte, tube-foot, body wall and gonad, and the highest expression level of AjCTL-2 in coelomocyte was about 4.2-fold (p sea cucumber. Copyright © 2018. Published by Elsevier Ltd.

  18. The 3D protein of duck hepatitis A virus type 1 binds to a viral genomic 3' UTR and shows RNA-dependent RNA polymerase activity.

    Science.gov (United States)

    Zhang, Yu; Cao, Qianda; Wang, Mingshu; Jia, Renyong; Chen, Shun; Zhu, Dekang; Liu, Mafeng; Sun, Kunfeng; Yang, Qiao; Wu, Ying; Zhao, Xinxin; Chen, Xiaoyue; Cheng, Anchun

    2017-12-01

    To explore the RNA-dependent RNA polymerase (RdRP) function of the 3D protein of duck hepatitis A virus type 1 (DHAV-1), the gene was cloned into the pET-32a(+) vector for prokaryotic expression. The 3' untranslated region (3' UTR) of DHAV-1 together with a T7 promoter was cloned into the pMD19-T vector for in vitro transcription of 3' UTR RNA, which was further used as a template in RNA-dependent RNA polymerization. In this study, three methods were applied to analyze the RdRP function of the 3D protein: (1) ammonium molybdate spectrophotometry to detect pyrophosphate produced during polymerization; (2) quantitative reverse transcription PCR (RT-qPCR) to investigate the changes in RNA quantity during polymerization; and (3) electrophoresis mobility shift assay to examine the interaction between the 3D protein and 3' UTR. The results showed the 3D protein was successfully expressed in bacteria culture supernatant in a soluble form, which could be purified by affinity chromatography. In 3D enzymatic activity assays, pyrophosphate and RNA were produced, the amounts of which increased based on approximative kinetics, and binding of the 3D protein to the 3' UTR was observed. These results indicate that prokaryotically expressed soluble DHAV-13D protein can bind to a viral genomic 3' UTR and exhibit RdRP activity.

  19. Identification of an osteoclast transcription factor that binds to the human T cell leukemia virus type I-long terminal repeat enhancer element.

    Science.gov (United States)

    Inoue, D; Santiago, P; Horne, W C; Baron, R

    1997-10-03

    Transgenic mice expressing human T cell leukemia virus type I (HTLV-I)-tax under the control of HTLV-I-long terminal repeat (LTR) promoter develop skeletal abnormalities with high bone turnover and myelofibrosis. In these animals, Tax is highly expressed in bone with a pattern of expression restricted to osteoclasts and spindle-shaped cells within the endosteal myelofibrosis. To test the hypothesis that lineage-specific transcription factors promote transgene expression from the HTLV-I-LTR in osteoclasts, we first examined tax expression in transgenic bone marrow cultures. Expression was dependent on 1alpha,25-dihydroxycholecalciferol and coincided with tartrate-resistant acid phosphatase (TRAP) expression, a marker of osteoclast differentiation. Furthermore, Tax was expressed in vitronectin receptor-positive mononuclear precursors as well as in mature osteoclast-like cells (OCLs). Consistent with our hypothesis, electrophoretic mobility shift assays revealed the presence of an OCL nuclear factor (NFOC-1) that binds to the LTR 21-base pair direct repeat, a region critical for the promoter activity. This binding is further enhanced by Tax. Since NFOC-1 is absent in macrophages and conserved in osteoclasts among species including human, such a factor may play a role in lineage determination and/or in expression of the differentiated osteoclast phenotype.

  20. Regional specific binding of [11C]RO 15 1788 to central type benzodiazepine receptors in human brain: quantitative evaluation by PET

    International Nuclear Information System (INIS)

    Pappata, S.; Samson, Y.; Chavoix, C.; Prenant, C.; Maziere, M.; Baron, J.C.

    1988-01-01

    The central type benzodiazepine receptors were studied in 17 healthy human subjects with 11 C-RO 15 1788 and positron emission tomography (PET). The brain regional distribution of the tracer in eight control studies performed after injection of trace doses of 11 C-RO 15 1788 was consistent with that of benzodiazepine receptors. Saturation studies with co-injected cold RO 15 1788 in the remaining subjects showed a dose-dependent decrease of brain radiotracer until full inhibition of specific binding was achieved with doses above 0.1 mg/kg (four studies). Based on the results, a simple method to estimate the specifically bound 11 C-RO 15 1788 regionally in a single PET study is proposed, using the data from the full-saturation studies as a stable estimate of the nondisplaceable radioligand concentration. Using this method, it was found that quasiequilibrium between the estimated specifically bound and nondisplaceable components was achieved at times equal to or longer than 20 min after tracer administration. The validity of this method was partly supported by further results, showing a good agreement between the regional specific binding so calculated and postmortem data of receptor density

  1. NpPDR1, a Pleiotropic Drug Resistance-Type ATP-Binding Cassette Transporter from Nicotiana plumbaginifolia, Plays a Major Role in Plant Pathogen Defense1

    Science.gov (United States)

    Stukkens, Yvan; Bultreys, Alain; Grec, Sébastien; Trombik, Tomasz; Vanham, Delphine; Boutry, Marc

    2005-01-01

    Nicotiana plumbaginifolia NpPDR1, a plasma membrane pleiotropic drug resistance-type ATP-binding cassette transporter formerly named NpABC1, has been suggested to transport the diterpene sclareol, an antifungal compound. However, direct evidence for a role of pleiotropic drug resistance transporters in the plant defense is still lacking. In situ immunolocalization and histochemical analysis using the gusA reporter gene showed that NpPDR1 was constitutively expressed in the whole root, in the leaf glandular trichomes, and in the flower petals. However, NpPDR1 expression was induced in the whole leaf following infection with the fungus Botrytis cinerea, and the bacteria Pseudomonas syringae pv tabaci, Pseudomonas fluorescens, and Pseudomonas marginalis pv marginalis, which do not induce a hypersensitive response in N. plumbaginifolia, whereas a weaker response was observed using P. syringae pv syringae, which does induce a hypersensitive response. Induced NpPDR1 expression was more associated with the jasmonic acid than the salicylic acid signaling pathway. These data suggest that NpPDR1 is involved in both constitutive and jasmonic acid-dependent induced defense. Transgenic plants in which NpPDR1 expression was prevented by RNA interference showed increased sensitivity to sclareol and reduced resistance to B. cinerea. These data show that NpPDR1 is involved in pathogen resistance and thus demonstrate a new role for the ATP-binding cassette transporter family. PMID:16126865

  2. NpPDR1, a pleiotropic drug resistance-type ATP-binding cassette transporter from Nicotiana plumbaginifolia, plays a major role in plant pathogen defense.

    Science.gov (United States)

    Stukkens, Yvan; Bultreys, Alain; Grec, Sébastien; Trombik, Tomasz; Vanham, Delphine; Boutry, Marc

    2005-09-01

    Nicotiana plumbaginifolia NpPDR1, a plasma membrane pleiotropic drug resistance-type ATP-binding cassette transporter formerly named NpABC1, has been suggested to transport the diterpene sclareol, an antifungal compound. However, direct evidence for a role of pleiotropic drug resistance transporters in the plant defense is still lacking. In situ immunolocalization and histochemical analysis using the gusA reporter gene showed that NpPDR1 was constitutively expressed in the whole root, in the leaf glandular trichomes, and in the flower petals. However, NpPDR1 expression was induced in the whole leaf following infection with the fungus Botrytis cinerea, and the bacteria Pseudomonas syringae pv tabaci, Pseudomonas fluorescens, and Pseudomonas marginalis pv marginalis, which do not induce a hypersensitive response in N. plumbaginifolia, whereas a weaker response was observed using P. syringae pv syringae, which does induce a hypersensitive response. Induced NpPDR1 expression was more associated with the jasmonic acid than the salicylic acid signaling pathway. These data suggest that NpPDR1 is involved in both constitutive and jasmonic acid-dependent induced defense. Transgenic plants in which NpPDR1 expression was prevented by RNA interference showed increased sensitivity to sclareol and reduced resistance to B. cinerea. These data show that NpPDR1 is involved in pathogen resistance and thus demonstrate a new role for the ATP-binding cassette transporter family.

  3. Type III restriction endonucleases are heterotrimeric: comprising one helicase–nuclease subunit and a dimeric methyltransferase that binds only one specific DNA

    Science.gov (United States)

    Butterer, Annika; Pernstich, Christian; Smith, Rachel M.; Sobott, Frank; Szczelkun, Mark D.; Tóth, Júlia

    2014-01-01

    Fundamental aspects of the biochemistry of Type III restriction endonucleases remain unresolved despite being characterized by numerous research groups in the past decades. One such feature is the subunit stoichiometry of these hetero-oligomeric enzyme complexes, which has important implications for the reaction mechanism. In this study, we present a series of results obtained by native mass spectrometry and size exclusion chromatography with multi-angle light scattering consistent with a 1:2 ratio of Res to Mod subunits in the EcoP15I, EcoPI and PstII complexes as the main holoenzyme species and a 1:1 stoichiometry of specific DNA (sDNA) binding by EcoP15I and EcoPI. Our data are also consistent with a model where ATP hydrolysis activated by recognition site binding leads to release of the enzyme from the site, dissociation from the substrate via a free DNA end and cleavage of the DNA. These results are discussed critically in the light of the published literature, aiming to resolve controversies and discuss consequences in terms of the reaction mechanism. PMID:24510100

  4. Bruton’s Tyrosine Kinase Phosphorylates DDX41 and Activates Its Binding of dsDNA and STING to Initiate Type 1 Interferon Response

    Directory of Open Access Journals (Sweden)

    Koon-Guan Lee

    2015-02-01

    Full Text Available The innate immune system senses cytosolic dsDNA and bacterial cyclic dinucleotides and initiates signaling via the adaptor STING to induce type 1 interferon (IFN response. We demonstrate here that BTK-deficient cells have impaired IFN-β production and TBK1/IRF3 activation when stimulated with agonists or infected with pathogens that activate STING signaling. BTK interacts with STING and DDX41 helicase. The kinase and SH3/SH2 interaction domains of BTK bind, respectively, the DEAD-box domain of DDX41 and transmembrane region of STING. BTK phosphorylates DDX41, and its kinase activities are critical for STING-mediated IFN-β production. We show that Tyr364 and Tyr414 of DDX41 are critical for its recognition of AT-rich DNA and binding to STING, and tandem mass spectrometry identifies Tyr414 as the BTK phosphorylation site. Modeling studies further indicate that phospho-Tyr414 strengthens DDX41’s interaction with STING. Hence, BTK plays a critical role in the activation of DDX41 helicase and STING signaling.

  5. A lack of association between vitamin D-binding protein and 25-hydroxyvitamin D concentrations in pediatric type 1 diabetes without microalbuminuria

    Directory of Open Access Journals (Sweden)

    Hwa Young Kim

    2017-12-01

    Full Text Available Purpose Vitamin D deficiency is reported to be more common in type 1 diabetes patients and might be associated with the increased urinary loss of vitamin D binding protein (VDBP consequent to impaired 25-hydroxyvitamin D (25(OHD circulation. We aimed to evaluate the possible increased urinary loss of VDBP, a correlation between VDBP and circulating 25(OHD level, and risk factors influencing low vitamin D level in pediatric type 1 diabetes patients without microalbuminuria. Methods This is a cross-sectional study of subjects who visited Seoul National University Children’s Hospital between January and March 2013. Forty-two type 1 diabetes patients and 29 healthy controls were included. Biochemical parameters including serum and urine VDBP concentrations were analyzed. Results There was no significant difference in the frequency of vitamin D deficiency or serum 25(OHD level between the 2 groups. The serum and urine VDBP concentrations did not show any difference between the 2 groups. Serum 25(OH D level did not correlate with serum or urine VDBP. Multivariate regression analysis revealed that daylight outdoor hours (β=2.948, P=0.003 and vitamin D intake (β=2.865, P=0.003 affected the 25(OHD level; the presence of type 1 diabetes or urinary VDBP excretion was not significant. Conclusions In pediatric type 1 diabetes patients, urinary VDBP excretion did not contribute to low serum 25(OHD level in the setting of normoalbuminuria. The factors associated with 25(OHD level during winter periods were daylight outdoor hours and vitamin D intake. Further studies including both micro- and macroalbuminuria patients with type 1 diabetes are warranted.

  6. Heparin/heparan sulfates bind to and modulate neuronal L-type (Cav1.2) voltage-dependent Ca2+ channels

    DEFF Research Database (Denmark)

    Garau, Gianpiero; Magotti, Paola; Heine, Martin

    2015-01-01

    Our previous studies revealed that L-type voltage-dependent Ca2+ channels (Cav1.2 L-VDCCs) are modulated by the neural extracellular matrix backbone, polyanionic glycan hyaluronic acid. Here we used isothermal titration calorimetry and screened a set of peptides derived from the extracellular......M), integrating their enthalpic and entropic binding contributions. Interaction between heparin and recombinant as well as native full-length neuronal Cav1.2α1 channels was confirmed using the heparin–agarose pull down assay. Whole cell patch clamp recordings in HEK293 cells transfected with neuronal Cav1.......2 channels revealed that enzymatic digestion of highly sulfated heparan sulfates with heparinase 1 affects neither voltage-dependence of channel activation nor the level of steady state inactivation, but did speed up channel inactivation. Treatment of hippocampal cultures with heparinase 1 reduced the firing...

  7. Type 2 diabetes is associated with altered NF-¿B DNA binding activity, JNK phosphorylation, and AMPK phosphorylation in skeletal muscle after LPS

    DEFF Research Database (Denmark)

    Andreasen, Anne Sofie; Kelly, Meghan; Berg, Ronan Martin Griffin

    2011-01-01

    Systemic inflammation is often associated with impaired glucose metabolism. We therefore studied the activation of inflammatory pathway intermediates that interfere with glucose uptake during systemic inflammation by applying a standardised inflammatory stimulus in vivo. After ethical approval......, informed consent and a thorough physical examination, 10 patients with type 2 diabetes and 10 participants with normal glucose tolerance (NGT) were given an intravenous bolus of E. coli lipopolysaccharide (LPS) of 0.3 ng/kg. Skeletal muscle biopsies and plasma were obtained at baseline and two, four...... and six hours after LPS. Nuclear factor (NF)-¿B p65 DNA binding activity measured by ELISA, tumor necrosis factor-a and interleukin-6 mRNA expression analysed by real time reverse transcription polymerase chain reaction, and abundance of inhibitor of NF-¿B (I¿B)a, phosphorylated c-Jun-N-terminal kinase...

  8. Suppression of type I interferon production by porcine epidemic diarrhea virus and degradation of CREB-binding protein by nsp1

    Energy Technology Data Exchange (ETDEWEB)

    Zhang, Qingzhan; Shi, Kaichuang; Yoo, Dongwan, E-mail: dyoo@illinois.edu

    2016-02-15

    Type I interferons (IFN-α/β) are the major components of the innate immune response of hosts, and in turn many viruses have evolved to modulate the host response during infection. We found that the IFN-β production was significantly suppressed during PEDV infection in cells. To identify viral IFN antagonists and to study their suppressive function, viral coding sequences for the entire structural and nonstructural proteins were cloned and expressed. Of 16 PEDV nonstructural proteins (nsps), nsp1, nsp3, nsp7, nsp14, nsp15 and nsp16 were found to inhibit the IFN-β and IRF3 promoter activities. The sole accessory protein ORF3, structure protein envelope (E), membrane (M), and nucleocapsid (N) protein were also shown to inhibit such activities. PEDV nsp1 did not interfere the IRF3 phosphorylation and nuclear translocation but interrupted the enhanceosome assembly of IRF3 and CREB-binding protein (CBP) by degrading CBP. A further study showed that the CBP degradation by nsp1 was proteasome-dependent. Our data demonstrate that PEDV modulates the host innate immune responses by degrading CBP and suppressing ISGs expression. - Highlights: • PEDV modulates the host innate immune system by suppressing the type I interferon production and ISGs expression. • Ten viral proteins were identified as IFN antagonists, and nsp1 was the most potent viral IFN antagonist. • PEDV nsp1 did not interfere the IRF3 phosphorylation and nuclear translocation but interrupted the enhanceosome assembly of IRF3 and CREB-binding protein (CBP). • PEDV nsp1 caused the CBP degradation in the nucleus, which may be the key mechanism for PEDV-mediated IFN downregulation.

  9. The structure of classical swine fever virus N(pro: a novel cysteine Autoprotease and zinc-binding protein involved in subversion of type I interferon induction.

    Directory of Open Access Journals (Sweden)

    Keerthi Gottipati

    Full Text Available Pestiviruses express their genome as a single polypeptide that is subsequently cleaved into individual proteins by host- and virus-encoded proteases. The pestivirus N-terminal protease (N(pro is a cysteine autoprotease that cleaves between its own C-terminus and the N-terminus of the core protein. Due to its unique sequence and catalytic site, it forms its own cysteine protease family C53. After self-cleavage, N(pro is no longer active as a protease. The released N(pro suppresses the induction of the host's type-I interferon-α/β (IFN-α/β response. N(pro binds interferon regulatory factor-3 (IRF3, the key transcriptional activator of IFN-α/β genes, and promotes degradation of IRF3 by the proteasome, thus preventing induction of the IFN-α/β response to pestivirus infection. Here we report the crystal structures of pestivirus N(pro. N(pro is structurally distinct from other known cysteine proteases and has a novel "clam shell" fold consisting of a protease domain and a zinc-binding domain. The unique fold of N(pro allows auto-catalysis at its C-terminus and subsequently conceals the cleavage site in the active site of the protease. Although many viruses interfere with type I IFN induction by targeting the IRF3 pathway, little information is available regarding structure or mechanism of action of viral proteins that interact with IRF3. The distribution of amino acids on the surface of N(pro involved in targeting IRF3 for proteasomal degradation provides insight into the nature of N(pro's interaction with IRF3. The structures thus establish the mechanism of auto-catalysis and subsequent auto-inhibition of trans-activity of N(pro, and its role in subversion of host immune response.

  10. The Breast Cancer-Associated Glycoforms of MUC1, MUC1-Tn and sialyl-Tn, Are Expressed in COSMC Wild-Type Cells and Bind the C-Type Lectin MGL

    DEFF Research Database (Denmark)

    Beatson, Richard; Maurstad, Gjertrud; Picco, Gianfranco

    2015-01-01

    Aberrant glycosylation occurs in the majority of human cancers and changes in mucin-type O-glycosylation are key events that play a role in the induction of invasion and metastases. These changes generate novel cancer-specific glyco-antigens that can interact with cells of the immune system through...... carbohydrate binding lectins. Two glyco-epitopes that are found expressed by many carcinomas are Tn (GalNAc-Ser/Thr) and STn (NeuAcα2,6GalNAc-Ser/Thr). These glycans can be carried on many mucin-type glycoproteins including MUC1. We show that the majority of breast cancers carry Tn within the same cell...

  11. Insights into the binding specificity of wild type and mutated wheat germ agglutinin towards Neu5Acα(2-3)Gal: a study by in silico mutations and molecular dynamics simulations.

    Science.gov (United States)

    Parasuraman, Ponnusamy; Murugan, Veeramani; Selvin, Jeyasigamani F A; Gromiha, M Michael; Fukui, Kazuhiko; Veluraja, Kasinadar

    2014-08-01

    Wheat germ agglutinin (WGA) is a plant lectin, which specifically recognizes the sugars NeuNAc and GlcNAc. Mutated WGA with enhanced binding specificity can be used as biomarkers for cancer. In silico mutations are performed at the active site of WGA to enhance the binding specificity towards sialylglycans, and molecular dynamics simulations of 20 ns are carried out for wild type and mutated WGAs (WGA1, WGA2, and WGA3) in complex with sialylgalactose to examine the change in binding specificity. MD simulations reveal the change in binding specificity of wild type and mutated WGAs towards sialylgalactose and bound conformational flexibility of sialylgalactose. The mutated polar amino acid residues Asn114 (S114N), Lys118 (G118K), and Arg118 (G118R) make direct and water mediated hydrogen bonds and hydrophobic interactions with sialylgalactose. An analysis of possible hydrogen bonds, hydrophobic interactions, total pair wise interaction energy between active site residues and sialylgalactose and MM-PBSA free energy calculation reveals the plausible binding modes and the role of water in stabilizing different binding modes. An interesting observation is that the binding specificity of mutated WGAs (cyborg lectin) towards sialylgalactose is found to be higher in double point mutation (WGA3). One of the substituted residues Arg118 plays a crucial role in sugar binding. Based on the interactions and energy calculations, it is concluded that the order of binding specificity of WGAs towards sialylgalactose is WGA3 > WGA1 > WGA2 > WGA. On comparing with the wild type, double point mutated WGA (WGA3) exhibits increased specificity towards sialylgalactose, and thus, it can be effectively used in targeted drug delivery and as biological cell marker in cancer therapeutics. Copyright © 2014 John Wiley & Sons, Ltd.

  12. F-Type Lectins: A Highly Diversified Family of Fucose-Binding Proteins with a Unique Sequence Motif and Structural Fold, Involved in Self/Non-Self-Recognition

    Directory of Open Access Journals (Sweden)

    Gerardo R. Vasta

    2017-11-01

    Full Text Available The F-type lectin (FTL family is one of the most recent to be identified and structurally characterized. Members of the FTL family are characterized by a fucose recognition domain [F-type lectin domain (FTLD] that displays a novel jellyroll fold (“F-type” fold and unique carbohydrate- and calcium-binding sequence motifs. This novel lectin family comprises widely distributed proteins exhibiting single, double, or greater multiples of the FTLD, either tandemly arrayed or combined with other structurally and functionally distinct domains, yielding lectin subunits of pleiotropic properties even within a single species. Furthermore, the extraordinary variability of FTL sequences (isoforms that are expressed in a single individual has revealed genetic mechanisms of diversification in ligand recognition that are unique to FTLs. Functions of FTLs in self/non-self-recognition include innate immunity, fertilization, microbial adhesion, and pathogenesis, among others. In addition, although the F-type fold is distinctive for FTLs, a structure-based search revealed apparently unrelated proteins with minor sequence similarity to FTLs that displayed the FTLD fold. In general, the phylogenetic analysis of FTLD sequences from viruses to mammals reveals clades that are consistent with the currently accepted taxonomy of extant species. However, the surprisingly discontinuous distribution of FTLDs within each taxonomic category suggests not only an extensive structural/functional diversification of the FTLs along evolutionary lineages but also that this intriguing lectin family has been subject to frequent gene duplication, secondary loss, lateral transfer, and functional co-option.

  13. Evaluation of lectin pathway activity and mannan-binding lectin levels in the course of pregnancy complicated by diabetes type 1, based on the genetic background.

    Science.gov (United States)

    Pertyńska Marczewska, Magdalena; Cedzyński, Maciej; Swierzko, Anna; Szala, Agnieszka; Sobczak, Małgorzata; Cypryk, Katarzyna; Wilczyński, Jan

    2009-01-01

    There are numerous indications that either mannan-binding lectin (MBL) deficiency or its excessive activity are associated with adverse pregnancy outcomes. High MBL concentrations and corresponding MBL2 genotypes were shown to be associated with microvascular complications in type 1 diabetes. The aim of this study was to evaluate levels of MBL and MBL-dependent activity of the lectin pathway (LP) of complement in the course of pregnancy in diabetic mothers, based on genetic background. These parameters were determined in samples from healthy non-pregnant (control), diabetic non-pregnant, healthy pregnant, and pregnant diabetic women. No significant differences in median MBL levels or LP activities were found in any study group compared to the control. However, statistically significant differences in MBL levels were noted during pregnancy between the 1st and 3rd trimesters in both healthy controls and pregnant diabetics. With regard to LP values, similar trends were evident, but statistically significant results were obtained only in the healthy pregnant group. When data analysis was confined to patients carrying the A/A (wild-type) MBL2 genotype, an increase in MBL level during pregnancy (in both healthy and diabetic pregnant women) was still observed. Similarly, LP activity increased during both healthy and diabetic pregnancies, significantly so for the former. Diabetes, an autoimmune disease, is a serious complication of pregnancy. Therefore, determination of MBL status might be beneficial in identifying type 1 diabetic patients who are at increased risk of developing both vascular complications and poor pregnancy outcomes.

  14. Human metabolites of synthetic cannabinoids JWH-018 and JWH-073 bind with high affinity and act as potent agonists at cannabinoid type-2 receptors

    International Nuclear Information System (INIS)

    Rajasekaran, Maheswari; Brents, Lisa K.; Franks, Lirit N.; Moran, Jeffery H.; Prather, Paul L.

    2013-01-01

    K2 or Spice is an emerging drug of abuse that contains synthetic cannabinoids, including JWH-018 and JWH-073. Recent reports indicate that monohydroxylated metabolites of JWH-018 and JWH-073 retain high affinity and activity at cannabinoid type-1 receptors (CB 1 Rs), potentially contributing to the enhanced toxicity of K2 compared to marijuana. Since the parent compounds also bind to cannabinoid type-2 receptors (CB 2 Rs), this study investigated the affinity and intrinsic activity of JWH-018, JWH-073 and several monohydroxylated metabolites at human CB 2 Rs (hCB 2 Rs). The affinity of cannabinoids for hCB 2 Rs was determined by competition binding studies employing CHO-hCB 2 membranes. Intrinsic activity of compounds was assessed by G-protein activation and adenylyl cyclase (AC)-inhibition in CHO-hCB 2 cells. JWH-073, JWH-018 and several of their human metabolites exhibit nanomolar affinity and act as potent agonists at hCB 2 Rs. Furthermore, a major omega hydroxyl metabolite of JWH-073 (JWH-073-M5) binds to CB 2 Rs with 10-fold less affinity than the parent molecule, but unexpectedly, is equipotent in regulating AC-activity when compared to the parent molecule. Finally, when compared to CP-55,940 and Δ 9 -tetrahydrocannabinol (Δ 9 -THC), JWH-018, JWH-018-M5 and JWH-073-M5 require significantly less CB 2 R occupancy to produce similar levels of AC-inhibition, indicating that these compounds may more efficiently couple CB 2 Rs to AC than the well characterized cannabinoid agonists examined. These results indicate that JWH-018, JWH-073 and several major human metabolites of these compounds exhibit high affinity and demonstrate distinctive signaling properties at CB 2 Rs. Therefore, future studies examining pharmacological and toxicological properties of synthetic cannabinoids present in K2 products should consider potential actions of these drugs at both CB 1 and CB 2 Rs. - Highlights: • JWH-018 and JWH-073 are synthetic cannabinoids present in abused K2

  15. Human metabolites of synthetic cannabinoids JWH-018 and JWH-073 bind with high affinity and act as potent agonists at cannabinoid type-2 receptors

    Energy Technology Data Exchange (ETDEWEB)

    Rajasekaran, Maheswari; Brents, Lisa K.; Franks, Lirit N. [Department of Pharmacology and Toxicology, University of Arkansas for Medical Sciences, Little Rock, AR 72205 (United States); Moran, Jeffery H. [Department of Pharmacology and Toxicology, University of Arkansas for Medical Sciences, Little Rock, AR 72205 (United States); Arkansas Department of Public Health, Public Health Laboratory, Little Rock, AR 72205 (United States); Prather, Paul L., E-mail: pratherpaull@uams.edu [Department of Pharmacology and Toxicology, University of Arkansas for Medical Sciences, Little Rock, AR 72205 (United States)

    2013-06-01

    K2 or Spice is an emerging drug of abuse that contains synthetic cannabinoids, including JWH-018 and JWH-073. Recent reports indicate that monohydroxylated metabolites of JWH-018 and JWH-073 retain high affinity and activity at cannabinoid type-1 receptors (CB{sub 1}Rs), potentially contributing to the enhanced toxicity of K2 compared to marijuana. Since the parent compounds also bind to cannabinoid type-2 receptors (CB{sub 2}Rs), this study investigated the affinity and intrinsic activity of JWH-018, JWH-073 and several monohydroxylated metabolites at human CB{sub 2}Rs (hCB{sub 2}Rs). The affinity of cannabinoids for hCB{sub 2}Rs was determined by competition binding studies employing CHO-hCB{sub 2} membranes. Intrinsic activity of compounds was assessed by G-protein activation and adenylyl cyclase (AC)-inhibition in CHO-hCB{sub 2} cells. JWH-073, JWH-018 and several of their human metabolites exhibit nanomolar affinity and act as potent agonists at hCB{sub 2}Rs. Furthermore, a major omega hydroxyl metabolite of JWH-073 (JWH-073-M5) binds to CB{sub 2}Rs with 10-fold less affinity than the parent molecule, but unexpectedly, is equipotent in regulating AC-activity when compared to the parent molecule. Finally, when compared to CP-55,940 and Δ{sup 9}-tetrahydrocannabinol (Δ{sup 9}-THC), JWH-018, JWH-018-M5 and JWH-073-M5 require significantly less CB{sub 2}R occupancy to produce similar levels of AC-inhibition, indicating that these compounds may more efficiently couple CB{sub 2}Rs to AC than the well characterized cannabinoid agonists examined. These results indicate that JWH-018, JWH-073 and several major human metabolites of these compounds exhibit high affinity and demonstrate distinctive signaling properties at CB{sub 2}Rs. Therefore, future studies examining pharmacological and toxicological properties of synthetic cannabinoids present in K2 products should consider potential actions of these drugs at both CB{sub 1} and CB{sub 2}Rs. - Highlights: • JWH-018

  16. A novel type of peptidoglycan-binding domain highly specific for amidated D-Asp cross-bridge, identified in Lactobacillus casei bacteriophage endolysins.

    Science.gov (United States)

    Regulski, Krzysztof; Courtin, Pascal; Kulakauskas, Saulius; Chapot-Chartier, Marie-Pierre

    2013-07-12

    Peptidoglycan hydrolases (PGHs) are responsible for bacterial cell lysis. Most PGHs have a modular structure comprising a catalytic domain and a cell wall-binding domain (CWBD). PGHs of bacteriophage origin, called endolysins, are involved in bacterial lysis at the end of the infection cycle. We have characterized two endolysins, Lc-Lys and Lc-Lys-2, identified in prophages present in the genome of Lactobacillus casei BL23. These two enzymes have different catalytic domains but similar putative C-terminal CWBDs. By analyzing purified peptidoglycan (PG) degradation products, we showed that Lc-Lys is an N-acetylmuramoyl-L-alanine amidase, whereas Lc-Lys-2 is a γ-D-glutamyl-L-lysyl endopeptidase. Remarkably, both lysins were able to lyse only Gram-positive bacterial strains that possess PG with D-Ala(4)→D-Asx-L-Lys(3) in their cross-bridge, such as Lactococcus casei, Lactococcus lactis, and Enterococcus faecium. By testing a panel of L. lactis cell wall mutants, we observed that Lc-Lys and Lc-Lys-2 were not able to lyse mutants with a modified PG cross-bridge, constituting D-Ala(4)→L-Ala-(L-Ala/L-Ser)-L-Lys(3); moreover, they do not lyse the L. lactis mutant containing only the nonamidated D-Asp cross-bridge, i.e. D-Ala(4)→D-Asp-L-Lys(3). In contrast, Lc-Lys could lyse the ampicillin-resistant E. faecium mutant with 3→3 L-Lys(3)-D-Asn-L-Lys(3) bridges replacing the wild-type 4→3 D-Ala(4)-D-Asn-L-Lys(3) bridges. We showed that the C-terminal CWBD of Lc-Lys binds PG containing mainly D-Asn but not PG with only the nonamidated D-Asp-containing cross-bridge, indicating that the CWBD confers to Lc-Lys its narrow specificity. In conclusion, the CWBD characterized in this study is a novel type of PG-binding domain targeting specifically the D-Asn interpeptide bridge of PG.

  17. Consortium for Osteogenesis Imperfecta Mutations in the Helical Domain of Type I Collagen: Regions Rich in Lethal Mutations Align With Collagen Binding Sites for Integrins and Proteoglycans

    Science.gov (United States)

    Marini, Joan C.; Forlino, Antonella; Cabral, Wayne A.; Barnes, Aileen M.; San Antonio, James D.; Milgrom, Sarah; Hyland, James C.; Körkkö, Jarmo; Prockop, Darwin J.; De Paepe, Anne; Coucke, Paul; Symoens, Sofie; Glorieux, Francis H.; Roughley, Peter J.; Lund, Alan M.; Kuurila-Svahn, Kaija; Hartikka, Heini; Cohn, Daniel H.; Krakow, Deborah; Mottes, Monica; Schwarze, Ulrike; Chen, Diana; Yang, Kathleen; Kuslich, Christine; Troendle, James; Dalgleish, Raymond; Byers, Peter H.

    2014-01-01

    Osteogenesis imperfecta (OI) is a generalized disorder of connective tissue characterized by fragile bones and easy susceptibility to fracture. Most cases of OI are caused by mutations in type I collagen. We have identified and assembled structural mutations in type I collagen genes (COL1A1 and COL1A2, encoding the proα1(I) and proα2(I) chains, respectively) that result in OI. Quantitative defects causing type I OI were not included. Of these 832 independent mutations, 682 result in substitution for glycine residues in the triple helical domain of the encoded protein and 150 alter splice sites. Distinct genotype–phenotype relationships emerge for each chain. One-third of the mutations that result in glycine substitutions in α1(I) are lethal, especially when the substituting residues are charged or have a branched side chain. Substitutions in the first 200 residues are nonlethal and have variable outcome thereafter, unrelated to folding or helix stability domains. Two exclusively lethal regions (helix positions 691–823 and 910–964) align with major ligand binding regions (MLBRs), suggesting crucial interactions of collagen monomers or fibrils with integrins, matrix metalloproteinases (MMPs), fibronectin, and cartilage oligomeric matrix protein (COMP). Mutations in COL1A2 are predominantly nonlethal (80%). Lethal substitutions are located in eight regularly spaced clusters along the chain, supporting a regional model. The lethal regions align with proteoglycan binding sites along the fibril, suggesting a role in fibril–matrix interactions. Recurrences at the same site in α2(I) are generally concordant for outcome, unlike α1(I). Splice site mutations comprise 20% of helical mutations identified in OI patients, and may lead to exon skipping, intron inclusion, or the activation of cryptic splice sites. Splice site mutations in COL1A1 are rarely lethal; they often lead to frameshifts and the mild type I phenotype. In α2(I), lethal exon skipping events are

  18. Relationship of dopamine type 2 receptor binding potential with fasting neuroendocrine hormones and insulin sensitivity in human obesity.

    Science.gov (United States)

    Dunn, Julia P; Kessler, Robert M; Feurer, Irene D; Volkow, Nora D; Patterson, Bruce W; Ansari, Mohammad S; Li, Rui; Marks-Shulman, Pamela; Abumrad, Naji N

    2012-05-01

    Midbrain dopamine (DA) neurons, which are involved with reward and motivation, are modulated by hormones that regulate food intake (insulin, leptin, and acyl ghrelin [AG]). We hypothesized that these hormones are associated with deficits in DA signaling in obesity. We assessed the relationships between fasting levels of insulin and leptin, and AG, BMI, and insulin sensitivity index (S(I)) with the availability of central DA type 2 receptor (D2R). We measured D2R availability using positron emission tomography and [(18)F]fallypride (radioligand that competes with endogenous DA) in lean (n = 8) and obese (n = 14) females. Fasting hormones were collected prior to scanning and S(I) was determined by modified oral glucose tolerance test. Parametric image analyses revealed associations between each metabolic measure and D2R. The most extensive findings were negative associations of AG with clusters involving the striatum and inferior temporal cortices. Regional regression analyses also found extensive negative relationships between AG and D2R in the caudate, putamen, ventral striatum (VS), amygdala, and temporal lobes. S(I) was negatively associated with D2R in the VS, while insulin was not. In the caudate, BMI and leptin were positively associated with D2R availability. The direction of associations of leptin and AG with D2R availability are consistent with their opposite effects on DA levels (decreasing and increasing, respectively). After adjusting for BMI, AG maintained a significant relationship in the VS. We hypothesize that the increased D2R availability in obese subjects reflects relatively reduced DA levels competing with the radioligand. Our findings provide evidence for an association between the neuroendocrine hormones and DA brain signaling in obese females.

  19. Vaccinia Virus Protein C6 Inhibits Type I IFN Signalling in the Nucleus and Binds to the Transactivation Domain of STAT2.

    Directory of Open Access Journals (Sweden)

    Jennifer H Stuart

    2016-12-01

    Full Text Available The type I interferon (IFN response is a crucial innate immune signalling pathway required for defense against viral infection. Accordingly, the great majority of mammalian viruses possess means to inhibit this important host immune response. Here we show that vaccinia virus (VACV strain Western Reserve protein C6, is a dual function protein that inhibits the cellular response to type I IFNs in addition to its published function as an inhibitor of IRF-3 activation, thereby restricting type I IFN production from infected cells. Ectopic expression of C6 inhibits the induction of interferon stimulated genes (ISGs in response to IFNα treatment at both the mRNA and protein level. C6 inhibits the IFNα-induced Janus kinase/signal transducer and activator of transcription (JAK/STAT signalling pathway at a late stage, downstream of STAT1 and STAT2 phosphorylation, nuclear translocation and binding of the interferon stimulated gene factor 3 (ISGF3 complex to the interferon stimulated response element (ISRE. Mechanistically, C6 associates with the transactivation domain of STAT2 and this might explain how C6 inhibits the type I IFN signalling very late in the pathway. During virus infection C6 reduces ISRE-dependent gene expression despite the presence of the viral protein phosphatase VH1 that dephosphorylates STAT1 and STAT2. The ability of a cytoplasmic replicating virus to dampen the immune response within the nucleus, and the ability of viral immunomodulators such as C6 to inhibit multiple stages of the innate immune response by distinct mechanisms, emphasizes the intricacies of host-pathogen interactions and viral immune evasion.

  20. Human 3α-hydroxysteroid dehydrogenase type 3: structural clues of 5α-DHT reverse binding and enzyme down-regulation decreasing MCF7 cell growth.

    Science.gov (United States)

    Zhang, Bo; Hu, Xiao-Jian; Wang, Xiao-Qiang; Thériault, Jean-François; Zhu, Dao-Wei; Shang, Peng; Labrie, Fernand; Lin, Sheng-Xiang

    2016-04-15

    Human 3α-HSD3 (3α-hydroxysteroid dehydrogenase type 3) plays an essential role in the inactivation of the most potent androgen 5α-DHT (5α-dihydrotestosterone). The present study attempts to obtain the important structure of 3α-HSD3 in complex with 5α-DHT and to investigate the role of 3α-HSD3 in breast cancer cells. We report the crystal structure of human 3α-HSD3·NADP(+)·A-dione (5α-androstane-3,17-dione)/epi-ADT (epiandrosterone) complex, which was obtained by co-crystallization with 5α-DHT in the presence of NADP(+) Although 5α-DHT was introduced during the crystallization, oxidoreduction of 5α-DHT occurred. The locations of A-dione and epi-ADT were identified in the steroid-binding sites of two 3α-HSD3 molecules per crystal asymmetric unit. An overlay showed that A-dione and epi-ADT were oriented upside-down and flipped relative to each other, providing structural clues for 5α-DHT reverse binding in the enzyme with the generation of different products. Moreover, we report the crystal structure of the 3α-HSD3·NADP(+)·4-dione (4-androstene-3,17-dione) complex. When a specific siRNA (100 nM) was used to suppress 3α-HSD3 expression without interfering with 3α-HSD4, which shares a highly homologous active site, the 5α-DHT concentration increased, whereas MCF7 cell growth was suppressed. The present study provides structural clues for 5α-DHT reverse binding within 3α-HSD3, and demonstrates for the first time that down-regulation of 3α-HSD3 decreases MCF7 breast cancer cell growth. © 2016 The Author(s). Published by Portland Press Limited on behalf of the Biochemical Society.

  1. A Purple Cupredoxin from Nitrosopumilus maritimus Containing a Mononuclear Type 1 Copper Center with an Open Binding Site

    Energy Technology Data Exchange (ETDEWEB)

    Hosseinzadeh, Parisa; Tian, Shiliang; Marshall, Nicholas M.; Hemp, James; Mullen, Timothy; Nilges, Mark J.; Gao, Yi-Gui; Robinson, Howard; Stahl, David A.; Gennis, Robert B.; Lu, Yi

    2016-05-25

    Mononuclear cupredoxin proteins usually contain a coordinately saturated type 1 copper (T1Cu) center and function exclusively as electron carriers. Here we report a cupredoxin isolated from the nitrifying archaeon Nitrosopumilus maritimus SCM1, called Nmar1307, that contains a T1Cu center with an open binding site containing water. It displays a deep purple color due to strong absorptions around 413 nm (1880 M–1 cm–1) and 558 nm (2290 M–1 cm–1) in the UV–vis electronic spectrum. EPR studies suggest the protein contains two Cu(II) species of nearly equal population, one nearly axial, with hyperfine constant A∥ = 98 × 10–4 cm–1, and another more rhombic, with a smaller A∥ value of 69 × 10–4 cm–1. The X-ray crystal structure at 1.6 Å resolution confirms that it contains a Cu atom coordinated by two His and one Cys in a trigonal plane, with an axial H2O at 2.25 Å. Both UV–vis absorption and EPR spectroscopic studies suggest that the Nmar1307 can oxidize NO to nitrite, an activity that is attributable to the high reduction potential (354 mV vs SHE) of the copper site. These results suggest that mononuclear cupredoxins can have a wide range of structural features, including an open binding site containing water, making this class of proteins even more versatile.

  2. Further Characterization of the UL37 Protein of Herpes Simplex Virus Type 1 and its Interaction with ICP8, the Major DNA-Binding Protein of Herpes Simplex Virus

    Science.gov (United States)

    1994-01-01

    Baringer, J.R. 1974. Recovery of herpes simplex virus from human sacral ganglions. N. Eng!. J. Med. 291:828-830. Baringer, J.R. 1976. The biology of herpes ...UL37 Protein of Herpes Simplex Virus Type 1 and its Interaction with [CPS, the Major DNA~Binding Protein of Herpes Simplex Virus" beyond brief...Protein of Herpes Simplex Virus Type 1 and its Interaction with [CPS, the Major DNA-Binding Protein of Herpes Simplex Virus Allen G. Albright Doctor of

  3. Cell-type specific role of the RNA-binding protein, NONO, in the DNA double-strand break response in the mouse testes.

    Science.gov (United States)

    Li, Shuyi; Shu, Feng-Jue; Li, Zhentian; Jaafar, Lahcen; Zhao, Shourong; Dynan, William S

    2017-03-01

    The tandem RNA recognition motif protein, NONO, was previously identified as a candidate DNA double-strand break (DSB) repair factor in a biochemical screen for proteins with end-joining stimulatory activity. Subsequent work showed that NONO and its binding partner, SFPQ, have many of the properties expected for bona fide repair factors in cell-based assays. Their contribution to the DNA damage response in intact tissue in vivo has not, however, been demonstrated. Here we compare DNA damage sensitivity in the testes of wild-type mice versus mice bearing a null allele of the NONO homologue (Nono gt ). In wild-type mice, NONO protein was present in Sertoli, peritubular myoid, and interstitial cells, with an increase in expression following induction of DNA damage. As expected for the product of an X-linked gene, NONO was not detected in germ cells. The Nono gt/0 mice had at most a mild testis developmental phenotype in the absence of genotoxic stress. However, following irradiation at sublethal, 2-4 Gy doses, Nono gt/0 mice displayed a number of indicators of radiosensitivity as compared to their wild-type counterparts. These included higher levels of persistent DSB repair foci, increased numbers of apoptotic cells in the seminiferous tubules, and partial degeneration of the blood-testis barrier. There was also an almost complete loss of germ cells at later times following irradiation, evidently arising as an indirect effect reflecting loss of stromal support. Results demonstrate a role for NONO protein in protection against direct and indirect biological effects of ionizing radiation in the whole animal. Copyright © 2017 Elsevier B.V. All rights reserved.

