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Sample records for c-myb binds mll

  1. c-Myb and GATA-3 cooperatively regulate IL-13 expression via CGRE and recruit MLL for histone modification of the IL-13 locus

    OpenAIRE

    Kozuka, Teruhiko; Sugita, Mayumi; Shetzline, Susan; Gewirtz, Alan M.; Nakata, Yuji

    2011-01-01

    The c-Myb and GATA-3 transcription factors play important roles in T-cell development. We recently reported that c-Myb, GATA-3, and Menin form a core transcription complex that regulates GATA-3 expression and ultimately Th2 cell development in human peripheral blood T-cells. However, c-Myb roles for Th2 cytokine expression were not demonstrated. Here, we report that c-Myb and GATA-3 cooperatively play an essential role in IL-13 expression though direct binding to a conserved GATA-3 response e...

  2. Regulation of FeLV-945 by c-Myb binding and CBP recruitment to the LTR

    Directory of Open Access Journals (Sweden)

    Finstad Samantha L

    2004-09-01

    Full Text Available Abstract Background Feline leukemia virus (FeLV induces degenerative, proliferative and malignant hematologic disorders in its natural host, the domestic cat. FeLV-945 is a viral variant identified as predominant in a cohort of naturally infected animals. FeLV-945 contains a unique sequence motif in the long terminal repeat (LTR comprised of a single copy of transcriptional enhancer followed by a 21-bp sequence triplicated in tandem. The LTR is precisely conserved among independent cases of multicentric lymphoma, myeloproliferative disease and anemia in animals from the cohort. The 21-bp triplication was previously shown to act as a transcriptional enhancer preferentially in hematopoietic cells and to confer a replicative advantage. The objective of the present study was to examine the molecular mechanism by which the 21-bp triplication exerts its influence and the selective advantage responsible for its precise conservation. Results Potential binding sites for the transcription factor, c-Myb, were identified across the repeat junctions of the 21-bp triplication. Such sites would not occur in the absence of the repeat; thus, a requirement for c-Myb binding to the repeat junctions of the triplication would exert a selective pressure to conserve its sequence precisely. Electrophoretic mobility shift assays demonstrated specific binding of c-Myb to the 21-bp triplication. Reporter gene assays showed that the triplication-containing LTR is responsive to c-Myb, and that responsiveness requires the presence of both c-Myb binding sites. Results further indicated that c-Myb in complex with the 21-bp triplication recruits the transcriptional co-activator, CBP, a regulator of normal hematopoiesis. FeLV-945 replication was shown to be positively regulated by CBP in a manner dependent on the presence of the 21-bp triplication. Conclusion Binding sites for c-Myb across the repeat junctions of the 21-bp triplication may account for its precise conservation in

  3. The DNA binding domain of c-Myb: over-expression and NMR characterization

    International Nuclear Information System (INIS)

    The DNA-binding domain of the Drosophila melanogaster c-Myb protein, 160 residues long, containing three conserved imperfect repeats of nearly 50 residues each has been over expressed in E. coli strain BL21(DE3). The protein is expressed to the extent of at least 20% of the total cellular protein. It has been purified by a four-step protocol developed in the laboratory. The protein has been characterized by various 2D NMR experiments. Several specific amino acids have been identified. A three-dimensional NMR spectrum has been recorded to achieve dispersion of cross-peaks into different 2D planes. (author). 19 refs., 6 figs., 1 tab

  4. GATA-1 and c-myb crosstalk during red blood cell differentiation through GATA-1 binding sites in the c-myb promoter

    Czech Academy of Sciences Publication Activity Database

    Bartůněk, Petr; Králová, Jarmila; Blendiger, G.; Dvořák, Michal; Zenke, M.

    2003-01-01

    Roč. 22, č. 13 (2003), s. 1927-1935. ISSN 0950-9232 R&D Projects: GA ČR GV301/98/K042 Institutional research plan: CEZ:AV0Z5052915 Keywords : GATA-1 * c-myb * erythropoiesis Subject RIV: EB - Genetics ; Molecular Biology Impact factor: 6.495, year: 2003

  5. c-Myb, Menin, GATA-3, and MLL form a dynamic transcription complex that plays a pivotal role in human T helper type 2 cell development

    OpenAIRE

    Nakata, Yuji; Brignier, Anne C.; Jin, Shenghao; Shen, Yuan; Rudnick, Stephen I.; Sugita, Mayumi; Gewirtz, Alan M.

    2010-01-01

    GATA-3 and c-Myb are core elements of a transcriptionally active complex essential for human Th2 cell development and maintenance. We report herein mechanistic details concerning the role of these transcription factors in human peripheral blood Th2 cell development. Silencing c-Myb in normal human naive CD4+ cells under Th2 cell-promoting conditions blocked up-regulation of GATA-3 and interleukin-4, and in effector/memory CD4+ T cells, decreased expression of GATA-3 and Th2 cytokines. In prim...

  6. c-myb stimulates cell growth by regulation of insulin-like growth factor (IGF) and IGF-binding protein-3 in K562 leukemia cells

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    Kim, Min-Sun; Kim, Sun-Young; Arunachalam, Sankarganesh [Department of Pediatrics, School of Medicine, Chonbuk National University, Jeonju 561-712 (Korea, Republic of); Hwang, Pyoung-Han [Department of Pediatrics, School of Medicine, Chonbuk National University, Jeonju 561-712 (Korea, Republic of); Research Institute of Clinical Medicine, School of Medicine, Chonbuk National University, Jeonju 561-712 (Korea, Republic of); Yi, Ho-Keun [Department of Biochemistry, School of Dentistry, Chonbuk National University, Jeonju 561-712 (Korea, Republic of); Nam, Sang-Yun [Department of Alternative Therapy, School of Alternative Medicine and Health Science, Jeonju University, Jeonju 561-712 (Korea, Republic of); Lee, Dae-Yeol, E-mail: leedy@chonbuk.ac.kr [Department of Pediatrics, School of Medicine, Chonbuk National University, Jeonju 561-712 (Korea, Republic of); Research Institute of Clinical Medicine, School of Medicine, Chonbuk National University, Jeonju 561-712 (Korea, Republic of)

    2009-07-17

    c-myb plays an important role in the regulation of cell growth and differentiation, and is highly expressed in immature hematopoietic cells. The human chronic myelogenous leukemia cell K562, highly expresses IGF-I, IGF-II, IGF-IR, and IGF-induced cellular proliferation is mediated by IGF-IR. To characterize the impact of c-myb on the IGF-IGFBP-3 axis in leukemia cells, we overexpressed c-myb using an adenovirus gene transfer system in K562 cells. The overexpression of c-myb induced cell proliferation, compared to control, and c-myb induced cell growth was inhibited by anti-IGF-IR antibodies. c-myb overexpression resulted in a significant increase in the expression of IGF-I, IGF-II, and IGF-IR, and a decrease in IGFBP-3 expression. By contrast, disruption of c-myb function by DN-myb overexpression resulted in significant reduction of IGF-I, IGF-II, IGF-IR, and elevation of IGFBP-3 expression. In addition, exogenous IGFBP-3 inhibited the proliferation of