  4. Backbone and sidechain 1H, 13C and 15N resonance assignments of the human brain-type fatty acid binding protein (FABP7) in its apo form and the holo forms binding to DHA, oleic acid, linoleic acid and elaidic acid

    DEFF Research Database (Denmark)

    Oeemig, Jesper S; Jørgensen, Mathilde L; Hansen, Mikka S

    2009-01-01

    In this manuscript, we present the backbone and side chain assignments of human brain-type fatty acid binding protein, also known as FABP7, in its apo form and in four different holo forms, bound to DHA, oleic acid, linoleic acid and elaidic acid.......In this manuscript, we present the backbone and side chain assignments of human brain-type fatty acid binding protein, also known as FABP7, in its apo form and in four different holo forms, bound to DHA, oleic acid, linoleic acid and elaidic acid....

  5. Spectroscopic Evidence for Covalent Binding of Sulfadiazine to Natural Soils via 1,4-nucleophilic addition (Michael Type Addition) studied by Spin Labeling ESR

    Science.gov (United States)

    Aleksandrova, Olga

    2015-04-01

    with different polarity. As shown by the spin labeling ESR experiment, molecules modeling SDZ were promptly bound to non-hydrolysable network of soil organic matter only via the aromatic amines that was accompanied by a prompt enlargement of humic particles binding aromatic amines, whereas binding of decomposition products of SDZ to humic acids of soil via the aliphatic amines was not observable. The ESR spectra obviously showed a single-phase process of covalent binding of the aromatic amines. Repeated washouts of labeled soil samples using distil water and ultrafiltration through the membrane of 5000 MWCO PES confirmed irreversible binding of the aromatic amines, and showed that via the aliphatic amines, binding of SDZ or decomposition products of SDZ to soil might also occur but reversibly and only to small soil molecules, which don't enter into the composition of non-hydrolysable part of soil organic matter. SL ESR experiments of different soils at the presence of Laccase highlighted that covalent binding of the aromatic amines to humic particles occurred in the specific hydrophobic areas of soil found as depleted in oxygen. All measured data evidenced that first, SDZ might be decomposed that allowed for measuring the same change of a paramagnetic signal of soil organic matter influenced by both aromatic and aliphatic amines as in the experiment of the interaction of soil with SDZ. Second, a decomposition product of SDZ with the aromatic amine might be bound to non-hydrolysable parts of soil organic matter under specific anaerobic conditions only via 1,4 - nucleophilic addition, Michael-type addition. Gulkowska, A., Thalmann, B., D., Hollender, J., & Krauss, M. (2014). Chemosphere, 107, 366 - 372. Müller, T., Rosendahl, I., Focks, A., Siemens, J., Klasmeier, J., & Matthies. (2013). Environmental Pollution, 172,180 - 185. Nowak, K.M., Miltner, A., Gehre, M., Schaeffer, A., & Kaestner, M. (2011). Environmental Science & Technology 45, 999 - 1006. Weber, E.J., Spidle

  6. Major groove binding track residues of the connection subdomain of human immunodeficiency virus type 1 reverse transcriptase enhance cDNA synthesis at high temperatures.

    Science.gov (United States)

    Matamoros, Tania; Barrioluengo, Verónica; Abia, David; Menéndez-Arias, Luis

    2013-12-23

    At high temperatures, RNA denaturation can improve the efficiency and specificity of reverse transcription. Refined structures and molecular models of HIV-1 reverse transcriptases (RTs) from phylogenetically distant clades (i.e., group M subtype B and group O) revealed a major interaction between the template-primer and the Arg³⁵⁸-Gly³⁵⁹-Ala³⁶⁰ triad in the large subunit of HIV-1M/B RT. However, fewer contacts were predicted for the equivalent Lys³⁵⁸-Ala³⁵⁹-Ser³⁶⁰ triad of HIV-1O RT and the nucleic acid. An engineered HIV-1O K358R/A359G/S360A RT showed increased cDNA synthesis efficiency above 68 °C, as determined by qualitative and quantitative reverse transcription polymerase chain reactions. In comparison with wild-type HIV-1O RT, the mutant enzyme showed higher thermal stability but retained wild-type RNase H activity. Mutations that increased the accuracy of HIV-1M/B RTs were tested in combination with the K358R/A359G/S360A triple mutation. Some of them (e.g., F61A, K65R, K65R/V75I, and V148I) had a negative effect on reverse transcription efficiency above 65 °C. RTs with improved DNA binding affinities also showed higher cDNA synthesis efficiencies at elevated temperatures. Two of the most thermostable RTs (i.e., mutants T69SSG/K358R/A359G/S360A and K358R/A359G/S360A/E478Q) showed moderately increased fidelity in forward mutation assays. Our results demonstrate that the triad of Arg³⁵⁸, Gly³⁵⁹, and Ala³⁶⁰ in the major groove binding track of HIV-1 RT is a major target for RT stabilization, and most relevant for improving reverse transcription efficiency at high temperatures.

  7. Serotonin storage pools in basophil leukemia and mast cells: characterization of two types of serotonin binding protein and radioautographic analysis of the intracellular distribution of [3H]serotonin

    International Nuclear Information System (INIS)

    Tamir, H.; Theoharides, T.C.; Gershon, M.D.; Askenase, P.W.

    1982-01-01

    The binding of serotonin to protein(s) derived from rat basophil leukemia (RBL) cells and mast cells was studied. Two types of serotonin binding protein in RBL cells was found. These proteins differed from one another in molecular weight and eluted in separate peaks from sephadex G-200 columns. Peak I protein (KD = 1.9 x 10 -6 M) was a glycoprotein that bound to concanavalin A (Con A); Peak II protein (KD 1 = 4.5 x 10 - 8 M; KD 2 = 3.9 x 10 -6 M) did not bind to Con A. Moreover, binding of [ 3 H]serotonin to protein of Peak I was sensitive to inhibition by reserpine, while binding of [ 3 H]serotonin to protein of Peak II resisted inhibition by that drug. Other differences between the two types of binding protein were found, the most significant of which was the far more vigorous conditions of homogenization required to extract Peak I than Peak II protein. Electron microscope radioautographic analysis of the intracellular distribution of [ 3 H] serotonin taken up in vitro by RBL cells or in vivo by murine mast cells indicated that essentially all of the labeled amine was located in cytoplasmic granules.No evidence for a pool in the cytosol was found and all granules were capable of becoming labeled. The presence of two types of intracellular serotonin binding proteins in these cells may indicate that there are two intracellular storage compartments for the amine. Both may be intragranular, but Peak I protein may be associated with the granular membrane while Peak II protein may be more free within the granular core. Different storage proteins may help to explain the differential release of amines from mast cell granules

  8. PDZ domain-binding motif of human T-cell leukemia virus type 1 Tax oncoprotein augments the transforming activity in a rat fibroblast cell line

    International Nuclear Information System (INIS)

    Hirata, Akira; Higuchi, Masaya; Niinuma, Akiko; Ohashi, Minako; Fukushi, Masaya; Oie, Masayasu; Akiyama, Tetsu; Tanaka, Yuetsu; Gejyo, Fumitake; Fujii, Masahiro

    2004-01-01

    While human T-cell leukemia virus type 1 (HTLV-1) is associated with the development of adult T-cell leukemia (ATL), HTLV-2 has not been reported to be associated with such malignant leukemias. HTLV-1 Tax1 oncoprotein transforms a rat fibroblast cell line (Rat-1) to form multiple large colonies in soft agar, and this activity is much greater than that of HTLV-2 Tax2. We have demonstrated here that the increased number of transformed colonies induced by Tax1 relative to Tax2 was mediated by a PDZ domain-binding motif (PBM) in Tax1, which is absent in Tax2. Tax1 PBM mediated the interaction of Tax1 with the discs large (Dlg) tumor suppressor containing PDZ domains, and the interaction correlated well with the transforming activities of Tax1 and the mutants. Through this interaction, Tax1 altered the subcellular localization of Dlg from the detergent-soluble to the detergent-insoluble fraction in a fibroblast cell line as well as in HTLV-1-infected T-cell lines. These results suggest that the interaction of Tax1 with PDZ domain protein(s) is critically involved in the transforming activity of Tax1, the activity of which may be a crucial factor in malignant transformation of HTLV-1-infected cells in vivo

  9. A Comparison of the Effects of Aerobic and Intense Exercise on the Type 2 Diabetes Mellitus Risk Marker Adipokines, Adiponectin and Retinol Binding Protein-4

    Directory of Open Access Journals (Sweden)

    Amy Phillips

    2014-01-01

    Full Text Available With a more sedentary population comes growing rates of obesity and increased type 2 diabetes mellitus (T2DM risk. Exercise generally induces positive changes in traditional T2DM risk markers such as lipids, glucose tolerance, and insulin sensitivity; however alterations in concentrations of many circulating cytokines and their respective receptors are also becoming apparent. These cytokines may be early-response health risk factors otherwise overlooked in traditional T2DM risk marker analysis. Plasma levels of two adipocyte-originating cytokines, adiponectin and retinol binding protein 4 (RBP-4, alter following exercise. Adiponectin has anti-inflammatory, anti-atherosclerotic, and anti-insulin resistance roles and its secretion increases with physical activity, whilst elevated RBP-4 leads to increased insulin resistance, and secretion decreases with increasing physical activity; thus these plasma adipokine levels alter favourably following exercise. Although current data are limited, they do suggest that the more intense the exercise, the greater the positive effect on plasma RBP-4 levels, whilst lower intensity aerobic exercise may positively improve adiponectin concentrations. Therefore short-duration, high intensity training may provide a time-efficient alternative to the recommended 150 min moderate aerobic exercise per week in providing positive changes in RBP-4 and other traditional T2DM risk markers and due to increased compliance give greater health benefits over the longer term.

  10. Hyaluronan and Hyaluronan-Binding Proteins Accumulate in Both Human Type 1 Diabetic Islets and Lymphoid Tissues and Associate With Inflammatory Cells in Insulitis

    Science.gov (United States)

    Bogdani, Marika; Johnson, Pamela Y.; Potter-Perigo, Susan; Nagy, Nadine; Day, Anthony J.; Bollyky, Paul L.

    2014-01-01

    Hyaluronan (HA) is an extracellular matrix glycosaminoglycan that is present in pancreatic islets, but little is known about its involvement in the development of human type 1 diabetes (T1D). We have evaluated whether pancreatic islets and lymphoid tissues of T1D and nondiabetic organ donors differ in the amount and distribution of HA and HA-binding proteins (hyaladherins), such as inter-α-inhibitor (IαI), versican, and tumor necrosis factor–stimulated gene-6 (TSG-6). HA was dramatically increased both within the islet and outside the islet endocrine cells, juxtaposed to islet microvessels in T1D. In addition, HA was prominent surrounding immune cells in areas of insulitis. IαI and versican were present in HA-rich areas of islets, and both molecules accumulated in diabetic islets and regions exhibiting insulitis. TSG-6 was observed within the islet endocrine cells and in inflammatory infiltrates. These patterns were only observed in tissues from younger donors with disease duration of <10 years. Furthermore, HA and IαI amassed in follicular germinal centers and in T-cell areas in lymph nodes and spleens in T1D patients compared with control subjects. Our observations highlight potential roles for HA and hyaladherins in the pathogenesis of diabetes. PMID:24677718

  11. Correlation between Heart-type Fatty Acid-binding Protein Gene Polymorphism and mRNA Expression with Intramuscular Fat in Baicheng-oil Chicken

    Directory of Open Access Journals (Sweden)

    Yong Wang

    2015-10-01

    Full Text Available This study aims to determine the polymorphism and mRNA expression pattern of the heart-type fatty acid-binding protein (H-FABP gene and their association with intramuscular fat (IMF content in the breast and leg muscles of Baicheng oil chicken (BOC. A total of 720 chickens, including 240 black Baicheng oil chicken (BBOC, 240 silky Baicheng oil chicken (SBOC, and 240 white Baicheng oil chicken (WBOC were raised. Three genotypes of H-FABP gene second extron following AA, AB, and BB were detected by polymerase chain reaction-single strand conformation polymorphism (PCR-SSCP strategy. The G939A site created AA genotype and G956A site created BB genotype. The content of IMF in AA genotype in breast muscle of BBOC was significantly higher than that of AB (p = 0.0176 and the genotype in leg muscle of WBOC was significantly higher than that of AB (p = 0.0145. The G939A site could be taken as genetic marker for higher IMF content selecting for breast muscle of BBOC and leg muscle of WBOC. The relative mRNA expression of H-FABP was measured by real-time PCR at 30, 60, 90, and 120 d. The IMF content significantly increased with age in both muscles. The mRNA expression level of H-FABP significantly decreased with age in both muscles of the three types of chickens. Moreover, a significant negative correlation between H-FABP abundance and IMF content in the leg muscles of WBOC (p = 0.035 was observed. The mRNA expression of H-FABP negatively correlated with the IMF content in both breast and leg muscles of BOC sat slaughter time.

  12. The capacity of Listeria monocytogenes mutants with in-frame deletions in putative ATP-binding cassette transporters to form biofilms and comparison with the wild type

    Directory of Open Access Journals (Sweden)

    Marina Ceruso

    2014-02-01

    Full Text Available Listeria monocytogenes (Lm is a food-borne pathogen responsible for human listeriosis, an invasive infection with high mortality rates. Lm has developed efficient strategies for survival under stress conditions such as starvation and wide variations in temperature, pH, and osmolarity. Therefore, Lm can survive in food under multiple stress conditions. Detailed studies to determine the mode of action of this pathogen for survival under stress conditions are important to control Lm in food. It has been shown that genes encoding for ATP-binding cassette (ABC transporters are induced in Lm in food, in particular under stress conditions. Previous studies showed that these genes are involved in sensitivity to nisin, acids, and salt. The aim of this study was to determine the involvement of some ABC transporters in biofilm formation. Therefore, deletion mutants of ABC transporter genes (LMOf2365_1875 and LMOf2365_1877 were created in Lm F2365, and then were compared to the wild type for their capacity to form biofilms. Lm strain F2365 was chosen as reference since the genome is fully sequenced and furthermore this strain is particularly involved in food-borne outbreaks of listeriosis. Our results showed that DLMOf2365_1875 had an increased capacity to form biofilms compared to the wild type, indicating that LMOf2365_1875 negatively regulates biofilm formation. A deeper knowledge on the ability to form biofilms in these mutants may help in the development of intervention strategies to control Lm in food and in the environment.

  13. Structure of the plasminogen kringle 4 binding calcium-free form of the C-type lectin-like domain of tetranectin

    DEFF Research Database (Denmark)

    Nielbo, Steen Günther; Thomsen, J.K.; Graversen, J. H.

    2004-01-01

    . A conserved proline, which was found to be in the cis conformation in holoTN3, is in apoTN3 predominantly in the trans conformation. Backbone dynamics indicate that, in apoTN3 especially, two of the three calcium-binding loops and two of the three K4-binding residues exhibit increased flexibility, whereas...

  14. Effects of Mutations and Ligands on the Thermostability of the l-Arginine/Agmatine Antiporter AdiC and Deduced Insights into Ligand-Binding of Human l-Type Amino Acid Transporters

    Directory of Open Access Journals (Sweden)

    Hüseyin Ilgü

    2018-03-01

    Full Text Available The l-arginine/agmatine transporter AdiC is a prokaryotic member of the SLC7 family, which enables pathogenic enterobacteria to survive the extremely acidic gastric environment. Wild-type AdiC from Escherichia coli, as well as its previously reported point mutants N22A and S26A, were overexpressed homologously and purified to homogeneity. A size-exclusion chromatography-based thermostability assay was used to determine the melting temperatures (Tms of the purified AdiC variants in the absence and presence of the selected ligands l-arginine (Arg, agmatine, l-arginine methyl ester, and l-arginine amide. The resulting Tms indicated stabilization of AdiC variants upon ligand binding, in which Tms and ligand binding affinities correlated positively. Considering results from this and previous studies, we revisited the role of AdiC residue S26 in Arg binding and proposed interactions of the α-carboxylate group of Arg exclusively with amide groups of the AdiC backbone. In the context of substrate binding in the human SLC7 family member l-type amino acid transporter-1 (LAT1; SLC7A5, an analogous role of S66 in LAT1 to S26 in AdiC is discussed based on homology modeling and amino acid sequence analysis. Finally, we propose a binding mechanism for l-amino acid substrates to LATs from the SLC7 family.

  15. Effects of Mutations and Ligands on the Thermostability of the l-Arginine/Agmatine Antiporter AdiC and Deduced Insights into Ligand-Binding of Human l-Type Amino Acid Transporters.

    Science.gov (United States)

    Ilgü, Hüseyin; Jeckelmann, Jean-Marc; Colas, Claire; Ucurum, Zöhre; Schlessinger, Avner; Fotiadis, Dimitrios

    2018-03-20

    The l-arginine/agmatine transporter AdiC is a prokaryotic member of the SLC7 family, which enables pathogenic enterobacteria to survive the extremely acidic gastric environment. Wild-type AdiC from Escherichia coli, as well as its previously reported point mutants N22A and S26A, were overexpressed homologously and purified to homogeneity. A size-exclusion chromatography-based thermostability assay was used to determine the melting temperatures ( T m s) of the purified AdiC variants in the absence and presence of the selected ligands l-arginine (Arg), agmatine, l-arginine methyl ester, and l-arginine amide. The resulting T m s indicated stabilization of AdiC variants upon ligand binding, in which T m s and ligand binding affinities correlated positively. Considering results from this and previous studies, we revisited the role of AdiC residue S26 in Arg binding and proposed interactions of the α-carboxylate group of Arg exclusively with amide groups of the AdiC backbone. In the context of substrate binding in the human SLC7 family member l-type amino acid transporter-1 (LAT1; SLC7A5), an analogous role of S66 in LAT1 to S26 in AdiC is discussed based on homology modeling and amino acid sequence analysis. Finally, we propose a binding mechanism for l-amino acid substrates to LATs from the SLC7 family.

  16. A platform to screen for C-type lectin receptor-binding carbohydrates and their potential for cell-specific targeting and immune modulation.

    Science.gov (United States)

    Maglinao, Maha; Eriksson, Magdalena; Schlegel, Mark K; Zimmermann, Stephanie; Johannssen, Timo; Götze, Sebastian; Seeberger, Peter H; Lepenies, Bernd

    2014-02-10

    Myeloid C-type lectin receptors (CLRs) in innate immunity represent a superfamily of pattern recognition receptors that recognize carbohydrate structures on pathogens and self-antigens. The primary interaction of an antigen-presenting cell and a pathogen shapes the following immune response. Therefore, the identification of CLR ligands that can either enhance or modulate the immune response is of interest. We have developed a screening platform based on glycan arrays to identify immune modulatory carbohydrate ligands of CLRs. A comprehensive library of CLRs was expressed by fusing the extracellular part of each respective CLR, the part containing the carbohydrate-recognition domain (CRD), to the Fc fragment of human IgG1 molecules. CLR-Fc fusion proteins display the CRD in a dimeric form, are properly glycosylated, and can be detected by a secondary antibody with a conjugated fluorophore. Thus, they are valuable tools for high-throughput screening. We were able to identify novel carbohydrate binders of CLRs using the glycan array technology. These CLR-binding carbohydrates were then covalently attached to the model antigen ovalbumin. The ovalbumin neoglycoconjugates were used in a dendritic cell/T cell co-culture assay to stimulate transgenic T cells in vitro. In addition, mice were immunized with these conjugates to analyze the immune modulatory properties of the CLR ligands in vivo. The CLR ligands induced an increased Th1 cytokine production in vitro and modulated the humoral response in vivo. The platform described here allows for the identification of CLR ligands, as well as the evaluation of each ligand's cell-specific targeting and immune modulatory properties. Copyright © 2013 Elsevier B.V. All rights reserved.

  17. The Ala54Thr Polymorphism of the Fatty Acid Binding Protein 2 Gene Modulates HDL Cholesterol in Mexican-Americans with Type 2 Diabetes

    Directory of Open Access Journals (Sweden)

    Lorena M. Salto

    2015-12-01

    Full Text Available The alanine to threonine amino acid substitution at codon 54 (Ala54Thr of the intestinal fatty acid binding protein (FABP2 has been associated with elevated levels of insulin and blood glucose as well as with dyslipidemia. The aim of this study was to characterize the effect of this FABP2 polymorphism in Mexican-Americans with type 2 diabetes (T2D in the context of a three-month intervention to determine if the polymorphism differentially modulates selected clinical outcomes. For this study, we genotyped 43 participant samples and performed post-hoc outcome analysis of the profile changes in fasting blood glucose, HbA1c, insulin, lipid panel and body composition, stratified by the Ala54Thr polymorphism. Our results show that the Thr54 allele carriers (those who were heterozygous or homozygous for the threonine-encoding allele had lower HDL cholesterol and higher triglyceride levels at baseline compared to the Ala54 homozygotes (those who were homozygous for the alanine-encoding allele. Both groups made clinically important improvements in lipid profiles and glycemic control as a response to the intervention. Whereas the Ala54 homozygotes decreased HDL cholesterol in the context of an overall total cholesterol decrease, Thr54 allele carriers increased HDL cholesterol as part of an overall total cholesterol decrease. We conclude that the Ala54Thr polymorphism of FABP2 modulates HDL cholesterol in Mexican-Americans with T2D and that Thr54 allele carriers may be responsive in interventions that include dietary changes.

  18. Omega 3 (peripheral type benzodiazepine binding) site distribution in the rat immune system: an autoradiographic study with the photoaffinity ligand [3H]PK 14105

    International Nuclear Information System (INIS)

    Benavides, J.; Dubois, A.; Dennis, T.; Hamel, E.; Scatton, B.

    1989-01-01

    The anatomical distribution of omega 3 (peripheral type benzodiazepine binding) sites in the immune system organs of the rat has been studied autoradiographically at both macroscopic and microscopic levels of resolution using either reversible or irreversible (UV irradiation) labeling with [ 3 H]PK 14105. In thymus sections, [ 3 H]PK 14105 labeled with high affinity (Kd, derived from saturation experiments = 10.8 nM) a single population of sites which possessed the pharmacological characteristics of omega 3 sites. In the thymus gland, higher omega 3 site densities were detected in the cortex than in the medulla; in these subregions, silver grains were associated to small (10-18 microns diameter) cells. In the spleen, omega 3 sites were more abundant in the white than in the red pulp. In the white pulp, silver grains were denser in the marginal zone than in the vicinity of the central artery and labeling was, as in the thymus, associated to small cytoplasm-poor cells. In the red pulp, omega 3 site associated silver grains were observed mainly in the Bilroth cords. In the lymph nodes, the medullary region showed a higher labeling than the surrounding follicles and paracortex. A significant accumulation of silver grains was observed in the lymph node medullary cords. In the intestine, Peyer patches were particularly enriched in omega 3 sites (especially in the periphery of the follicles). The distribution of omega 3 sites in the immune system organs suggests a preferential labeling of cells of T and monocytic lineages. This is consistent with the proposed immunoregulatory properties of some omega 3 site ligands

  19. Association of heart-type fatty acid-binding protein with cardiovascular risk factors and all-cause mortality in the general population: the Takahata study.

    Directory of Open Access Journals (Sweden)

    Yoichiro Otaki

    Full Text Available Despite many recent advances in medicine, preventing the development of cardiovascular diseases remains a challenge. Heart-type fatty acid-binding protein (H-FABP is a marker of ongoing myocardial damage and has been reported to be a useful indicator for future cardiovascular events. However, it remains to be determined whether H-FABP can predict all-cause and cardiovascular deaths in the general population.This longitudinal cohort study included 3,503 subjects who participated in a community-based health checkup with a 7-year follow-up. Serum H-FABP was measured in registered subjects. The results demonstrated that higher H-FABP levels were associated with increasing numbers of cardiovascular risk factors, including hypertension, diabetes mellitus, obesity, and metabolic syndrome. There were 158 deaths during the follow-up period, including 50 cardiovascular deaths. Deceased subjects had higher H-FABP levels compared to surviving subjects. Multivariate Cox proportional hazard regression analysis revealed that H-FABP is an independent predictor of all-cause and cardiovascular deaths after adjustments for confounding factors. Subjects were divided into four quartiles according to H-FABP level, and Kaplan-Meier analysis demonstrated that the highest H-FABP quartile was associated with the greatest risks for all-cause and cardiovascular deaths. Net reclassification index and integrated discrimination index were significantly increased by addition of H-FABP to cardiovascular risk factors.H-FABP level was increased in association with greater numbers of cardiovascular risk factors and was an independent risk factor for all-cause and cardiovascular deaths. H-FABP could be a useful indicator for the early identification of high-risk subjects in the general population.

  20. TANK-Binding Kinase 1 (TBK1 Isoforms Negatively Regulate Type I Interferon Induction by Inhibiting TBK1-IRF3 Interaction and IRF3 Phosphorylation

    Directory of Open Access Journals (Sweden)

    Yi Wei Hu

    2018-01-01

    Full Text Available TANK-binding kinase 1 (TBK1 is an important serine/threonine-protein kinase that mediates phosphorylation and nuclear translocation of IRF3, which contributes to induction of type I interferons (IFNs in the innate antiviral response. In mammals, TBK1 spliced isoform negatively regulates the virus-triggered IFN-β signaling pathway by disrupting the interaction between retinoic acid-inducible gene I (RIG-I and mitochondria antiviral-signaling protein (MAVS. However, it is still unclear whether alternative splicing patterns and the function of TBK1 isoform(s exist in teleost fish. In this study, we identify two alternatively spliced isoforms of TBK1 from zebrafish, termed TBK1_tv1 and TBK1_tv2. Both TBK1_tv1 and TBK1_tv2 contain an incomplete STKc_TBK1 domain. Moreover, the UBL_TBK1_like domain is also missing for TBK1_tv2. TBK1_tv1 and TBK1_tv2 are expressed in zebrafish larvae. Overexpression of TBK1_tv1 and TBK1_tv2 inhibits RIG-I-, MAVS-, TBK1-, and IRF3-mediated activation of IFN promoters in response to spring viremia of carp virus infection. Also, TBK1_tv1 and TBK1_tv2 inhibit expression of IFNs and IFN-stimulated genes induced by MAVS and TBK1. Mechanistically, TBK1_tv1 and TBK1_tv2 competitively associate with TBK1 and IRF3 to disrupt the formation of a functional TBK1-IRF3 complex, impeding the phosphorylation of IRF3 mediated by TBK1. Collectively, these results demonstrate that TBK1 spliced isoforms are dominant negative regulators in the RIG-I/MAVS/TBK1/IRF3 antiviral pathway by targeting the functional TBK1-IRF3 complex formation. Identification and functional characterization of piscine TBK1 spliced isoforms may contribute to understanding the role of TBK1 expression in innate antiviral response.

  1. Electrochemical and circular dichroism spectroscopic evidence of two types of interaction between [Ru(NH3)(6)](3+) and an elongated thrombin binding aptamer G-quadruplex

    Czech Academy of Sciences Publication Activity Database

    De Rache, A.; Kejnovská, Iva; Buess-Herman, C.; Doneux, T.

    2015-01-01

    Roč. 179, OCT 2015 (2015), s. 84-92 ISSN 0013-4686 Institutional support: RVO:68081707 Keywords : Biosensors * Thrombin binding aptamer * Hexaammineruthenium Subject RIV: BO - Biophysics Impact factor: 4.803, year: 2015

  2. The roles of the RIIβ linker and N-terminal cyclic nucleotide-binding domain in determining the unique structures of the type IIβ protein kinase A: a small angle x-ray and neutron scattering study.

    Science.gov (United States)

    Blumenthal, Donald K; Copps, Jeffrey; Smith-Nguyen, Eric V; Zhang, Ping; Heller, William T; Taylor, Susan S

    2014-10-10

    Protein kinase A (PKA) is ubiquitously expressed and is responsible for regulating many important cellular functions in response to changes in intracellular cAMP concentrations. The PKA holoenzyme is a tetramer (R2:C2), with a regulatory subunit homodimer (R2) that binds and inhibits two catalytic (C) subunits; binding of cAMP to the regulatory subunit homodimer causes activation of the catalytic subunits. Four different R subunit isoforms exist in mammalian cells, and these confer different structural features, subcellular localization, and biochemical properties upon the PKA holoenzymes they form. The holoenzyme containing RIIβ is structurally unique in that the type IIβ holoenzyme is much more compact than the free RIIβ homodimer. We have used small angle x-ray scattering and small angle neutron scattering to study the solution structure and subunit organization of a holoenzyme containing an RIIβ C-terminal deletion mutant (RIIβ(1-280)), which is missing the C-terminal cAMP-binding domain to better understand the structural organization of the type IIβ holoenzyme and the RIIβ domains that contribute to stabilizing the holoenzyme conformation. Our results demonstrate that compaction of the type IIβ holoenzyme does not require the C-terminal cAMP-binding domain but rather involves large structural rearrangements within the linker and N-terminal cyclic nucleotide-binding domain of the RIIβ homodimer. The structural rearrangements are significantly greater than seen previously with RIIα and are likely to be important in mediating short range and long range interdomain and intersubunit interactions that uniquely regulate the activity of the type IIβ isoform of PKA. © 2014 by The American Society for Biochemistry and Molecular Biology, Inc.

  3. DIFFERENTIAL BINDING OF HUMAN INTERLEUKIN-1 (IL-1) RECEPTOR ANTAGONIST TO NATURAL AND RECOMBINANT SOLUBLE AND CELLULAR IL-1 TYPE-I RECEPTORS

    DEFF Research Database (Denmark)

    Svenson, Morten; Nedergaard, Susanne; Heegaard, Peter M. H.

    1995-01-01

    antagonist (IL-1ra). Recombinant soluble human IL-1RI expressed in COS cells (sIL-1RI) consists of the extracellular part of the receptor and binds all three known IL-1 species but preferentially to IL-1ra. We further characterized the sizes and binding of IL-1raBF and sIL-1RI to IL-1ra by polyacrylamide gel...... electrophoresis in the presence of sodium dodecylsulfate, ligand binding interference analyses, N-glycosidase treatment, concanavalin A affinity chromatography, and with the use of monoclonal antibodies (mAb) to human recombinant IL-1ra. We also evaluated the binding of IL-1ra to cellular IL-1RI on MRC5...... binding of both molecules to IL-1ra. Both factors blocked binding of IL-1ra to cellular IL-1RI, as did mAb to IL-1ra, but the sites on IL-1ra which bound to the mAb, and to IL-1raBF and sIL-1RI, differed. We conclude that there are important differences between the natural and recombinant forms of soluble...

  4. An ice-binding and tandem beta-sandwich domain-containing protein in Shewanella frigidimarina is a potential new type of ice adhesin.

    Science.gov (United States)

    Vance, Tyler D R; Graham, Laurie A; Davies, Peter L

    2018-04-01

    Out of the dozen different ice-binding protein (IBP) structures known, the DUF3494 domain is the most widespread, having been passed many times between prokaryotic and eukaryotic microorganisms by horizontal gene transfer. This ~25-kDa β-solenoid domain with an adjacent parallel α-helix is most commonly associated with an N-terminal secretory signal peptide. However, examples of the DUF3494 domain preceded by tandem Bacterial Immunoglobulin-like (BIg) domains are sometimes found, though uncharacterized. Here, we present one such protein (SfIBP_1) from the Antarctic bacterium Shewanella frigidimarina. We have confirmed and characterized the ice-binding activity of its ice-binding domain using thermal hysteresis measurements, fluorescent ice plane affinity analysis, and ice recrystallization inhibition assays. X-ray crystallography was used to solve the structure of the SfIBP_1 ice-binding domain, to further characterize its ice-binding surface and unique method of stabilizing or 'capping' the ends of the solenoid structure. The latter is formed from the interaction of two loops mediated by a combination of tandem prolines and electrostatic interactions. Furthermore, given their domain architecture and membrane association, we propose that these BIg-containing DUF3494 IBPs serve as ice-binding adhesion proteins that are capable of adsorbing their host bacterium onto ice. Submitted new structure to the Protein Data Bank (PDB: 6BG8). © 2018 Federation of European Biochemical Societies.

  5. Analysis of a two-domain binding site for the urokinase-type plasminogen activator-plasminogen activator inhibitor-1 complex in low-density-lipoprotein-receptor-related protein.

    Science.gov (United States)

    Andersen, O M; Petersen, H H; Jacobsen, C; Moestrup, S K; Etzerodt, M; Andreasen, P A; Thøgersen, H C

    2001-07-01

    The low-density-lipoprotein-receptor (LDLR)-related protein (LRP) is composed of several classes of domains, including complement-type repeats (CR), which occur in clusters that contain binding sites for a multitude of different ligands. Each approximately 40-residue CR domain contains three conserved disulphide linkages and an octahedral Ca(2+) cage. LRP is a scavenging receptor for ligands from extracellular fluids, e.g. alpha(2)-macroglobulin (alpha(2)M)-proteinase complexes, lipoprotein-containing particles and serine proteinase-inhibitor complexes, like the complex between urokinase-type plasminogen activator (uPA) and the plasminogen activator inhibitor-1 (PAI-1). In the present study we analysed the interaction of the uPA-PAI-1 complex with an ensemble of fragments representing a complete overlapping set of two-domain fragments accounting for the ligand-binding cluster II (CR3-CR10) of LRP. By ligand blotting, solid-state competition analysis and surface-plasmon-resonance analysis, we demonstrate binding to multiple CR domains, but show a preferential interaction between the uPA-PAI-1 complex and a two-domain fragment comprising CR domains 5 and 6 of LRP. We demonstrate that surface-exposed aspartic acid and tryptophan residues at identical positions in the two homologous domains, CR5 and CR6 (Asp(958,CR5), Asp(999,CR6), Trp(953,CR5) and Trp(994,CR6)), are critical for the binding of the complex as well as for the binding of the receptor-associated protein (RAP) - the folding chaperone/escort protein required for transport of LRP to the cell surface. Accordingly, the present work provides (1) an identification of a preferred binding site within LRP CR cluster II; (2) evidence that the uPA-PAI-1 binding site involves residues from two adjacent protein domains; and (3) direct evidence identifying specific residues as important for the binding of uPA-PAI-1 as well as for the binding of RAP.

  6. A 48 kDa collagen-binding phosphoprotein isolated from bovine aortic endothelial cells interacts with the collagenous domain, but not the globular domain, of collagen type IV.

    Science.gov (United States)

    Yannariello-Brown, J; Madri, J A

    1990-01-15

    We have identified collagen-binding proteins in detergent extracts of metabolically labelled bovine aortic endothelial cells (BAEC) by collagen type IV-Sepharose affinity chromatography. The major collagen type IV-binding protein identified by SDS/PAGE had a molecular mass of 48 kDa, which we term the 'collagen-binding 48 kDa protein' (CB48). The pI of CB48 was 8.0-8.3 in a two-dimensional gel system, running non-equilibrium pH gel electrophoresis in the first dimension and SDS/PAGE in the second dimension. Under these conditions CB48 separated into two major (a and b) and one minor isoform (c); a was the most basic of the three isoforms. Two-dimensional chymotryptic peptide maps derived from each individual isoform were virtually identical. The charge differences between the isoforms were due in part to differential H3(32)PO4 incorporation by the protein. CB48 bound to intact collagen type IV and the collagenous region of collagen type IV, but not to the globular NC1 domain. Cell-surface labelling and indirect immunofluorescence experiments localized the bulk of CB48 intracellularly in the endoplasmic reticulum Golgi region, with a minor population of molecules on the cell surface. A specific rabbit polyclonal anti-CB48 serum did not inhibit the attachment or spreading of BAEC to collagen type IV in an 'in vitro' adhesion assay, suggesting that the cell-surface population of CB48 is not involved in BAEC adhesion. We conclude that CB48 is a collagen-binding phosphoprotein that interacts with the collagenous domain of collagen type IV and may be involved in intracellular transport of collagen molecules.

  7. In vitro and in vivo Analysis of the Binding of the C Terminus of the HDL Receptor Scavenger Receptor Class B type I (SR-BI) to the PDZ1 Domain of its Cytoplasmic Adaptor Protein PDZK1

    Energy Technology Data Exchange (ETDEWEB)

    O Kocher; G Birrane; K Tsukamoto; S Fenske; A Yesilaltay; R Pal; K Daniels; J Ladias; M Krieger

    2011-12-31

    The PDZ1 domain of the four PDZ domain-containing protein PDZK1 has been reported to bind the C terminus of the HDL receptor scavenger receptor class B, type I (SR-BI), and to control hepatic SR-BI expression and function. We generated wild-type (WT) and mutant murine PDZ1 domains, the mutants bearing single amino acid substitutions in their carboxylate binding loop (Lys(14)-Xaa(4)-Asn(19)-Tyr-Gly-Phe-Phe-Leu(24)), and measured their binding affinity for a 7-residue peptide corresponding to the C terminus of SR-BI ((503)VLQEAKL(509)). The Y20A and G21Y substitutions abrogated all binding activity. Surprisingly, binding affinities (K(d)) of the K14A and F22A mutants were 3.2 and 4.0 ?M, respectively, similar to 2.6 ?M measured for the WT PDZ1. To understand these findings, we determined the high resolution structure of WT PDZ1 bound to a 5-residue sequence from the C-terminal SR-BI ((505)QEAKL(509)) using x-ray crystallography. In addition, we incorporated the K14A and Y20A substitutions into full-length PDZK1 liver-specific transgenes and expressed them in WT and PDZK1 knock-out mice. In WT mice, the transgenes did not alter endogenous hepatic SR-BI protein expression (intracellular distribution or amount) or lipoprotein metabolism (total plasma cholesterol, lipoprotein size distribution). In PDZK1 knock-out mice, as expected, the K14A mutant behaved like wild-type PDZK1 and completely corrected their hepatic SR-BI and plasma lipoprotein abnormalities. Unexpectedly, the 10-20-fold overexpressed Y20A mutant also substantially, but not completely, corrected these abnormalities. The results suggest that there may be an additional site(s) within PDZK1 that bind(s) SR-BI and mediate(s) productive SR-BI-PDZK1 interaction previously attributed exclusively to the canonical binding of the C-terminal SR-BI to PDZ1.

  8. Structures of a bi-functional Kunitz-type STI family inhibitor of serine and aspartic proteases: Could the aspartic protease inhibition have evolved from a canonical serine protease-binding loop?

    Science.gov (United States)

    Guerra, Yasel; Valiente, Pedro A; Pons, Tirso; Berry, Colin; Rudiño-Piñera, Enrique

    2016-08-01

    Bi-functional inhibitors from the Kunitz-type soybean trypsin inhibitor (STI) family are glycosylated proteins able to inhibit serine and aspartic proteases. Here we report six crystal structures of the wild-type and a non-glycosylated mutant of the bifunctional inhibitor E3Ad obtained at different pH values and space groups. The crystal structures show that E3Ad adopts the typical β-trefoil fold of the STI family exhibiting some conformational changes due to pH variations and crystal packing. Despite the high sequence identity with a recently reported potato cathepsin D inhibitor (PDI), three-dimensional structures obtained in this work show a significant conformational change in the protease-binding loop proposed for aspartic protease inhibition. The E3Ad binding loop for serine protease inhibition is also proposed, based on structural similarity with a novel non-canonical conformation described for the double-headed inhibitor API-A from the Kunitz-type STI family. In addition, structural and sequence analyses suggest that bifunctional inhibitors of serine and aspartic proteases from the Kunitz-type STI family are more similar to double-headed inhibitor API-A than other inhibitors with a canonical protease-binding loop. Copyright © 2016. Published by Elsevier Inc.

  9. Expression of Ulex europaeus agglutinin I lectin-binding sites in squamous cell carcinomas and their absence in basal cell carcinomas. Indicator of tumor type and differentiation.

    Science.gov (United States)

    Heng, M C; Fallon-Friedlander, S; Bennett, R

    1992-06-01

    Lectins bind tightly to carbohydrate moieties on cell surfaces. Alterations in lectin binding have been reported to accompany epidermal cell differentiation, marking alterations in membrane sugars during this process. The presence of UEA I (Ulex europaeus agglutinin I) L-fucose-specific lectin-binding sites has been used as a marker for terminally differentiated (committed) keratinocytes. In this article, we report the presence of UEA-I-binding sites on squamous keratinocytes of well-differentiated squamous cell carcinomas, with patchy loss of UEA I positivity on poorly differentiated cells of squamous cell carcinomas, suggesting a possible use for this technique in the rapid assessment of less differentiated areas within the squamous cell tumor. The absence of UEA-I-binding sites on basal cell carcinomas may be related to an inability of cells comprising this tumor to convert the L-D-pyranosyl moiety on basal cells to the L-fucose moiety, resulting in an inability of basal cell carcinoma cell to undergo terminal differentiation into a committed keratinocyte.

  10. Structures of holo wild-type human cellular retinol-binding protein II (hCRBPII) bound to retinol and retinal.

    Science.gov (United States)

    Nossoni, Zahra; Assar, Zahra; Yapici, Ipek; Nosrati, Meisam; Wang, Wenjing; Berbasova, Tetyana; Vasileiou, Chrysoula; Borhan, Babak; Geiger, James

    2014-12-01

    Cellular retinol-binding proteins (CRBPs) I and II, which are members of the intracellular lipid-binding protein (iLBP) family, are retinoid chaperones that are responsible for the intracellular transport and delivery of both retinol and retinal. Although structures of retinol-bound CRBPI and CRBPII are known, no structure of a retinal-bound CRBP has been reported. In addition, the retinol-bound human CRBPII (hCRBPII) structure shows partial occupancy of a noncanonical conformation of retinol in the binding pocket. Here, the structure of retinal-bound hCRBPII and the structure of retinol-bound hCRBPII with retinol fully occupying the binding pocket are reported. It is further shown that the retinoid derivative seen in both the zebrafish CRBP and the hCRBPII structures is likely to be the product of flux-dependent and wavelength-dependent X-ray damage during data collection. The structures of retinoid-bound CRBPs are compared and contrasted, and rationales for the differences in binding affinities for retinal and retinol are provided.

  11. Heart-type fatty acid-binding protein (H-FABP) as an early diagnostic biomarker in patients with acute chest pain.

    Science.gov (United States)

    Vupputuri, Anjith; Sekhar, Saritha; Krishnan, Sajitha; Venugopal, K; Natarajan, K U

    2015-01-01

    Heart-type fatty acid-binding protein (H-FABP) is an emerging biomarker, which was found to be sensitive for the early diagnosis of acute myocardial infarction (AMI). We prospectively investigated the usefulness of H-FABP determination for the evaluation of acute chest pain in patients arriving at the emergency department. Fifty-four patients presenting with acute ischemic chest pain were evaluated. H-FABP was estimated at admission using latex-enhanced immunoturbidimetric assay. Serial cardiac troponin I (cTnI), creatinine kinase-MB (CK-MB) determination, ischemia workup with stress testing, and/or coronary angiogram (CAG) were performed according to standard protocols. The sensitivity and specificity of H-FABP was 89.7% and 68%, for cTnI it was 62.1% and 100%, and for CK-MB it was 44.8% and 92%, respectively for diagnosis of AMI. The sensitivity of H-FABP was found to be far superior to initial cTnI and CK-MB, for those seen within 6h (100% vs. 46.1%, 33% respectively). On further evaluation of patients with positive H-FABP and negative cTnI, 71.4% of the patients had significant lesion on CAG, indicating ischemic cause of H-FABP elevation. Six patients with normal cTnI and CK-MB with high H-FABP had ST elevation on subsequent ECGs and were taken for primary angioplasty. H-FABP is a highly sensitive biomarker for the early diagnosis of AMI. H-FABP as early marker and cTnI as late marker would be the ideal combination to cover the complete diagnostic window for AMI. Detection of myocardial injury by H-FABP may also be applied in patients with unstable angina. H-FABP can also be used as a marker for early detection of STEMI before the ECG changes become apparent. Copyright © 2015 Cardiological Society of India. Published by Elsevier B.V. All rights reserved.

  12. Point-of-care heart-type fatty acid binding protein versus high-sensitivity troponin T testing in emergency patients at high risk for acute coronary syndrome.

    Science.gov (United States)

    Kellens, Sebastiaan; Verbrugge, Frederik H; Vanmechelen, Maxime; Grieten, Lars; Van Lierde, Johan; Dens, Joseph; Vrolix, Mathias; Vandervoort, Pieter

    2016-04-01

    High-sensitivity cardiac troponin testing is used to detect myocardial damage in patients with acute chest pain. Heart-type fatty acid binding protein (H-FABP) may be an alternative, available as point-of-care test. Patients (n=203) referred by general practitioners for suspected acute coronary syndrome or presenting with typical chest pain and one major cardiovascular risk factor at the emergency department were prospectively included in a single-centre cohort study. High-sensitivity cardiac troponin T (hs-TnT) and point-of-care H-FABP testing were concomitantly performed at admission and after 6h. Maximal hs-TnT levels above the 99th percentile were observed in 152 patients (75%) with 127 (63%) fulfilling criteria for myocardial infarction. Upon admission, hs-TnT and H-FABP were associated with an area under the curve (95% CI) of 0.83 (0.77-0.89) and 0.79 (0.73-0.85), respectively, to predict myocardial infarction, which increased to 0.93 (0.90-0.97) and 0.88 (0.84-0.93), respectively, after 6h. The diagnostic accuracy for non-ST-segment elevation myocardial infarction was somewhat lower with an area under the curve (95% CI) of 0.80 (0.72-0.87), 0.90 (0.84-0.96), 0.73 (0.64-0.81) and 0.77 (0.67-0.86), respectively. When assessment was performed within 3h of chest pain onset, diagnostic accuracy of H-FABP versus hs-TnT was similar. Each standard deviation increase in admission H-FABP was associated with a 68% relative risk increase of all-cause mortality (p-value=0.027) during 666 ± 155 days of follow-up. Point-of-care H-FABP testing has lower diagnostic accuracy compared with hs-TnT assessment in patients with high pre-test acute coronary syndrome probability, but might be of interest when assessment is possible early after chest pain onset. © The European Society of Cardiology 2015.

  13. The Manchester Acute Coronary Syndromes (MACS) decision rule: validation with a new automated assay for heart-type fatty acid binding protein.

    Science.gov (United States)

    Body, Richard; Burrows, Gillian; Carley, Simon; Lewis, Philip S

    2015-10-01

    The Manchester Acute Coronary Syndromes (MACS) decision rule may enable acute coronary syndromes to be immediately 'ruled in' or 'ruled out' in the emergency department. The rule incorporates heart-type fatty acid binding protein (h-FABP) and high sensitivity troponin T levels. The rule was previously validated using a semiautomated h-FABP assay that was not practical for clinical implementation. We aimed to validate the rule with an automated h-FABP assay that could be used clinically. In this prospective diagnostic cohort study we included patients presenting to the emergency department with suspected cardiac chest pain. Serum drawn on arrival was tested for h-FABP using an automated immunoturbidimetric assay (Randox) and high sensitivity troponin T (Roche). The primary outcome, a diagnosis of acute myocardial infarction (AMI), was adjudicated based on 12 h troponin testing. A secondary outcome, major adverse cardiac events (MACE; death, AMI, revascularisation or new coronary stenosis), was determined at 30 days. Of the 456 patients included, 78 (17.1%) had AMI and 97 (21.3%) developed MACE. Using the automated h-FABP assay, the MACS rule had the same C-statistic for MACE as the original rule (0.91; 95% CI 0.88 to 0.92). 18.9% of patients were identified as 'very low risk' and thus eligible for immediate discharge with no missed AMIs and a 2.3% incidence of MACE (n=2, both coronary stenoses). 11.1% of patients were classed as 'high-risk' and had a 92.0% incidence of MACE. Our findings validate the performance of a refined MACS rule incorporating an automated h-FABP assay, facilitating use in clinical settings. The effectiveness of this refined rule should be verified in an interventional trial prior to implementation. UK CRN 8376. Published by the BMJ Publishing Group Limited. For permission to use (where not already granted under a licence) please go to http://group.bmj.com/group/rights-licensing/permissions.

  14. Effects of intraperitoneal insulin versus subcutaneous insulin administration on sex hormone-binding globulin concentrations in patients with type 1 diabetes mellitus

    Directory of Open Access Journals (Sweden)

    M Boering

    2016-06-01

    Full Text Available Aims Elevated sex hormone-binding globulin (SHBG concentrations have been described in patients with type 1 diabetes mellitus (T1DM, probably due to low portal insulin concentrations. We aimed to investigate whether the route of insulin administration, continuous intraperitoneal insulin infusion (CIPII, or subcutaneous (SC, influences SHBG concentrations among T1DM patients. Methods Post hoc analysis of SHBG in samples derived from a randomized, open-labeled crossover trial was carried out in 20 T1DM patients: 50% males, mean age 43 (±13 years, diabetes duration 23 (±11 years, and hemoglobin A1c (HbA1c 8.7 (±1.1 (72 (±12 mmol/mol. As secondary outcomes, testosterone, 17-β-estradiol, luteinizing hormone (LH, and follicle-stimulating hormone (FSH were analyzed. Results Estimated mean change in SHBG was −10.3nmol/L (95% CI: −17.4, −3.2 during CIPII and 3.7nmol/L (95% CI: −12.0, 4.6 during SC insulin treatment. Taking the effect of treatment order into account, the difference in SHBG between therapies was −6.6nmol/L (95% CI: −17.5, 4.3; −12.7nmol/L (95% CI: −25.1, −0.4 for males and −1.7nmol/L (95% CI: −24.6, 21.1 for females, respectively. Among males, SHBG and testosterone concentrations changed significantly during CIPII; −15.8nmol/L (95% CI: −24.2, −7.5 and −8.3nmol/L (95% CI: −14.4, −2.2, respectively. The difference between CIPII and SC insulin treatment was also significant for change in FSH 1.2U/L (95% CI: 0.1, 2.2 among males. Conclusions SHBG concentrations decreased significantly during CIPII treatment. Moreover, the difference in change between CIPII and SC insulin therapy was significant for SHBG and FSH among males. These findings support the hypothesis that portal insulin administration influences circulating SHBG and sex steroids.

  15. Analysis of the roles of E6 binding to E6TP1 and nuclear localization in the human papillomavirus type 31 life cycle

    International Nuclear Information System (INIS)

    Lee, Choongho; Wooldridge, Tonia R.; Laimins, Laimonis A.

    2007-01-01

    The E6 oncoproteins of high-risk human papillomaviruses provide important functions not only for malignant transformation but also in the productive viral life cycle. E6 proteins have been shown to bind to a number of cellular factors, but only a limited number of analyses have investigated the effects of these interactions on the viral life cycle. In this study, we investigated the consequences of HPV 31 E6 binding to E6TP1, a putative Rap1 GAP protein. HPV 16 E6 has been shown to bind as well as induce the rapid turnover of E6TP1, and similar effects were observed with HPV 31 E6. Mutation of amino acid 128 in HPV 31 E6 was found to abrogate the ability to bind and degrade E6TP1 but did not alter binding to another α-helical domain protein, E6AP. When HPV 31 genomes containing mutations at amino acid 128 were transfected into human keratinocytes, the viral DNAs were not stably maintained as episomes indicating the importance of this residue for pathogenesis. Many E6 binding partners including E6TP1 are cytoplasmic proteins, but E6 has been also reported to be localized to the nucleus. We therefore investigated the importance of E6 localization to the nucleus in the viral life cycle. Using a fusion of E6 to Green Fluorescent Protein, we mapped one component of the nuclear localization sequences to residues 121 to 124 of HPV 31 E6. Mutation of these residues in the context of the HPV 31 genome abrogated the ability for episomes to be stably maintained and impaired the ability to extend the life span of cells. These studies identify two activities of HPV 31 E6 that are important for its function in the viral life cycle and for extension of cell life span

  16. Effect of Multiple Mutations in the Hemoglobin- and Hemoglobin-Haptoglobin-Binding Proteins, HgpA, HgpB, and HgpC, of Haemophilus influenzae Type b

    OpenAIRE

    Morton, Daniel J.; Whitby, Paul W.; Jin, Hongfan; Ren, Zhen; Stull, Terrence L.

    1999-01-01

    Haemophilus influenzae requires heme for growth and can utilize hemoglobin and hemoglobin-haptoglobin as heme sources. We previously identified two hemoglobin- and hemoglobin-haptoglobin-binding proteins, HgpA and HgpB, in H. influenzae HI689. Insertional mutation of hgpA and hgpB, either singly or together, did not abrogate the ability to utilize or bind either hemoglobin or the hemoglobin-haptoglobin complex. A hemoglobin affinity purification method was used to isolate a protein of approxi...

  17. The calmodulin-binding, short linear motif, NSCaTE is conserved in L-type channel ancestors of vertebrate Cav1.2 and Cav1.3 channels.

    Directory of Open Access Journals (Sweden)

    Valentina Taiakina

    Full Text Available NSCaTE is a short linear motif of (xWxxx(I or Lxxxx, composed of residues with a high helix-forming propensity within a mostly disordered N-terminus that is conserved in L-type calcium channels from protostome invertebrates to humans. NSCaTE is an optional, lower affinity and calcium-sensitive binding site for calmodulin (CaM which competes for CaM binding with a more ancient, C-terminal IQ domain on L-type channels. CaM bound to N- and C- terminal tails serve as dual detectors to changing intracellular Ca(2+ concentrations, promoting calcium-dependent inactivation of L-type calcium channels. NSCaTE is absent in some arthropod species, and is also lacking in vertebrate L-type isoforms, Cav1.1 and Cav1.4 channels. The pervasiveness of a methionine just downstream from NSCaTE suggests that L-type channels could generate alternative N-termini lacking NSCaTE through the choice of translational start sites. Long N-terminus with an NSCaTE motif in L-type calcium channel homolog LCav1 from pond snail Lymnaea stagnalis has a faster calcium-dependent inactivation than a shortened N-termini lacking NSCaTE. NSCaTE effects are present in low concentrations of internal buffer (0.5 mM EGTA, but disappears in high buffer conditions (10 mM EGTA. Snail and mammalian NSCaTE have an alpha-helical propensity upon binding Ca(2+-CaM and can saturate both CaM N-terminal and C-terminal domains in the absence of a competing IQ motif. NSCaTE evolved in ancestors of the first animals with internal organs for promoting a more rapid, calcium-sensitive inactivation of L-type channels.

  18. Label-Free LC-MS Profiling of Skeletal Muscle Reveals Heart-Type Fatty Acid Binding Protein as a Candidate Biomarker of Aerobic Capacity

    Directory of Open Access Journals (Sweden)

    Zulezwan A. Malik

    2013-12-01

    Full Text Available Two-dimensional gel electrophoresis provides robust comparative analysis of skeletal muscle, but this technique is laborious and limited by its inability to resolve all proteins. In contrast, orthogonal separation by SDS-PAGE and reverse-phase liquid chromatography (RPLC coupled to mass spectrometry (MS affords deep mining of the muscle proteome, but differential analysis between samples is challenging due to the greater level of fractionation and the complexities of quantifying proteins based on the abundances of their tryptic peptides. Here we report simple, semi-automated and time efficient (i.e., 3 h per sample proteome profiling of skeletal muscle by 1-dimensional RPLC electrospray ionisation tandem MS. Solei were analysed from rats (n = 5, in each group bred as either high- or low-capacity runners (HCR and LCR, respectively that exhibited a 6.4-fold difference (1,625 ± 112 m vs. 252 ± 43 m, p < 0.0001 in running capacity during a standardized treadmill test. Soluble muscle proteins were extracted, digested with trypsin and individual biological replicates (50 ng of tryptic peptides subjected to LC-MS profiling. Proteins were identified by triplicate LC-MS/MS analysis of a pooled sample of each biological replicate. Differential expression profiling was performed on relative abundances (RA of parent ions, which spanned three orders of magnitude. In total, 207 proteins were analysed, which encompassed almost all enzymes of the major metabolic pathways in skeletal muscle. The most abundant protein detected was type I myosin heavy chain (RA = 5,843 ± 897 and the least abundant protein detected was heat shock 70 kDa protein (RA = 2 ± 0.5. Sixteen proteins were significantly (p < 0.05 more abundant in HCR muscle and hierarchal clustering of the profiling data highlighted two protein subgroups, which encompassed proteins associated with either the respiratory chain or fatty acid oxidation. Heart-type fatty acid binding protein (FABPH was 1

  19. Identification and Characterization of the UL37 Protein of Herpes Simplex Virus Type 1 and Demonstration that it Interacts with ICP8, the Major DNA Binding Protein of Herpes Simplex Virus

    Science.gov (United States)

    1992-10-20

    R . 1974 . Recovery of herpes simplex virus from human sacral gangl ions. N. Engl. J. Med. 291 :828-830. Baringer, J.R . 1975. Herpes simplex virus...AII’I fORCE MEDICAL C(NTEIt Title of Dissertation : "Ideatification and Characterization of the UL37 Protein of Herpes Simplex Virus Type 1 and...Demonstration that It Interacts with reps. the Major DNA Binding Protein of Herpes Simplex Virus" Name of Candidate: Lisa Shelton Doctor of

  20. Mutations in the putative zinc-binding motif of UL52 demonstrate a complex interdependence between the UL5 and UL52 subunits of the human herpes simplex virus type 1 helicase/primase complex.

    Science.gov (United States)

    Chen, Yan; Carrington-Lawrence, Stacy D; Bai, Ping; Weller, Sandra K

    2005-07-01

    Herpes simplex virus type 1 (HSV-1) encodes a heterotrimeric helicase-primase (UL5/8/52) complex. UL5 contains seven motifs found in helicase superfamily 1, and UL52 contains conserved motifs found in primases. The contributions of each subunit to the biochemical activities of the complex, however, remain unclear. We have previously demonstrated that a mutation in the putative zinc finger at UL52 C terminus abrogates not only primase but also ATPase, helicase, and DNA-binding activities of a UL5/UL52 subcomplex, indicating a complex interdependence between the two subunits. To test this hypothesis and to further investigate the role of the zinc finger in the enzymatic activities of the helicase-primase, a series of mutations were constructed in this motif. They differed in their ability to complement a UL52 null virus: totally defective, partial complementation, and potentiating. In this study, four of these mutants were studied biochemically after expression and purification from insect cells infected with recombinant baculoviruses. All mutants show greatly reduced primase activity. Complementation-defective mutants exhibited severe defects in ATPase, helicase, and DNA-binding activities. Partially complementing mutants displayed intermediate levels of these activities, except that one showed a wild-type level of helicase activity. These data suggest that the UL52 zinc finger motif plays an important role in the activities of the helicase-primase complex. The observation that mutations in UL52 affected helicase, ATPase, and DNA-binding activities indicates that UL52 binding to DNA via the zinc finger may be necessary for loading UL5. Alternatively, UL5 and UL52 may share a DNA-binding interface.

  1. Inhibition of Binding of the AB5-Type Enterotoxins LT-I and Cholera Toxin to Ganglioside GM1 by Galactose-Rich Dietary Components

    NARCIS (Netherlands)

    Becker, P.M.; Widjaja-Greefkes, H.C.A.; Wikselaar, van P.G.

    2010-01-01

    Cholera, travelers' diarrhea, or colibacillosis in pigs can possibly be prevented or attenuated by dietary provision of competitive inhibitors that react with the GM1-binding sites of the enterotoxins cholera toxin (CT), human Escherichia coli heat-labile enterotoxin of serogroup I (LTh-I), and

  2. Incident microalbuminuria and complement factor mannan-binding lectin-associated protein 19 in people with newly diagnosed type 1 diabetes

    DEFF Research Database (Denmark)

    Ostergaard, J A; Thiel, S; Hoffmann-Petersen, I T

    2017-01-01

    BACKGROUND: Evidence links the lectin pathway of complement activation to diabetic kidney disease. Upon carbohydrate-recognition by pattern-recognition molecules, e.g., mannan-binding lectin (MBL), the MBL-associated serine protease (MASP-2) is activated and initiates the complement cascade. The ...

  3. The Second Transmembrane Domain of the Human Type 1 Angiotensin II Receptor Participates in the Formation of the Ligand Binding Pocket and Undergoes Integral Pivoting Movement during the Process of Receptor Activation*

    Science.gov (United States)

    Domazet, Ivana; Holleran, Brian J.; Martin, Stéphane S.; Lavigne, Pierre; Leduc, Richard; Escher, Emanuel; Guillemette, Gaétan

    2009-01-01

    The octapeptide hormone angiotensin II (AngII) exerts a wide variety of cardiovascular effects through the activation of the angiotensin II type-1 (AT1) receptor, which belongs to the G protein-coupled receptor superfamily. Like other G protein-coupled receptors, the AT1 receptor possesses seven transmembrane domains that provide structural support for the formation of the ligand-binding pocket. In order to identify those residues in the second transmembrane domain (TMD2) that contribute to the formation of the binding pocket of the AT1 receptor, we used the substituted cysteine accessibility method. All of the residues within the Leu-70 to Trp-94 region were mutated one at a time to a cysteine, and, after expression in COS-7 cells, the mutant receptors were treated with the sulfhydryl-specific alkylating agent methanethiosulfonate-ethylammonium (MTSEA). MTSEA reacts selectively with water-accessible, free sulfhydryl groups of endogenous or introduced point mutation cysteines. If a cysteine is found in the binding pocket, the covalent modification will affect the binding kinetics of the ligand. MTSEA substantially decreased the binding affinity of D74C-AT1, L81C-AT1, A85C-AT1, T88C-AT1, and A89C-AT1 mutant receptors, which suggests that these residues orient themselves within the water-accessible binding pocket of the AT1 receptor. Interestingly, this pattern of acquired MTSEA sensitivity was altered for TMD2 reporter cysteines engineered in a constitutively active N111G-AT1 receptor background. Indeed, mutant D74C-N111G-AT1 became insensitive to MTSEA, whereas mutant L81C-N111G-AT1 lost some sensitivity and mutant V86C-N111G-AT1 became sensitive to MTSEA. Our results suggest that constitutive activation of the AT1 receptor causes TMD2 to pivot, bringing the top of TMD2 closer to the binding pocket and pushing the bottom of TMD2 away from the binding pocket. PMID:19276075

  4. The second transmembrane domain of the human type 1 angiotensin II receptor participates in the formation of the ligand binding pocket and undergoes integral pivoting movement during the process of receptor activation.

    Science.gov (United States)

    Domazet, Ivana; Holleran, Brian J; Martin, Stéphane S; Lavigne, Pierre; Leduc, Richard; Escher, Emanuel; Guillemette, Gaétan

    2009-05-01

    The octapeptide hormone angiotensin II (AngII) exerts a wide variety of cardiovascular effects through the activation of the angiotensin II type-1 (AT(1)) receptor, which belongs to the G protein-coupled receptor superfamily. Like other G protein-coupled receptors, the AT(1) receptor possesses seven transmembrane domains that provide structural support for the formation of the ligand-binding pocket. In order to identify those residues in the second transmembrane domain (TMD2) that contribute to the formation of the binding pocket of the AT(1) receptor, we used the substituted cysteine accessibility method. All of the residues within the Leu-70 to Trp-94 region were mutated one at a time to a cysteine, and, after expression in COS-7 cells, the mutant receptors were treated with the sulfhydryl-specific alkylating agent methanethiosulfonate-ethylammonium (MTSEA). MTSEA reacts selectively with water-accessible, free sulfhydryl groups of endogenous or introduced point mutation cysteines. If a cysteine is found in the binding pocket, the covalent modification will affect the binding kinetics of the ligand. MTSEA substantially decreased the binding affinity of D74C-AT(1), L81C-AT(1), A85C-AT(1), T88C-AT(1), and A89C-AT(1) mutant receptors, which suggests that these residues orient themselves within the water-accessible binding pocket of the AT(1) receptor. Interestingly, this pattern of acquired MTSEA sensitivity was altered for TMD2 reporter cysteines engineered in a constitutively active N111G-AT(1) receptor background. Indeed, mutant D74C-N111G-AT(1) became insensitive to MTSEA, whereas mutant L81C-N111G-AT(1) lost some sensitivity and mutant V86C-N111G-AT(1) became sensitive to MTSEA. Our results suggest that constitutive activation of the AT(1) receptor causes TMD2 to pivot, bringing the top of TMD2 closer to the binding pocket and pushing the bottom of TMD2 away from the binding pocket.

  5. 5-(Piperidin-4-yl)-3-Hydroxypyrazole: A Novel Scaffold for Probing the Orthosteric γ-Aminobutyric Acid Type A Receptor Binding Site

    DEFF Research Database (Denmark)

    Krall, Jacob; Kongstad, Kenneth Thermann; Nielsen, Birgitte

    2014-01-01

    A series of bioisosteric N1- and N2-substituted 5-(piperidin-4-yl)-3-hydroxypyrazole analogues of the partial GABAAR agonists 4-PIOL and 4-PHP have been designed, synthesized, and characterized pharmacologically. The unsubstituted 3-hydroxypyrazole analogue of 4-PIOL (2 a; IC50∼300 μM) is a weak...... indicate that the N1-substituted analogues of 4-PIOL and 4-PHP, 2 a–k, and previously reported 3-substituted 4-PHP analogues share a common binding mode to the orthosteric binding site in the receptor. Interestingly, the core scaffold of the N2-substituted analogues of 4-PIOL and 4-PHP, 3 b...

  6. Detection of a nuclear, EBNA-type antigen in apparently EBNA-negative Herpesvirus papio (HVP)-transformed lymphoid lines by the acid-fixed nuclear binding technique.

    Science.gov (United States)

    Ohno, S; Luka, J; Falk, L; Klein, G

    1977-12-15

    In agreement with the findings of previous authors, we could not detect a virally determined nuclear antigen in Herpesvirus papio (HVP)-transformed baboon lymphoid lines by anticomplementary staining in situ, as for EBNA. However, by means of our recently developed acid-fixed nuclear binding technique an EBNA-like antigen could be readily demonstrated, after extraction from both producer and non-producer lines. We propose to designate the antigen as HUPNA. It can be detected by a human anti-EBNA antibody, suggesting cross-reactivity, if not identity, between EBNA and HUPNA. HVP-DNA carrying non-producer lines, negative for in situ ACIF stainability but capable of yielding HUPNA by the nuclear binding technique, can be superinfected with EBV, with brilliant EBNA expression as the result, suggesting that the defective in situ staining is a property associated with the baboon HVP, rather than the baboon lymphoid cell per se.

  7. Affinity crosslinking of /sup 125/I-human beta-endorphin to cell lines possessing either mu or delta type opioid binding sites

    Energy Technology Data Exchange (ETDEWEB)

    Keren, O.; Gioannini, T.L.; Hiller, J.M.; Simon, E.J.

    1986-01-01

    Affinity crosslinking of human /sup 125/I-beta-Endorphin to cell lines possessing either mu or delta binding sites was carried out. Autoradiography of SDS-PAGE gels from these crosslinked cell lines revealed that these two sites contain major peptide subunits that differ in molecular size. This confirms our earlier finding in mammalian brain which demonstrated separate and distinct subunits for mu and delta opioid receptors.

  8. Effect of structural modulation and thickness of a graphene overlayer on the binding energy of the Rashba-type surface state of Ir(111)

    International Nuclear Information System (INIS)

    Sánchez-Barriga, J; Marchenko, D; Rader, O; Varykhalov, A; Bihlmayer, G; Wortmann, D

    2013-01-01

    The Ir(111) surface is known to host a surface state with a giant spin–orbit splitting due to the Rashba effect. This surface state is stable even in air when Ir is protected with an epitaxial graphene overlayer. In the present paper, we reveal an effect allowing one to tune the binding energy of this spin-split surface state up and down and demonstrate the practical application of this effect by two different approaches. The first approach is related to a decoration of the moiré pattern of single-layer graphene on Ir(111) by self-assembled nanoclusters of different compositions. The clusters locally pin graphene to the Ir substrate and enhance the amplitude of its structural corrugation, which, in turn, leads to an increase in the surface state binding energy. The second approach is related to the synthesis of few-layer graphene on Ir(111) by segregation of carbon. Additional graphene layers induce a shift of the Ir surface state towards lower binding energies and bring it almost to the Fermi level. Based on density functional calculations performed for the graphene/Ir(111) system, we show that in both cases the effect causing the binding energy shifts is intimately related to the distance between graphene and the Ir surface, which is subject to change due to deposition of clusters or by increasing the amount of graphene overlayers. In contrast, the observed spin–orbit splitting of the Ir(111) surface state remains remarkably robust and constant in all cases. Our theoretical analysis reveals that such stability can be explained by the localization properties of the Ir surface state that is a deep surface resonance. (paper)

  9. The RNA binding G-patch domain in retroviral protease is important for infectivity and D-type morphogenesis of Mason-Pfizer monkey virus

    Czech Academy of Sciences Publication Activity Database

    Bauerová, Helena; Štokrová, Jitka; Stříšovský, Kvido; Hunter, E.; Ruml, Tomáš; Pichová, Iva

    2005-01-01

    Roč. 280, č. 51 (2005), s. 42106-42112 ISSN 0021-9258 R&D Projects: GA MŠk(CZ) 1M0508; GA MŠk(CZ) 1M0520 Institutional research plan: CEZ:AV0Z40550506; CEZ:AV0Z50520514 Keywords : retroviral protease * RNA binding domain * M-PMV * infectivity * assembly Subject RIV: CE - Biochemistry Impact factor: 5.854, year: 2005

  10. Binding energy, phonon spectra and thermodynamic properties of elements with type structures A1 (Al, Cu), A2 (V, Ti2), A3 (Mg, Tiβ), A4 (Si, Sn)

    International Nuclear Information System (INIS)

    Sirota, N.N.; Soshnina, T.M.; Sirota, I.M.; Sokolovskij, T.D.

    2001-01-01

    One calculated dependences of binding energy on spacing between the nearest atoms of Al and Cu elements with A 1 type structure, of V and Ti α elements with A 2 type structure, of Mg and Ti β elements with A 3 type structure, Si and Sn elements with A 4 type structure. To calculate one applied the methods based on the Thomas-Fermi statistic theory of atom. The derived dependences were approximated using the expression in the form of the Mie-Grueneisen potential. On the basis of the Born-von-Karman model of solid body one calculated the phonon spectra using which one determined temperature dependences of specific heat, free and internal energy of the investigated elements. The calculated values of energy of atomization, equilibrium closest interatomic spacing and temperature dependences of specific heat are in compliance with the experimental data [ru

  11. Protein structural studies by paramagnetic solid-state NMR spectroscopy aided by a compact cyclen-type Cu(II) binding tag

    Energy Technology Data Exchange (ETDEWEB)

    Sengupta, Ishita; Gao, Min; Arachchige, Rajith J.; Nadaud, Philippe S. [The Ohio State University, Department of Chemistry and Biochemistry (United States); Cunningham, Timothy F.; Saxena, Sunil [University of Pittsburgh, Department of Chemistry (United States); Schwieters, Charles D. [National Institutes of Health, Center for Information Technology (United States); Jaroniec, Christopher P., E-mail: jaroniec@chemistry.ohio-state.edu [The Ohio State University, Department of Chemistry and Biochemistry (United States)

    2015-01-15

    Paramagnetic relaxation enhancements (PREs) are a rich source of structural information in protein solid-state NMR spectroscopy. Here we demonstrate that PRE measurements in natively diamagnetic proteins are facilitated by a thiol-reactive compact, cyclen-based, high-affinity Cu{sup 2+} binding tag, 1-[2-(pyridin-2-yldisulfanyl)ethyl]-1,4,7,10-tetraazacyclododecane (TETAC), that overcomes the key shortcomings associated with the use of larger, more flexible metal-binding tags. Using the TETAC–Cu{sup 2+} K28C mutant of B1 immunoglobulin-binding domain of protein G as a model, we find that amino acid residues located within ∼10 Å of the Cu{sup 2+} center experience considerable transverse PREs leading to severely attenuated resonances in 2D {sup 15}N–{sup 13}C correlation spectra. For more distant residues, electron–nucleus distances are accessible via quantitative measurements of longitudinal PREs, and we demonstrate such measurements for {sup 15}N–Cu{sup 2+} distances up to ∼20 Å.

  12. AHM1, a Novel Type of Nuclear Matrix–Localized, MAR Binding Protein with a Single AT Hook and a J Domain–Homologous Region

    Science.gov (United States)

    Morisawa, Gaku; Han-yama, Atsushi; Moda, Ichiro; Tamai, Atsushi; Iwabuchi, Masaki; Meshi, Tetsuo

    2000-01-01

    Interactions between the nuclear matrix and special regions of chromosomal DNA called matrix attachment regions (MARs) have been implicated in various nuclear functions. We have identified a novel protein from wheat, AT hook–containing MAR binding protein1 (AHM1), that binds preferentially to MARs. A multidomain protein, AHM1 has the special combination of a J domain–homologous region and a Zn finger–like motif (a J-Z array) and an AT hook. For MAR binding, the AT hook at the C terminus was essential, and an internal portion containing the Zn finger–like motif was additionally required in vivo. AHM1 was found in the nuclear matrix fraction and was localized in the nucleoplasm. AHM1 fused to green fluorescent protein had a speckled distribution pattern inside the nucleus. AHM1 is most likely a nuclear matrix component that functions between intranuclear framework and MARs. J-Z arrays can be found in a group of (hypothetical) proteins in plants, which may share some functions, presumably to recruit specific Hsp70 partners as co-chaperones. PMID:11041885

  13. Maize DRE-binding proteins DBF1 and DBF2 are involved in rab17 regulation through the drought-responsive element in an ABA-dependent pathway.

    Science.gov (United States)

    Kizis, Dimosthenis; Pagès, Montserrat

    2002-06-01

    The abscisic acid-responsive gene rab17 of maize is expressed during late embryogenesis, and is induced by ABA and desiccation in embryo and vegetative tissues. ABRE and DRE cis-elements are involved in regulation of the gene by ABA and drought. Using yeast one-hybrid screening, we isolated two cDNAs encoding two new DRE-binding proteins, designated DBF1 and DBF2, that are members of the AP2/EREBP transcription factor family. Analysis of mRNA accumulation profiles showed that DBF1 is induced during maize embryogenesis and after desiccation, NaCl and ABA treatments in plant seedlings, whereas the DBF2 mRNA is not induced. DNA-binding preferences of DBFs were analysed by electrophoretic mobility shift assays, and showed that both DBF1 and DBF2 bound to the wild-type DRE2 element, but not to the DRE2 mutant or to the DRE1 element which differs only in a single nucleotide. Transactivation activity using particle bombardment showed that DBF1 functioned as activator of DRE2-dependent transcription of rab17 promoter by ABA, whereas DBF2 overexpression had a repression action downregulating not only the basal promoter activity, but also the ABA effect. These results show that ABA plays a role in the regulation of DBF activity, and suggests the existence of an ABA-dependent pathway for the regulation of genes through the C-repeat/DRE element.

  14. Tight-binding study of the hole subband structure properties of p-type delta-doped quantum wells in Si by using a Thomas-Fermi-Dirac potential

    International Nuclear Information System (INIS)

    Rodriguez-Vargas, I; Madrigal-Melchor, J; Vlaev, S J

    2009-01-01

    We present the hole subband structure of p-type delta-doped single, double, multiple and superlattice quantum wells in Si. We use the first neighbors sp 3 s' tight-binding approximation including spin for the hole level structure analysis. The parameters of the tight-binding hamiltonian were taken from Klimeck et al. [Klimeck G, Bowen R C, Boykin T B, Salazar-Lazaro C, Cwik T A and Stoica A 2000 Superlattice. Microst. 27 77], first neighbors parameters that give realiable results for the valence band of Si. The calculations are based on a scheme previously proposed and applied to delta-doped quantum well systems [Vlaev S J and Gaggero-Sager L M 1998 Phys. Rev. B 58 1142]. The scheme relies on the incorporation of the delta-doped quantum well potential in the diagonal terms of the tight-binding hamiltonian. We give a detail description of the delta-doped quantum well structures, this is, we study the hole subband structure behavior as a function of the impurity density, the interwell distance of the doped planes and the superlattice period. We also compare our results with the available theoretical and experimental data, obtaining a reasonable agreement.

  15. Tight-binding study of the hole subband structure properties of p-type delta-doped quantum wells in Si by using a Thomas-Fermi-Dirac potential

    Energy Technology Data Exchange (ETDEWEB)

    Rodriguez-Vargas, I; Madrigal-Melchor, J; Vlaev, S J, E-mail: isaac@planck.reduaz.m [Unidad Academica de Fisica, Universidad Autonoma de Zacatecas, Calzada Solidaridad Esquina Con Paseo La Bufa S/N, 98060 Zacatecas, ZAC. (Mexico)

    2009-05-01

    We present the hole subband structure of p-type delta-doped single, double, multiple and superlattice quantum wells in Si. We use the first neighbors sp{sup 3}s' tight-binding approximation including spin for the hole level structure analysis. The parameters of the tight-binding hamiltonian were taken from Klimeck et al. [Klimeck G, Bowen R C, Boykin T B, Salazar-Lazaro C, Cwik T A and Stoica A 2000 Superlattice. Microst. 27 77], first neighbors parameters that give realiable results for the valence band of Si. The calculations are based on a scheme previously proposed and applied to delta-doped quantum well systems [Vlaev S J and Gaggero-Sager L M 1998 Phys. Rev. B 58 1142]. The scheme relies on the incorporation of the delta-doped quantum well potential in the diagonal terms of the tight-binding hamiltonian. We give a detail description of the delta-doped quantum well structures, this is, we study the hole subband structure behavior as a function of the impurity density, the interwell distance of the doped planes and the superlattice period. We also compare our results with the available theoretical and experimental data, obtaining a reasonable agreement.

  16. Mutation of the dengue virus type 2 envelope protein heparan sulfate binding sites or the domain III lateral ridge blocks replication in Vero cells prior to membrane fusion

    International Nuclear Information System (INIS)

    Roehrig, John T.; Butrapet, Siritorn; Liss, Nathan M.; Bennett, Susan L.; Luy, Betty E.; Childers, Thomas; Boroughs, Karen L.; Stovall, Janae L.; Calvert, Amanda E.; Blair, Carol D.; Huang, Claire Y.-H.

    2013-01-01

    Using an infectious cDNA clone we engineered seven mutations in the putative heparan sulfate- and receptor-binding motifs of the envelope protein of dengue virus serotype 2, strain 16681. Four mutant viruses, KK122/123EE, E202K, G304K, and KKK305/307/310EEE, were recovered following transfection of C6/36 cells. A fifth mutant, KK291/295EE, was recovered from C6/36 cells with a compensatory E295V mutation. All mutants grew in and mediated fusion of virus-infected C6/36 cells, but three of the mutants, KK122/123EE, E202K, G304K, did not grow in Vero cells without further modification. Two Vero cell lethal mutants, KK291/295EV and KKK307/307/310EEE, failed to replicate in DC-SIGN-transformed Raji cells and did not react with monoclonal antibodies known to block DENV attachment to Vero cells. Additionally, both mutants were unable to initiate negative-strand vRNA synthesis in Vero cells by 72 h post-infection, suggesting that the replication block occurred prior to virus-mediated membrane fusion. - Highlights: • Heparan sulfate- and receptor-binding motifs of DENV2 envelope protein were mutated. • Four mutant viruses were isolated—all could fuse C6/36 cells. • Two of these mutants were lethal in Vero cells without further modification. • Lethal mutations were KK291/295EV and KKK305/307/310EEE. • Cell attachment was implicated as the replication block for both mutants

  17. Acquisition of human-type receptor binding specificity by new H5N1 influenza virus sublineages during their emergence in birds in Egypt.

    Directory of Open Access Journals (Sweden)

    Yohei Watanabe

    2011-05-01

    Full Text Available Highly pathogenic avian influenza A virus subtype H5N1 is currently widespread in Asia, Europe, and Africa, with 60% mortality in humans. In particular, since 2009 Egypt has unexpectedly had the highest number of human cases of H5N1 virus infection, with more than 50% of the cases worldwide, but the basis for this high incidence has not been elucidated. A change in receptor binding affinity of the viral hemagglutinin (HA from α2,3- to α2,6-linked sialic acid (SA is thought to be necessary for H5N1 virus to become pandemic. In this study, we conducted a phylogenetic analysis of H5N1 viruses isolated between 2006 and 2009 in Egypt. The phylogenetic results showed that recent human isolates clustered disproportionally into several new H5 sublineages suggesting that their HAs have changed their receptor specificity. Using reverse genetics, we found that these H5 sublineages have acquired an enhanced binding affinity for α2,6 SA in combination with residual affinity for α2,3 SA, and identified the amino acid mutations that produced this new receptor specificity. Recombinant H5N1 viruses with a single mutation at HA residue 192 or a double mutation at HA residues 129 and 151 had increased attachment to and infectivity in the human lower respiratory tract but not in the larynx. These findings correlated with enhanced virulence of the mutant viruses in mice. Interestingly, these H5 viruses, with increased affinity to α2,6 SA, emerged during viral diversification in bird populations and subsequently spread to humans. Our findings suggested that emergence of new H5 sublineages with α2,6 SA specificity caused a subsequent increase in human H5N1 influenza virus infections in Egypt, and provided data for understanding the virus's pandemic potential.

  18. Mutation of the dengue virus type 2 envelope protein heparan sulfate binding sites or the domain III lateral ridge blocks replication in Vero cells prior to membrane fusion

    Energy Technology Data Exchange (ETDEWEB)

    Roehrig, John T., E-mail: jtr1@cdc.gov [Division of Vector-Borne Diseases, Centers for Disease Control and Prevention, Fort Collins, CO 80521 (United States); Butrapet, Siritorn; Liss, Nathan M. [Division of Vector-Borne Diseases, Centers for Disease Control and Prevention, Fort Collins, CO 80521 (United States); Bennett, Susan L. [Arthropod-borne and Infectious Diseases Laboratory, Department of Microbiology, Immunology, and Pathology, Colorado State University, Fort Collins, CO 80523 (United States); Luy, Betty E.; Childers, Thomas; Boroughs, Karen L.; Stovall, Janae L.; Calvert, Amanda E. [Division of Vector-Borne Diseases, Centers for Disease Control and Prevention, Fort Collins, CO 80521 (United States); Blair, Carol D. [Arthropod-borne and Infectious Diseases Laboratory, Department of Microbiology, Immunology, and Pathology, Colorado State University, Fort Collins, CO 80523 (United States); Huang, Claire Y.-H. [Division of Vector-Borne Diseases, Centers for Disease Control and Prevention, Fort Collins, CO 80521 (United States)

    2013-07-05

    Using an infectious cDNA clone we engineered seven mutations in the putative heparan sulfate- and receptor-binding motifs of the envelope protein of dengue virus serotype 2, strain 16681. Four mutant viruses, KK122/123EE, E202K, G304K, and KKK305/307/310EEE, were recovered following transfection of C6/36 cells. A fifth mutant, KK291/295EE, was recovered from C6/36 cells with a compensatory E295V mutation. All mutants grew in and mediated fusion of virus-infected C6/36 cells, but three of the mutants, KK122/123EE, E202K, G304K, did not grow in Vero cells without further modification. Two Vero cell lethal mutants, KK291/295EV and KKK307/307/310EEE, failed to replicate in DC-SIGN-transformed Raji cells and did not react with monoclonal antibodies known to block DENV attachment to Vero cells. Additionally, both mutants were unable to initiate negative-strand vRNA synthesis in Vero cells by 72 h post-infection, suggesting that the replication block occurred prior to virus-mediated membrane fusion. - Highlights: • Heparan sulfate- and receptor-binding motifs of DENV2 envelope protein were mutated. • Four mutant viruses were isolated—all could fuse C6/36 cells. • Two of these mutants were lethal in Vero cells without further modification. • Lethal mutations were KK291/295EV and KKK305/307/310EEE. • Cell attachment was implicated as the replication block for both mutants.

  19. NHE1 inhibition by amiloride- and benzoylguanidine-type compounds. Inhibitor binding loci deduced from chimeras of NHE1 homologues with endogenous differences in inhibitor sensitivity

    DEFF Research Database (Denmark)

    Pedersen, Stine F; King, Scott A; Nygaard, Eva B

    2007-01-01

    NHE1). Although highly homologous to the amiloride- and HOE-sensitive human NHE1 (hNHE1), AtNHE1 is insensitive to HOE-type and PaNHE1 to both amiloride- and HOE-type compounds. Here we generated chimeras to "knock in" amiloride and HOE sensitivity to PaNHE1, and we thereby identified several NHE1...

  20. Pictorial binding: endeavor to classify

    Directory of Open Access Journals (Sweden)

    Zinchenko S.

    2015-01-01

    Full Text Available The article is devoted to the classification of bindings of the 1-19th centuries with a unique and untypical book binding decoration technique (encaustic, tempera and oil paintings. Analysis of design features, materials and techniques of art decoration made it possible to identify them as a separate type - pictorial bindings and divide them into four groups. The first group consists of Coptic bindings, decorated with icon-painting images in encaustic technique. The second group is made up of leather Western bindings of the 13-14th centuries, which have the decoration and technique of ornamentation close to iconography. The third group involves parchment bindings, ornamentation technique of which is closer to the miniature. The last group comprises bindings of East Slavic origin of the 15-19th centuries, decorated with icon-painting pictures made in the technique of tempera or oil painting. The proposed classification requires further basic research as several specific kinds of bindings have not yet been investigated

  1. Detection of heart-type fatty acid-binding protein (h-FABP) using piezoresistive polymer microcantilevers functionalized by a dry method

    Science.gov (United States)

    Agarwal, Dilip Kumar; Prasad, Abhinav; Vinchurkar, Madhuri; Gandhi, Sahir; Prabhakar, Deepika; Mukherji, Soumyo; Rao, V. Ramgopal

    2018-03-01

    Piezoresistive microcantilever-based sensor platform is being used for the last two decades due to their low cost, rapid response and label-free detection system. In this work, we are reporting a microfabricated piezoresistive SU-8/carbon black (polymer cantilever)-based sensor platform for the detection of a clinically important early-stage cardiac marker, i.e., fatty acid-binding protein. It is a most preferred cardiac marker for the diagnosis of acute myocardial infarction. The embodiment of the sensor is a SU-8 microcantilever chip with an integrated nanoparticle composite (carbon black) as a piezoresistor for on-chip electrical transduction. Prior to improving the sensing and susceptibility towards the specific target biomolecule (i.e., h-FABP), the fabricated SU-8 polymer cantilevers were subjected to tailored functionalization. This includes the use of an in-house dry method of hot wire chemical vapour deposition technique to graft amine groups onto the SU-8 surface. The surface-modified microcantilevers were further integrated with a polydimethylsiloxane liquid flow cell and connected externally with an electrical read-out system. Immobilization of the antibody corresponding to the marker protein on the microcantilever surface and subsequent recording of the signal generated upon the antibody-antigen interaction were carried out inside the liquid flow cell. Using our optimized immobilization protocol with this experimental set-up, we were successfully able to detect h-FABP concentration as low as 100 ng/ml.

  2. Sociality and the telencephalic distribution of corticotrophin-releasing factor, urocortin 3, and binding sites for CRF type 1 and type 2 receptors: A comparative study of eusocial naked mole-rats and solitary Cape mole-rats.

    Science.gov (United States)

    Coen, Clive W; Kalamatianos, Theodosis; Oosthuizen, Maria K; Poorun, Ravi; Faulkes, Christopher G; Bennett, Nigel C

    2015-11-01

    Various aspects of social behavior are influenced by the highly conserved corticotrophin-releasing factor (CRF) family of peptides and receptors in the mammalian telencephalon. This study has mapped and compared the telencephalic distribution of the CRF receptors, CRF1 and CRF2 , and two of their ligands, CRF and urocortin 3, respectively, in African mole-rat species with diametrically opposed social behavior. Naked mole-rats live in large eusocial colonies that are characterized by exceptional levels of social cohesion, tolerance, and cooperation in burrowing, foraging, defense, and alloparental care for the offspring of the single reproductive female. Cape mole-rats are solitary; they tolerate conspecifics only fleetingly during the breeding season. The telencephalic sites at which the level of CRF1 binding in naked mole-rats exceeds that in Cape mole-rats include the basolateral amygdaloid nucleus, hippocampal CA3 subfield, and dentate gyrus; in contrast, the level is greater in Cape mole-rats in the shell of the nucleus accumbens and medial habenular nucleus. For CRF2 binding, the sites with a greater level in naked mole-rats include the basolateral amygdaloid nucleus and dentate gyrus, but the septohippocampal nucleus, lateral septal nuclei, amygdalostriatal transition area, bed nucleus of the stria terminalis, and medial habenular nucleus display a greater level in Cape mole-rats. The results are discussed with reference to neuroanatomical and behavioral studies of various species, including monogamous and promiscuous voles. By analogy with findings in those species, we speculate that the abundance of CRF1 binding in the nucleus accumbens of Cape mole-rats reflects their lack of affiliative behavior. © 2015 Wiley Periodicals, Inc.

  3. Reformulated tight binding calculation for band discontinuity at CdTe/Hg {sub x}Cd{sub 1-x}Te heterointerfaces and their type I-type III transitions

    Energy Technology Data Exchange (ETDEWEB)

    Ekpunobi, A.J. [Department of Physics and Industrial Physics, Nnamdi Azikiwe University, P.M.B. 5025, Awka, Anambra State (Nigeria)

    2005-02-25

    A recently reformulated tight binding method is used to calculate the valence band discontinuity at the CdTe/Hg {sub x}Cd{sub 1-x}Te interface in the s{sup 2}p{sup 2} configuration. The calculated valence band discontinuity of 0.31 eV at CdTe/HgTe interface is in good agreement with self-consistent calculation and accepted experimental value. Calculations were extended to alloy interfaces, which enabled the investigation of the band-offset problem at the transition point. Both valence band discontinuity ratio and conduction band discontinuity ratio show inflexions at the transition point.

  4. Retinol Binding Protein-4 Is Associated with TNF-α and Not Insulin Resistance in Subjects with Type 2 Diabetes Mellitus and Coronary Heart Disease

    Directory of Open Access Journals (Sweden)

    Nasser M. Al-Daghri

    2009-01-01

    Full Text Available We studied the association between RBP4 and various markers related to insulin resistance and diabetic complications as well as inflammatory markers in Saudi population suffering from type 2 diabetes and coronary heart disease. Patients with type 2 diabetes were divided into 3 groups according to the type of treatment and involvement of coronary artery disease. Serum RBP4, TNF-α, insulin, CRP, resistin, leptin and adiponectin were analysed in all samples. RBP4 levels increased significantly in the group of diabetic subjects treated with oral hypoglycemic agents and diabetic patients with coronary heart disease (30.2 ± 11.8; 33.4 ± 13.6 respectively, while there was no significant change in the other group for diabetic subjects on low-carbohydrate diet (25.1 ± 10.9 compared to control group (22.6 ± 9.5. RPB4 levels were positively correlated with TNF-α in the group of diabetic subjects on oral hypoglycemic agents and diabetic patients with coronary heart disease (r = 0.52, P < 0.05; r = 0.58, P < 0.05 respectively. No correlations were found between RBP4 levels and insulin resistance in all studied groups. Our findings suggest that serum RBP4 levels is associated with pro-inflammatory cytokine (TNF-α and is not associated with insulin resistance among patients with type 2 diabetes and coronary heart disease.

  5. The neck region of the C-type lectin DC-SIGN regulates its surface spatiotemporal organization and virus-binding capacity on antigen presenting cells

    NARCIS (Netherlands)

    Manzo, C.; Torreno-Pina, J.A.; Joosten, B.; Reinieren-Beeren, I.; Gualda, E.J.; Loza-Alvarez, P.; Figdor, Carl; Garcia Parajo, M.F.; Cambi, A.

    2012-01-01

    The C-type lectin DC-SIGN expressed on dendritic cells (DCs) facilitates capture and internalization of a plethora of different pathogens. Although it is known that DC-SIGN organizes in nanoclusters at the surface of DCs, the molecular mechanisms responsible for this well defined nanopatterning and

  6. The Neck Region of the C-type Lectin DC-SIGN Regulates Its Surface Spatiotemporal Organization and Virus-binding Capacity on Antigen-presenting Cells

    NARCIS (Netherlands)

    Manzo, C.; Torreno-Pina, J.A.; Joosten, B.; Reinieren-Beeren, I.; Gualda, E.J.; Loza-Alvarez, P.; Figdor, C.G.; Garcia-Parajo, M.F.; Cambi, A.

    2012-01-01

    The C-type lectin DC-SIGN expressed on dendritic cells (DCs) facilitates capture and internalization of a plethora of different pathogens. Although it is known that DC-SIGN organizes in nanoclusters at the surface of DCs, the molecular mechanisms responsible for this well defined nanopatterning and

  7. Bile salt-stimulated lipase from human milk binds DC-SIGN and inhibits human immunodeficiency virus type 1 transfer to CD4+ T cells

    NARCIS (Netherlands)

    Naarding, Marloes A.; Dirac, Annette M.; Ludwig, Irene S.; Speijer, Dave; Lindquist, Susanne; Vestman, Eva-Lotta; Stax, Martijn J.; Geijtenbeek, Teunis B. H.; Pollakis, Georgios; Hernell, Olle; Paxton, William A.

    2006-01-01

    A wide range of pathogens, including human immunodeficiency virus type 1 (HIV-1), hepatitis C virus, Ebola virus, cytomegalovirus, dengue virus, Mycobacterium, Leishmania, and Helicobacter pylori, can interact with dendritic cell (DC)-specific ICAM3-grabbing nonintegrin (DC-SIGN), expressed on DCs

  8. Scavenger Receptor Class B Type I Mediates Biliary Cholesterol Secretion Independent of ATP-Binding Cassette Transporter g5/g8 in Mice

    NARCIS (Netherlands)

    Wiersma, Harmen; Gatti, Alberto; Nijstad, Niels; Elferink, Ronald P. J. Oude; Kuipers, Folkert; Tietge, Uwe J. F.

    2009-01-01

    Scavenger receptor class B type I (SR-BI) mediates selective uptake of cholesterol from high-density lipoprotein (HDL) particles by the liver and influences biliary cholesterol secretion. However, it is not dear, if this effect is direct or indirect. The aim of this study was to determine the impact

  9. Structural requirements for the binding of tRNA Lys3 to reverse transcriptase of the human immunodeficiency virus type 1

    NARCIS (Netherlands)

    Oude Essink, B. B.; Das, A. T.; Berkhout, B.

    1995-01-01

    Reverse transcription of the human immunodeficiency virus type 1 (HIV-1) RNA genome is primed by the cellular tRNA Lys3 molecule. Packaging of this tRNA primer during virion assembly is thought to be mediated by specific interactions with the reverse transcriptase (RT) protein. Portions of the tRNA

  10. Insulin binding to individual rat skeletal muscles

    International Nuclear Information System (INIS)

    Koerker, D.J.; Sweet, I.R.; Baskin, D.G.

    1990-01-01

    Studies of insulin binding to skeletal muscle, performed using sarcolemmal membrane preparations or whole muscle incubations of mixed muscle or typical red (soleus, psoas) or white [extensor digitorum longus (EDL), gastrocnemius] muscle, have suggested that red muscle binds more insulin than white muscle. We have evaluated this hypothesis using cryostat sections of unfixed tissue to measure insulin binding in a broad range of skeletal muscles; many were of similar fiber-type profiles. Insulin binding per square millimeter of skeletal muscle slice was measured by autoradiography and computer-assisted densitometry. We found a 4.5-fold range in specific insulin tracer binding, with heart and predominantly slow-twitch oxidative muscles (SO) at the high end and the predominantly fast-twitch glycolytic (FG) muscles at the low end of the range. This pattern reflects insulin sensitivity. Evaluation of displacement curves for insulin binding yielded linear Scatchard plots. The dissociation constants varied over a ninefold range (0.26-2.06 nM). Binding capacity varied from 12.2 to 82.7 fmol/mm2. Neither binding parameter was correlated with fiber type or insulin sensitivity; e.g., among three muscles of similar fiber-type profile, the EDL had high numbers of low-affinity binding sites, whereas the quadriceps had low numbers of high-affinity sites. In summary, considerable heterogeneity in insulin binding was found among hindlimb muscles of the rat, which can be attributed to heterogeneity in binding affinities and the numbers of binding sites. It can be concluded that a given fiber type is not uniquely associated with a set of insulin binding parameters that result in high or low binding

  11. Common variants of the liver fatty acid binding protein gene influence the risk of type 2 diabetes and insulin resistance in Spanish population.

    Directory of Open Access Journals (Sweden)

    Maria Luisa Mansego

    Full Text Available SUMMARY: The main objective was to evaluate the association between SNPs and haplotypes of the FABP1-4 genes and type 2 diabetes, as well as its interaction with fat intake, in one general Spanish population. The association was replicated in a second population in which HOMA index was also evaluated. METHODS: 1217 unrelated individuals were selected from a population-based study [Hortega study: 605 women; mean age 54 y; 7.8% with type 2 diabetes]. The replication population included 805 subjects from Segovia, a neighboring region of Spain (446 females; mean age 52 y; 10.3% with type 2 diabetes. DM2 mellitus was defined in a similar way in both studies. Fifteen SNPs previously associated with metabolic traits or with potential influence in the gene expression within the FABP1-4 genes were genotyped with SNPlex and tested. Age, sex and BMI were used as covariates in the logistic regression model. RESULTS: One polymorphism (rs2197076 and two haplotypes of the FABP-1 showed a strong association with the risk of DM2 in the original population. This association was further confirmed in the second population as well as in the pooled sample. None of the other analyzed variants in FABP2, FABP3 and FABP4 genes were associated. There was not a formal interaction between rs2197076 and fat intake. A significant association between the rs2197076 and the haplotypes of the FABP1 and HOMA-IR was also present in the replication population. CONCLUSIONS: The study supports the role of common variants of the FABP-1 gene in the development of type 2 diabetes in Caucasians.

  12. DNA-binding site of major regulatory protein alpha 4 specifically associated with promoter-regulatory domains of alpha genes of herpes simplex virus type 1.

    OpenAIRE

    Kristie, T M; Roizman, B

    1986-01-01

    Herpes simplex virus type 1 genes form at least five groups (alpha, beta 1, beta 2, gamma 1, and gamma 2) whose expression is coordinately regulated and sequentially ordered in a cascade fashion. Previous studies have shown that functional alpha 4 gene product is essential for the transition from alpha to beta protein synthesis and have suggested that alpha 4 gene expression is autoregulatory. We have previously reported that labeled DNA fragments containing promoter-regulatory domains of thr...

  13. A novel-type phosphatidylinositol phosphate-interactive, Ca-binding protein PCaP1 in Arabidopsis thaliana: stable association with plasma membrane and partial involvement in stomata closure.

    Science.gov (United States)

    Nagata, Chisako; Miwa, Chika; Tanaka, Natsuki; Kato, Mariko; Suito, Momoe; Tsuchihira, Ayako; Sato, Yori; Segami, Shoji; Maeshima, Masayoshi

    2016-05-01

    The Ca(2+)-binding protein-1 (PCaP1) of Arabidopsis thaliana is a new type protein that binds to phosphatidylinositol phosphates and Ca(2+)-calmodulin complex as well as free Ca(2+). Although biochemical properties, such as binding to ligands and N-myristoylation, have been revealed, the intracellular localization, tissue and cell specificity, integrity of membrane association and physiological roles of PCaP1 are unknown. We investigated the tissue and intracellular distribution of PCaP1 by using transgenic lines expressing PCaP1 linked with a green fluorescence protein (GFP) at the carboxyl terminus of PCaP1. GFP fluorescence was obviously detected in most tissues including root, stem, leaf and flower. In these tissues, PCaP1-GFP signal was observed predominantly in the plasma membrane even under physiological stress conditions but not in other organelles. The fluorescence was detected in the cytosol when the 25-residue N-terminal sequence was deleted from PCaP1 indicating essential contribution of N-myristoylation to the plasma membrane anchoring. Fluorescence intensity of PCaP1-GFP in roots was slightly decreased in seedlings grown in medium supplemented with high concentrations of iron for 1 week and increased in those grown with copper. In stomatal guard cells, PCaP1-GFP was strictly, specifically localized to the plasma membrane at the epidermal-cell side but not at the pore side. A T-DNA insertion mutant line of PCaP1 did not show marked phenotype in a life cycle except for well growth under high CO2 conditions. However, stomata of the mutant line did not close entirely even in high osmolarity, which usually induces stomata closure. These results suggest that PCaP1 is involved in the stomatal movement, especially closure process, in leaves and response to excessive copper in root and leaf as a mineral nutrient as a physiological role.

  14. External validation of heart-type fatty acid binding protein, high-sensitivity cardiac troponin, and electrocardiography as rule-out for acute myocardial infarction.

    Science.gov (United States)

    Van Hise, Christopher B; Greenslade, Jaimi H; Parsonage, William; Than, Martin; Young, Joanna; Cullen, Louise

    2018-02-01

    To externally validate a clinical decision rule incorporating heart fatty acid binding protein (h-FABP), high-sensitivity troponin (hs-cTn) and electrocardiogram (ECG) for the detection of acute myocardial infarction (AMI) on presentation to the Emergency Department. We also investigated whether this clinical decision rule improved identification of AMI over algorithms incorporating hs-cTn and ECG only. This study included data from 789 patients from the Brisbane ADAPT cohort and 441 patients from the Christchurch TIMI RCT cohort. The primary outcome was index AMI. Sensitivity, specificity, positive predictive value and negative predictive value were used to assess the diagnostic accuracy of the algorithms. 1230 patients were recruited, including 112 (9.1%) with AMI. The algorithm including h-FABP and hs-cTnT had 100% sensitivity and 32.4% specificity. The algorithm utilising h-FABP and hs-cTnI had similar sensitivity (99.1%) and higher specificity (43.4%). The hs-cTnI and hs-cTnT algorithms without h-FABP both had a sensitivity of 98.2%; a result that was not significantly different from either algorithm incorporating h-FABP. Specificity was higher for the hs-cTnI algorithm (68.1%) compared to the hs-cTnT algorithm (33.0%). The specificity of the algorithm incorporating hs-cTnI alone was also significantly higher than both of the algorithms incorporating h-FABP (p<0.01). For patients presenting to the Emergency Department with chest pain, an algorithm incorporating h-FABP, hs-cTn and ECG has high accuracy and can rule out up to 40% of patients. An algorithm incorporating only hs-cTn and ECG has similar sensitivity and may rule out a higher proportion of patients. Each of the algorithms can be used to safely identify patients as low risk for AMI on presentation to the Emergency Department. Copyright © 2017 The Canadian Society of Clinical Chemists. All rights reserved.

  15. The C proteins of human parainfluenza virus type 1 block IFN signaling by binding and retaining Stat1 in perinuclear aggregates at the late endosome.

    Directory of Open Access Journals (Sweden)

    Henrick Schomacker

    Full Text Available Interferons (IFNs play a crucial role in the antiviral immune response. Whereas the C proteins of wild-type human parainfluenza virus type 1 (WT HPIV1 inhibit both IFN-β induction and signaling, a HPIV1 mutant encoding a single amino acid substitution (F170S in the C proteins is unable to block either host response. Here, signaling downstream of the type 1 IFN receptor was examined in Vero cells to define at what stage WT HPIV1 can block, and F170S HPIV1 fails to block, IFN signaling. WT HPIV1 inhibited phosphorylation of both Stat1 and Stat2, and this inhibition was only slightly reduced for F170S HPIV1. Degradation of Stat1 or Stat2 was not observed. The HPIV1 C proteins were found to accumulate in the perinuclear space, often forming large granules, and co-localized with Stat1 and the cation-independent mannose 6-phosphate receptor (M6PR that is a marker for late endosomes. Upon stimulation with IFN-β, both the WT and F170S C proteins remained in the perinuclear space, but only the WT C proteins prevented Stat1 translocation to the nucleus. In addition, WT HPIV1 C proteins, but not F170S C proteins, co-immunoprecipitated both phosphorylated and unphosphorylated Stat1. Our findings suggest that the WT HPIV1 C proteins form a stable complex with Stat1 in perinuclear granules that co-localize with M6PR, and that this direct interaction between the WT HPIV1 C proteins and Stat1 is the basis for the ability of HPIV1 to inhibit IFN signaling. The F170S mutation in HPIV1 C did not prevent perinuclear co-localization with Stat1, but apparently weakened this interaction such that, upon IFN stimulation, Stat1 was translocated to the nucleus to induce an antiviral response.

  16. The In Vivo DNA Binding Properties of Wild-Type and Mutant p53 Proteins in Mammary Cell Lines During the Course of Cell Cycle.

    Science.gov (United States)

    1996-08-01

    that my statement of work (SOW) for the current project omitted many of the tasks that had to be carried out in order to get the lab up and running...that we knew that we could stabilize wild-type p53 in ML-1 cells along with the possibility of being able to get an excellent elutriation profile with...nuclear protein extract was immunoprecipitated with PAb421 cross-linked to ProteinA -Sepharose beads and analysed by SDS-PAGE Western blot analysis with

  17. The C-type lectin receptor SIGNR3 binds to fungi present in commensal microbiota and influences immune regulation in experimental colitis

    Directory of Open Access Journals (Sweden)

    Magdalena eEriksson

    2013-07-01

    Full Text Available Inflammatory bowel disease is a condition of acute and chronic inflammation of the gut. An important factor contributing to pathogenesis is a dysregulated mucosal immunity against commensal bacteria and fungi. Host pattern recognition receptors sense commensals in the gut and are involved in maintaining the balance between controlled responses to pathogens and overwhelming innate immune activation. C-type lectin receptors (CLRs are pattern recognition receptors recognizing glycan structures on pathogens and self-antigens. Here we examined the role of the murine CLR SIGNR3 in the recognition of commensals and its involvement in intestinal immunity. SIGNR3 is the closest murine homologue of the human DC-SIGN receptor recognizing similar carbohydrate ligands such as terminal fucose or high-mannose glycans. We discovered that SIGNR3 recognizes fungi present in the commensal microbiota. To analyze if this interaction impacts the intestinal immunity against microbiota, the dextran sulfate sodium (DSS-induced colitis model was employed. SIGNR3-/- mice exhibited an increased weight loss associated with more severe colitis symptoms compared to wild-type control mice. The increased inflammation in SIGNR3-/- mice was accompanied by a higher level of TNF-α in colon. Our findings demonstrate for the first time that SIGNR3 recognizes intestinal fungi and has an immune regulatory role in colitis.

  18. A radioligand immunoassay for 1,25-dihydroxyvitamin D3 receptors using monoclonal antibody: detection of a phenotypic receptor variant in vitamin D-dependency rickets (type II) which does not bind hormone

    International Nuclear Information System (INIS)

    Pike, J.W.; Dokoh, Shigeharu; Liberman, U.A.; Eil, C.; Haussler, M.R.; Marx, S.J.

    1984-01-01

    Vitamin D-dependency rickets, type II (VDDRII), is a well recognized heritable disorder characterized by peripheral target organ resistance to 1,25-dihydroxyvitamin D 3 (1,25(OH) 2 D 3 ), the hormonally active form of the vitamin. Recently, cultured skin fibroblasts obtained from a number of patients with VDDRII have been utilized to characterize the underlying molecular defects associated with this malady. Recently monoclonal antibodies to the vitamin D receptor have been generated, and a radioligand immunoassay (RLIA) for the detection of this molecule has been developed which is independent of its hormone-binding capacity. This report describes the application of the immunoassay in the detection of receptor-like molecules in fibroblasts derived from patients with VDDRII. The results indicate that the molecule is generally present in all patients, and provides a mechanism for individual responsiveness to pharmacologic treatment with vitamin D 3 metabolites. 8 refs.; 3 figs.; 1 table

  19. Human chimera-type galectin-3: defining the critical tail length for high-affinity glycoprotein/cell surface binding and functional competition with galectin-1 in neuroblastoma cell growth regulation.

    Science.gov (United States)

    Kopitz, Jürgen; Vértesy, Sabine; André, Sabine; Fiedler, Sabine; Schnölzer, Martina; Gabius, Hans-Joachim

    2014-09-01

    Many human proteins have a modular design with receptor and structural domains. Using adhesion/growth-regulatory galectin-3 as model, we describe an interdisciplinary strategy to define the functional significance of its tail established by nine non-triple helical collagen-like repeats (I-IX) and the N-terminal peptide. Genetic engineering with sophisticated mass spectrometric product analysis provided the tools for biotesting, i.e. eight protein variants with different degrees of tail truncation. Evidently,various aspects of galectin-3 activity (cis binding and cell bridging) are affected by tail shortening in a different manner. Thus, this combined approach reveals an unsuspected complexity of structure-function relationship, encouraging further application beyond this chimera-type galectin. Copyright © 2014 Elsevier Masson SAS. All rights reserved.

  20. The relation between lattice parameters of NaCl type U, Np and Pu compounds and their magnetic and binding properties

    International Nuclear Information System (INIS)

    Ohmichi, Toshihiko; Arai, Yasuo

    1992-01-01

    The lattice parameters of NaCl type uranium, neptunium and plutonium compounds are analyzed using Pauling equation. The numbers of bonding electrons and localized electrons of the compounds are calculated using the single bond radius by Pauling for metalloids and assumed ones for the actinides. The number of bonding electrons per unit bonding length can be correlated linearly with the uranium compounds. The number of localized electrons is found to have a speculative relation with magnetic properties, in particular magnetic moments in ordered state; excluding some compounds, the magnetic moments increase at a gradient of 45 degrees with increasing localized electrons in the range up to about 2.5 and then decrease to zero value roughly at a same gradient with increasing localized electrons. (author)

  1. The Yersinia enterocolitica type three secretion chaperone SycO is integrated into the Yop regulatory network and binds to the Yop secretion protein YscM1

    Directory of Open Access Journals (Sweden)

    Heesemann Jürgen

    2007-07-01

    Full Text Available Abstract Background Pathogenic yersiniae (Y. pestis, Y. pseudotuberculosis, Y. enterocolitica share a virulence plasmid encoding a type three secretion system (T3SS. This T3SS comprises more than 40 constituents. Among these are the transport substrates called Yops (Yersinia outer proteins, the specific Yop chaperones (Sycs, and the Ysc (Yop secretion proteins which form the transport machinery. The effectors YopO and YopP are encoded on an operon together with SycO, the chaperone of YopO. The characterization of SycO is the focus of this study. Results We have established the large-scale production of recombinant SycO in its outright form. We confirm that Y. enterocolitica SycO forms homodimers which is typical for Syc chaperones. SycO overproduction in Y. enterocolitica decreases secretion of Yops into the culture supernatant suggesting a regulatory role of SycO in type III secretion. We demonstrate that in vitro SycO interacts with YscM1, a negative regulator of Yop expression in Y. enterocolitica. However, the SycO overproduction phenotype was not mediated by YscM1, YscM2, YopO or YopP as revealed by analysis of isogenic deletion mutants. Conclusion We present evidence that SycO is integrated into the regulatory network of the Yersinia T3SS. Our picture of the Yersinia T3SS interactome is supplemented by identification of the SycO/YscM1 interaction. Further, our results suggest that at least one additional interaction partner of SycO has to be identified.

  2. High dietary fat-induced obesity in Wistar rats and type 2 diabetes in nonobese Goto-Kakizaki rats differentially affect retinol binding protein 4 expression and vitamin A metabolism.

    Science.gov (United States)

    Shirai, Tomomi; Shichi, Yuta; Sato, Miyuki; Tanioka, Yuri; Furusho, Tadasu; Ota, Toru; Tadokoro, Tadahiro; Suzuki, Tsukasa; Kobayashi, Ken-Ichi; Yamamoto, Yuji

    2016-03-01

    Obesity is a major risk factor for type 2 diabetes, which is caused mainly by insulin resistance. Retinol binding protein 4 (RBP4) is the only specific transport protein for retinol in the serum. RBP4 level is increased in the diabetic state and high-fat condition, indicating that retinol metabolism may be affected under these conditions. However, the precise effect of diabetes and high fat-induced obesity on retinol metabolism is unknown. In this study, we examined differences in retinol metabolite levels in rat models of diet-induced obesity and type 2 diabetes (Goto-Kakizaki [GK] rat). Four-week-old male Wistar and GK rats were given either a control diet (AIN-93G) or a high-fat diet (HFD, 40% fat kJ). After 15 weeks of feeding, the RBP4 levels increased by 2-fold in the serum of GK rats but not HFD-fed rats. The hepatic retinol concentration of HFD-fed rats was approximately 50% that of the controls (P type 2 diabetes mellitus. Copyright © 2016 Elsevier Inc. All rights reserved.

  3. To bind or not to bind? Different temporal binding effects from voluntary pressing and releasing actions.

    Science.gov (United States)

    Zhao, Ke; Chen, Yu-Hsin; Yan, Wen-Jing; Fu, Xiaolan

    2013-01-01

    Binding effect refers to the perceptual attraction between an action and an outcome leading to a subjective compression of time. Most studies investigating binding effects exclusively employ the "pressing" action without exploring other types of actions. The present study addresses this issue by introducing another action, releasing action or the voluntary lifting of the finger/wrist, to investigate the differences between voluntary pressing and releasing actions. Results reveal that releasing actions led to robust yet short-lived temporal binding effects, whereas pressing condition had steady temporal binding effects up to super-seconds. The two actions also differ in sensitivity to changes in temporal contiguity and contingency, which could be attributed to the difference in awareness of action. Extending upon current models of "willed action," our results provide insights from a temporal point of view and support the concept of a dual system consisting of predictive motor control and top-down mechanisms.

  4. Overexpression and purification of U24 from human herpesvirus type-6 in E. coli: unconventional use of oxidizing environments with a maltose binding protein-hexahistine dual tag to enhance membrane protein yield

    Directory of Open Access Journals (Sweden)

    Straus Suzana K

    2011-06-01

    Full Text Available Abstract Background Obtaining membrane proteins in sufficient quantity for biophysical study and biotechnological applications has been a difficult task. Use of the maltose binding protein/hexahistidine dual tag system with E.coli as an expression host is emerging as a high throughput method to enhance membrane protein yield, solubility, and purity, but fails to be effective for certain proteins. Optimizing the variables in this system to fine-tune for efficiency can ultimately be a daunting task. To identify factors critical to success in this expression system, we have selected to study U24, a novel membrane protein from Human Herpesvirus type-6 with potent immunosuppressive ability and a possible role in the pathogenesis of the disease multiple sclerosis. Results We expressed full-length U24 as a C-terminal fusion to a maltose binding protein/hexahistidine tag and examined the effects of temperature, growth medium type, cell strain type, oxidizing vs. reducing conditions and periplasmic vs. cytoplasmic expression location. Temperature appeared to have the greatest effect on yield; at 37°C full-length protein was either poorly expressed (periplasm or degraded (cytoplasm whereas at 18°C, expression was improved especially in the periplasm of C41(DE3 cells and in the cytoplasm of oxidizing Δtrx/Δgor mutant strains, Origami 2 and SHuffle. Expression of the fusion protein in these strains were estimated to be 3.2, 5.3 and 4.3 times greater, respectively, compared to commonly-used BL21(DE3 cells. We found that U24 is isolated with an intramolecular disulfide bond under these conditions, and we probed whether this disulfide bond was critical to high yield expression of full-length protein. Expression analysis of a C21SC37S cysteine-free mutant U24 demonstrated that this disulfide was not critical for full-length protein expression, but it is more likely that strained metabolic conditions favour factors which promote protein expression. This

  5. Cyclic Di-GMP Binding by an Assembly ATPase (PilB2) and Control of Type IV Pilin Polymerization in the Gram-Positive Pathogen Clostridium perfringens.

    Science.gov (United States)

    Hendrick, William A; Orr, Mona W; Murray, Samantha R; Lee, Vincent T; Melville, Stephen B

    2017-05-15

    The Gram-positive pathogen Clostridium perfringens possesses type IV pili (TFP), which are extracellular fibers that are polymerized from a pool of pilin monomers in the cytoplasmic membrane. Two proteins that are essential for pilus functions are an assembly ATPase (PilB) and an inner membrane core protein (PilC). Two homologues each of PilB and PilC are present in C. perfringens , called PilB1/PilB2 and PilC1/PilC2, respectively, along with four pilin proteins, PilA1 to PilA4. The gene encoding PilA2, which is considered the major pilin based on previous studies, is immediately downstream of the pilB2 and pilC2 genes. Purified PilB2 had ATPase activity, bound zinc, formed hexamers even in the absence of ATP, and bound the second messenger molecule cyclic di-GMP (c-di-GMP). Circular dichroism spectroscopy of purified PilC2 indicated that it retained its predicted degree of alpha-helical secondary structure. Even though no direct interactions between PilB2 and PilC2 could be detected in vivo or in vitro even in the presence of c-di-GMP, high levels of expression of a diguanylate cyclase from C. perfringens (CPE1788) stimulated polymerization of PilA2 in a PilB2- and PilC2-dependent manner. These results suggest that PilB2 activity is controlled by c-di-GMP levels in vivo but that PilB2-PilC2 interactions are either transitory or of low affinity, in contrast to results reported previously from in vivo studies of the PilB1/PilC1 pair in which PilC1 was needed for polar localization of PilB1. This is the first biochemical characterization of a c-di-GMP-dependent assembly ATPase from a Gram-positive bacterium. IMPORTANCE Type IV pili (TFP) are protein fibers involved in important bacterial functions, including motility, adherence to surfaces and host cells, and natural transformation. All clostridia whose genomes have been sequenced show evidence of the presence of TFP. The genetically tractable species Clostridium perfringens was used to study proteins involved in

  6. Purification and characterization of Cc-Lec, C-type lactose-binding lectin: A platelet aggregation and blood-clotting inhibitor from Cerastes cerastes venom.

    Science.gov (United States)

    Samah, Saoud; Fatah, Chérifi; Jean-Marc, Berjeaud; Safia, Kellou-Taîri; Fatima, Laraba-Djebari

    2017-09-01

    In this study, we reported for the first time the biochemical and structural characterization of Cc-Lec, a C-type lectin purified from Cerastes cerastes venom by affinity chromatography. This lectin was homogeneous by SDS-PAGE, and was shown to be a 34 271.59Da polypeptide by Electrospray mass spectrometry MS-ES-TOF. Its identified sequence of 160 amino acids corresponding to one subunit, revealed a high identity with other related proteins. Cc-Lec modeled 3D structure appeared as homodimer cross-linked by one disulfide bridge. Cc-Lec exhibited a calcium dependent hemagglutinating activity against human group O erythrocytes. Cc-Lec inhibited platelet aggregation induced by ADP, arachidonic acid or fibrinogen suggesting its interaction with their specific receptors namely P2Y1 and/or P2Y12, GPIIb/IIIa and TPα respectively. Cc-Lec was not lethal for mice until 10mg/kg administered by i.p. route. The lectin displayed a lasting anticoagulation on mice plasma even two days post-injection. This anticoagulation seems to be related to its interaction with coagulation factors Xa and IXa. Therefore, Cc-Lec prevented FXa amidolytic activity with Km=4.3310 -4 μg/mL and ki=14.4μg/mL. It seems to interact with these targets through CRD domain which could make it a good target as a pharmacological promising molecule in thrombosis diagnosis and therapy. Copyright © 2017 Elsevier B.V. All rights reserved.

  7. Association of angiotensin-converting enzyme (ACE) and fatty acid binding protein 2 (FABP2) genes polymorphism with type 2 diabetes mellitus in Northern India.

    Science.gov (United States)

    Raza, Syed Tasleem; Fatima, Jalees; Ahmed, Faisal; Abbas, Shania; Zaidi, Zeashan Haider; Singh, Seema; Mahdi, Farzana

    2014-12-01

    Type 2 diabetes mellitus (T2DM) is growing in an epidemic manner across the world with an expected doubling of the incidence to millions of affected individuals in the last decades. At present, adequate data are not available regarding the ACE and FABP2 polymorphisms and their susceptibility with T2DM cases in the North Indian population. Thus we conceived the need for further study of ACE (I/D) and FABP2 (Ala54Thr) genes polymorphism and its susceptibility to T2DM in the North Indian population. In this study, a total of 300 subjects (including 190 T2DM cases and 110 controls) participated. ACE and FABP2 gene polymorphisms in the cases and controls were evaluated by polymerase chain reaction and restriction fragment length polymorphism. The frequencies of ACE I/I, I/D and D/D genotypes in T2DM cases and controls were 28.73%, 55.17%, 16.09% and 13.63%, 57.95%, 28.40%, respectively. The frequencies of FABP2 Ala54Ala, Ala54Thr and Thr54Thr in T2DM cases were 18.39%, 66.66%, 14.94% and 22.72%, 61.36%, 15.90% in controls, respectively. ACE I/I genotype was significantly more frequent in cases as compared to controls (p = 0.003, χ(2) = 9.13). It appears that the ACE I/I genotype frequency was significantly higher in the T2DM cases as compared to the controls. © The Author(s) 2013.

  8. Label-Free LC-MS Profiling of Skeletal Muscle Reveals Heart-Type Fatty Acid Binding Protein as a Candidate Biomarker of Aerobic Capacity.

    Science.gov (United States)

    Malik, Zulezwan Ab; Cobley, James N; Morton, James P; Close, Graeme L; Edwards, Ben J; Koch, Lauren G; Britton, Steven L; Burniston, Jatin G

    2013-12-01

    Two-dimensional gel electrophoresis provides robust comparative analysis of skeletal muscle, but this technique is laborious and limited by its inability to resolve all proteins. In contrast, orthogonal separation by SDS-PAGE and reverse-phase liquid chromatography (RPLC) coupled to mass spectrometry (MS) affords deep mining of the muscle proteome, but differential analysis between samples is challenging due to the greater level of fractionation and the complexities of quantifying proteins based on the abundances of their tryptic peptides. Here we report simple, semi-automated and time efficient ( i.e ., 3 h per sample) proteome profiling of skeletal muscle by 1-dimensional RPLC electrospray ionisation tandem MS. Solei were analysed from rats (n = 5, in each group) bred as either high- or low-capacity runners (HCR and LCR, respectively) that exhibited a 6.4-fold difference (1,625 ± 112 m vs . 252 ± 43 m, p ions, which spanned three orders of magnitude. In total, 207 proteins were analysed, which encompassed almost all enzymes of the major metabolic pathways in skeletal muscle. The most abundant protein detected was type I myosin heavy chain (RA = 5,843 ± 897) and the least abundant protein detected was heat shock 70 kDa protein (RA = 2 ± 0.5). Sixteen proteins were significantly ( p ion (551.21 m/z ) of the doubly-charged peptide SLGVGFATR (454.19 m/z ) of residues 23-31 of FABPH. SRM was conducted on technical replicates of each biological sample and exhibited a coefficient of variation of 20%. The abundance of FABPH measured by SRM was 2.84-fold greater ( p = 0.0095) in HCR muscle. In addition, SRM of FABPH was performed in vastus lateralis samples of young and elderly humans with different habitual activity levels (collected during a previous study) finding FABPH abundance was 2.23-fold greater ( p = 0.0396) in endurance-trained individuals regardless of differences in age. In summary, our findings in HCR/LCR rats provide protein-level confirmation for

  9. The Type IV Pilus Assembly ATPase PilB of Myxococcus xanthus Interacts with the Inner Membrane Platform Protein PilC and the Nucleotide-binding Protein PilM.

    Science.gov (United States)

    Bischof, Lisa Franziska; Friedrich, Carmen; Harms, Andrea; Søgaard-Andersen, Lotte; van der Does, Chris

    2016-03-25

    Type IV pili (T4P) are ubiquitous bacterial cell surface structures, involved in processes such as twitching motility, biofilm formation, bacteriophage infection, surface attachment, virulence, and natural transformation. T4P are assembled by machinery that can be divided into the outer membrane pore complex, the alignment complex that connects components in the inner and outer membrane, and the motor complex in the inner membrane and cytoplasm. Here, we characterize the inner membrane platform protein PilC, the cytosolic assembly ATPase PilB of the motor complex, and the cytosolic nucleotide-binding protein PilM of the alignment complex of the T4P machinery ofMyxococcus xanthus PilC was purified as a dimer and reconstituted into liposomes. PilB was isolated as a monomer and bound ATP in a non-cooperative manner, but PilB fused to Hcp1 ofPseudomonas aeruginosaformed a hexamer and bound ATP in a cooperative manner. Hexameric but not monomeric PilB bound to PilC reconstituted in liposomes, and this binding stimulated PilB ATPase activity. PilM could only be purified when it was stabilized by a fusion with a peptide corresponding to the first 16 amino acids of PilN, supporting an interaction between PilM and PilN(1-16). PilM-N(1-16) was isolated as a monomer that bound but did not hydrolyze ATP. PilM interacted directly with PilB, but only with PilC in the presence of PilB, suggesting an indirect interaction. We propose that PilB interacts with PilC and with PilM, thus establishing the connection between the alignment and the motor complex. © 2016 by The American Society for Biochemistry and Molecular Biology, Inc.

  10. Bovine papillomavirus type 2 (BPV-2) E5 oncoprotein binds to the subunit D of the V₁-ATPase proton pump in naturally occurring urothelial tumors of the urinary bladder of cattle.

    Science.gov (United States)

    Roperto, Sante; Russo, Valeria; Borzacchiello, Giuseppe; Urraro, Chiara; Lucà, Roberta; Esposito, Iolanda; Riccardi, Marita Georgia; Raso, Cinzia; Gaspari, Marco; Ceccarelli, Dora Maria; Galasso, Rocco; Roperto, Franco

    2014-01-01

    Active infection by bovine papillomavirus type 2 (BPV-2) was documented for fifteen urinary bladder tumors in cattle. Two were diagnosed as papillary urothelial neoplasm of low malignant potential (PUNLMP), nine as papillary and four as invasive urothelial cancers. In all cancer samples, PCR analysis revealed a BPV-2-specific 503 bp DNA fragment. E5 protein, the major oncoprotein of the virus, was shown both by immunoprecipitation and immunohistochemical analysis. E5 was found to bind to the activated (phosphorylated) form of the platelet derived growth factor β receptor. PDGFβR immunoprecipitation from bladder tumor samples and from normal bladder tissue used as control revealed a protein band which was present in the pull-down from bladder cancer samples only. The protein was identified with mass spectrometry as "V₁-ATPase subunit D", a component of the central stalk of the V₁-ATPase vacuolar pump. The subunit D was confirmed in this complex by coimmunoprecipitation investigations and it was found to colocalize with the receptor. The subunit D was also shown to be overexpressed by Western blot, RT-PCR and immunofluorescence analyses. Immunoprecipitation and immunofluorescence also revealed that E5 oncoprotein was bound to the subunit D. For the first time, a tri-component complex composed of E5/PDGFβR/subunit D has been documented in vivo. Previous in vitro studies have shown that the BPV-2 E5 oncoprotein binds to the proteolipid c ring of the V₀-ATPase sector. We suggest that the E5/PDGFβR/subunit D complex may perturb proteostasis, organelle and cytosol homeostasis, which can result in altered protein degradation and in autophagic responses.

  11. Myxoma Virus dsRNA Binding Protein M029  Inhibits the Type I IFN-Induced Antiviral State in a  Highly Species-Specific Fashion.

    Science.gov (United States)

    Rahman, Masmudur M; McFadden, Grant

    2017-02-02

    Myxoma virus (MYXV) is Leporipoxvirus that possesses a specific rabbit-restricted host tropism but exhibits a much broader  cellular host range in cultured cells. MYXV is able to efficiently  block all aspects of the type I interferon (IFN)-induced  antiviral  state  in rabbit cells, partially in  human  cells  and  very  poorly  in  mouse  cells.  The mechanism(s) of this species-specific inhibition of  type I IFN-induced antiviral state is not well understood. Here we demonstrate that MYXV encoded  protein  M029, a truncated relative of the vaccinia virus (VACV) E3 double-stranded RNA (dsRNA)  binding  protein  that  inhibits  protein  kinase  R (PKR),  can  also  antagonize the type I IFN-induced  antiviral state in a highly species-specific manner. In cells pre-treated with type I IFN prior to  infection,  MYXV  exploits  M029  to  overcome  the  induced  antiviral  state completely in rabbit cells,  partially  in  human  cells,  but  not at all in mouse cells. However, in cells pre-infected with MYXV,  IFN-induced  signaling  is fully  inhibited  even  in the  absence  of M029 in cells from all three species,  suggesting  that  other  MYXV  protein(s)  apart  from  M029  block  IFN  signaling  in  a  speciesindependent  manner.  We  also  show  that  the  antiviral  state  induced in rabbit, human or mouse cells  by  type  I IFN  can  inhibit M029-knockout MYXV even when PKR is genetically knocked-out, suggesting  that  M029  targets  other  host  proteins  for  this  antiviral state inhibition. Thus, the MYXV  dsRNA  binding  protein  M029  not  only  antagonizes  PKR  from  multiple  species  but  also blocks the  type I IFN antiviral state independently of PKR in a highly species-specific fashion.

  12. Estradiol up-regulates L-type Ca2+ channels via membrane-bound estrogen receptor/phosphoinositide-3-kinase/Akt/cAMP response element-binding protein signaling pathway.

    Science.gov (United States)

    Yang, Xiaoyan; Mao, Xiaofang; Xu, Gao; Xing, Shasha; Chattopadhyay, Ansuman; Jin, Si; Salama, Guy

    2018-05-01

    In long QT syndrome type 2, women are more prone than men to the lethal arrhythmia torsades de pointes. We previously reported that 17β-estradiol (E2) up-regulates L-type Ca 2+ channels and current (I Ca,L ) (∼30%) in rabbit ventricular myocytes by a classic genomic mechanism mediated by estrogen receptor-α (ERα). In long QT syndrome type 2 (I Kr blockade or bradycardia), the higher Ca 2+ influx via I Ca,L causes Ca 2+ overload, spontaneous sarcoplasmic reticulum Ca 2+ release, and reactivation of I Ca,L that triggers early afterdepolarizations and torsades de pointes. The purpose of this study was to investigate the molecular mechanisms whereby E2 up-regulates I Ca,L , which are poorly understood. H9C2 and rat myocytes were incubated with E2 ± ER antagonist, or inhibitors of downstream transcription factors, for 24 hours, followed by western blots of Cav1.2α1C and voltage-clamp measurements of I Ca,L . Incubation of H9C2 cells with E2 (10-100 nM) increased I Ca,L density and Cav1.2α1C expression, which were suppressed by the ER antagonist ICI182,780 (1 μM). Enhanced I Ca,L and Cav1.2α1C expression by E2 was suppressed by inhibitors of phosphoinositide-3-kinase (Pi3K) (30 μM LY294002; P L via plasma membrane ER and by activating Pi3K, Akt, and CREB signaling. The promoter regions of the CACNA1C gene (human-rabbit-rat) contain adjacent/overlapping binding sites for p-CREB and ERα, which suggests a synergistic regulation by these pathways. Copyright © 2018 Heart Rhythm Society. Published by Elsevier Inc. All rights reserved.

  13. Bitopic Ligands and Metastable Binding Sites

    DEFF Research Database (Denmark)

    Fronik, Philipp; Gaiser, Birgit I; Sejer Pedersen, Daniel

    2017-01-01

    of orthosteric binding sites. Bitopic ligands have been employed to address the selectivity problem by combining (linking) an orthosteric ligand with an allosteric modulator, theoretically leading to high-affinity subtype selective ligands. However, it remains a challenge to identify suitable allosteric binding...... that have been reported to date, this type of bitopic ligands would be composed of two identical pharmacophores. Herein, we outline the concept of bitopic ligands, review metastable binding sites, and discuss their potential as a new source of allosteric binding sites....

  14. Binding energies of cluster ions

    International Nuclear Information System (INIS)

    Parajuli, R.; Matt, S.; Scheier, P.; Echt, O.; Stamatovic, A.; Maerk, T.D.

    2002-01-01

    The binding energy of charged clusters may be measured by analyzing the kinetic energy released in the metastable decay of mass selected parent ions. Using finite heat bath theory to determine the binding energies of argon, neon, krypton, oxygen and nitrogen from their respective average kinetic energy released were carried out. A high-resolution double focussing two-sector mass spectrometer of reversed Nier-Johnson type geometry was used. MIKE ( mass-analysed ion kinetic energy) were measured to investigate decay reactions of mass-selected ions. For the inert gases neon (Ne n + ), argon (Ar n + ) and krypton (Kr n + ), it is found that the binding energies initially decrease with increasing size n and then level off at a value above the enthalpy of vaporization of the condensed phase. Oxygen cluster ions shown a characteristic dependence on cluster size (U-shape) indicating a change in the metastable fragmentation mechanism when going from the dimer to the decamer ion. (nevyjel)

  15. 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 ...

  16. Modulation of type I interferon induction by porcine reproductive and respiratory syndrome virus and degradation of CREB-binding protein by non-structural protein 1 in MARC-145 and HeLa cells

    International Nuclear Information System (INIS)

    Kim, Oekyung; Sun Yan; Lai, Frances W.; Song Cheng; Yoo, Dongwan

    2010-01-01

    Porcine reproductive and respiratory syndrome (PRRS) is an emerged disease of swine characterized by negligible response of type I IFNs and viral persistence. We show that the PRRSV non-structural protein 1 (Nsp1) is the viral component responsible for modulation of IFN response. Nsp1 blocked dsRNA-induced IRF3 and IFN promoter activities. Nsp1 did not block phosphorylation and nuclear translocation of IRF3 but inhibited IRF3 association with CREB-binding protein (CBP) in the nucleus. While IRF3 was stable, CBP was degraded, and CBP degradation was proteasome-dependent, suggesting that CBP degradation is not due to the protease activity of Nsp1 but an intermediary is involved. Our data suggest that the Nsp1-mediated CBP degradation inhibits the recruitment of CBP for enhanceosome assembly, leading to the block of IFN response. CBP degradation is a novel strategy for viral evasion from the host response, and Nsp1 may form a new class of viral antagonists for IFN modulation.

  17. The association between insulin-like growth factor 1 (IGF-1), IGF-binding proteins (IGFBPs), and the carboxyterminal propeptide of type I procollagen (PICP) in pre- and postmenopausal women with rheumatoid arthritis.

    Science.gov (United States)

    Szeremeta, A; Jura-Półtorak, A; Komosińska-Vassev, K; Zoń-Giebel, A; Kapołka, D; Olczyk, K

    2017-05-01

    To assess the association between plasma levels of the insulin-like growth factor (IGF) system including IGF-1, IGF-binding proteins (IGFBPs) including IGFBP-1, total (t-)IGFBP-3 and functional (f-)IGFBP-3, and the carboxyterminal propeptide of type I procollagen (PICP) in pre- and postmenopausal women with rheumatoid arthritis (RA). Plasma concentrations of IGF-1, IGFBP-1, t-IGFBP-3, f-IGFBP-3, and PICP were measured by immunoassay. No significant difference was observed in plasma IGF-1 levels between pre- and postmenopausal subjects. Plasma levels of IGFBP-1 were elevated in RA. PICP and f-IGFBP-3 were greatly affected by menopausal status. Of the three IGFBPs tested, only f-IGFBP-3 plasma levels in RA women correlated negatively with age and disease duration. A positive correlation was demonstrated between PICP and erythrocyte sedimentation rate (ESR) in RA. Moreover, there was no correlation between PICP and IGF-1 and any of the IGFBPs in RA women. Considerable disruption of the IGF system in RA was found to be related to disease activity and duration. Changes in the IGF-IGFBP axis and PICP levels were different in pre- and postmenopausal women with RA. Elevated plasma PICP concentrations may indicate an increased rate of bone formation in postmenopausal RA women. Additionally, the observed changes in the IGF/IGFBP system did not affect bone formation during RA.

  18. 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

  19. 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...

  20. СHARACTERISTICS OF THE HEART FATTY ACID-BINDING PROTEIN, INTERLEUKIN-6 AND INTERLEUKIN-8 AS ALTERNATIVE MARKERS OF DIABETIC NEPHROPATHY PROGRESSION IN PATIENTS WITH TYPE 1 DIABETES MELLITUS

    Directory of Open Access Journals (Sweden)

    Yu. A. Ryzhikova

    2015-01-01

    Full Text Available The aim of this work was to study the levels of the heart fatty acid-binding protein (h-FABP, interleukin6 (IL-6 and interleukin-8 (IL-8, in diabetic nephropathy (DN in patients with type 1 diabetes mellitus (T1DM. Material and methods. We examined 87 patients aged 18 to 54 with T1DM within the study group. 30 patients with type 1 diabetes were diagnosed with normoalbuminuria, 29 patients – with microalbuminuria and 28 patients – with proteinuria. The control group consisted of 24 healthy donor aged 22 to 29. The comparison group included 22 patients aged 20 to 42 with verified diagnosis of essential arterial hypertension (AH without carbohydrate metabolism disorders. The daily urinary albumin excretion was determined by immunoturbidimetric technique. 30 patients with type 1 diabetes were diagnosed with normoalbuminuria, 29 patients – with microalbuminuria and 28 patients with proteinuria.Calculation of glomerular filtration rate was performed according to the Hoek formula with the use of cystatinС serum concentrations. Contents of h-FABP, IL-6 and cystatin C in serum and h-FABP, IL-8 inurine were determined by enzyme-linked immunosorbent assay. Results. Analysis of the h-FABP content in serum showed that the concentration of this marker in individuals with T1DM was higher than in patients of the control group and the comparison group. Analysis of the h-FABP content in the urine revealed that individuals with essential hypertension showed an increased level of h-FABP while patients with T1DM demonstrated the highest concentration of h-FABP. The concentration of IL-6 inindividuals with T1DM and in individuals with AH significantly exceeded the control values. The contents of h-FABP and IL-6 inserum and h-FABP and IL-8 inurine increased with the progression of DN and reached maximum in individuals of the proteinuria subgroup. At the same time, the levels of h-FABP and IL-8 inthe urine of patients in the microalbuminuria (MAU subgroup were

  1. Verification of an immunoturbidimetric assay for heart-type fatty acid-binding protein (H-FABP) on a clinical chemistry platform and establishment of the upper reference limit.

    Science.gov (United States)

    Da Molin, Simona; Cappellini, Fabrizio; Falbo, Rosanna; Signorini, Stefano; Brambilla, Paolo

    2014-11-01

    Heart-type fatty acid-binding protein (H-FABP) is an early biomarker of cardiac injury. Randox Laboratories developed an immunoturbidimetric H-FABP assay for non-proprietary automated clinical chemistry analysers that could be useful in the emergency department. We verified the analytical performances claimed by Randox Laboratories on Roche Cobas 6000 clinical chemistry platform in use in our laboratory, and we defined our own 99th percentile upper reference limit for H-FABP. For the verification of method performances, we used pools of spared patient samples from routine and two levels of quality control material, while samples for the reference value study were collected from 545 blood donors. Following CLSI guidelines we verified limit of blank (LOB), limit of detection (LOD), limit of quantitation (LOQ), repeatability and within-laboratory precision, trueness, linearity, and the stability of H-FABP in EDTA over 24h. The LOQ (3.19 μg/L) was verified with a CV% of 10.4. The precision was verified for the low (mean 5.88 μg/L, CV=6.7%), the medium (mean 45.28 μg/L, CV=3.0%), and the high concentration (mean 88.81 μg/L, CV=4.0%). The trueness was verified as well as the linearity over the indicated measurement interval of 0.747-120 μg/L. The H-FABP in EDTA samples is stable throughout 24h both at room temperature and at 4 °C. The H-FABP 99th percentile upper reference limit for all subjects (3.60 μg/L, 95% CI 3.51-3.77) is more appropriate than gender-specific ones that are not statistically different. Copyright © 2014 The Canadian Society of Clinical Chemists. Published by Elsevier Inc. All rights reserved.

  2. The PorX response regulator of the Porphyromonas gingivalis PorXY two-component system does not directly regulate the Type IX secretion genes but binds the PorL subunit.

    Directory of Open Access Journals (Sweden)

    Maxence S Vincent

    2016-08-01

    Full Text Available The Type IX secretion system (T9SS is a versatile multi-protein complex restricted to bacteria of the Bacteriodetes phylum and responsible for the secretion of surface attachment of diverse proteins that participate to S-layer formation, gliding motility or pathogenesis. The T9SS is poorly characterized but a number of proteins involved in the assembly of the secretion apparatus in the oral pathogen Porphyromonas gingivalis have been identified based on genome substractive analyses. Among these proteins, PorY and PorX encode typical two-component system (TCS sensor and CheY-like response regulator respectively. Although the porX and porY genes do not localize at the same genetic locus, it has been proposed that PorXY form a bona fide TCS. Deletion of the porX in P. gingivalis causes a slight decrease of the expression of a number of other T9SS genes, including sov, porT, porP, porK, porL, porM, porN and porY. Here, we show that PorX and the soluble cytoplasmic domain of PorY interact. Using electrophoretic mobility shift, DNA-protein co-purification and heterologous host expression assays, we showed that PorX does not bind and does not directly regulate expression of the T9SS genes. Finally, we show that PorX interacts with the cytoplasmic domain of PorL, a component of the T9SS membrane core complex and propose that the CheY-like PorX protein might be involved in the dynamics of the T9SS.

  3. The PorX Response Regulator of the Porphyromonas gingivalis PorXY Two-Component System Does Not Directly Regulate the Type IX Secretion Genes but Binds the PorL Subunit

    Science.gov (United States)

    Vincent, Maxence S.; Durand, Eric; Cascales, Eric

    2016-01-01

    The Type IX secretion system (T9SS) is a versatile multi-protein complex restricted to bacteria of the Bacteriodetes phylum and responsible for the secretion or cell surface exposition of diverse proteins that participate to S-layer formation, gliding motility or pathogenesis. The T9SS is poorly characterized but a number of proteins involved in the assembly of the secretion apparatus in the oral pathogen Porphyromonas gingivalis have been identified based on genome substractive analyses. Among these proteins, PorY, and PorX encode typical two-component system (TCS) sensor and CheY-like response regulator respectively. Although the porX and porY genes do not localize at the same genetic locus, it has been proposed that PorXY form a bona fide TCS. Deletion of porX in P. gingivalis causes a slight decrease of the expression of a number of other T9SS genes, including sov, porT, porP, porK, porL, porM, porN, and porY. Here, we show that PorX and the soluble cytoplasmic domain of PorY interact. Using electrophoretic mobility shift, DNA-protein co-purification and heterologous host expression assays, we demonstrate that PorX does not bind T9SS gene promoters and does not directly regulate expression of the T9SS genes. Finally, we show that PorX interacts with the cytoplasmic domain of PorL, a component of the T9SS membrane core complex and propose that the CheY-like PorX protein might be involved in the dynamics of the T9SS. PMID:27630829

  4. Human immunodeficiency virus type 1 enhancer-binding protein 3 is essential for the expression of asparagine-linked glycosylation 2 in the regulation of osteoblast and chondrocyte differentiation.

    Science.gov (United States)

    Imamura, Katsuyuki; Maeda, Shingo; Kawamura, Ichiro; Matsuyama, Kanehiro; Shinohara, Naohiro; Yahiro, Yuhei; Nagano, Satoshi; Setoguchi, Takao; Yokouchi, Masahiro; Ishidou, Yasuhiro; Komiya, Setsuro

    2014-04-04

    Human immunodeficiency virus type 1 enhancer-binding protein 3 (Hivep3) suppresses osteoblast differentiation by inducing proteasomal degradation of the osteogenesis master regulator Runx2. In this study, we tested the possibility of cooperation of Hivep1, Hivep2, and Hivep3 in osteoblast and/or chondrocyte differentiation. Microarray analyses with ST-2 bone stroma cells demonstrated that expression of any known osteochondrogenesis-related genes was not commonly affected by the three Hivep siRNAs. Only Hivep3 siRNA promoted osteoblast differentiation in ST-2 cells, whereas all three siRNAs cooperatively suppressed differentiation in ATDC5 chondrocytes. We further used microarray analysis to identify genes commonly down-regulated in both MC3T3-E1 osteoblasts and ST-2 cells upon knockdown of Hivep3 and identified asparagine-linked glycosylation 2 (Alg2), which encodes a mannosyltransferase residing on the endoplasmic reticulum. The Hivep3 siRNA-mediated promotion of osteoblast differentiation was negated by forced Alg2 expression. Alg2 suppressed osteoblast differentiation and bone formation in cultured calvarial bone. Alg2 was immunoprecipitated with Runx2, whereas the combined transfection of Runx2 and Alg2 interfered with Runx2 nuclear localization, which resulted in suppression of Runx2 activity. Chondrocyte differentiation was promoted by Hivep3 overexpression, in concert with increased expression of Creb3l2, whose gene product is the endoplasmic reticulum stress transducer crucial for chondrogenesis. Alg2 silencing suppressed Creb3l2 expression and chondrogenesis of ATDC5 cells, whereas infection of Alg2-expressing virus promoted chondrocyte maturation in cultured cartilage rudiments. Thus, Alg2, as a downstream mediator of Hivep3, suppresses osteogenesis, whereas it promotes chondrogenesis. To our knowledge, this study is the first to link a mannosyltransferase gene to osteochondrogenesis.

  5. The PDZ domain binding motif (PBM) of human T-cell leukemia virus type 1 Tax can be substituted by heterologous PBMs from viral oncoproteins during T-cell transformation.

    Science.gov (United States)

    Aoyagi, Tomoya; Takahashi, Masahiko; Higuchi, Masaya; Oie, Masayasu; Tanaka, Yuetsu; Kiyono, Tohru; Aoyagi, Yutaka; Fujii, Masahiro

    2010-04-01

    Several tumor viruses, such as human T-cell leukemia virus (HTLV), human papilloma virus (HPV), human adenovirus, have high-oncogenic and low-oncogenic subtypes, and such subtype-specific oncogenesis is associated with the PDZ-domain binding motif (PBM) in their transforming proteins. HTLV-1, the causative agent of adult T-cell leukemia, encodes Tax1 with PBM as a transforming protein. The Tax1 PBM was substituted with those from other oncoviruses, and the transforming activity was examined. Tax1 mutants with PBM from either HPV-16 E6 or adenovirus type 9 E4ORF1 are fully active in the transformation of a mouse T-cell line from interleukin-2-dependent growth into independent growth. Interestingly, one such Tax1 PBM mutant had an extra amino acid insertion derived from E6 between PBM and the rest of Tax1, thus suggesting that the amino acid sequences of the peptides between PBM and the rest of Tax1 and the numbers only slightly affect the function of PBM in the transformation. Tax1 and Tax1 PBM mutants interacted with tumor suppressors Dlg1 and Scribble with PDZ-domains. Unlike E6, Tax1 PBM mutants as well as Tax1 did not or minimally induced the degradations of Dlg1 and Scribble, but instead induced their subcellular translocation from the detergent-soluble fraction into the insoluble fraction, thus suggesting that the inactivation mechanism of these tumor suppressor proteins is distinct. The present results suggest that PBMs of high-risk oncoviruses have a common function(s) required for these three tumor viruses to transform cells, which is likely associated with the subtype-specific oncogenesis of these tumor viruses.

  6. Suspected acute coronary syndrome in the emergency room: Limited added value of heart type fatty acid binding protein point of care or ELISA tests: The FAME-ER (Fatty Acid binding protein in Myocardial infarction Evaluation in the Emergency Room) study.

    Science.gov (United States)

    Bank, Ingrid Em; Dekker, Marieke S; Hoes, Arno W; Zuithoff, Nicolaas Pa; Verheggen, Peter Whm; de Vrey, Evelyn A; Wildbergh, Thierry X; Timmers, Leo; de Kleijn, Dominique Pv; Glatz, Jan Fc; Mosterd, Arend

    2016-08-01

    Timely recognition of acute coronary syndrome remains a challenge as many biomarkers, including troponin, remain negative in the first hours following the onset of chest pain. We assessed the diagnostic accuracy of heart-type fatty acid binding protein (H-FABP), a cardiac biomarker with potential value immediately post symptom onset. Prospective monocentre diagnostic accuracy study of H-FABP bedside point of care (CardioDetect®) and ELISA tests in acute coronary syndrome suspected patients presenting within 24 hours of symptom onset to the emergency department, in addition to clinical findings, electrocardiography and the currently recommended biomarker high sensitivity troponin-T (hs-cTnT). The final diagnosis of acute coronary syndrome was adjudicated by two independent cardiologists, blinded to H-FABP results. Acute coronary syndrome was diagnosed in 149 (32.9%) of 453 unselected patients with suspected acute coronary syndrome (56% men, mean age 62.6 years). Negative predictive values were similar for H-FABP point of care and ELISA tests (79% vs. 78% respectively), but inferior to initial hs-cTnT (negative predictive value 86%). The addition of H-FABP point of care results to hs-cTnT increased the negative predictive value to 89%. In a multivariable logistic regression model, H-FABP point of care and ELISA tests yielded relevant diagnostic information in addition to clinical findings and ECG (likelihood ratio test pacute coronary syndrome presenting to the emergency department, H-FABP testing improves diagnostic accuracy in addition to clinical findings and electrocardiography. H-FABP, however, has no additional diagnostic value when hs-cTnT measurements are also available. © The European Society of Cardiology 2015.

  7. (TH) diazepam binding to human granulocytes

    Energy Technology Data Exchange (ETDEWEB)

    Bond, P.A.; Cundall, R.L.; Rolfe, B.

    1985-07-08

    (TH)-diazepam binds to sites on human granulocyte membranes, with little or no binding to platelets or lymphocytes. These (TH)-diazepam binding sites are of the peripheral type, being strongly inhibited by R05-4864 (Ki=6.23nM) but only weakly by clonazepam (Ki=14 M). Binding of (TH) diazepam at 0 is saturable, specific and stereoselective. Scatchard analysis indicates a single class of sites with Bmax of 109 +/- 17f moles per mg of protein and K/sub D/ of 3.07 +/- 0.53nM. Hill plots of saturation experiments gave straight lines with a mean Hill coefficient of 1.03 +/- 0.014. Binding is time dependent and reversible and it varies linearly with granulocyte protein concentration over the range 0.025-0.300 mg of protein. 11 references, 3 figures, 1 table.

  8. Complement receptors type 1 (CR1, CD35) and 2 (CR2, CD21) cooperate in the binding of hydrolyzed complement factor 3 (C3i) to human B lymphocytes

    DEFF Research Database (Denmark)

    Leslie, Robert Graham Quinton; Prodinger, Wolfgang Maria; Nielsen, Claus Henrik

    2003-01-01

    The C3b-binding receptor, CR1/CD35, supports CR2/CD21-mediated activation of complement by human B lymphocytes, possibly by associating with CR2 to promote or stabilize the binding of hydrolyzed C3 (C3i), the primary component of the AP convertase, C3i-Bb. To evaluate this hypothesis, we examined...... the uptake kinetics and binding equilibria for C3i dimer interaction with human blood cells in the absence and presence of CR1- and CR2-blocking mAb. C3i displayed dual uptake kinetics to B lymphocytes, comprising of rapid binding to CR1 and slower binding to CR2. The forward rate constants (k(1)) for CR1...... and CR2, operating independently, differed ca. 9-fold (k(1)=193+/-9.4 and 22.2+/-6.0 x 10(3) M(-1)s(-1), respectively). Equilibrium binding of C3i to B lymphocytes was also complex, varying in strength by ca. 13-fold over the C3i concentration range examined. The maximum association constant (K(a, max...

  9. Fundamental considerations in ski binding analysis.

    Science.gov (United States)

    Mote, C D; Hull, M L

    1976-01-01

    should be able to release his bindings in every mode by simply pulling or twisting his foot outward. If that cannot be done without injury, the skier has identified for himself one type of fall that will result in injury. 8. And lastly, a little advice from Ben Franklin--"Carelessness does more harm than a want of knowledge."

  10. Feature Binding in Zebrafish

    Directory of Open Access Journals (Sweden)

    P Neri

    2012-07-01

    Full Text Available Binding operations are primarily ascribed to cortex or similarly complex avian structures. My experiments show that the zebrafish, a lower vertebrate lacking cortex, supports visual feature binding of form and motion for the purpose of social behavior. These results challenge the notion that feature binding may require highly evolved neural structures and demonstrate that the nervous system of lower vertebrates can afford unexpectedly complex computations.

  11. Mannose-Binding Lectin Binds to Amyloid Protein and Modulates Inflammation

    Directory of Open Access Journals (Sweden)

    Mykol Larvie

    2012-01-01

    Full Text Available Mannose-binding lectin (MBL, a soluble factor of the innate immune system, is a pattern recognition molecule with a number of known ligands, including viruses, bacteria, and molecules from abnormal self tissues. In addition to its role in immunity, MBL also functions in the maintenance of tissue homeostasis. We present evidence here that MBL binds to amyloid β peptides. MBL binding to other known carbohydrate ligands is calcium-dependent and has been attributed to the carbohydrate-recognition domain, a common feature of other C-type lectins. In contrast, we find that the features of MBL binding to Aβ are more similar to the reported binding characteristics of the cysteine-rich domain of the unrelated mannose receptor and therefore may involve the MBL cysteine-rich domain. Differences in MBL ligand binding may contribute to modulation of inflammatory response and may correlate with the function of MBL in processes such as coagulation and tissue homeostasis.

  12. Binding of corroded ions to human saliva.

    Science.gov (United States)

    Mueller, H J

    1985-05-01

    Employing equilibrium dialysis, the binding abilities of Cu, Al, Co and Cr ions from corroded Cu-Al and Co-Cr dental casting alloys towards human saliva and two of its gel chromatographic fractions were determined. Results indicate that both Cu and Co bind to human saliva i.e. 0.045 and 0.027 mg/mg protein, respectively. Besides possessing the largest binding ability, Cu also possessed the largest binding capacity. The saturation of Cu binding was not reached up to the limit of 0.35 mg protein/ml employed in the tests, while Co reached full saturation at about 0.2 mg protein/ml. Chromium showed absolutely no binding to human saliva while Al ions did not pass through the dialysis membranes. Compared to the binding with solutions that were synthetically made up to contain added salivary-type proteins, it is shown that the binding to human saliva is about 1 order of magnitude larger, at least for Cu ions.

  13. Melanin-binding radiopharmaceuticals

    International Nuclear Information System (INIS)

    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

  14. Competitive protein binding assay

    International Nuclear Information System (INIS)

    Kaneko, Toshio; Oka, Hiroshi

    1975-01-01

    The measurement of cyclic GMP (cGMP) by competitive protein binding assay was described and discussed. The principle of binding assay was represented briefly. Procedures of our method by binding protein consisted of preparation of cGMP binding protein, selection of 3 H-cyclic GMP on market, and measurement procedures. In our method, binding protein was isolated from the chrysalis of silk worm. This method was discussed from the points of incubation medium, specificity of binding protein, the separation of bound cGMP from free cGMP, and treatment of tissue from which cGMP was extracted. cGMP existing in the tissue was only one tenth or one scores of cGMP, and in addition, cGMP competed with cGMP in binding with binding protein. Therefore, Murad's technique was applied to the isolation of cGMP. This method provided the measurement with sufficient accuracy; the contamination by cAMP was within several per cent. (Kanao, N.)

  15. Regulation of adipocyte 11β-hydroxysteroid dehydrogenase type 1 (11β-HSD1 by CCAAT/enhancer-binding protein (C/EBP β isoforms, LIP and LAP.

    Directory of Open Access Journals (Sweden)

    Cristina L Esteves

    Full Text Available 11β-Hydroxysteroid dehydrogenase type 1 (11β-HSD1 catalyses intracellular regeneration of active glucocorticoids, notably in liver and adipose tissue. 11β-HSD1 is increased selectively in adipose tissue in human obesity, a change implicated in the pathogenesis of metabolic syndrome. With high fat (HF-feeding, adipose tissue 11β-HSD1 is down-regulated in mice, plausibly to counteract metabolic disease. Transcription of 11β-HSD1 is directly regulated by members of the CCAAT/enhancer binding protein (C/EBP family. Here we show that while total C/EBPβ in adipose tissue is unaltered by HF diet, the ratio of the C/EBPβ isoforms liver-enriched inhibitor protein (LIP and liver-enriched activator protein (LAP (C/EBPβ-LIP:LAP is increased in subcutaneous adipose. This may cause changes in 11β-HSD1 expression since genetically modified C/EBPβ((+/L mice, with increased C/EBPβ-LIP:LAP ratio, have decreased subcutaneous adipose 11β-HSD1 mRNA levels, whereas C/EBPβ(ΔuORF mice, with decreased C/EBPβ-LIP:LAP ratio, show increased subcutaneous adipose 11β-HSD1. C/EBPβ-LIP:LAP ratio is regulated by endoplasmic reticulum (ER stress and mTOR signalling, both of which are altered in obesity. In 3T3-L1 adipocytes, 11β-HSD1 mRNA levels were down-regulated following induction of ER stress by tunicamycin but were up-regulated following inhibition of mTOR by rapamycin. These data point to a central role for C/EBPβ and its processing to LIP and LAP in transcriptional regulation of 11β-HSD1 in adipose tissue. Down-regulation of 11β-HSD1 by increased C/EBPβ-LIP:LAP in adipocytes may be part of a nutrient-sensing mechanism counteracting nutritional stress generated by HF diet.

  16. Facts about Type 2

    Medline Plus

    Full Text Available ... Diabetes Research & Practice Home We Are Research Leaders World's Largest Diabetes Meeting Recent Advances Type 1 Research ... survey-loading').remove(); $('#emailsurvey').hide(); $('#emailsurveythanks').show(); } } }; /* bind any forms with the "luminateApi" class */ luminateExtend.api.bind(); }); })( ...

  17. Datin, a yeast poly(dA:dT)-binding protein, behaves as an activator of the wild-type ILV1 promoter and interacts synergistically with Reb1p

    DEFF Research Database (Denmark)

    Moreira, José Manuel Alfonso; Remacle, J E; Kielland-Brandt, Morten

    1998-01-01

    A cis-acting element required for GCN4-independent basal-level transcription of ILV1 was previously identified in our laboratories as a binding site for the REB1 protein (Reb1p). Further deletion analysis of the ILV1 promoter region identified a second element also required for GCN4-independent...... basal-level ILV1 expression. This second element is an A.T-rich tract (26 As out of 32 nucleotides) situated 15 bp downstream of the Reb1p-binding site. Deletion of both the Reblp site and the poly(dA:dT) element totally eliminates basal activity of the ILV1 promoter. We show that the two elements act...... synergistically to control ILV1 expression and that the synergistic effect is distance dependent. We demonstrate that (i) datin (Dat1p), the only known poly (dA:dT)-binding protein in yeast, specifically binds to the ILV1 poly(dA:dT) element in vitro; (ii) Dat1p functions as a trans-activating factor in the ILV1...

  18. SHBG (Sex Hormone Binding Globulin)

    Science.gov (United States)

    ... Links Patient Resources For Health Professionals Subscribe Search Sex Hormone Binding Globulin (SHBG) Send Us Your Feedback ... As Testosterone-estrogen Binding Globulin TeBG Formal Name Sex Hormone Binding Globulin This article was last reviewed ...

  19. Tissue types (image)

    Science.gov (United States)

    ... are 4 basic types of tissue: connective tissue, epithelial tissue, muscle tissue, and nervous tissue. Connective tissue supports ... binds them together (bone, blood, and lymph tissues). Epithelial tissue provides a covering (skin, the linings of the ...

  20. Endocytosis of Integrin-Binding Human Picornaviruses

    Directory of Open Access Journals (Sweden)

    Pirjo Merilahti

    2012-01-01

    Full Text Available Picornaviruses that infect humans form one of the largest virus groups with almost three hundred virus types. They include significant enteroviral pathogens such as rhino-, polio-, echo-, and coxsackieviruses and human parechoviruses that cause wide range of disease symptoms. Despite the economic importance of picornaviruses, there are no antivirals. More than ten cellular receptors are known to participate in picornavirus infection, but experimental evidence of their role in cellular infection has been shown for only about twenty picornavirus types. Three enterovirus types and one parechovirus have experimentally been shown to bind and use integrin receptors in cellular infection. These include coxsackievirus A9 (CV-A9, echovirus 9, and human parechovirus 1 that are among the most common and epidemic human picornaviruses and bind to αV-integrins via RGD motif that resides on virus capsid. In contrast, echovirus 1 (E-1 has no RGD and uses integrin α2β1 as cellular receptor. Endocytosis of CV-A9 has recently been shown to occur via a novel Arf6- and dynamin-dependent pathways, while, contrary to collagen binding, E-1 binds inactive β1 integrin and enters via macropinocytosis. In this paper, we review what is known about receptors and endocytosis of integrin-binding human picornaviruses.

  1. Monoclonal antibody OKB7, which identifies the 14OKd complement receptor type 2 (CR/sub 2/), also identifies a 72Kd secreted fragment of CR/sub 2/ that contains the C3d-binding site

    Energy Technology Data Exchange (ETDEWEB)

    Myones, B.L.; Ross, G.D.

    1986-03-05

    CR/sub 2/ is a 140-145Kd glycoprotein expressed on B lymphocytes which binds both C3d and Epstein-Barr virus (EBV). OKB7, an IgG/sub 2a/ monoclonal antibody to CR/sub 2/, blocks C3d and EBV binding, while HB-5, another monoclonal IgG/sub 2a/ anti-CR/sub 2/, does not. A 72Kd C3d-binding glycoprotein (gp72), isolated from Raji cell media, was previously thought to be CR/sub 2/ because a polyclonal rabbit anti-gp72 inhibited EC3d rosettes. ELISA assay demonstrated that OKB7, but not HB-5, bound to purified gp72 fixed to microtiter wells. Insoluble and soluble gp72 blocked Raji cell uptake of /sup 125/I-labeled OKB7, but not labeled anti-B2 or HB-5. Rabbit anti-gp72 immunoprecipitated bands at 140Kd and 72Kd from /sup 125/I-labelled and solubilized B cell membranes. Culture media from Raji cells grown in the presence /sup 3/H-labeled amino acids was sequentially immunoprecipitated by irrelevant antibody, OKB7, and HB-5. A single 72Kd radiolabeled band was demonstrated only with OKB7, and this was identical to that produced by the immunoprecipitation of /sup 125/I-labeled gp72 with rabbit anti-gp72. Thus, OKB7, which identifies the 140Kd CR/sub 2/ molecule, also identifies a 72Kd shed fragment of CR/sub 2/ isolated from Raji cell media, which contains the C3d-binding site.

  2. Monoclonal antibody OKB7, which identifies the 14OKd complement receptor type 2 (CR2), also identifies a 72Kd secreted fragment of CR2 that contains the C3d-binding site

    International Nuclear Information System (INIS)

    Myones, B.L.; Ross, G.D.

    1986-01-01

    CR 2 is a 140-145Kd glycoprotein expressed on B lymphocytes which binds both C3d and Epstein-Barr virus (EBV). OKB7, an IgG/sub 2a/ monoclonal antibody to CR 2 , blocks C3d and EBV binding, while HB-5, another monoclonal IgG/sub 2a/ anti-CR 2 , does not. A 72Kd C3d-binding glycoprotein (gp72), isolated from Raji cell media, was previously thought to be CR 2 because a polyclonal rabbit anti-gp72 inhibited EC3d rosettes. ELISA assay demonstrated that OKB7, but not HB-5, bound to purified gp72 fixed to microtiter wells. Insoluble and soluble gp72 blocked Raji cell uptake of 125 I-labeled OKB7, but not labeled anti-B2 or HB-5. Rabbit anti-gp72 immunoprecipitated bands at 140Kd and 72Kd from 125 I-labelled and solubilized B cell membranes. Culture media from Raji cells grown in the presence 3 H-labeled amino acids was sequentially immunoprecipitated by irrelevant antibody, OKB7, and HB-5. A single 72Kd radiolabeled band was demonstrated only with OKB7, and this was identical to that produced by the immunoprecipitation of 125 I-labeled gp72 with rabbit anti-gp72. Thus, OKB7, which identifies the 140Kd CR 2 molecule, also identifies a 72Kd shed fragment of CR 2 isolated from Raji cell media, which contains the C3d-binding site

  3. CARBOHYDRATE-CONTAINING COMPOUNDS WHICH BIND TO CARBOHYDRATE BINDING RECEPTORS

    DEFF Research Database (Denmark)

    1995-01-01

    Carbohydrate-containing compounds which contain saccharides or derivatives thereof and which bind to carbohydrate binding receptors are useful in pharmaceutical products for treatment of inflammatory diseases and other diseases.......Carbohydrate-containing compounds which contain saccharides or derivatives thereof and which bind to carbohydrate binding receptors are useful in pharmaceutical products for treatment of inflammatory diseases and other diseases....

  4. Replacement of the V3 domain in the surface subunit of the feline immunodeficiency virus envelope glycoprotein with the equivalent region of a T cell-tropic human immunodeficiency virus type 1 results in a chimeric surface protein that efficiently binds to CXCR4.

    Science.gov (United States)

    González, Silvia A; Falcón, Juan I; Affranchino, José L

    2014-03-01

    Feline immunodeficiency virus (FIV) and the T cell-tropic strains of human immunodeficiency virus type 1 (HIV-1) share the use of the chemokine receptor CXCR4 for cell entry. To study this process further we developed a cell surface binding assay based on the expression of a soluble version of the FIV SU C-terminally tagged with the influenza virus hemagglutinin epitope (HA). The specificity of the assay was demonstrated by the following evidence: (1) the SU-HA protein bound to HeLa cells that express CXCR4 but not to MDCK cells that lack this chemokine receptor; and (2) binding of the SU-HA to HeLa cells was blocked by incubation with the CXCR4 antagonist AMD3100 as well as with the anti-CXCR4 monoclonal antibody (MAb) 12G5. Deletion of the V3 region from the FIV SU glycoprotein abolished its ability to bind CXCR4-expressing cells. Remarkably, substitution of the V3 domain of the FIV SU by the equivalent region of the HIV-1 NL4-3 isolate resulted in efficient cell surface binding of the chimeric SU protein to CXCR4. Moreover, transfection of MDCK cells with a plasmid encoding human CXCR4 allowed the association of the chimeric SU-HA glycoprotein to the transfected cells. Interestingly, while cell binding of the chimeric FIV-HIV SU was inhibited by an anti-HIV-1 V3 MAb, its association with CXCR4 was found to be resistant to AMD3100. Of note, the chimeric FIV-HIV Env glycoprotein was capable of promoting CXCR4-dependent cell-to-cell fusion.

  5. Sugar-Binding Profiles of Chitin-Binding Lectins from the Hevein Family: A Comprehensive Study

    Directory of Open Access Journals (Sweden)

    Yoko Itakura

    2017-05-01

    Full Text Available Chitin-binding lectins form the hevein family in plants, which are defined by the presence of single or multiple structurally conserved GlcNAc (N-acetylglucosamine-binding domains. Although they have been used as probes for chito-oligosaccharides, their detailed specificities remain to be investigated. In this study, we analyzed six chitin-binding lectins, DSA, LEL, PWM, STL, UDA, and WGA, by quantitative frontal affinity chromatography. Some novel features were evident: WGA showed almost comparable affinity for pyridylaminated chitotriose and chitotetraose, while LEL and UDA showed much weaker affinity, and DSA, PWM, and STL had no substantial affinity for the former. WGA showed selective affinity for hybrid-type N-glycans harboring a bisecting GlcNAc residue. UDA showed extensive binding to high-mannose type N-glycans, with affinity increasing with the number of Man residues. DSA showed the highest affinity for highly branched N-glycans consisting of type II LacNAc (N-acetyllactosamine. Further, multivalent features of these lectins were investigated by using glycoconjugate and lectin microarrays. The lectins showed substantial binding to immobilized LacNAc as well as chito-oligosaccharides, although the extents to which they bound varied among them. WGA showed strong binding to heavily sialylated glycoproteins. The above observations will help interpret lectin-glycoprotein interactions in histochemical studies and glyco-biomarker investigations.

  6. Five of Five VHHs Neutralizing Poliovirus Bind the Receptor-Binding Site.

    Science.gov (United States)

    Strauss, Mike; Schotte, Lise; Thys, Bert; Filman, David J; Hogle, James M

    2016-01-13

    Nanobodies, or VHHs, that recognize poliovirus type 1 have previously been selected and characterized as candidates for antiviral agents or reagents for standardization of vaccine quality control. In this study, we present high-resolution cryo-electron microscopy reconstructions of poliovirus with five neutralizing VHHs. All VHHs bind the capsid in the canyon at sites that extensively overlap the poliovirus receptor-binding site. In contrast, the interaction involves a unique (and surprisingly extensive) surface for each of the five VHHs. Five regions of the capsid were found to participate in binding with all five VHHs. Four of these five regions are known to alter during the expansion of the capsid associated with viral entry. Interestingly, binding of one of the VHHs, PVSS21E, resulted in significant changes of the capsid structure and thus seems to trap the virus in an early stage of expansion. We describe the cryo-electron microscopy structures of complexes of five neutralizing VHHs with the Mahoney strain of type 1 poliovirus at resolutions ranging from 3.8 to 6.3Å. All five VHHs bind deep in the virus canyon at similar sites that overlap extensively with the binding site for the receptor (CD155). The binding surfaces on the VHHs are surprisingly extensive, but despite the use of similar binding surfaces on the virus, the binding surface on the VHHs is unique for each VHH. In four of the five complexes, the virus remains essentially unchanged, but for the fifth there are significant changes reminiscent of but smaller in magnitude than the changes associated with cell entry, suggesting that this VHH traps the virus in a previously undescribed early intermediate state. The neutralizing mechanisms of the VHHs and their potential use as quality control agents for the end game of poliovirus eradication are discussed. Copyright © 2016, American Society for Microbiology. All Rights Reserved.

  7. Cognitive and neuropsychological underpinnings of relational and conjunctive working memory binding across age.

    Science.gov (United States)

    van Geldorp, Bonnie; Parra, Mario A; Kessels, Roy P C

    2015-01-01

    The ability to form associations (i.e., binding) is critical for memory formation. Recent studies suggest that aging specifically affects relational binding (associating separate features) but not conjunctive binding (integrating features within an object). Possibly, this dissociation may be driven by the spatial nature of the studies so far. Alternatively, relational binding may simply require more attentional resources. We assessed relational and conjunctive binding in three age groups and we included an interfering task (i.e., an articulatory suppression task). Binding was examined in a working memory (WM) task using non-spatial features: shape and colour. Thirty-one young adults (mean age = 22.35), 30 middle-aged adults (mean age = 54.80) and 30 older adults (mean age = 70.27) performed the task. Results show an effect of type of binding and an effect of age but no interaction between type of binding and age. The interaction between type of binding and interference was significant. These results indicate that aging affects relational binding and conjunctive binding similarly. However, relational binding is more susceptible to interference than conjunctive binding, which suggests that relational binding may require more attentional resources. We suggest that a general decline in WM resources associated with frontal dysfunction underlies age-related deficits in WM binding.

  8. Binding of bufuralol, dextromethorphan, and 3,4-methylenedioxymethylamphetamine to wild-type and F120A mutant cytochrome P450 2D6 studied by resonance Raman spectroscopy

    International Nuclear Information System (INIS)

    Bonifacio, Alois; Keizers, Peter H.J.; Commandeur, Jan N.M.; Vermeulen, Nico P.E.; Robert, Bruno; Gooijer, Cees; Zwan, Gert van der

    2006-01-01

    Cytochrome P450 2D6 (CYP2D6) is one of the most important drug-metabolizing enzymes in humans. Resonance Raman data, reported for First time for CYP2D6, show that the CYP2D6 heme is found to be in a six-coordinated low-spin state in the absence of substrates, and it is perturbed to different extents by bufuralol, dextromethorphan, and 3,4-methylenedioxymethylamphetamine (MDMA). Dextromethorphan and MDMA induce in CYP2D6 a significant amount of five-coordinated high-spin heme species and reduce the polarity of its heme-pocket, whereas bufuralol does not. Spectra of the F120A mutant CYP2D6 suggest that Phe 12 is involved in substrate-binding of dextromethorphan and MDMA, being responsible for the spectral differences observed between these two compounds and bufuralol. These differences could be explained postulating a different substrate mobility for each compound in the CYP2D6 active site, consistently with the role previously suggested for Phe 12 in binding dextromethorphan and MDMA

  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. Cellulose binding domain proteins

    Science.gov (United States)

    Shoseyov, Oded; Shpiegl, Itai; Goldstein, Marc; Doi, Roy

    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.

  11. Binding and Bulgarian

    NARCIS (Netherlands)

    Schürcks-Grozeva, Lilia Lubomirova

    2003-01-01

    In haar proefschrift analyseert Lilia Schürcks de anaforische verschijnselen in de Bulgaarse taal. Het gaat dan om wederkerende aspecten, uitgedrukt bij woorden als ‘zich’ en ‘elkaar’. De situatie in het Bulgaars blijkt moeilijk in te passen in de klassieke Binding Theory van Noam Chomsky. Bron: RUG

  12. Maltose-binding protein effectively stabilizes the partially closed conformation of the ATP-binding cassette transporter MalFGK2

    KAUST Repository

    Weng, Jingwei; Gu, Shuo; Gao, Xin; Huang, Xuhui; Wang, Wenning

    2017-01-01

    Maltose transporter MalFGK2 is a type-I importer in the ATP-binding cassette (ABC) transporter superfamily. Upon the binding of its periplasmic binding protein, MalE, the ATPase activity of MalFGK2 can be greatly enhanced. Crystal structures of the MalFGK2-MalE-maltose complex in a so-called

  13. Maltose-binding protein effectively stabilizes the partially closed conformation of the ATP-binding cassette transporter MalFGK2

    KAUST Repository

    Weng, Jingwei

    2017-02-23

    Maltose transporter MalFGK2 is a type-I importer in the ATP-binding cassette (ABC) transporter superfamily. Upon the binding of its periplasmic binding protein, MalE, the ATPase activity of MalFGK2 can be greatly enhanced. Crystal structures of the MalFGK2-MalE-maltose complex in a so-called

  14. Probing the binding of coumarins and cyclothialidines to DNA gyrase

    DEFF Research Database (Denmark)

    Kampranis, S C; Gormley, N A; Tranter, R

    1999-01-01

    B and coumarin and cyclothialidine drugs and made mutations by site-directed mutagenesis. We used proteolysis as a probe of drug binding to wild-type and mutant proteins. Limited proteolysis of gyrase revealed that binding of these antibiotics is associated with a characteristic proteolytic fingerprint......, suggesting a drug-induced conformational change. The ability of the mutants to bind the drugs was studied by testing their ability to induce the coumarin-associated proteolytic signature and to bind to a novobiocin-affinity column. To analyze further the interaction of the drugs with gyrase, we studied...

  15. Unexpected Binding Mode of a Potent Indeno[1,2-b]indole-Type Inhibitor of Protein Kinase CK2 Revealed by Complex Structures with the Catalytic Subunit CK2α and Its Paralog CK2α′

    Directory of Open Access Journals (Sweden)

    Jennifer Hochscherf

    2017-12-01

    Full Text Available Protein kinase CK2, a member of the eukaryotic protein kinase superfamily, is associated with cancer and other human pathologies and thus an attractive drug target. The indeno[1,2-b]indole scaffold is a novel lead structure to develop ATP-competitive CK2 inhibitors. Some indeno[1,2-b]indole-based CK2 inhibitors additionally obstruct ABCG2, an ABC half transporter overexpressed in breast cancer and co-responsible for drug efflux and resistance. Comprehensive derivatization studies revealed substitutions of the indeno[1,2-b]indole framework that boost either the CK2 or the ABCG2 selectivity or even support the dual inhibition potential. The best indeno[1,2-b]indole-based CK2 inhibitor described yet (IC50 = 25 nM is 5-isopropyl-4-(3-methylbut-2-enyl-oxy-5,6,7,8-tetrahydroindeno[1,2-b]indole-9,10-dione (4p. Herein, we demonstrate the membrane permeability of 4p and describe co-crystal structures of 4p with CK2α and CK2α′, the paralogs of human CK2 catalytic subunit. As expected, 4p occupies the narrow, hydrophobic ATP site of CK2α/CK2α′, but surprisingly with a unique orientation: its hydrophobic substituents point towards the solvent while its two oxo groups are hydrogen-bonded to a hidden water molecule. An equivalent water molecule was found in many CK2α structures, but never as a critical mediator of ligand binding. This unexpected binding mode is independent of the interdomain hinge/helix αD region conformation and of the salt content in the crystallization medium.

  16. The Tomato Nucleotide-binding Leucine-rich Repeat Immune Receptor I-2 Couples DNA-binding to Nucleotide-binding Domain Nucleotide Exchange*

    Science.gov (United States)

    Fenyk, Stepan; Dixon, Christopher H.; Gittens, William H.; Townsend, Philip D.; Sharples, Gary J.; Pålsson, Lars-Olof; Takken, Frank L. W.; Cann, Martin J.

    2016-01-01

    Plant nucleotide-binding leucine-rich repeat (NLR) proteins enable plants to recognize and respond to pathogen attack. Previously, we demonstrated that the Rx1 NLR of potato is able to bind and bend DNA in vitro. DNA binding in situ requires its genuine activation following pathogen perception. However, it is unknown whether other NLR proteins are also able to bind DNA. Nor is it known how DNA binding relates to the ATPase activity intrinsic to NLR switch function required to immune activation. Here we investigate these issues using a recombinant protein corresponding to the N-terminal coiled-coil and nucleotide-binding domain regions of the I-2 NLR of tomato. Wild type I-2 protein bound nucleic acids with a preference of ssDNA ≈ dsDNA > ssRNA, which is distinct from Rx1. I-2 induced bending and melting of DNA. Notably, ATP enhanced DNA binding relative to ADP in the wild type protein, the null P-loop mutant K207R, and the autoactive mutant S233F. DNA binding was found to activate the intrinsic ATPase activity of I-2. Because DNA binding by I-2 was decreased in the presence of ADP when compared with ATP, a cyclic mechanism emerges; activated ATP-associated I-2 binds to DNA, which enhances ATP hydrolysis, releasing ADP-bound I-2 from the DNA. Thus DNA binding is a general property of at least a subset of NLR proteins, and NLR activation is directly linked to its activity at DNA. PMID:26601946

  17. The Tomato Nucleotide-binding Leucine-rich Repeat Immune Receptor I-2 Couples DNA-binding to Nucleotide-binding Domain Nucleotide Exchange.

    Science.gov (United States)

    Fenyk, Stepan; Dixon, Christopher H; Gittens, William H; Townsend, Philip D; Sharples, Gary J; Pålsson, Lars-Olof; Takken, Frank L W; Cann, Martin J

    2016-01-15

    Plant nucleotide-binding leucine-rich repeat (NLR) proteins enable plants to recognize and respond to pathogen attack. Previously, we demonstrated that the Rx1 NLR of potato is able to bind and bend DNA in vitro. DNA binding in situ requires its genuine activation following pathogen perception. However, it is unknown whether other NLR proteins are also able to bind DNA. Nor is it known how DNA binding relates to the ATPase activity intrinsic to NLR switch function required to immune activation. Here we investigate these issues using a recombinant protein corresponding to the N-terminal coiled-coil and nucleotide-binding domain regions of the I-2 NLR of tomato. Wild type I-2 protein bound nucleic acids with a preference of ssDNA ≈ dsDNA > ssRNA, which is distinct from Rx1. I-2 induced bending and melting of DNA. Notably, ATP enhanced DNA binding relative to ADP in the wild type protein, the null P-loop mutant K207R, and the autoactive mutant S233F. DNA binding was found to activate the intrinsic ATPase activity of I-2. Because DNA binding by I-2 was decreased in the presence of ADP when compared with ATP, a cyclic mechanism emerges; activated ATP-associated I-2 binds to DNA, which enhances ATP hydrolysis, releasing ADP-bound I-2 from the DNA. Thus DNA binding is a general property of at least a subset of NLR proteins, and NLR activation is directly linked to its activity at DNA. © 2016 by The American Society for Biochemistry and Molecular Biology, Inc.

  18. Synthesis and anion binding properties of porphyrins and related compounds

    KAUST Repository

    Figueira, Flá vio; Rodrigues, Joã o M M; Farinha, Andreia; Cavaleiro, José A S; Tomé , Joã o P C

    2016-01-01

    promising. In this review, we summarize the most recent developments in anion binding studies while outlining the strategies that may be used to synthesize and functionalize these type of macrocycles. © 2016 World Scientific Publishing Company.

  19. DNABP: Identification of DNA-Binding Proteins Based on Feature Selection Using a Random Forest and Predicting Binding Residues.

    Science.gov (United States)

    Ma, Xin; Guo, Jing; Sun, Xiao

    2016-01-01

    DNA-binding proteins are fundamentally important in cellular processes. Several computational-based methods have been developed to improve the prediction of DNA-binding proteins in previous years. However, insufficient work has been done on the prediction of DNA-binding proteins from protein sequence information. In this paper, a novel predictor, DNABP (DNA-binding proteins), was designed to predict DNA-binding proteins using the random forest (RF) classifier with a hybrid feature. The hybrid feature contains two types of novel sequence features, which reflect information about the conservation of physicochemical properties of the amino acids, and the binding propensity of DNA-binding residues and non-binding propensities of non-binding residues. The comparisons with each feature demonstrated that these two novel features contributed most to the improvement in predictive ability. Furthermore, to improve the prediction performance of the DNABP model, feature selection using the minimum redundancy maximum relevance (mRMR) method combined with incremental feature selection (IFS) was carried out during the model construction. The results showed that the DNABP model could achieve 86.90% accuracy, 83.76% sensitivity, 90.03% specificity and a Matthews correlation coefficient of 0.727. High prediction accuracy and performance comparisons with previous research suggested that DNABP could be a useful approach to identify DNA-binding proteins from sequence information. The DNABP web server system is freely available at http://www.cbi.seu.edu.cn/DNABP/.

  20. An Electrostatic Funnel in the GABA-Binding Pathway.

    Directory of Open Access Journals (Sweden)

    Timothy S Carpenter

    2016-04-01

    Full Text Available The γ-aminobutyric acid type A receptor (GABAA-R is a major inhibitory neuroreceptor that is activated by the binding of GABA. The structure of the GABAA-R is well characterized, and many of the binding site residues have been identified. However, most of these residues are obscured behind the C-loop that acts as a cover to the binding site. Thus, the mechanism by which the GABA molecule recognizes the binding site, and the pathway it takes to enter the binding site are both unclear. Through the completion and detailed analysis of 100 short, unbiased, independent molecular dynamics simulations, we have investigated this phenomenon of GABA entering the binding site. In each system, GABA was placed quasi-randomly near the binding site of a GABAA-R homology model, and atomistic simulations were carried out to observe the behavior of the GABA molecules. GABA fully entered the binding site in 19 of the 100 simulations. The pathway taken by these molecules was consistent and non-random; the GABA molecules approach the binding site from below, before passing up behind the C-loop and into the binding site. This binding pathway is driven by long-range electrostatic interactions, whereby the electrostatic field acts as a 'funnel' that sweeps the GABA molecules towards the binding site, at which point more specific atomic interactions take over. These findings define a nuanced mechanism whereby the GABAA-R uses the general zwitterionic features of the GABA molecule to identify a potential ligand some 2 nm away from the binding site.

  1. Synthesis of iron oxide nanoparticles in Listeria innocua Dps (DNA-binding protein from starved cells): a study with the wild-type protein and a catalytic centre mutant.

    Science.gov (United States)

    Ceci, Pierpaolo; Chiancone, Emilia; Kasyutich, Oksana; Bellapadrona, Giuliano; Castelli, Lisa; Fittipaldi, Maria; Gatteschi, Dante; Innocenti, Claudia; Sangregorio, Claudio

    2010-01-11

    A comparative analysis of the magnetic properties of iron oxide nanoparticles grown in the cavity of the DNA-binding protein from starved cells of the bacterium Listeria innocua, LiDps, and of its triple-mutant lacking the catalytic ferroxidase centre, LiDps-tm, is presented. TEM images and static and dynamic magnetic and electron magnetic resonance (EMR) measurements reveal that, under the applied preparation conditions, namely alkaline pH, high temperature (65 degrees C), exclusion of oxygen, and the presence of hydrogen peroxide, maghemite and/or magnetite nanoparticles with an average diameter of about 3 nm are mineralised inside the cavities of both LiDps and LiDps-tm. The magnetic nanoparticles (MNPs) thus formed show similar magnetic properties, with superparamagnetic behaviour above 4.5 K and a large magnetic anisotropy. Interestingly, in the EMR spectra an absorption at half-field is observed, which can be considered as a manifestation of the quantum behaviour of the MNPs. These results indicate that Dps proteins can be advantageously used for the production of nanomagnets at the interface between molecular clusters and traditional MNPs and that the presence of the ferroxidase centre, though increasing the efficiency of nanoparticle formation, does not affect the nature and fine structure of the MNPs. Importantly, the self-organisation of MNP-containing Dps on HRTEM grids suggests that Dps-enclosed MNPs can be deposited on surfaces in an ordered fashion.

  2. Hexa-histidin tag position influences disulfide structure but not binding behavior of in vitro folded N-terminal domain of rat corticotropin-releasing factor receptor type 2a

    OpenAIRE

    Klose, Jana; Wendt, Norbert; Kubald, Sybille; Krause, Eberhard; Fechner, Klaus; Beyermann, Michael; Bienert, Michael; Rudolph, Rainer; Rothemund, Sven

    2004-01-01

    The oxidative folding, particularly the arrangement of disulfide bonds of recombinant extracellular N-terminal domains of the corticotropin-releasing factor receptor type 2a bearing five cysteines (C2 to C6), was investigated. Depending on the position of a His-tag, two types of disulfide patterns were found. In the case of an N-terminal His-tag, the disulfide bonds C2–C3 and C4–C6 were found, leaving C5 free, whereas the C-terminal position of the His-tag led to the disulfide pattern C2–C5 a...

  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. Optical Binding of Nanowires

    Czech Academy of Sciences Publication Activity Database

    Simpson, Stephen Hugh; Zemánek, Pavel; Marago, O.M.; Jones, P.H.; Hanna, S.

    2017-01-01

    Roč. 17, č. 6 (2017), s. 3485-3492 ISSN 1530-6984 R&D Projects: GA ČR GB14-36681G Grant - others:AV ČR(CZ) CNR-16-12 Program:Bilaterální spolupráce Institutional support: RVO:68081731 Keywords : optical binding nanowires * Brownian motion * self-organization * non-equilibrium thermodynamics * non-equilibrium steady state * spin-orbit coupling * emergent phenomena Subject RIV: BH - Optics, Masers, Lasers OBOR OECD: Optics (including laser optics and quantum optics) Impact factor: 12.712, year: 2016

  5. cAMP response element binding protein1 is essential for activation of steroyl co-enzyme a desaturase 1 (Scd1 in mouse lung type II epithelial cells.

    Directory of Open Access Journals (Sweden)

    Nisha Antony

    Full Text Available Cyclic AMP Response Element-Binding Protein 1 (Creb1 is a transcription factor that mediates cyclic adenosine 3', 5'-monophosphate (cAMP signalling in many tissues. Creb1(-/- mice die at birth due to respiratory failure and previous genome-wide microarray analysis of E17.5 Creb1(-/- fetal mouse lung identified important Creb1-regulated gene targets during lung development. The lipogenic enzymes stearoyl-CoA desaturase 1 (Scd1 and fatty acid synthase (Fasn showed highly reduced gene expression in Creb1(-/- lungs. We therefore hypothesized that Creb1 plays a crucial role in the transcriptional regulation of genes involved in pulmonary lipid biosynthetic pathways during lung development. In this study we confirmed that Scd1 and Fasn mRNA levels were down regulated in the E17.5 Creb1(-/- mouse lung while the lipogenic-associated transcription factors SrebpF1, C/ebpα and Pparγ were increased. In vivo studies using germline (Creb1(-/- and lung epithelial-specific (Creb1(EpiΔ/Δ Creb1 knockout mice showed strongly reduced Scd1, but not Fasn gene expression and protein levels in lung epithelial cells. In vitro studies using mouse MLE-15 epithelial cells showed that forskolin-mediated activation of Creb1 increased both Scd1 gene expression and protein synthesis. Additionally, MLE15 cells transfected with a dominant-negative ACreb vector blocked forskolin-mediated stimulation of Scd1 gene expression. Lipid profiling in MLE15 cells showed that dominant-negative ACreb suppressed forskolin-induced desaturation of ether linked lipids to produce plasmalogens, as well as levels of phosphatidylethanolamine, ceramide and lysophosphatidylcholine. Taken together these results demonstrate that Creb1 is essential for the induction and maintenance of Scd1 in developing fetal mouse lung epithelial cells.

  6. The pattern-recognition molecule mannan-binding lectin (MBL) in the pathophysiology of diabetic nephropathy

    DEFF Research Database (Denmark)

    Axelgaard, Esben; Thiel, Steffen; Hansen, Troels Krarup

    to carbohydrates of both specific type and density, which thus provides sufficient binding avidity. The character of MBL binding-sites on host cells remain unknown, but it is speculated that altered protein glycation in diabetes permits MBL binding. Based on new studies using MBL/double knockout C57bl/6j mice, we...

  7. Surface Passivation in Empirical Tight Binding

    OpenAIRE

    He, Yu; Tan, Yaohua; Jiang, Zhengping; Povolotskyi, Michael; Klimeck, Gerhard; Kubis, Tillmann

    2015-01-01

    Empirical Tight Binding (TB) methods are widely used in atomistic device simulations. Existing TB methods to passivate dangling bonds fall into two categories: 1) Method that explicitly includes passivation atoms is limited to passivation with atoms and small molecules only. 2) Method that implicitly incorporates passivation does not distinguish passivation atom types. This work introduces an implicit passivation method that is applicable to any passivation scenario with appropriate parameter...

  8. IGF binding proteins.

    Science.gov (United States)

    Bach, Leon A

    2017-12-18

    Insulin-like growth factor binding proteins (IGFBPs) 1-6 bind IGFs but not insulin with high affinity. They were initially identified as serum carriers and passive inhibitors of IGF actions. However, subsequent studies showed that, although IGFBPs inhibit IGF actions in many circumstances, they may also potentiate these actions. IGFBPs are widely expressed in most tissues, and they are flexible endocrine and autocrine/paracrine regulators of IGF activity, which is essential for this important physiological system. More recently, individual IGFBPs have been shown to have IGF-independent actions. Mechanisms underlying these actions include (i) interaction with non-IGF proteins in compartments including the extracellular space and matrix, the cell surface and intracellularly; (ii) interaction with and modulation of other growth factor pathways including EGF, TGF- and VEGF; and (iii) direct or indirect transcriptional effects following nuclear entry of IGFBPs. Through these IGF-dependent and IGF-independent actions, IGFBPs modulate essential cellular processes including proliferation, survival, migration, senescence, autophagy and angiogenesis. They have been implicated in a range of disorders including malignant, metabolic, neurological and immune diseases. A more complete understanding of their cellular roles may lead to the development of novel IGFBP-based therapeutic opportunities.

  9. The necessity of connection structures in neural models of variable binding.

    Science.gov (United States)

    van der Velde, Frank; de Kamps, Marc

    2015-08-01

    In his review of neural binding problems, Feldman (Cogn Neurodyn 7:1-11, 2013) addressed two types of models as solutions of (novel) variable binding. The one type uses labels such as phase synchrony of activation. The other ('connectivity based') type uses dedicated connections structures to achieve novel variable binding. Feldman argued that label (synchrony) based models are the only possible candidates to handle novel variable binding, whereas connectivity based models lack the flexibility required for that. We argue and illustrate that Feldman's analysis is incorrect. Contrary to his conclusion, connectivity based models are the only viable candidates for models of novel variable binding because they are the only type of models that can produce behavior. We will show that the label (synchrony) based models analyzed by Feldman are in fact examples of connectivity based models. Feldman's analysis that novel variable binding can be achieved without existing connection structures seems to result from analyzing the binding problem in a wrong frame of reference, in particular in an outside instead of the required inside frame of reference. Connectivity based models can be models of novel variable binding when they possess a connection structure that resembles a small-world network, as found in the brain. We will illustrate binding with this type of model with episode binding and the binding of words, including novel words, in sentence structures.

  10. Two unique ligand-binding clamps of Rhizopus oryzae starch binding domain for helical structure disruption of amylose.

    Directory of Open Access Journals (Sweden)

    Ting-Ying Jiang

    Full Text Available The N-terminal starch binding domain of Rhizopus oryzae glucoamylase (RoSBD has a high binding affinity for raw starch. RoSBD has two ligand-binding sites, each containing a ligand-binding clamp: a polyN clamp residing near binding site I is unique in that it is expressed in only three members of carbohydrate binding module family 21 (CBM21 members, and a Y32/F58 clamp located at binding site II is conserved in several CBMs. Here we characterized different roles of these sites in the binding of insoluble and soluble starches using an amylose-iodine complex assay, atomic force microscopy, isothermal titration calorimetry, site-directed mutagenesis, and structural bioinformatics. RoSBD induced the release of iodine from the amylose helical cavity and disrupted the helical structure of amylose type III, thereby significantly diminishing the thickness and length of the amylose type III fibrils. A point mutation in the critical ligand-binding residues of sites I and II, however, reduced both the binding affinity and amylose helix disruption. This is the first molecular model for structure disruption of the amylose helix by a non-hydrolytic CBM21 member. RoSBD apparently twists the helical amylose strands apart to expose more ligand surface for further SBD binding. Repeating the process triggers the relaxation and unwinding of amylose helices to generate thinner and shorter amylose fibrils, which are more susceptible to hydrolysis by glucoamylase. This model aids in understanding the natural roles of CBMs in protein-glycan interactions and contributes to potential molecular engineering of CBMs.

  11. Localization of gonadotropin binding sites in human ovarian neoplasms

    International Nuclear Information System (INIS)

    Nakano, R.; Kitayama, S.; Yamoto, M.; Shima, K.; Ooshima, A.

    1989-01-01

    The binding of human luteinizing hormone and human follicle-stimulating hormone to ovarian tumor biopsy specimens from 29 patients was analyzed. The binding sites for human luteinizing hormone were demonstrated in one tumor of epithelial origin (mucinous cystadenoma) and in one of sex cord-stromal origin (theca cell tumor). The binding sites for human follicle-stimulating hormone were found in three tumors of epithelial origin (serous cystadenoma and mucinous cystadenoma) and in two of sex cord-stromal origin (theca cell tumor and theca-granulosa cell tumor). The surface-binding autoradiographic study revealed that the binding sites for gonadotropins were localized in the stromal tissue. The results suggest that gonadotropic hormones may play a role in the growth and differentiation of a certain type of human ovarian neoplasms

  12. Association of variants in the sterol regulatory element-binding factor 1 (SREBF1) gene with type 2 diabetes, glycemia, and insulin resistance: a study of 15,734 Danish subjects

    DEFF Research Database (Denmark)

    Grarup, Niels; Stender-Petersen, Kirstine L; Andersson, Ehm A

    2008-01-01

    alleles of rs2297508, rs11868035, and rs1889018 (linkage disequilibrium R(2) = 0.6-0.8) associated with a modestly increased risk of type 2 diabetes (rs2297508: OR 1.17 [95% CI 1.05-1.30], P = 0.003), which was confirmed in meta-analyses of all published studies (rs2297508 G-allele: 1.08 [1.03-1.14] per.......03). Furthermore, the diabetes-associated alleles associated with a modestly increased A1C level in the population-based Inter99 of middle-aged subjects and in the ADDITION study of high-risk individuals (P = 0.006 and P = 0.008, respectively). CONCLUSIONS: We associate sequence variation in SREBF1 with a modestly...

  13. Visually Relating Gene Expression and in vivo DNA Binding Data

    Energy Technology Data Exchange (ETDEWEB)

    Huang, Min-Yu; Mackey, Lester; Ker?,; nen, Soile V. E.; Weber, Gunther H.; Jordan, Michael I.; Knowles, David W.; Biggin, Mark D.; Hamann, Bernd

    2011-09-20

    Gene expression and in vivo DNA binding data provide important information for understanding gene regulatory networks: in vivo DNA binding data indicate genomic regions where transcription factors are bound, and expression data show the output resulting from this binding. Thus, there must be functional relationships between these two types of data. While visualization and data analysis tools exist for each data type alone, there is a lack of tools that can easily explore the relationship between them. We propose an approach that uses the average expression driven by multiple of ciscontrol regions to visually relate gene expression and in vivo DNA binding data. We demonstrate the utility of this tool with examples from the network controlling early Drosophila development. The results obtained support the idea that the level of occupancy of a transcription factor on DNA strongly determines the degree to which the factor regulates a target gene, and in some cases also controls whether the regulation is positive or negative.

  14. Genome Binding and Gene Regulation by Stem Cell Transcription Factors

    NARCIS (Netherlands)

    J.H. Brandsma (Johan)

    2016-01-01

    markdownabstractNearly all cells of an individual organism contain the same genome. However, each cell type transcribes a different set of genes due to the presence of different sets of cell type-specific transcription factors. Such transcription factors bind to regulatory regions such as promoters

  15. Factors Affecting the Binding of a Recombinant Heavy Metal-Binding Domain (CXXC motif Protein to Heavy Metals

    Directory of Open Access Journals (Sweden)

    Kamala Boonyodying

    2012-06-01

    Full Text Available A number of heavy metal-binding proteins have been used to study bioremediation. CXXC motif, a metal binding domain containing Cys-X-X-Cys motif, has been identified in various organisms. These proteins are capable of binding various types of heavy metals. In this study, heavy metal binding domain (CXXC motif recombinant protein encoded from mcsA gene of S. aureus were cloned and overexpressed in Escherichia coli. The factors involved in the metal-binding activity were determined in order to analyze the potential of recombinant protein for bioremediation. A recombinant protein can be bound to Cd2+, Co2+, Cu2+ and Zn2+. The thermal stability of a recombinant protein was tested, and the results showed that the metal binding activity to Cu2+ and Zn2+ still exist after treating the protein at 85ºC for 30 min. The temperature and pH that affected the metal binding activity was tested and the results showed that recombinant protein was still bound to Cu2+ at 65ºC, whereas a pH of 3-7 did not affect the metal binding E. coli harboring a pRset with a heavy metal-binding domain CXXC motif increased the resistance of heavy metals against CuCl2 and CdCl2. This study shows that metal binding domain (CXXC motif recombinant protein can be effectively bound to various types of heavy metals and may be used as a potential tool for studying bioremediation.

  16. Analysis of leukocyte binding to depletion filters: role of passive binding, interaction with platelets, and plasma components.

    Science.gov (United States)

    Henschler, R; Rüster, B; Steimle, A; Hansmann, H L; Walker, W; Montag, T; Seifried, E

    2005-08-01

    Since limited knowledge exists on the mechanisms which regulate cell binding to leukocyte removal filter surfaces, we investigated the binding patterns of leukocytes to individual layers of leukocyte depletion filters. After passage of 1 unit of whole blood, blotting of isolated filter layers on glass slides or elution of cells from filter layers revealed that most leukocytes were located within the first 10 of a total of 28 filter layers, peaking at layers 6 to 8, with granulocytes binding on average to earlier filter layers than lymphocytes. Leukocytes preincubated with inhibitors of actin activation showed unchanged distribution between filter layers, suggesting that cytoskeletal activation does not significantly contribute to their binding. When leukocytes were directly incubated with single filter layers, binding of up to 30% of input cells was recorded in the absence of Ca(2+). Immunohistological analyses showed colocalization of platelets and leukocytes, with co-clustering of platelets and leukocytes. Monocytes and to some degree lymphocytes but not granulocytes competed with platelets for filter binding. Precoating of filter layers with individual plasma components showed that hyaluronic acid, plasma type fibronectin, and fibrinogen all increased the binding of leukocytes compared with albumin coating. In conclusion, leukocytes can bind passively to filters in a process which does not require Ca(2+), which is independent of cytoskeletal activation and which may depend on individual plasma components. These results are of importance when new selective cell enrichment or depletion strategies through specific filters are envisaged.

  17. Tetrodotoxin- and tributyltin-binding abilities of recombinant pufferfish saxitoxin and tetrodotoxin binding proteins of Takifugu rubripes.

    Science.gov (United States)

    Satone, Hina; Nonaka, Shohei; Lee, Jae Man; Shimasaki, Yohei; Kusakabe, Takahiro; Kawabata, Shun-Ichiro; Oshima, Yuji

    2017-01-01

    We investigated the ability of recombinant pufferfish saxitoxin and tetrodotoxin binding protein types 1 and 2 of Takifugu rubripes (rTrub.PSTBP1 and rTrub.PSTBP2) to bind to tetrodotoxin (TTX) and tributyltin. Both rTrub.PSTBPs bound to tributyltin in an ultrafiltration binding assay but lost this ability on heat denaturation. In contrast, only rTrub.PSTBP2 bound to TTX even heat denaturation. This result suggests that the amino acid sequence of PSTBP2 may be contributed for its affinity for TTX. Copyright © 2016 Elsevier Ltd. All rights reserved.

  18. The role of attention in item-item binding in visual working memory.

    Science.gov (United States)

    Peterson, Dwight J; Naveh-Benjamin, Moshe

    2017-09-01

    An important yet unresolved question regarding visual working memory (VWM) relates to whether or not binding processes within VWM require additional attentional resources compared with processing solely the individual components comprising these bindings. Previous findings indicate that binding of surface features (e.g., colored shapes) within VWM is not demanding of resources beyond what is required for single features. However, it is possible that other types of binding, such as the binding of complex, distinct items (e.g., faces and scenes), in VWM may require additional resources. In 3 experiments, we examined VWM item-item binding performance under no load, articulatory suppression, and backward counting using a modified change detection task. Binding performance declined to a greater extent than single-item performance under higher compared with lower levels of concurrent load. The findings from each of these experiments indicate that processing item-item bindings within VWM requires a greater amount of attentional resources compared with single items. These findings also highlight an important distinction between the role of attention in item-item binding within VWM and previous studies of long-term memory (LTM) where declines in single-item and binding test performance are similar under divided attention. The current findings provide novel evidence that the specific type of binding is an important determining factor regarding whether or not VWM binding processes require attention. (PsycINFO Database Record (c) 2017 APA, all rights reserved).

  19. Gamma-aminobutyric acid-modulated benzodiazepine binding sites in bacteria

    International Nuclear Information System (INIS)

    Lummis, S.C.R.; Johnston, G.A.R.; Nicoletti, G.; Holan, G.

    1991-01-01

    Benzodiazepine binding sites, which were once considered to exist only in higher vertebrates, are here demonstrated in the bacteria E. coli. The bacterial [ 3 H]diazepam binding sites are modulated by GABA; the modulation is dose dependent and is reduced at high concentrations. The most potent competitors of E.Coli [ 3 H]diazepam binding are those that are active in displacing [ 3 H]benzodiazepines from vertebrate peripheral benzodiazepine binding sites. These vertebrate sites are not modulated by GABA, in contrast to vertebrate neuronal benzodiazepine binding sites. The E.coli benzodiazepine binding sites therefore differ from both classes of vertebrate benzodiazepine binding sites; however the ligand spectrum and GABA-modulatory properties of the E.coli sites are similar to those found in insects. This intermediate type of receptor in lower species suggests a precursor for at least one class of vertebrate benzodiazepine binding sites may have existed

  20. 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 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...

  1. Investigation of arc repressor DNA-binding specificity by comparative molecular dynamics simulations.

    Science.gov (United States)

    Song, Wei; Guo, Jun-Tao

    2015-01-01

    Transcription factors regulate gene expression through binding to specific DNA sequences. How transcription factors achieve high binding specificity is still not well understood. In this paper, we investigated the role of protein flexibility in protein-DNA-binding specificity by comparative molecular dynamics (MD) simulations. Protein flexibility has been considered as a key factor in molecular recognition, which is intrinsically a dynamic process involving fine structural fitting between binding components. In this study, we performed comparative MD simulations on wild-type and F10V mutant P22 Arc repressor in both free and complex conformations. The F10V mutant has lower DNA-binding specificity though both the bound and unbound main-chain structures between the wild-type and F10V mutant Arc are highly similar. We found that the DNA-binding motif of wild-type Arc is structurally more flexible than the F10V mutant in the unbound state, especially for the six DNA base-contacting residues in each dimer. We demonstrated that the flexible side chains of wild-type Arc lead to a higher DNA-binding specificity through forming more hydrogen bonds with DNA bases upon binding. Our simulations also showed a possible conformational selection mechanism for Arc-DNA binding. These results indicate the important roles of protein flexibility and dynamic properties in protein-DNA-binding specificity.

  2. Protein-binding RNA aptamers affect molecular interactions distantly from their binding sites.

    Directory of Open Access Journals (Sweden)

    Daniel M Dupont

    Full Text Available Nucleic acid aptamer selection is a powerful strategy for the development of regulatory agents for molecular intervention. Accordingly, aptamers have proven their diligence in the intervention with serine protease activities, which play important roles in physiology and pathophysiology. Nonetheless, there are only a few studies on the molecular basis underlying aptamer-protease interactions and the associated mechanisms of inhibition. In the present study, we use site-directed mutagenesis to delineate the binding sites of two 2´-fluoropyrimidine RNA aptamers (upanap-12 and upanap-126 with therapeutic potential, both binding to the serine protease urokinase-type plasminogen activator (uPA. We determine the subsequent impact of aptamer binding on the well-established molecular interactions (plasmin, PAI-1, uPAR, and LRP-1A controlling uPA activities. One of the aptamers (upanap-126 binds to the area around the C-terminal α-helix in pro-uPA, while the other aptamer (upanap-12 binds to both the β-hairpin of the growth factor domain and the kringle domain of uPA. Based on the mapping studies, combined with data from small-angle X-ray scattering analysis, we construct a model for the upanap-12:pro-uPA complex. The results suggest and highlight that the size and shape of an aptamer as well as the domain organization of a multi-domain protein such as uPA, may provide the basis for extensive sterical interference with protein ligand interactions considered distant from the aptamer binding site.

  3. [3]tetrahydrotrazodone binding. Association with serotonin binding sites

    International Nuclear Information System (INIS)

    Kendall, D.A.; Taylor, D.P.; Enna, S.J.

    1983-01-01

    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

  4. Protein binding of psychotropic agents

    International Nuclear Information System (INIS)

    Hassan, H.A.

    1990-01-01

    Based upon fluorescence measurements, protein binding of some psychotropic agents (chlorpromazine, promethazine, and trifluoperazine) to human IgG and HSA was studied in aqueous cacodylate buffer, PH7. The interaction parameters determined from emission quenching of the proteins. The interaction parameters determined include the equilibrium constant (K), calculated from equations derived by Borazan and coworkers, the number of binding sites (n) available to the monomer molecules on a single protein molecule. The results revealed a high level of affinity, as reflected by high values of K, and the existence of specific binding sites, since a limited number of n values are obtained. 39 tabs.; 37 figs.; 83 refs

  5. Solid-Binding Peptides in Biomedicine.

    Science.gov (United States)

    Care, Andrew; Bergquist, Peter L; Sunna, Anwar

    2017-01-01

    Some peptides are able to bind to inorganic materials such as silica and gold. Over the past decade, Solid-binding peptides (SBPs) have been used increasingly as molecular building blocks in nanobiotechnology. These peptides show selectivity and bind with high affinity to a diverse range of inorganic surfaces e.g. metals, metal oxides, metal compounds, magnetic materials, semiconductors, carbon materials, polymers and minerals. They can be used in applications such as protein purification and synthesis, assembly and the functionalization of nanomaterials. They offer simple and versatile bioconjugation methods that can increase biocompatibility and also direct the immobilization and orientation of nanoscale entities onto solid supports without impeding their functionality. SBPs have been employed in numerous nanobiotechnological applications such as the controlled synthesis of nanomaterials and nanostructures, formation of hybrid biomaterials, immobilization of functional proteins and improved nanomaterial biocompatibility. With advances in nanotechnology, a multitude of novel nanomaterials have been designed and synthesized for diagnostic and therapeutic applications. New approaches have been developed recently to exert a greater control over bioconjugation and eventually, over the optimal and functional display of biomolecules on the surfaces of many types of solid materials. In this chapter we describe SBPs and highlight some selected examples of their potential applications in biomedicine.

  6. Superresolution microscopy with transient binding.

    Science.gov (United States)

    Molle, Julia; Raab, Mario; Holzmeister, Susanne; Schmitt-Monreal, Daniel; Grohmann, Dina; He, Zhike; Tinnefeld, Philip

    2016-06-01

    For single-molecule localization based superresolution, the concentration of fluorescent labels has to be thinned out. This is commonly achieved by photophysically or photochemically deactivating subsets of molecules. Alternatively, apparent switching of molecules can be achieved by transient binding of fluorescent labels. Here, a diffusing dye yields bright fluorescent spots when binding to the structure of interest. As the binding interaction is weak, the labeling is reversible and the dye ligand construct diffuses back into solution. This approach of achieving superresolution by transient binding (STB) is reviewed in this manuscript. Different realizations of STB are discussed and compared to other localization-based superresolution modalities. We propose the development of labeling strategies that will make STB a highly versatile tool for superresolution microscopy at highest resolution. Copyright © 2015 Elsevier Ltd. All rights reserved.

  7. Exploring the binding sites and binding mechanism for hydrotrope encapsulated griseofulvin drug on γ-tubulin protein.

    Directory of Open Access Journals (Sweden)

    Shubhadip Das

    Full Text Available The protein γ-tubulin plays an important role in centrosomal clustering and this makes it an attractive therapeutic target for treating cancers. Griseofulvin, an antifungal drug, has recently been used to inhibit proliferation of various types of cancer cells. It can also affect the microtubule dynamics by targeting the γ-tubulin protein. So far, the binding pockets of γ-tubulin protein are not properly identified and the exact mechanism by which the drug binds to it is an area of intense speculation and research. The aim of the present study is to investigate the binding mechanism and binding affinity of griseofulvin on γ-tubulin protein using classical molecular dynamics simulations. Since the drug griseofulvin is sparingly soluble in water, here we also present a promising approach for formulating and achieving delivery of hydrophobic griseofulvin drug via hydrotrope sodium cumene sulfonate (SCS cluster. We observe that the binding pockets of γ-tubulin protein are mainly formed by the H8, H9 helices and S7, S8, S14 strands and the hydrophobic interactions between the drug and γ-tubulin protein drive the binding process. The release of the drug griseofulvin from the SCS cluster is confirmed by the coordination number analysis. We also find hydrotrope-induced alteration of the binding sites of γ-tubulin protein and the weakening of the drug-protein interactions.

  8. Synthesis of Sulochrin-125I and Its Binding Affinity as α-Glucosidase Inhibitor using Radioligand Binding Assay (RBA Method

    Directory of Open Access Journals (Sweden)

    W. Lestari

    2014-04-01

    Full Text Available Most of diabetics patients have type 2 diabetes mellitus or non insulin dependent diabetes mellitus. Treatment type 2 diabetes mellitus can be done by inhibiting α-glucosidase enzyme which converts carbohydrates into glucose. Sulochrin is one of the potential compounds which can inhibit the function of α-glucosidase enzyme. This study was carried out to obtain data of sulochrin binding with α-glucosidase enzyme as α-glucosidase inhibitor using Radioligand Binding Assay (RBA method. Primary reagent required in RBA method is labeled radioactive ligand (radioligand. In this study, the radioligand was sulochrin-125I and prior to sulochrin-125I synthesis, the sulochrin-I was synthesized. Sulochrin-I and sulochrin-125I were synthesized and their bindings were studied using Radioligand Binding Assay method. Sulochrin-I was synthesized with molecular formula C17H15O7I and molecular weight 457.9940. Sulochrin-125I was synthesized from sulochrin-I by isotope exchange method. From the RBA method, dissociation constant (Kd and maximum binding (Bmax were obtained 26.316 nM and Bmax 9.302 nM respectively. This low Kd indicated that sulochrin was can bind to α-glucosidase

  9. Predicting Flavin and Nicotinamide Adenine Dinucleotide-Binding Sites in Proteins Using the Fragment Transformation Method

    Directory of Open Access Journals (Sweden)

    Chih-Hao Lu

    2015-01-01

    Full Text Available We developed a computational method to identify NAD- and FAD-binding sites in proteins. First, we extracted from the Protein Data Bank structures of proteins that bind to at least one of these ligands. NAD-/FAD-binding residue templates were then constructed by identifying binding residues through the ligand-binding database BioLiP. The fragment transformation method was used to identify structures within query proteins that resembled the ligand-binding templates. By comparing residue types and their relative spatial positions, potential binding sites were identified and a ligand-binding potential for each residue was calculated. Setting the false positive rate at 5%, our method predicted NAD- and FAD-binding sites at true positive rates of 67.1% and 68.4%, respectively. Our method provides excellent results for identifying FAD- and NAD-binding sites in proteins, and the most important is that the requirement of conservation of residue types and local structures in the FAD- and NAD-binding sites can be verified.

  10. Binding of collagens to an enterotoxigenic strain of Escherichia coli

    International Nuclear Information System (INIS)

    Visai, L.; Speziale, P.; Bozzini, S.

    1990-01-01

    An enterotoxigenic strain of Escherichia coli, B34289c, has been shown to bind the N-terminal region of fibronectin with high affinity. We now report that this strain also binds collagen. The binding of 125I-labeled type II collagen to bacteria was time dependent and reversible. Bacteria expressed a limited number of collagen receptors (2.2 x 10(4) per cell) and bound collagen with a Kd of 20 nM. All collagen types tested (I to V) as well as all tested cyanogen bromide-generated peptides [alpha 1(I)CB2, alpha 1(I)CB3, alpha 1(I)CB7, alpha 1(I)CB8, and alpha 2(I)CB4] were recognized by bacterial receptors, as demonstrated by the ability of these proteins to inhibit the binding of 125I-labeled collagen to bacteria. Of several unlabeled proteins tested in competition experiments, fibronectin and its N-terminal region strongly inhibited binding of the radiolabeled collagen to E. coli cells. Conversely, collagen competed with an 125I-labeled 28-kilodalton fibronectin fragment for bacterial binding. Collagen bound to bacteria could be displaced by excess amounts of either unlabeled fibronectin or its N-terminal fragment. Similarly, collagen could displace 125I-labeled N-terminal peptide of fibronectin bound to the bacterial cell surface. Bacteria grown at 41 degrees C or in the presence of glucose did not express collagen or fibronectin receptors. These results indicate the presence of specific binding sites for collagen on the surface of E. coli cells and furthermore that the collagen and fibronectin binding sites are located in close proximity, possibly on the same structure

  11. Object-Oriented Type Inference

    DEFF Research Database (Denmark)

    Schwartzbach, Michael Ignatieff; Palsberg, Jens

    1991-01-01

    We present a new approach to inferring types in untyped object-oriented programs with inheritance, assignments, and late binding. It guarantees that all messages are understood, annotates the program with type information, allows polymorphic methods, and can be used as the basis of an op...

  12. MARs Wars: heterogeneity and clustering of DNA-binding domains in the nuclear matrix

    Directory of Open Access Journals (Sweden)

    Ioudinkova E. S.

    2009-12-01

    Full Text Available Aim. CO326 is a chicken nuclear scaffold/matrix attachment region (MAR associated with the nuclear matrix in several types of chicken cells. It contains a binding site for a sequence-specific DNA-binding protein, F326. We have studied its interaction with the nuclear matrix. Methods. We have used an in vitro MAR assay with isolated matrices from chicken HD3 cells. Results. We have found that an oligonucleotide binding site for the F326 inhibits binding of the CO326 to the nuclear matrix. At the same time, the binding of heterologous MARs is enhanced. Conclusions. Taken together, these data suggest that there exist several classes of MARs and MAR-binding domains and that the MAR-binding proteins may be clustered in the nuclear matrix.

  13. A mosquito hemolymph odorant-binding protein family member specifically binds juvenile hormone

    Energy Technology Data Exchange (ETDEWEB)

    Kim, Il Hwan; Pham, Van; Jablonka, Willy; Goodman, Walter G.; Ribeiro, José M. C.; Andersen, John F.

    2017-07-27

    Juvenile hormone (JH) is a key regulator of insect development and reproduction. In adult mosquitoes, it is essential for maturation of the ovary and normal male reproductive behavior, but how JH distribution and activity is regulated after secretion is unclear. Here, we report a new type of specific JH-binding protein, given the name mosquito juvenile hormone-binding protein (mJHBP), which circulates in the hemolymph of pupal and adult Aedes aegypti males and females. mJHBP is a member of the odorant-binding protein (OBP) family, and orthologs are present in the genomes of Aedes, Culex, and Anopheles mosquito species. Using isothermal titration calorimetry, we show that mJHBP specifically binds JH II and JH III but not eicosanoids or JH derivatives. mJHBP was crystallized in the presence of JH III and found to have a double OBP domain structure reminiscent of salivary “long” D7 proteins of mosquitoes. We observed that a single JH III molecule is contained in the N-terminal domain binding pocket that is closed in an apparent conformational change by a C-terminal domain-derived α-helix. The electron density for the ligand indicated a high occupancy of the natural 10R enantiomer of JH III. Of note, mJHBP is structurally unrelated to hemolymph JHBP from lepidopteran insects. A low level of expression of mJHBP in Ae. aegypti larvae suggests that it is primarily active during the adult stage where it could potentially influence the effects of JH on egg development, mating behavior, feeding, or other processes.

  14. A mosquito hemolymph odorant-binding protein family member specifically binds juvenile hormone.

    Science.gov (United States)

    Kim, Il Hwan; Pham, Van; Jablonka, Willy; Goodman, Walter G; Ribeiro, José M C; Andersen, John F

    2017-09-15

    Juvenile hormone (JH) is a key regulator of insect development and reproduction. In adult mosquitoes, it is essential for maturation of the ovary and normal male reproductive behavior, but how JH distribution and activity is regulated after secretion is unclear. Here, we report a new type of specific JH-binding protein, given the name mosquito juvenile hormone-binding protein (mJHBP), which circulates in the hemolymph of pupal and adult Aedes aegypti males and females. mJHBP is a member of the odorant-binding protein (OBP) family, and orthologs are present in the genomes of Aedes , Culex , and Anopheles mosquito species. Using isothermal titration calorimetry, we show that mJHBP specifically binds JH II and JH III but not eicosanoids or JH derivatives. mJHBP was crystallized in the presence of JH III and found to have a double OBP domain structure reminiscent of salivary "long" D7 proteins of mosquitoes. We observed that a single JH III molecule is contained in the N-terminal domain binding pocket that is closed in an apparent conformational change by a C-terminal domain-derived α-helix. The electron density for the ligand indicated a high occupancy of the natural 10 R enantiomer of JH III. Of note, mJHBP is structurally unrelated to hemolymph JHBP from lepidopteran insects. A low level of expression of mJHBP in Ae. aegypti larvae suggests that it is primarily active during the adult stage where it could potentially influence the effects of JH on egg development, mating behavior, feeding, or other processes.

  15. A novel Zea mays ssp. mexicana L. MYC-type ICE-like transcription factor gene ZmmICE1, enhances freezing tolerance in transgenic Arabidopsis thaliana.

    Science.gov (United States)

    Lu, Xiang; Yang, Lei; Yu, Mengyuan; Lai, Jianbin; Wang, Chao; McNeil, David; Zhou, Meixue; Yang, Chengwei

    2017-04-01

    The annual Zea mays ssp. mexicana L., a member of the teosinte group, is a close wild relative of maize and thus can be effectively used in maize improvement. In this study, an ICE-like gene, ZmmICE1, was isolated from a cDNA library of RNA-Seq from cold-treated seedling tissues of Zea mays ssp. mexicana L. The deduced protein of ZmmICE1 contains a highly conserved basic helix-loop-helix (bHLH) domain and C-terminal region of ICE-like proteins. The ZmmICE1 protein localizes to the nucleus and shows sumoylation when expressed in an Escherichia coli reconstitution system. In addition, yeast one hybrid assays indicated that ZmmICE1 has transactivation activities. Moreover, ectopic expression of ZmmICE1 in the Arabidopsis ice1-2 mutant increased freezing tolerance. The ZmmICE1 overexpressed plants showed lower electrolyte leakage (EL), reduced contents of malondialdehyde (MDA). The expression of downstream cold related genes of Arabidopsis C-repeat-binding factors (AtCBF1, AtCBF2 and AtCBF3), cold-responsive genes (AtCOR15A and AtCOR47), kinesin-1 member gene (AtKIN1) and responsive to desiccation gene (AtRD29A) was significantly induced when compared with wild type under low temperature treatment. Taken together, these results indicated that ZmmICE1 is the homolog of Arabidopsis inducer of CBF expression genes (AtICE1/2) and plays an important role in the regulation of freezing stress response. Copyright © 2017 Elsevier Masson SAS. All rights reserved.

  16. Targeting Human Cancer by a Glycosaminoglycan Binding Malaria Protein

    DEFF Research Database (Denmark)

    Salanti, Ali; Clausen, Thomas M.; Agerbæk, Mette Ø.

    2015-01-01

    Plasmodium falciparum engineer infected erythrocytes to present the malarial protein, VAR2CSA, which binds a distinct type chondroitin sulfate (CS) exclusively expressed in the placenta. Here, we show that the same CS modification is present on a high proportion of malignant cells and that it can...

  17. Ligand-binding sites in human serum amyloid P component

    DEFF Research Database (Denmark)

    Heegaard, N.H.H.; Heegaard, Peter M. H.; Roepstorff, P.

    1996-01-01

    Amyloid P component (AP) is a naturally occurring glycoprotein that is found in serum and basement membranes, AP is also a component of all types of amyloid, including that found in individuals who suffer from Alzheimer's disease and Down's syndrome. Because AP has been found to bind strongly...

  18. Some comments about the binding interpretation of the EMC effect

    International Nuclear Information System (INIS)

    Chanfray, G.

    1990-01-01

    The effect of binding on the nuclear structure function F 2A , deduced from effective Hartree-Fock theory is much smaller than the result obtained from a microscopic approach including correlations. We show that the contradiction between these two types of conventional nuclear approaches is to a large extent, only apparent since they do not refer to the same degrees of freedom

  19. Metal binding by food components

    DEFF Research Database (Denmark)

    Tang, Ning

    for zinc binding by the investigated amino acids, peptides and proteins. The thiol group or imidazole group containing amino acids, peptides and proteins which exhibited strong zinc binding ability were further selected for interacting with zinc salts in relation to zinc absorption. The interactions...... between the above selected food components and zinc citrate or zinc phytate will lead to the enhanced solubility of zinc citrate or zinc phytate. The main driving force for this observed solubility enhancement is the complex formation between zinc and investigated food components as revealed by isothermal...... titration calorimetry and quantum mechanical calculations. This is due to the zinc binding affinity of the relatively softer ligands (investigated food components) will become much stronger than citrate or phytate when they present together in aqueous solution. This mechanism indicates these food components...

  20. Ion Binding Energies Determining Functional Transport of ClC Proteins

    Science.gov (United States)

    Yu, Tao; Guo, Xu; Zou, Xian-Wu; Sang, Jian-Ping

    2014-06-01

    The ClC-type proteins, a large family of chloride transport proteins ubiquitously expressed in biological organisms, have been extensively studied for decades. Biological function of ClC proteins can be reflected by analyzing the binding situation of Cl- ions. We investigate ion binding properties of ClC-ec1 protein with the atomic molecular dynamics simulation approach. The calculated electrostatic binding energy results indicate that Cl- at the central binding site Scen has more binding stability than the internal binding site Sint. Quantitative comparison between the latest experimental heat release data isothermal titration calorimetry (ITC) and our calculated results demonstrates that chloride ions prefer to bind at Scen than Sint in the wild-type ClC-ec1 structure and prefer to bind at Sext and Scen than Sint in mutant E148A/E148Q structures. Even though the chloride ions make less contribution to heat release when binding to Sint and are relatively unstable in the Cl- pathway, they are still part contributors for the Cl- functional transport. This work provides a guide rule to estimate the importance of Cl- at the binding sites and how chloride ions have influences on the function of ClC proteins.

  1. 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.

  2. Skyrmions with low binding energies

    International Nuclear Information System (INIS)

    Gillard, Mike; Harland, Derek; Speight, Martin

    2015-01-01

    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

  3. 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.

  4. CIVAC CV-01 type pressure gage for middle vacuum pumps

    International Nuclear Information System (INIS)

    Olaru, Grigore; Aculai, Agustin

    1997-01-01

    The digital display pressure gage CIVAC CV-01 measures absolute pressures in vacuum range of 10 2 - 10 -3 mbar in any installation or equipment generating or using low pressures. It uses a transducer type PIRANI, model TR-02. It is a portable device, easy to exploit and handle. It is applied in research, chemistry, metallurgical industry, mechanical engineering. The system of coupling the transducer to the enclosure where the pressure is to be measured is of type ISO-PNEUROP with flange, collar and adjusting ring with a DN 25 ring gasket. The technical and functional features are: - Measuring error: ± 35% of real conventional value ; - Response time: 20 ms; - Number of measuring points: 1; - Output signal: 0 - 10 V d.c.; - Repeatability error: 5%; - Max weight: 1,600 Kg; Size: 91 x 117 x 187 mm. (authors)

  5. A urokinase receptor-associated protein with specific collagen binding properties

    DEFF Research Database (Denmark)

    Behrendt, N; Jensen, O N; Engelholm, L H

    2000-01-01

    membrane-bound lectin with hitherto unknown function. The human cDNA was cloned and sequenced. The protein, designated uPARAP, is a member of the macrophage mannose receptor protein family and contains a putative collagen-binding (fibronectin type II) domain in addition to 8 C-type carbohydrate recognition...... domains. It proved capable of binding strongly to a single type of collagen, collagen V. This collagen binding reaction at the exact site of plasminogen activation on the cell may lead to adhesive functions as well as a contribution to cellular degradation of collagen matrices....

  6. 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.

  7. An N-terminal Region of Mot-2 Binds to p53 In Vitro

    Directory of Open Access Journals (Sweden)

    Sunil C. Kaul

    2001-01-01

    Full Text Available The mouse mot-2 protein was earlier shown to bind to the tumor suppressor protein, p53. The mot-2 binding site of p53 was mapped to C-terminal amino acid residues 312–352, which includes the cytoplasmic sequestration domain. In the present study, we have found that both mot-1 and mot-2 bind to p53 in vitro. By using His-tagged deletion mutant proteins, the p53-binding domain of mot-2 was mapped to its Nterminal amino acid residues 253–282, which are identical in mot-1 and mot-2 proteins. Some peptides containing the p53-binding region of mot-2 were able to compete with the full-length protein for p53 binding. The data provided rationale for in vitro binding of mot-1 and mot-2 proteins to p53 and supported the conclusion that inability of mot-1 protein to bind p53 in vivo depends on secondary structure or its binding to other cellular factors. Most interestingly, the p53-binding region of mot-2 was common to its MKT-077, a cationic dye that exhibits antitumor activity, binding region. Therefore it is most likely that MKT-077-induced nuclear translocation and restoration of wild-type p53 function in transformed cells takes place by a competitional mechanism.

  8. THE USE OF DEDICATED PEPTIDE LIBRARIES PERMITS THE DISCOVERY OF HIGH-AFFINITY BINDING PEPTIDES

    NARCIS (Netherlands)

    DEKOSTER, HS; AMONS, R; BENCKHUIJSEN, WE; FEIJLBRIEF, M; SCHELLEKENS, GA; DRIJFHOUT, JW

    1995-01-01

    The motif for peptide binding to monoclonal antibody mAb A16, which is known to be directed against glycoprotein D of Herpes simplex virus type 1, was determined using two dedicated peptide libraries. As a starting point for this study we used an A-16 binding lead sequence, which had previously been

  9. Guanylate kinase domains of the MAGUK family scaffold proteins as specific phospho-protein-binding modules

    OpenAIRE

    Zhu, Jinwei; Shang, Yuan; Xia, Caihao; Wang, Wenning; Wen, Wenyu; Zhang, Mingjie

    2011-01-01

    Membrane-associated guanylate kinases (MAGUK) family proteins contain an inactive guanylate kinase (GK) domain, whose function has been elusive. Here, this domain is revealed as a new type of phospho-peptide-binding module, in which the GMP-binding site has evolved to accommodate phospho-serines or -threonines.

  10. Possible heterogeneity of centres of binding 1,8-ANS in molecules of oxygenated hemoglobin

    International Nuclear Information System (INIS)

    Parul', D.A.; Bokut', S.B.; Milyutin, A.A.; Petrov, E.P.; Nemkovich, N.A.; Sobchuk, A.N.; Dzhagarov, B.M.

    1999-01-01

    Forming of oxygenated hemoglobin is one of effects of ionizing radiation action on organism. It was revealed heterogeneity of centers of binding of 1,8-ANS in intact molecule of oxygenated hemoglobin. Two types of binding centers possible reflect the existence of two regions in protein molecule with different accessibility to molecules of water

  11. Cellulose binding domain fusion proteins

    Science.gov (United States)

    Shoseyov, Oded; Shpiegl, Itai; Goldstein, Marc A.; Doi, Roy H.

    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.

  12. When is protein binding important?

    Science.gov (United States)

    Heuberger, Jules; Schmidt, Stephan; Derendorf, Hartmut

    2013-09-01

    The present paper is an ode to a classic citation by Benet and Hoener (2002. Clin Pharm Ther 71(3):115-121). The now classic paper had a huge impact on drug development and the way the issue of protein binding is perceived and interpreted. Although the authors very clearly pointed out the limitations and underlying assumptions for their delineations, these are too often overlooked and the classic paper's message is misinterpreted by broadening to cases that were not intended. Some members of the scientific community concluded from the paper that protein binding is not important. This was clearly not intended by the authors, as they finished their paper with a paragraph entitled: "When is protein binding important?" Misinterpretation of the underlying assumptions in the classic work can result in major pitfalls in drug development. Therefore, we revisit the topic of protein binding with the intention of clarifying when clinically relevant changes should be considered during drug development. Copyright © 2013 Wiley Periodicals, Inc.

  13. Normalization by evaluation with typed abstract syntax

    DEFF Research Database (Denmark)

    Danvy, Olivier; Rhiger, Morten; Rose, Kristoffer H.

    2001-01-01

    In higher-order abstract syntax, the variables and bindings of an object language are represented by variables and bindings of a meta-language. Let us consider the simply typed λ-calculus as object language and Haskell as meta-language. For concreteness, we also throw in integers and addition, bu...

  14. Dominant Alcohol-Protein Interaction via Hydration-Enabled Enthalpy-Driven Binding Mechanism

    Science.gov (United States)

    Chong, Yuan; Kleinhammes, Alfred; Tang, Pei; Xu, Yan; Wu, Yue

    2015-01-01

    Water plays an important role in weak associations of small drug molecules with proteins. Intense focus has been on binding-induced structural changes in the water network surrounding protein binding sites, especially their contributions to binding thermodynamics. However, water is also tightly coupled to protein conformations and dynamics, and so far little is known about the influence of water-protein interactions on ligand binding. Alcohols are a type of low-affinity drugs, and it remains unclear how water affects alcohol-protein interactions. Here, we present alcohol adsorption isotherms under controlled protein hydration using in-situ NMR detection. As functions of hydration level, Gibbs free energy, enthalpy, and entropy of binding were determined from the temperature dependence of isotherms. Two types of alcohol binding were found. The dominant type is low-affinity nonspecific binding, which is strongly dependent on temperature and the level of hydration. At low hydration levels, this nonspecific binding only occurs above a threshold of alcohol vapor pressure. An increased hydration level reduces this threshold, with it finally disappearing at a hydration level of h~0.2 (g water/g protein), gradually shifting alcohol binding from an entropy-driven to an enthalpy-driven process. Water at charged and polar groups on the protein surface was found to be particularly important in enabling this binding. Although further increase in hydration has smaller effects on the changes of binding enthalpy and entropy, it results in significant negative change in Gibbs free energy due to unmatched enthalpy-entropy compensation. These results show the crucial role of water-protein interplay in alcohol binding. PMID:25856773

  15. Absence of serum growth hormone binding protein in patients with growth hormone receptor deficiency (Laron dwarfism)

    Energy Technology Data Exchange (ETDEWEB)

    Daughaday, W.H.; Trivedi, B.

    1987-07-01

    It has recently been recognized that human serum contains a protein that specifically binds human growth hormone (hGH). This protein has the same restricted specificity for hGH as the membrane-bound GH receptor. To determine whether the GH-binding protein is a derivative of, or otherwise related to, the GH receptor, the authors have examined the serum of three patients with Laron-type dwarfism, a condition in which GH refractoriness has been attributed to a defect in the GH receptor. The binding of /sup 125/I-labeled hGH incubated with serum has been measured after gel filtration of the serum through an Ultrogel AcA 44 minicolumn. Results are expressed as percent of specifically bound /sup 125/I-hGH and as specific binding relative to that of a reference serum after correction is made for endogenous GH. The mean +/- SEM of specific binding of sera from eight normal adults (26-46 years of age) was 21.6 +/- 0.45%, and the relative specific binding was 101.1 +/- 8.6%. Sera from 11 normal children had lower specific binding of 12.5 +/- 1.95% and relative specific binding of 56.6 +/- 9.1%. Sera from three children with Laron-type dwarfism lacked any demonstrable GH binding, whereas sera from 10 other children with other types of nonpituitary short stature had normal relative specific binding. They suggest that the serum GH-binding protein is a soluble derivative of the GH receptor. Measurement of the serum GH-binding protein may permit recognition of other abnormalities of the GH receptor.

  16. Absence of serum growth hormone binding protein in patients with growth hormone receptor deficiency (Laron dwarfism)

    International Nuclear Information System (INIS)

    Daughaday, W.H.; Trivedi, B.

    1987-01-01

    It has recently been recognized that human serum contains a protein that specifically binds human growth hormone (hGH). This protein has the same restricted specificity for hGH as the membrane-bound GH receptor. To determine whether the GH-binding protein is a derivative of, or otherwise related to, the GH receptor, the authors have examined the serum of three patients with Laron-type dwarfism, a condition in which GH refractoriness has been attributed to a defect in the GH receptor. The binding of 125 I-labeled hGH incubated with serum has been measured after gel filtration of the serum through an Ultrogel AcA 44 minicolumn. Results are expressed as percent of specifically bound 125 I-hGH and as specific binding relative to that of a reference serum after correction is made for endogenous GH. The mean +/- SEM of specific binding of sera from eight normal adults (26-46 years of age) was 21.6 +/- 0.45%, and the relative specific binding was 101.1 +/- 8.6%. Sera from 11 normal children had lower specific binding of 12.5 +/- 1.95% and relative specific binding of 56.6 +/- 9.1%. Sera from three children with Laron-type dwarfism lacked any demonstrable GH binding, whereas sera from 10 other children with other types of nonpituitary short stature had normal relative specific binding. They suggest that the serum GH-binding protein is a soluble derivative of the GH receptor. Measurement of the serum GH-binding protein may permit recognition of other abnormalities of the GH receptor

  17. Structural Changes of Creatine Kinase upon Substrate Binding

    OpenAIRE

    Forstner, Michael; Kriechbaum, Manfred; Laggner, Peter; Wallimann, Theo

    1998-01-01

    Small-angle x-ray scattering was used to investigate structural changes upon binding of individual substrates or a transition state analog complex (TSAC; Mg-ADP, creatine, and KNO3) to creatine kinase (CK) isoenzymes (dimeric muscle-type (M)-CK and octameric mitochondrial (Mi)-CK) and monomeric arginine kinase (AK). Considerable changes in the shape and the size of the molecules occurred upon binding of Mg-nucleotide or TSAC. The radius of gyration of Mi-CK was reduced from 55.6 A (free enzym...

  18. On Anaphora and the Binding Principles in Categorial Grammar

    Science.gov (United States)

    Morrill, Glyn; Valentín, Oriol

    In type logical categorial grammar the analysis of an expression is a resource-conscious proof. Anaphora represents a particular challenge to this approach in that the antecedent resource is multiplied in the semantics. This duplication, which corresponds logically to the structural rule of contraction, may be treated lexically or syntactically. Furthermore, anaphora is subject to constraints, which Chomsky (1981) formulated as Binding Principles A, B, and C. In this paper we consider English anaphora in categorial grammar including reference to the binding principles. We invoke displacement calculus, modal categorial calculus, categorial calculus with limited contraction, and entertain addition of negation as failure.

  19. Heat-Labile Enterotoxin: Beyond G M1 Binding

    Directory of Open Access Journals (Sweden)

    Benjamin Mudrak

    2010-06-01

    Full Text Available Enterotoxigenic Escherichia coli (ETEC is a significant source of morbidity and mortality worldwide. One major virulence factor released by ETEC is the heat-labile enterotoxin LT, which is structurally and functionally similar to cholera toxin. LT consists of five B subunits carrying a single catalytically active A subunit. LTB binds the monosialoganglioside GM1, the toxin’s host receptor, but interactions with A-type blood sugars and E. coli lipopolysaccharide have also been identified within the past decade. Here, we review the regulation, assembly, and binding properties of the LT B-subunit pentamer and discuss the possible roles of its numerous molecular interactions.

  20. Acyl-CoA binding protein and epidermal barrier function

    DEFF Research Database (Denmark)

    Bloksgaard, Maria; Neess, Ditte; Færgeman, Nils J

    2014-01-01

    The acyl-CoA binding protein (ACBP) is a 10kDa intracellular protein expressed in all eukaryotic species and mammalian tissues investigated. It binds acyl-CoA esters with high specificity and affinity and is thought to act as an intracellular transporter of acyl-CoA esters between different...... includes tousled and greasy fur, development of alopecia and scaling of the skin with age. Furthermore, epidermal barrier function is compromised causing a ~50% increase in transepidermal water loss relative to that of wild type mice. Lipidomic analyses indicate that this is due to significantly reduced...

  1. Reflection-Based Python-C++ Bindings

    International Nuclear Information System (INIS)

    Generowicz, Jacek; Lavrijsen, Wim T.L.P.; Marino, Massimo; Mato, Pere

    2004-01-01

    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

  2. Variation in the number of EPIYA-C repeats in CagA protein from Colombian Helicobacter pylori strains and its ability to induce hummingbird phenotype in gastric epithelial cells Variación en el número de repeticiones EPIYA-C en la proteína CagA de aislamientos colombianos de Helicobacter pylori y su capacidad para inducir fenotipo colibrí en células epiteliales gástricas

    Directory of Open Access Journals (Sweden)

    María Mercedes Bravo

    2010-08-01

    Full Text Available Introduction. Studies using Western Helicobacter pylori strains have shown that a risk factor for gastric cancer is the number of EPIYA-C motifs in the cytotoxin-associated A protein. CagA is delivered into epithelial cells, where it becomes tyrosine phosphorylated in their EPIYA repeats and induces cytoskeleton rearrangements.
    Objectives. The objective of this study was to evaluate H. pylori cagA positive strains isolated from Colombian patients with gastroduodenal diseases for the number of EPIYA-C repeats in cagA and their ability to induce cytoskeleton rearrangements in epithelial cells.
    Materials and methods. We analyzed the 3' EPIYA repeats region of cagA by PCR in 93 H. pylori cagA positive strains from 49 patients with gastritis, 17 with gastric cancer, and 24 with duodenal ulcer. AGS cells exposed to the various H. pylori isolates were evaluated for rearrangements in their cytoskeleton.
    Results. Strains with one EPIYA-C were the most frequent in gastritis and duodenal ulcer patients. Strains with three EPIYA-C were mainly found in gastric cancer. We found a significantly higher risk of gastric cancer for individuals infected with strains harboring three EPIYA-C motifs (OR=12.4, CI95%: 2.32-66.3. Strains from gastric cancer showed significantly higher percentages of induction of cytoskeleton rearrangements in comparison with those from gastritis (p Mann-Whitney<0.005.
    Conclusions. H. pylori strains with three EPIYA-C repeats can confer an increased risk of cancer to infected individuals.Introducción. En los aislamientos de Helicobacter pylori del hemisferio occidental, se ha observado que el número de repeticiones EPIYA-C en la proteína CagA es un factor de riesgo para cáncer gástrico. La proteína CagA es introducida en la célula epitelial y, posteriormente, es fosforilada en las tirosinas presentes en los motivos EPIYA e induce rearreglos en el citoesqueleto.
    Objetivos. Nuestro propósito fue evaluar el n

  3. Cyclophilin B binding to platelets supports calcium-dependent adhesion to collagen.

    Science.gov (United States)

    Allain, F; Durieux, S; Denys, A; Carpentier, M; Spik, G

    1999-08-01

    We have recently reported that cyclophilin B (CyPB), a secreted cyclosporine-binding protein, could bind to T lymphocytes through interactions with two types of binding sites. The first ones, referred to as type I, involve interactions with the conserved domain of CyPB and promote the endocytosis of surface-bound ligand, while the second type of binding sites, termed type II, are represented by glycosaminoglycans (GAG). Here, we further investigated the interactions of CyPB with blood cell populations. In addition to lymphocytes, CyPB was found to interact mainly with platelets. The binding is specific, with a dissociation constant (kd) of 9 +/- 3 nmol/L and the number of sites estimated at 960 +/- 60 per cell. Platelet glycosaminoglycans are not required for the interactions, but the binding is dramatically reduced by active cyclosporine derivatives. We then analyzed the biologic effects of CyPB and found a significant increase in platelet adhesion to collagen. Concurrently, CyPB initiates a transmembranous influx of Ca(2+) and induces the phosphorylation of the P-20 light chains of myosin. Taken together, the present results demonstrate for the first time that extracellular CyPB specifically interacts with platelets through a functional receptor related to the lymphocyte type I binding sites and might act by regulating the activity of a receptor-operated membrane Ca(2+) channel.

  4. In vitro binding of puroindolines to wheat starch granules

    DEFF Research Database (Denmark)

    Sørensen, Helle Aagaard; Darlington, H.F.; Shewry, P.R.

    2001-01-01

    Puroindoline (pin) preparations made from flours of hard and soft wheats contained a mixture of pin-a, 0.19/0.53 alpha -amylase inhibitor, and purothionins. Starch granule preparations from the same cultivars were treated with proteinase to remove surface proteins and incubated with solutions...... of the pin preparations. Binding of pin-a and purothionins but not the 0.19/0.53 inhibitor was observed with no apparent differences between the behavior of the pin preparations or starch granule preparations from hard or soft types. No binding was observed when several other proteins (bovine serum albumin......, total albumins, a commercial preparation of wheat alpha -amylase inhibitors, and barley beta -amylase) were incubated with the starch granules under the same conditions, indicating that in vitro binding can be used to study specific starch granule and protein interactions....

  5. Drosophila DNA-Binding Proteins in Polycomb Repression

    Directory of Open Access Journals (Sweden)

    Maksim Erokhin

    2018-01-01

    Full Text Available The formation of individual gene expression patterns in different cell types is required during differentiation and development of multicellular organisms. Polycomb group (PcG proteins are key epigenetic regulators responsible for gene repression, and dysregulation of their activities leads to developmental abnormalities and diseases. PcG proteins were first identified in Drosophila, which still remains the most convenient system for studying PcG-dependent repression. In the Drosophila genome, these proteins bind to DNA regions called Polycomb response elements (PREs. A major role in the recruitment of PcG proteins to PREs is played by DNA-binding factors, several of which have been characterized in detail. However, current knowledge is insufficient for comprehensively describing the mechanism of this process. In this review, we summarize and discuss the available data on the role of DNA-binding proteins in PcG recruitment to chromatin.

  6. Surface Passivation in Empirical Tight Binding

    Science.gov (United States)

    He, Yu; Tan, Yaohua; Jiang, Zhengping; Povolotskyi, Michael; Klimeck, Gerhard; Kubis, Tillmann

    2016-03-01

    Empirical Tight Binding (TB) methods are widely used in atomistic device simulations. Existing TB methods to passivate dangling bonds fall into two categories: 1) Method that explicitly includes passivation atoms is limited to passivation with atoms and small molecules only. 2) Method that implicitly incorporates passivation does not distinguish passivation atom types. This work introduces an implicit passivation method that is applicable to any passivation scenario with appropriate parameters. This method is applied to a Si quantum well and a Si ultra-thin body transistor oxidized with SiO2 in several oxidation configurations. Comparison with ab-initio results and experiments verifies the presented method. Oxidation configurations that severely hamper the transistor performance are identified. It is also shown that the commonly used implicit H atom passivation overestimates the transistor performance.

  7. Kinetics of fatty acid binding ability of glycated human serum albumin

    Indian Academy of Sciences (India)

    Unknown

    1-anilino-8-naphtharene sulphonic acid; diabetes, dissociation constant; fatty acids binding; fluorescence displacement ... thought to play an important role in the complications of ..... concentration of serum fatty acid level in type 2 diabetes,.

  8. Probing protein phosphatase substrate binding

    DEFF Research Database (Denmark)

    Højlys-Larsen, Kim B.; Sørensen, Kasper Kildegaard; Jensen, Knud Jørgen

    2012-01-01

    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...... 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......, without the need for phosphopeptide mimics or phosphatase inhibitors. As no proven ILKAP substrates were available, we selected phosphopeptide substrates among known PP2Cδ substrates including the protein kinases: p38, ATM, Chk1, Chk2 and RSK2 and synthesized directly on PEGA solid supports through a BAL...

  9. How Native and Alien Metal Cations Bind ATP: Implications for Lithium as a Therapeutic Agent

    Science.gov (United States)

    Dudev, Todor; Grauffel, Cédric; Lim, Carmay

    2017-02-01

    Adenosine triphosphate (ATP), the major energy currency of the cell, exists in solution mostly as ATP-Mg. Recent experiments suggest that Mg2+ interacts with the highly charged ATP triphosphate group and Li+ can co-bind with the native Mg2+ to form ATP-Mg-Li and modulate the neuronal purine receptor response. However, it is unclear how the negatively charged ATP triphosphate group binds Mg2+ and Li+ (i.e. which phosphate group(s) bind Mg2+/Li+) and how the ATP solution conformation depends on the type of metal cation and the metal-binding mode. Here, we reveal the preferred ATP-binding mode of Mg2+/Li+ alone and combined: Mg2+ prefers to bind ATP tridentately to each of the three phosphate groups, but Li+ prefers to bind bidentately to the terminal two phosphates. We show that the solution ATP conformation depends on the cation and its binding site/mode, but it does not change significantly when Li+ binds to Mg2+-loaded ATP. Hence, ATP-Mg-Li, like Mg2+-ATP, can fit in the ATP-binding site of the host enzyme/receptor, activating specific signaling pathways.

  10. Ligand binding by PDZ domains

    DEFF Research Database (Denmark)

    Chi, Celestine N.; Bach, Anders; Strømgaard, Kristian

    2012-01-01

    , for example, are particularly rich in these domains. The general function of PDZ domains is to bring proteins together within the appropriate cellular compartment, thereby facilitating scaffolding, signaling, and trafficking events. The many functions of PDZ domains under normal physiological as well...... as pathological conditions have been reviewed recently. In this review, we focus on the molecular details of how PDZ domains bind their protein ligands and their potential as drug targets in this context....

  11. Calcium binding by dietary fibre

    International Nuclear Information System (INIS)

    James, W.P.T.; Branch, W.J.; Southgate, D.A.T.

    1978-01-01

    Dietary fibre from plants low in phytate bound calcium in proportion to its uronic-acid content. This binding by the non-cellulosic fraction of fibre reduces the availability of calcium for small-intestinal absorption, but the colonic microbial digestion of uronic acids liberates the calcium. Thus the ability to maintain calcium balance on high-fibre diets may depend on the adaptive capacity on the colon for calcium. (author)

  12. Making Type Inference Practical

    DEFF Research Database (Denmark)

    Schwartzbach, Michael Ignatieff; Oxhøj, Nicholas; Palsberg, Jens

    1992-01-01

    We present the implementation of a type inference algorithm for untyped object-oriented programs with inheritance, assignments, and late binding. The algorithm significantly improves our previous one, presented at OOPSLA'91, since it can handle collection classes, such as List, in a useful way. Abo......, the complexity has been dramatically improved, from exponential time to low polynomial time. The implementation uses the techniques of incremental graph construction and constraint template instantiation to avoid representing intermediate results, doing superfluous work, and recomputing type information....... Experiments indicate that the implementation type checks as much as 100 lines pr. second. This results in a mature product, on which a number of tools can be based, for example a safety tool, an image compression tool, a code optimization tool, and an annotation tool. This may make type inference for object...

  13. Rigidification of the autolysis loop enhances Na(+) binding to thrombin.

    Science.gov (United States)

    Pozzi, Nicola; Chen, Raymond; Chen, Zhiwei; Bah, Alaji; Di Cera, Enrico

    2011-11-01

    Binding of Na(+) to thrombin ensures high activity toward physiological substrates and optimizes the procoagulant and prothrombotic roles of the enzyme in vivo. Under physiological conditions of pH and temperature, the binding affinity of Na(+) is weak due to large heat capacity and enthalpy changes associated with binding, and the K(d)=80 mM ensures only 64% saturation of the site at the concentration of Na(+) in the blood (140 mM). Residues controlling Na(+) binding and activation have been identified. Yet, attempts to improve the interaction of Na(+) with thrombin and possibly increase catalytic activity under physiological conditions have so far been unsuccessful. Here we report how replacement of the flexible autolysis loop of human thrombin with the homologous rigid domain of the murine enzyme results in a drastic (up to 10-fold) increase in Na(+) affinity and a significant improvement in the catalytic activity of the enzyme. Rigidification of the autolysis loop abolishes the heat capacity change associated with Na(+) binding observed in the wild-type and also increases the stability of thrombin. These findings have general relevance to protein engineering studies of clotting proteases and trypsin-like enzymes. Copyright © 2011 Elsevier B.V. All rights reserved.

  14. Rigidification of the autolysis loop enhances Na+ binding to thrombin

    Science.gov (United States)

    Pozzi, Nicola; Chen, Raymond; Chen, Zhiwei; Bah, Alaji; Di Cera, Enrico

    2011-01-01

    Binding of Na+ to thrombin ensures high activity toward physiological substrates and optimizes the procoagulant and prothrombotic roles of the enzyme in vivo. Under physiological conditions of pH and temperature, the binding affinity of Na+ is weak due to large heat capacity and enthalpy changes associated with binding, and the Kd=80 mM ensures only 64% saturation of the site at the concentration of Na+ in the blood (140 mM). Residues controlling Na+ binding and activation have been identified. Yet, attempts to improve the interaction of Na+ with thrombin and possibly increase catalytic activity under physiological conditions have so far been unsuccessful. Here we report how replacement of the flexible autolysis loop of human thrombin with the homologous rigid domain of the murine enzyme results in a drastic (up to 10-fold) increase in Na+ affinity and a significant improvement in the catalytic activity of the enzyme. Rigidification of the autolysis loop abolishes the heat capacity change associated with Na+ binding observed in the wild-type and also increases the stability of thrombin. These findings have general relevance to protein engineering studies of clotting proteases and trypsin-like enzymes. PMID:21536369

  15. Steroid production and estrogen binding in flowers of Gladiolus

    International Nuclear Information System (INIS)

    Adler, J.H.; Wolfe, G.R.; Janik, J.R.

    1987-01-01

    The bioconversion of 3 H-cholesterol to steroids was examined in excised tissue from the pistils and bracts of Gladiolus. Ovary-ovule and stigma-style tissues produce a compound with chromatographic properties on reverse phase HPLC similar to 17β-estradiol (E 2 ). The stigma-style fraction also produced a compound that chromatographed similarly to progesterone. Bracts and the oxidation controls produced no radiolabeled compounds which were chromatographically similar to E 2 . An endogenous E 2 binding protein was partially characterized from the ovules. The protein binds E 2 , estriol, and diethylstilbesterol whereas testosterone and progesterone do not bind. The total specific binding capacities in the cytosolic and nuclear fractions are 1.6 and 2.2 femtomoles of estradiol per mg of tissue. The dissociation constant is 1.1 x 10 -9 M -1 for both subcellular fractions. The protein-estradiol complex has a sedimentation coefficient of 4.7 +/- 0.1S. The tissue specific biosynthesis of estrogens and the presence of a steroid binding protein similar to a Type 1 estrogen receptor found in mammals is suggestive of a role for steroids in pistil ontogeny

  16. Material Binding Peptides for Nanotechnology

    Directory of Open Access Journals (Sweden)

    Urartu Ozgur Safak Seker

    2011-02-01

    Full Text Available Remarkable progress has been made to date in the discovery of material binding peptides and their utilization in nanotechnology, which has brought new challenges and opportunities. Nowadays phage display is a versatile tool, important for the selection of ligands for proteins and peptides. This combinatorial approach has also been adapted over the past decade to select material-specific peptides. Screening and selection of such phage displayed material binding peptides has attracted great interest, in particular because of their use in nanotechnology. Phage display selected peptides are either synthesized independently or expressed on phage coat protein. Selected phage particles are subsequently utilized in the synthesis of nanoparticles, in the assembly of nanostructures on inorganic surfaces, and oriented protein immobilization as fusion partners of proteins. In this paper, we present an overview on the research conducted on this area. In this review we not only focus on the selection process, but also on molecular binding characterization and utilization of peptides as molecular linkers, molecular assemblers and material synthesizers.

  17. 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.

  18. Anion binding in biological systems

    International Nuclear Information System (INIS)

    Feiters, Martin C; Meyer-Klaucke, Wolfram; Kostenko, Alexander V; Soldatov, Alexander V; Leblanc, Catherine; Michel, Gurvan; Potin, Philippe; Kuepper, Frithjof C; Hollenstein, Kaspar; Locher, Kaspar P; Bevers, Loes E; Hagedoorn, Peter-Leon; Hagen, Wilfred R

    2009-01-01

    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 L 3 (2p 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.

  19. 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.

  20. Assessing the model transferability for prediction of transcription factor binding sites based on chromatin accessibility.

    Science.gov (United States)

    Liu, Sheng; Zibetti, Cristina; Wan, Jun; Wang, Guohua; Blackshaw, Seth; Qian, Jiang

    2017-07-27

    Computational prediction of transcription factor (TF) binding sites in different cell types is challenging. Recent technology development allows us to determine the genome-wide chromatin accessibility in various cellular and developmental contexts. The chromatin accessibility profiles provide useful information in prediction of TF binding events in various physiological conditions. Furthermore, ChIP-Seq analysis was used to determine genome-wide binding sites for a range of different TFs in multiple cell types. Integration of these two types of genomic information can improve the prediction of TF binding events. We assessed to what extent a model built upon on other TFs and/or other cell types could be used to predict the binding sites of TFs of interest. A random forest model was built using a set of cell type-independent features such as specific sequences recognized by the TFs and evolutionary conservation, as well as cell type-specific features derived from chromatin accessibility data. Our analysis suggested that the models learned from other TFs and/or cell lines performed almost as well as the model learned from the target TF in the cell type of interest. Interestingly, models based on multiple TFs performed better than single-TF models. Finally, we proposed a universal model, BPAC, which was generated using ChIP-Seq data from multiple TFs in various cell types. Integrating chromatin accessibility information with sequence information improves prediction of TF binding.The prediction of TF binding is transferable across TFs and/or cell lines suggesting there are a set of universal "rules". A computational tool was developed to predict TF binding sites based on the universal "rules".

  1. Different mechanisms are involved in the antibody mediated inhibition of ligand binding to the urokinase receptor

    DEFF Research Database (Denmark)

    List, K; Høyer-Hansen, G; Rønne, E

    1999-01-01

    Certain monoclonal antibodies are capable of inhibiting the biological binding reactions of their target proteins. At the molecular level, this type of effect may be brought about by completely different mechanisms, such as competition for common binding determinants, steric hindrance or interfer......Certain monoclonal antibodies are capable of inhibiting the biological binding reactions of their target proteins. At the molecular level, this type of effect may be brought about by completely different mechanisms, such as competition for common binding determinants, steric hindrance......) can be employed as a highly useful tool to characterize the inhibitory mechanism of specific antagonist antibodies. Two inhibitory antibodies against uPAR, mAb R3 and mAb R5, were shown to exhibit competitive and non-competitive inhibition, respectively, of ligand binding to the receptor. The former...

  2. Evolutionarily conserved bias of amino-acid usage refines the definition of PDZ-binding motif

    Directory of Open Access Journals (Sweden)

    Launey Thomas

    2011-06-01

    Full Text Available Abstract Background The interactions between PDZ (PSD-95, Dlg, ZO-1 domains and PDZ-binding motifs play central roles in signal transductions within cells. Proteins with PDZ domains bind to PDZ-binding motifs almost exclusively when the motifs are located at the carboxyl (C- terminal ends of their binding partners. However, it remains little explored whether PDZ-binding motifs show any preferential location at the C-terminal ends of proteins, at genome-level. Results Here, we examined the distribution of the type-I (x-x-S/T-x-I/L/V or type-II (x-x-V-x-I/V PDZ-binding motifs in proteins encoded in the genomes of five different species (human, mouse, zebrafish, fruit fly and nematode. We first established that these PDZ-binding motifs are indeed preferentially present at their C-terminal ends. Moreover, we found specific amino acid (AA bias for the 'x' positions in the motifs at the C-terminal ends. In general, hydrophilic AAs were favored. Our genomics-based findings confirm and largely extend the results of previous interaction-based studies, allowing us to propose refined consensus sequences for all of the examined PDZ-binding motifs. An ontological analysis revealed that the refined motifs are functionally relevant since a large fraction of the proteins bearing the motif appear to be involved in signal transduction. Furthermore, co-precipitation experiments confirmed two new protein interactions predicted by our genomics-based approach. Finally, we show that influenza virus pathogenicity can be correlated with PDZ-binding motif, with high-virulence viral proteins bearing a refined PDZ-binding motif. Conclusions Our refined definition of PDZ-binding motifs should provide important clues for identifying functional PDZ-binding motifs and proteins involved in signal transduction.

  3. The heparin-binding site in tetranectin is located in the N-terminal region and binding does not involve the carbohydrate recognition domain.

    Science.gov (United States)

    Lorentsen, R H; Graversen, J H; Caterer, N R; Thogersen, H C; Etzerodt, M

    2000-04-01

    Tetranectin is a homotrimeric plasma and extracellular-matrix protein that binds plasminogen and complex sulphated polysaccharides including heparin. In terms of primary and tertiary structure, tetranectin is related to the collectin family of Ca(2+)-binding C-type lectins. Tetranectin is encoded in three exons. Exon 3 encodes the carbohydrate recognition domain, which binds to kringle 4 in plasminogen at low levels of Ca(2+). Exon 2 encodes an alpha-helix, which is necessary and sufficient to govern the trimerization of tetranectin by assembling into a triple-helical coiled-coil structural element. Here we show that the heparin-binding site in tetranectin resides not in the carbohydrate recognition domain but within the N-terminal region, comprising the 16 amino acid residues encoded by exon 1. In particular, the lysine residues in the decapeptide segment KPKKIVNAKK (tetranectin residues 6-15) are shown to be of primary importance in heparin binding.

  4. Object-Oriented Type Systems

    DEFF Research Database (Denmark)

    Schwartzbach, Michael Ignatieff; Palsberg, Jens

    binding. Existing languages employ different type systems, and it can be difficult to compare, evaluate and improve them, since there is currently no uniform theory for such languages. This book provides such a theory. The authors review the type systems of Simula, Smalltalk, C++ and Eiffel and present......Object-Oriented Type Systems Jens Palsberg and Michael I. Schwartzbach Aarhus University, Denmark Type systems are required to ensure reliability and efficiency of software. For object-oriented languages, typing is an especially challenging problem because of inheritance, assignment, and late...... a type system that generalizes and explains them. The theory is based on an idealized object-oriented language called BOPL (Basic Object Programming Language), containing common features of the above languages. A type system, type inference algorithm, and typings of inheritance and genericity...

  5. Crystallographic structure and substrate-binding interactions of the molybdate-binding protein of the phytopathogen Xanthomonas axonopodis pv. citri.

    Science.gov (United States)

    Balan, Andrea; Santacruz-Pérez, Carolina; Moutran, Alexandre; Ferreira, Luís Carlos Souza; Neshich, Goran; Gonçalves Barbosa, João Alexandre Ribeiro

    2008-02-01

    In Xanthomonas axonopodis pv. citri (Xac or X. citri), the modA gene codes for a periplasmic protein (ModA) that is capable of binding molybdate and tungstate as part of the ABC-type transporter required for the uptake of micronutrients. In this study, we report the crystallographic structure of the Xac ModA protein with bound molybdate. The Xac ModA structure is similar to orthologs with known three-dimensional structures and consists of two nearly symmetrical domains separated by a hinge region where the oxyanion-binding site lies. Phylogenetic analysis of different ModA orthologs based on sequence alignments revealed three groups of molybdate-binding proteins: bacterial phytopathogens, enterobacteria and soil bacteria. Even though the ModA orthologs are segregated into different groups, the ligand-binding hydrogen bonds are mostly conserved, except for Archaeglobus fulgidus ModA. A detailed discussion of hydrophobic interactions in the active site is presented and two new residues, Ala38 and Ser151, are shown to be part of the ligand-binding pocket.

  6. Solute-vacancy binding in aluminum

    International Nuclear Information System (INIS)

    Wolverton, C.

    2007-01-01

    Previous efforts to understand solute-vacancy binding in aluminum alloys have been hampered by a scarcity of reliable, quantitative experimental measurements. Here, we report a large database of solute-vacancy binding energies determined from first-principles density functional calculations. The calculated binding energies agree well with accurate measurements where available, and provide an accurate predictor of solute-vacancy binding in other systems. We find: (i) some common solutes in commercial Al alloys (e.g., Cu and Mg) possess either very weak (Cu), or even repulsive (Mg), binding energies. Hence, we assert that some previously reported large binding energies for these solutes are erroneous. (ii) Large binding energies are found for Sn, Cd and In, confirming the proposed mechanism for the reduced natural aging in Al-Cu alloys containing microalloying additions of these solutes. (iii) In addition, we predict that similar reduction in natural aging should occur with additions of Si, Ge and Au. (iv) Even larger binding energies are found for other solutes (e.g., Pb, Bi, Sr, Ba), but these solutes possess essentially no solubility in Al. (v) We have explored the physical effects controlling solute-vacancy binding in Al. We find that there is a strong correlation between binding energy and solute size, with larger solute atoms possessing a stronger binding with vacancies. (vi) Most transition-metal 3d solutes do not bind strongly with vacancies, and some are even energetically strongly repelled from vacancies, particularly for the early 3d solutes, Ti and V

  7. Polymeric competitive protein binding adsorbents for radioassay

    International Nuclear Information System (INIS)

    Adams, R.J.

    1976-01-01

    Serum protein comprising specific binding proteins such as antibodies, B 12 intrinsic factor, thyroxin binding globulin and the like may be copolymerized with globulin constituents of serum by the action of ethylchloroformate to form readily packed insoluble precipitates which, following purification as by washing, are eminently suited for employment as competitive binding protein absorbents in radioassay procedures. 10 claims, no drawings

  8. A peek into tropomyosin binding and unfolding on the actin filament.

    Directory of Open Access Journals (Sweden)

    Abhishek Singh

    that local disorder is a property typical of coiled coil binding sites and proteins that have multiple binding partners, of which tropomyosin is one type.

  9. Ubiquinone accumulates in the mitochondria of yeast mutated in the ubiquinone binding protein, Qcr8p

    International Nuclear Information System (INIS)

    Hagerman, Ruth A.; Waring, Natashya J.; Willis, Richard A.; Hagerman, Ann E.

    2006-01-01

    In Saccharomyces cerevisiae, the trans-membrane helix of Qcr8p, the ubiquinone binding protein of complex III, contributes to the Q binding site. In wild-type cells, residue 62 of the helix is non-polar (proline). Substitution of proline 62 with a polar, uncharged residue does not impair the ability of the cells to respire, complex III assembly is unaffected, ubiquinone occupancy of the Q binding site is unchanged, and mitochondrial ubiquinone levels are in the wild-type range. Substitution with a +1 charged residue is associated with partial respiratory competence, impaired complex III assembly, and loss of cytochrome b. Although ubiquinone occupancy of the Q binding site is similar to wild-type, total mitochondrial ubiquinone doubled in these mutants. Mutants with a +2 charged substitution at position 62 are unable to respire. These results suggest that the accumulation of ubiquinone in the mitochondria may be a compensatory mechanism for impaired electron transport at cytochrome b

  10. The binding mechanism of a peptidic cyclic serine protease inhibitor

    DEFF Research Database (Denmark)

    Jiang, Longguang; Svane, Anna Sigrid P.; Sørensen, Hans Peter

    2011-01-01

    Serine proteases are classical objects for studies of catalytic and inhibitory mechanisms as well as interesting as therapeutic targets. Since small-molecule serine protease inhibitors generally suffer from specificity problems, peptidic inhibitors, isolated from phage-displayed peptide libraries......, have attracted considerable attention. Here, we have investigated the mechanism of binding of peptidic inhibitors to serine protease targets. Our model is upain-1 (CSWRGLENHRMC), a disulfide-bond-constrained competitive inhibitor of human urokinase-type plasminogen activator with a noncanonical...... inhibitory mechanism and an unusually high specificity. Using a number of modified variants of upain-1, we characterised the upain-1-urokinase-type plasminogen activator complex using X-ray crystal structure analysis, determined a model of the peptide in solution by NMR spectroscopy, and analysed binding...

  11. Type-safe pattern combinators

    DEFF Research Database (Denmark)

    Rhiger, Morten

    2009-01-01

    Macros still haven't made their way into typed higher-order programming languages such as Haskell and Standard ML. Therefore, to extend the expressiveness of Haskell or Standard ML, one must express new linguistic features in terms of functions that fit within the static type systems of these lan...... of these languages. This is particularly challenging when introducing features that span across multiple types and that bind variables. We address this challenge by developing, in a step by step manner, mechanisms for encoding patterns and pattern matching in Haskell in a type-safe way....

  12. Aptamer-Conjugated Calcium Phosphate Nanoparticles for Reducing Diabetes Risk via Retinol Binding Protein 4 Inhibition.

    Science.gov (United States)

    Torabi, Raheleh; Ghourchian, Hedayatollah; Amanlou, Massoud; Pasalar, Parvin

    2017-06-01

    Inhibition of the binding of retinol to its carrier, retinol binding protein 4, is a new strategy for treating type 2 diabetes; for this purpose, we have provided an aptamer-functionalized multishell calcium phosphate nanoparticle. First, calcium phosphate nanoparticles were synthesized and conjugated to the aptamer. The cytotoxicity of nanoparticles releases the process of aptamer from nanoparticles and their inhibition function of binding retinol to retinol binding protein 4. After synthesizing and characterizing the multishell calcium phosphate nanoparticles and observing the noncytotoxicity of conjugate, the optimum time (48 hours) and the pH (7.4) for releasing the aptamer from the nanoparticles was determined. The half-maximum inhibitory concentration (IC 50 ) value for inhibition of retinol binding to retinol binding protein 4 was 210 femtomolar (fmol). The results revealed that the aptamer could prevent connection between retinol and retinol binding protein 4 at a very low IC 50 value (210 fmol) compared to other reported inhibitors. It seems that this aptamer could be used as an efficient candidate not only for decreasing the insulin resistance in type 2 diabetes, but also for inhibiting the other retinol binding protein 4-related diseases. Copyright © 2017 Diabetes Canada. Published by Elsevier Inc. All rights reserved.

  13. Oscillatory mechanisms of process binding in memory.

    Science.gov (United States)

    Klimesch, Wolfgang; Freunberger, Roman; Sauseng, Paul

    2010-06-01

    A central topic in cognitive neuroscience is the question, which processes underlie large scale communication within and between different neural networks. The basic assumption is that oscillatory phase synchronization plays an important role for process binding--the transient linking of different cognitive processes--which may be considered a special type of large scale communication. We investigate this question for memory processes on the basis of different types of oscillatory synchronization mechanisms. The reviewed findings suggest that theta and alpha phase coupling (and phase reorganization) reflect control processes in two large memory systems, a working memory and a complex knowledge system that comprises semantic long-term memory. It is suggested that alpha phase synchronization may be interpreted in terms of processes that coordinate top-down control (a process guided by expectancy to focus on relevant search areas) and access to memory traces (a process leading to the activation of a memory trace). An analogous interpretation is suggested for theta oscillations and the controlled access to episodic memories. Copyright (c) 2009 Elsevier Ltd. All rights reserved.

  14. EPR study of manganese(II) binding to 55'-ATP, hemoglobin, and hemocyanin

    Energy Technology Data Exchange (ETDEWEB)

    Chang, S.S. (Duquesne Univ., Pittsburgh); Li, N.C.; Pratt, D.W.

    1975-01-01

    Several divalent metal ions affect the oxygen affinity of hemoglobin and hemocyanin. It is important, therefore, to understand the nature of metal-ion binding to these proteins. By comparing the EPR spectra of Mn(II), 0.001 M, in the absence and presence of carboxyhemoglobin or Limulus oxyhemocyanin (pH 7.3, Trizma buffer), the number of Mn binding sites, n, and the binding constant, K, can be determined. For carboxyhemoglobin, HbCO, we find 0.5 Mn binding sites per heme, K = 450 M/sup -1/. Each hemoglobin tetramer therefore binds two manganous ions suggesting that Mn(II), like Cu(II), may bind preferentially to one of the two types of subunits in hemoglobin. For hemocyanin, HcO/sub 2/, we find n = 5.8, K = 1.55 x 10/sup 3/ M/sup -1/. Each oxyhemocyanine therefore binds approximately six manganous ions, and the binding constant is three times larger than that for HbCO. We have also carried out similar experiments on 5'-ATP, and on solutions of HbCO and ATP containing McCl/sub 2/ or ZnCl/sub 2/. Zn(II) effectively competes with Mn(II) in binding hemoglobin and ATP, whereas Mg(II) does not, in accord with expectations from data on oxygen affinity of hemoglobin. (auth)

  15. Location and nature of calcium-binding sites in salivary acidic proline-rich phosphoproteins

    International Nuclear Information System (INIS)

    Bennick, A.; McLaughlin, A.C.; Grey, A.A.; Madapallimattam, G.

    1981-01-01

    The location of the calcium-binding sites in the human acidic proline-rich proteins, salivary proteins A and C, was determined by equilibrium dialysis of the tryptic peptides with buffers containing 45 Ca. All the calcium-binding sites are located in the NH 2 -terminal tryptic peptide (TX peptide). The nature of the calcium binding sites in the TX peptide and native salivary proteins A and C, as well as dephosphorylated proteins was compared. Two types of sites can be distinguished in peptide TX. Type I sites have an apparent dissociation constant (K) of 38 μM and are responsible for the binding of 2.6 mol of Ca/mol of peptide. The corresponding figures for Type II sites are 780 μM and 5.3 mol of Ca/mol of peptide. In the native proteins, the amount of calcium bound at the type II sites decreases to 3.9 mol of Ca/mol of proteins A and C and K increases to 1100 μM. The amount of calcium bound at type I sites decreases to 1.5 mol/mol of protein A and 0.6 mol/mol of protein C, but there is no change in K. Dephosphorylation affects the calcium binding at both types of sites. The experiments indicate that the COOH-terminal parts of the native proteins affect the number and the nature of the protein calcium-binding sites. Proton and phosphorous NMR data demonstrate that β-COOH in aspartic acid, as well as phosphoserine, are part of the calcium-binding sites. The difference in calcium binding to salivary proteins A and C may be due at least partially to differences in the environment of one or more aspartic acids

  16. A molecular dynamics investigation of CDK8/CycC and ligand binding: conformational flexibility and implication in drug discovery

    Science.gov (United States)

    Cholko, Timothy; Chen, Wei; Tang, Zhiye; Chang, Chia-en A.

    2018-05-01

    Abnormal activity of cyclin-dependent kinase 8 (CDK8) along with its partner protein cyclin C (CycC) is a common feature of many diseases including colorectal cancer. Using molecular dynamics (MD) simulations, this study determined the dynamics of the CDK8-CycC system and we obtained detailed breakdowns of binding energy contributions for four type-I and five type-II CDK8 inhibitors. We revealed system motions and conformational changes that will affect ligand binding, confirmed the essentialness of CycC for inclusion in future computational studies, and provide guidance in development of CDK8 binders. We employed unbiased all-atom MD simulations for 500 ns on twelve CDK8-CycC systems, including apoproteins and protein-ligand complexes, then performed principal component analysis (PCA) and measured the RMSF of key regions to identify protein dynamics. Binding pocket volume analysis identified conformational changes that accompany ligand binding. Next, H-bond analysis, residue-wise interaction calculations, and MM/PBSA were performed to characterize protein-ligand interactions and find the binding energy. We discovered that CycC is vital for maintaining a proper conformation of CDK8 to facilitate ligand binding and that the system exhibits motion that should be carefully considered in future computational work. Surprisingly, we found that motion of the activation loop did not affect ligand binding. Type-I and type-II ligand binding is driven by van der Waals interactions, but electrostatic energy and entropic penalties affect type-II binding as well. Binding of both ligand types affects protein flexibility. Based on this we provide suggestions for development of tighter-binding CDK8 inhibitors and offer insight that can aid future computational studies.

  17. Blood typing

    Science.gov (United States)

    ... detect these minor antigens. It is done before transfusions, except in emergency situations. Alternative Names Cross matching; Rh typing; ABO blood typing; Blood group; Anemia - immune hemolytic blood type; ...

  18. 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.

  19. Characterization of the Organic Component of Low-Molecular-Weight Chromium-Binding Substance and Its Binding of Chromium123

    Science.gov (United States)

    Chen, Yuan; Watson, Heather M.; Gao, Junjie; Sinha, Sarmistha Halder; Cassady, Carolyn J.; Vincent, John B.

    2011-01-01

    Chromium was proposed to be an essential element over 50 y ago and was shown to have therapeutic potential in treating the symptoms of type 2 diabetes; however, its mechanism of action at a molecular level is unknown. One chromium-binding biomolecule, low-molecular weight chromium-binding substance (LMWCr or chromodulin), has been found to be biologically active in in vitro assays and proposed as a potential candidate for the in vivo biologically active form of chromium. Characterization of the organic component of LMWCr has proven difficult. Treating bovine LMWCr with trifluoroacetic acid followed by purification on a graphite powder micro-column generates a heptapeptide fragment of LMWCr. The peptide sequence of the fragment was analyzed by MS and tandem MS (MS/MS and MS/MS/MS) using collision-induced dissociation and post-source decay. Two candidate sequences, pEEEEGDD and pEEEGEDD (where pE is pyroglutamate), were identified from the MS/MS experiments; additional tandem MS suggests the sequence is pEEEEGDD. The N-terminal glutamate residues explain the inability to sequence LMWCr by the Edman method. Langmuir isotherms and Hill plots were used to analyze the binding constants of chromic ions to synthetic peptides similar in composition to apoLMWCr. The sequence pEEEEGDD was found to bind 4 chromic ions per peptide with nearly identical cooperativity and binding constants to those of apoLMWCr. This work should lead to further studies elucidating or eliminating a potential role for LMWCr in treating the symptoms of type 2 diabetes and other conditions resulting from improper carbohydrate and lipid metabolism. PMID:21593351

  20. N-Acetylgalactosaminyltransferase 14, a novel insulin-like growth factor binding protein-3 binding partner

    International Nuclear Information System (INIS)

    Wu, Chen; Yao, Guangyin; Zou, Minji; Chen, Guangyu; Wang, Min; Liu, Jingqian; Wang, Jiaxi; Xu, Donggang

    2007-01-01

    Insulin-like growth factor binding protein-3 (IGFBP-3) is known to inhibit cell proliferation and induce apoptosis in IGF-dependent and IGF-independent manners, but the mechanism underlying IGF-independent effects is not yet clear. In a yeast two-hybrid assay, IGFBP-3 was used as the bait to screen a human fetal liver cDNA library for it interactors that may potentially mediate IGFBP-3-regulated functions. N-Acetylgalactosaminyltransferase 14 (GalNAc-T14), a member of the GalNAc-Tases family, was identified as a novel IGFBP-3 binding partner. This interaction involved the ricin-type beta-trefoil domain of GalNAc-T14. The interaction between IGFBP-3 and GalNAc-T14 was reconfirmed in vitro and in vivo, using GST pull-down, co-immunoprecipitation and mammalian two-hybrid assays. Our findings may provide new clues for further study on the mechanism behind the IGF-independent effects of IGFBP-3 promoting apoptosis. The role of GalNAc-T14 as an intracellular mediator of the effects of IGFBP-3 need to be verified in future studies

  1. Klebsiella Typing

    DEFF Research Database (Denmark)

    Hansen, D S; Skov, R; Benedí, J.V.

    2002-01-01

    OBJECTIVE: To compare pulsed-field gel electrophoresis (PFGE) typing and O:K-serotyping of Klebsiella in two different epidemiological settings. METHODS: One hundred and four bacteremia isolates without known epidemiological relation and 47 isolates from an outbreak in a neonatal intensive care...... unit (NICU) were K-typed by countercurrent immunoelectrophoresis (CCIE), O-typed by an inhibition enzyme-linked immunosorbent assay method, and typed by pulsed-field gel electrophoresis (PFGE) using the restriction enzyme XbaI. RESULTS: Typing data for the 104 bacteremia isolates were compared...... with regard to typability, number of types, maximum number of isolates per type, and the Discriminative Index (DI). O-typing combined with K-typing (DI 0.98) as O:K-serotyping (DI 0.99) gave a very discriminative typing system, whereas O-typing alone was not very discriminative (DI 0.76). PFGE (DI 1...

  2. Ropizine concurrently enhances and inhibits [3H] dextromethorpan binding to different structures of the guinea pig brain: Autoradiographic evidence for multiple binding sites

    International Nuclear Information System (INIS)

    Canoll, P.D.; Smith, P.R.; and Musacchio, J.M.

    1990-01-01

    Ropizine produces a simultaneous enhancement and inhibition of [ 3 H] dextromethorphan (DM) high-affinity binding to different areas of the guinea pig brain. These results imply that there are two distinct types of high-affinity [ 3 H]DM binding sites, which are present in variable proportions in different brain structures. The ropizine-enhances [ 3 H]DM binding type was preferentially inhibited by (+)-pentazocine. This is consistent with the presumption that the (+)-pentazocine-sensitive site is identical with the common site for DM and 3-(-3-Hydroxphenyl)-N-(1-propyl)piperidine ((+)-3-PPP). The second binding type, which is inhibited by ropizine and is not so sensitive to (+)- pentazocine, has not been fully characterized. This study demonstrates that the biphasic effects to ropizine are due, at least in part, to the effects of ropizine on two different types of [ 3 H]DM binding sites. However, this study does not rule out that the common DM/(+)-3-PPP site also might be inhibited by higher concentrations of ropizine

  3. Structural and binding studies of SAP-1 protein with heparin.

    Science.gov (United States)

    Yadav, Vikash K; Mandal, Rahul S; Puniya, Bhanwar L; Kumar, Rahul; Dey, Sharmistha; Singh, Sarman; Yadav, Savita

    2015-03-01

    SAP-1 is a low molecular weight cysteine protease inhibitor (CPI) which belongs to type-2 cystatins family. SAP-1 protein purified from human seminal plasma (HuSP) has been shown to inhibit cysteine and serine proteases and exhibit interesting biological properties, including high temperature and pH stability. Heparin is a naturally occurring glycosaminoglycan (with varied chain length) which interacts with a number of proteins and regulates multiple steps in different biological processes. As an anticoagulant, heparin enhances inhibition of thrombin by the serpin antithrombin III. Therefore, we have employed surface plasmon resonance (SPR) to improve our understanding of the binding interaction between heparin and SAP-1 (protease inhibitor). SPR data suggest that SAP-1 binds to heparin with a significant affinity (KD = 158 nm). SPR solution competition studies using heparin oligosaccharides showed that the binding of SAP-1 to heparin is dependent on chain length. Large oligosaccharides show strong binding affinity for SAP-1. Further to get insight into the structural aspect of interactions between SAP-1 and heparin, we used modelled structure of the SAP-1 and docked with heparin and heparin-derived polysaccharides. The results suggest that a positively charged residue lysine plays important role in these interactions. Such information should improve our understanding of how heparin, present in the reproductive tract, regulates cystatins activity. © 2014 John Wiley & Sons A/S.

  4. Quantitative autoradiography of [3H]ouabain binding sites in rat brain

    International Nuclear Information System (INIS)

    Spyropoulos, A.C.; Rainbow, T.C.

    1984-01-01

    In vitro quantitative autoradiography was used to localize in rat brain binding sites for [ 3 H]ouabain, an inhibitor of the Na + ,K + -ATPase. High levels of [ 3 H]ouabain sites were found in the superior and inferior colliculi, the mammillary nucleus, the interpeduncular nucleus, and in various divisions of the olfactory, auditory and somatomotor systems. The heterogeneous distribution of [ 3 H]ouabain binding closely parallels the regional brain glucose consumption as determined by the [ 14 C]deoxyglucose method. Lesion studies of the rat hippocampus using the excitotoxin, ibotenic acid, showed both a marked decrease of neuronal cell types on the injected side and a corresponding decrease in [ 3 H]ouabain binding, indicating that some of the [ 3 H]ouabain binding sites are localized to neurons. The close correlation between [ 3 H]ouabain binding and regional glucose utilization provides further evidence for a linkage between glucose utilization and the neuronal Na + ,K + -ATPase. (Auth.)

  5. 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....... This method of detection was used to determine the distribution of SHBG phenotypes in healthy controls of both sexes and in five different pathological conditions characterized by changes in the SHBG level or endocrine disturbances (malignant and benign ovarian neoplasms, hirsutism, liver cirrhosis...... on the experimental values. Differences in SHBG phenotypes do not appear to have any clinical significance and no sex difference was found in the SHBG phenotype distribution....

  6. DNA Binding Hydroxyl Radical Probes.

    Science.gov (United States)

    Tang, Vicky J; Konigsfeld, Katie M; Aguilera, Joe A; Milligan, Jamie R

    2012-01-01

    The hydroxyl radical is the primary mediator of DNA damage by the indirect effect of ionizing radiation. It is a powerful oxidizing agent produced by the radiolysis of water and is responsible for a significant fraction of the DNA damage associated with ionizing radiation. There is therefore an interest in the development of sensitive assays for its detection. The hydroxylation of aromatic groups to produce fluorescent products has been used for this purpose. We have examined four different chromophores which produce fluorescent products when hydroxylated. Of these, the coumarin system suffers from the fewest disadvantages. We have therefore examined its behavior when linked to a cationic peptide ligand designed to bind strongly to DNA.

  7. Asymmetric cation-binding catalysis

    DEFF Research Database (Denmark)

    Oliveira, Maria Teresa; Lee, Jiwoong

    2017-01-01

    The employment of metal salts is quite limited in asymmetric catalysis, although it would provide an additional arsenal of safe and inexpensive reagents to create molecular functions with high optical purity. Cation chelation by polyethers increases the salts' solubility in conventional organic...... solvents, thus increasing their applicability in synthesis. The expansion of this concept to chiral polyethers led to the emergence of asymmetric cation-binding catalysis, where chiral counter anions are generated from metal salts, particularly using BINOL-based polyethers. Alkali metal salts, namely KF...... highly enantioselective silylation reactions in polyether-generated chiral environments, and leading to a record-high turnover in asymmetric organocatalysis. This can lead to further applications by the asymmetric use of other inorganic salts in various organic transformations....

  8. Lectin binding profiles of SSEA-4 enriched, pluripotent human embryonic stem cell surfaces

    Science.gov (United States)

    Venable, Alison; Mitalipova, Maisam; Lyons, Ian; Jones, Karen; Shin, Soojung; Pierce, Michael; Stice, Steven

    2005-01-01

    Background Pluripotent human embryonic stem cells (hESCs) have the potential to form every cell type in the body. These cells must be appropriately characterized prior to differentiation studies or when defining characteristics of the pluripotent state. Some developmentally regulated cell surface antigens identified by monoclonal antibodies in a variety of species and stem cell types have proven to be side chains of membrane glycolipids and glycoproteins. Therefore, to examine hESC surfaces for other potential pluripotent markers, we used a panel of 14 lectins, which were chosen based on their specificity for a variety of carbohydrates and carbohydrate linkages, along with stage specific embryonic antigen-4 (SSEA-4), to determine binding quantitation by flow cytometry and binding localization in adherent colonies by immunocytochemistry. Results Enriching cells for SSEA-4 expression increased the percentage of SSEA-4 positive cells to 98–99%. Using enriched high SSEA-4-expressing hESCs, we then analyzed the binding percentages of selected lectins and found a large variation in binding percentages ranging from 4% to 99% binding. Lycopersicon (tomato)esculetum lectin (TL), Ricinus communis agglutinin (RCA), and Concanavalin A (Con A) bound to SSEA-4 positive regions of hESCs and with similar binding percentages as SSEA-4. In contrast, we found Dolichos biflorus agglutinin (DBA) and Lotus tetragonolobus lectin (LTL) did not bind to hESCs while Phaseolus vulgaris leuco-agglutinin (PHA-L), Vicia villosa agglutinin (VVA), Ulex europaeus agglutinin (UEA), Phaseolus vulgaris erythro-agglutinin (PHA-E), and Maackia amurensis agglutinin (MAA) bound partially to hESCs. These binding percentages correlated well with immunocytochemistry results. Conclusion Our results provide information about types of carbohydrates and carbohydrate linkages found on pluripotent hESC surfaces. We propose that TL, RCA and Con A may be used as markers that are associated with the pluripotent

  9. Lectin binding profiles of SSEA-4 enriched, pluripotent human embryonic stem cell surfaces

    Directory of Open Access Journals (Sweden)

    Shin Soojung

    2005-07-01

    Full Text Available Abstract Background Pluripotent human embryonic stem cells (hESCs have the potential to form every cell type in the body. These cells must be appropriately characterized prior to differentiation studies or when defining characteristics of the pluripotent state. Some developmentally regulated cell surface antigens identified by monoclonal antibodies in a variety of species and stem cell types have proven to be side chains of membrane glycolipids and glycoproteins. Therefore, to examine hESC surfaces for other potential pluripotent markers, we used a panel of 14 lectins, which were chosen based on their specificity for a variety of carbohydrates and carbohydrate linkages, along with stage specific embryonic antigen-4 (SSEA-4, to determine binding quantitation by flow cytometry and binding localization in adherent colonies by immunocytochemistry. Results Enriching cells for SSEA-4 expression increased the percentage of SSEA-4 positive cells to 98–99%. Using enriched high SSEA-4-expressing hESCs, we then analyzed the binding percentages of selected lectins and found a large variation in binding percentages ranging from 4% to 99% binding. Lycopersicon (tomatoesculetum lectin (TL, Ricinus communis agglutinin (RCA, and Concanavalin A (Con A bound to SSEA-4 positive regions of hESCs and with similar binding percentages as SSEA-4. In contrast, we found Dolichos biflorus agglutinin (DBA and Lotus tetragonolobus lectin (LTL did not bind to hESCs while Phaseolus vulgaris leuco-agglutinin (PHA-L, Vicia villosa agglutinin (VVA, Ulex europaeus agglutinin (UEA, Phaseolus vulgaris erythro-agglutinin (PHA-E, and Maackia amurensis agglutinin (MAA bound partially to hESCs. These binding percentages correlated well with immunocytochemistry results. Conclusion Our results provide information about types of carbohydrates and carbohydrate linkages found on pluripotent hESC surfaces. We propose that TL, RCA and Con A may be used as markers that are associated with the

  10. The helical structure of DNA facilitates binding

    International Nuclear Information System (INIS)

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

    2016-01-01

    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. (paper)

  11. Involvement of two classes of binding sites in the interactions of cyclophilin B with peripheral blood T-lymphocytes.

    Science.gov (United States)

    Denys, A; Allain, F; Carpentier, M; Spik, G

    1998-12-15

    Cyclophilin B (CyPB) is a cyclosporin A (CsA)-binding protein, mainly associated with the secretory pathway, and is released in biological fluids. We recently reported that CyPB specifically binds to T-lymphocytes and promotes enhanced incorporation of CsA. The interactions with cellular binding sites involved, at least in part, the specific N-terminal extension of the protein. In this study, we intended to specify further the nature of the CyPB-binding sites on peripheral blood T-lymphocytes. We first provide evidence that the CyPB binding to heparin-Sepharose is prevented by soluble sulphated glycosaminoglycans (GAG), raising the interesting possibility that such interactions may occur on the T-cell surface. We then characterized CyPB binding to T-cell surface GAG and found that these interactions involved the N-terminal extension of CyPB, but not its conserved CsA-binding domain. In addition, we determined the presence of a second CyPB binding site, which we termed a type I site, in contrast with type II for GAG interactions. The two binding sites exhibit a similar affinity but the expression of the type I site was 3-fold lower. The conclusion that CyPB binding to the type I site is distinct from the interactions with GAG was based on the findings that it was (1) resistant to NaCl wash and GAG-degrading enzyme treatments, (2) reduced in the presence of CsA or cyclophilin C, and (3) unmodified in the presence of either the N-terminal peptide of CyPB or protamine. Finally, we showed that the type I binding sites were involved in an endocytosis process, supporting the hypothesis that they may correspond to a functional receptor for CyPB.

  12. Flow-cytometric determination of high-density-lipoprotein binding sites on human leukocytes

    International Nuclear Information System (INIS)

    Schmitz, G.; Wulf, G.; Bruening, T.A.; Assmann, G.

    1987-01-01

    In this method, leukocytes were isolated from 6 mL of EDTA-blood by density-gradient centrifugation and subsequently incubated with rhodamine isothiocyanate (RITC)-conjugated high-density lipoproteins (HDL). The receptor-bound conjugate particles were determined by fluorescent flow cytometry and compared with 125 I-labeled HDL binding data for the same cells. Human granulocytes express the highest number of HDL binding sites (9.4 x 10(4)/cell), followed by monocytes (7.3 x 10(4)/cell) and lymphocytes (4.0 x 10(4)/cell). Compared with conventional analysis of binding of 125 I-labeled HDL in tissue-culture dishes, the present determination revealed significantly lower values for nonspecific binding. In competition studies, the conjugate competes for the same binding sites as 125 I-labeled HDL. With the use of tetranitromethane-treated HDL3, which fails to compete for the HDL receptor sites while nonspecific binding is not affected, we could clearly distinguish between 37 degrees C surface binding and specific 37 degrees C uptake of RITC-HDL3, confirming that the HDL receptor leads bound HDL particles into an intracellular pathway rather than acting as a docking type of receptor. Patients with familial dysbetalipoproteinemia showed a significantly higher number of HDL binding sites in the granulocyte population but normal in lymphocytes and monocytes, indicating increased uptake of cholesterol-containing lipoproteins. In patients with familial hypercholesterolemia, HDL binding was increased in all three cell types, indicating increased cholesterol uptake and increased cholesterol synthesis. The present method allows rapid determination of HDL binding sites in leukocytes from patients with various forms of hyper- and dyslipoproteinemias

  13. Lead-Binding Proteins: A Review

    Directory of Open Access Journals (Sweden)

    Harvey C. Gonick

    2011-01-01

    Full Text Available Lead-binding proteins are a series of low molecular weight proteins, analogous to metallothionein, which segregate lead in a nontoxic form in several organs (kidney, brain, lung, liver, erythrocyte. Whether the lead-binding proteins in every organ are identical or different remains to be determined. In the erythrocyte, delta-aminolevulinic acid dehydratase (ALAD isoforms have commanded the greatest attention as proteins and enzymes that are both inhibitable and inducible by lead. ALAD-2, although it binds lead to a greater degree than ALAD-1, appears to bind lead in a less toxic form. What may be of greater significance is that a low molecular weight lead-binding protein, approximately 10 kDa, appears in the erythrocyte once blood lead exceeds 39 μg/dL and eventually surpasses the lead-binding capacity of ALAD. In brain and kidney of environmentally exposed humans and animals, a cytoplasmic lead-binding protein has been identified as thymosin β4, a 5 kDa protein. In kidney, but not brain, another lead-binding protein has been identified as acyl-CoA binding protein, a 9 kDa protein. Each of these proteins, when coincubated with liver ALAD and titrated with lead, diminishes the inhibition of ALAD by lead, verifying their ability to segregate lead in a nontoxic form.

  14. Drug binding properties of neonatal albumin

    DEFF Research Database (Denmark)

    Brodersen, R; Honoré, B

    1989-01-01

    Neonatal and adult albumin was isolated by gel chromatography on Sephacryl S-300, from adult and umbilical cord serum, respectively. Binding of monoacetyl-diamino-diphenyl sulfone, warfarin, sulfamethizole, and diazepam was studied by means of equilibrium dialysis and the binding data were analyzed...... by the method of several acceptable fitted curves. It was found that the binding affinity to neonatal albumin is less than to adult albumin for monoacetyl-diamino-diphenyl sulfone and warfarin. Sulfamethizole binding to the neonatal protein is similarly reduced when more than one molecule of the drug is bound...

  15. Retinoid-binding proteins: similar protein architectures bind similar ligands via completely different ways.

    Directory of Open Access Journals (Sweden)

    Yu-Ru Zhang

    Full Text Available BACKGROUND: Retinoids are a class of compounds that are chemically related to vitamin A, which is an essential nutrient that plays a key role in vision, cell growth and differentiation. In vivo, retinoids must bind with specific proteins to perform their necessary functions. Plasma retinol-binding protein (RBP and epididymal retinoic acid binding protein (ERABP carry retinoids in bodily fluids, while cellular retinol-binding proteins (CRBPs and cellular retinoic acid-binding proteins (CRABPs carry retinoids within cells. Interestingly, although all of these transport proteins possess similar structures, the modes of binding for the different retinoid ligands with their carrier proteins are different. METHODOLOGY/PRINCIPAL FINDINGS: In this work, we analyzed the various retinoid transport mechanisms using structure and sequence comparisons, binding site analyses and molecular dynamics simulations. Our results show that in the same family of proteins and subcellular location, the orientation of a retinoid molecule within a binding protein is same, whereas when different families of proteins are considered, the orientation of the bound retinoid is completely different. In addition, none of the amino acid residues involved in ligand binding is conserved between the transport proteins. However, for each specific binding protein, the amino acids involved in the ligand binding are conserved. The results of this study allow us to propose a possible transport model for retinoids. CONCLUSIONS/SIGNIFICANCE: Our results reveal the differences in the binding modes between the different retinoid-binding proteins.

  16. Site-directed alkylation of multiple opioid receptors. I. Binding selectivity

    International Nuclear Information System (INIS)

    James, I.F.; Goldstein, A.

    1984-01-01

    A method for measuring and expressing the binding selectivity of ligands for mu, delta, and kappa opioid binding sites is reported. Radioligands are used that are partially selective for these sites in combination with membrane preparations enriched in each site. Enrichment was obtained by treatment of membranes with the alkylating agent beta-chlornaltrexamine in the presence of appropriate protecting ligands. After enrichment for mu receptors, [ 3 H] dihydromorphine bound to a single type of site as judged by the slope of competition binding curves. After enrichment for delta or kappa receptors, binding sites for [ 3 H] [D-Ala2, D-Leu5]enkephalin and [3H]ethylketocyclazocine, respectively, were still not homogeneous. There were residual mu sites in delta-enriched membranes but no evidence for residual mu or delta sites in kappa-enriched membranes were found. This method was used to identify ligands that are highly selective for each of the three types of sites

  17. A tool for calculating binding-site residues on proteins from PDB structures

    Directory of Open Access Journals (Sweden)

    Hu Jing

    2009-08-01

    Full Text Available Abstract Background In the research on protein functional sites, researchers often need to identify binding-site residues on a protein. A commonly used strategy is to find a complex structure from the Protein Data Bank (PDB that consists of the protein of interest and its interacting partner(s and calculate binding-site residues based on the complex structure. However, since a protein may participate in multiple interactions, the binding-site residues calculated based on one complex structure usually do not reveal all binding sites on a protein. Thus, this requires researchers to find all PDB complexes that contain the protein of interest and combine the binding-site information gleaned from them. This process is very time-consuming. Especially, combing binding-site information obtained from different PDB structures requires tedious work to align protein sequences. The process becomes overwhelmingly difficult when researchers have a large set of proteins to analyze, which is usually the case in practice. Results In this study, we have developed a tool for calculating binding-site residues on proteins, TCBRP http://yanbioinformatics.cs.usu.edu:8080/ppbindingsubmit. For an input protein, TCBRP can quickly find all binding-site residues on the protein by automatically combining the information obtained from all PDB structures that consist of the protein of interest. Additionally, TCBRP presents the binding-site residues in different categories according to the interaction type. TCBRP also allows researchers to set the definition of binding-site residues. Conclusion The developed tool is very useful for the research on protein binding site analysis and prediction.

  18. FRET-based binding assay between a fluorescent cAMP analogue and a cyclic nucleotide-binding domain tagged with a CFP.

    Science.gov (United States)

    Romero, Francisco; Santana-Calvo, Carmen; Sánchez-Guevara, Yoloxochitl; Nishigaki, Takuya

    2017-09-01

    The cyclic nucleotide-binding domain (CNBD) functions as a regulatory domain of many proteins involved in cyclic nucleotide signalling. We developed a straightforward and reliable binding assay based on intermolecular fluorescence resonance energy transfer (FRET) between an adenosine-3', 5'-cyclic monophosphate analogue labelled with fluorescein and a recombinant CNBD of human EPAC1 tagged with a cyan fluorescence protein (CFP). The high FRET efficiency of this method (~ 80%) allowed us to perform several types of binding experiments with nanomolar range of sample using conventional equipment. In addition, the CFP tag on the CNBD enabled us to perform a specific binding experiment using an unpurified protein. Considering these advantages, this technique is useful to study poorly characterized CNBDs. © 2017 Federation of European Biochemical Societies.

  19. Serum levels of mannan-binding lectin in chickens prior to and during experimental infection with avian infectious bronchitis virus

    DEFF Research Database (Denmark)

    Juul-Madsen, H.R.; Munch, M.; Handberg, Kurt

    2003-01-01

    Mannan-binding lectin (MBL) is a glycoprotein and a member of the C-type lectin super family, the collectin family, and the acute phase protein family. The MBL exerts its function by directly binding to microbial surfaces through its carbohydrate recognition domains, followed by direct opsonizati...

  20. Caveolin-1-mediated apolipoprotein A-I membrane binding sites are not required for cholesterol efflux.

    Directory of Open Access Journals (Sweden)

    Soazig Le Lay

    Full Text Available Caveolin-1 (Cav1, a structural protein required for the formation of invaginated membrane domains known as caveolae, has been implicated in cholesterol trafficking and homeostasis. Here we investigated the contribution of Cav1 to apolipoprotein A-I (apoA-I cell surface binding and intracellular processing using mouse embryonic fibroblasts (MEFs derived from wild type (WT or Cav1-deficient (Cav1(-/- animals. We found that cells expressing Cav1 have 2.6-fold more apoA-I binding sites than Cav1(-/- cells although these additional binding sites are not associated with detergent-free lipid rafts. Further, Cav1-mediated binding targets apoA-I for internalization and degradation and these processes are not correlated to cholesterol efflux. Despite lower apoA-I binding, cholesterol efflux from Cav1(-/- MEFs is 1.7-fold higher than from WT MEFs. Stimulation of ABCA1 expression with an LXR agonist enhances cholesterol efflux from both WT and Cav1(-/- cells without increasing apoA-I surface binding or affecting apoA-I processing. Our results indicate that there are at least two independent lipid binding sites for apoA-I; Cav1-mediated apoA-I surface binding and uptake is not linked to cholesterol efflux, indicating that membrane domains other than caveolae regulate ABCA1-mediated cholesterol efflux.

  1. Bilirubin Binding to PPARα Inhibits Lipid Accumulation

    Science.gov (United States)

    Stec, David E.; John, Kezia; Trabbic, Christopher J.; Luniwal, Amarjit; Hankins, Michael W.; Baum, Justin

    2016-01-01

    Numerous clinical and population studies have demonstrated that increased serum bilirubin levels protect against cardiovascular and metabolic diseases such as obesity and diabetes. Bilirubin is a potent antioxidant, and the beneficial actions of moderate increases in plasma bilirubin have been thought to be due to the antioxidant effects of this bile pigment. In the present study, we found that bilirubin has a new function as a ligand for PPARα. We show that bilirubin can bind directly to PPARα and increase transcriptional activity. When we compared biliverdin, the precursor to bilirubin, on PPARα transcriptional activation to known PPARα ligands, WY 14,643 and fenofibrate, it showed that fenofibrate and biliverdin have similar activation properties. Treatment of 3T3-L1 adipocytes with biliverdin suppressed lipid accumulation and upregulated PPARα target genes. We treated wild-type and PPARα KO mice on a high fat diet with fenofibrate or bilirubin for seven days and found that both signal through PPARα dependent mechanisms. Furthermore, the effect of bilirubin on lowering glucose and reducing body fat percentage was blunted in PPARα KO mice. These data demonstrate a new function for bilirubin as an agonist of PPARα, which mediates the protection from adiposity afforded by moderate increases in bilirubin. PMID:27071062

  2. Analysis of electric moments of RNA-binding proteins: implications for mechanism and prediction

    Directory of Open Access Journals (Sweden)

    Sarai Akinori

    2011-02-01

    Full Text Available Abstract Background Protein-RNA interactions play important role in many biological processes such as gene regulation, replication, protein synthesis and virus assembly. Although many structures of various types of protein-RNA complexes have been determined, the mechanism of protein-RNA recognition remains elusive. We have earlier shown that the simplest electrostatic properties viz. charge, dipole and quadrupole moments, calculated from backbone atomic coordinates of proteins are biased relative to other proteins, and these quantities can be used to identify DNA-binding proteins. Closely related, RNA-binding proteins are investigated in this study. In particular, discrimination between various types of RNA-binding proteins, evolutionary conservation of these bulk electrostatic features and effect of conformational changes by complex formation are investigated. Basic binding mechanism of a putative RNA-binding protein (HI1333 from Haemophilus influenza is suggested as a potential application of this study. Results We found that similar to DNA-binding proteins (DBPs, RNA-binding proteins (RBPs also show significantly higher values of electric moments. However, higher moments in RBPs are found to strongly depend on their functional class: proteins binding to ribosomal RNA (rRNA constitute the only class with all three of the properties (charge, dipole and quadrupole moments being higher than control proteins. Neural networks were trained using leave-one-out cr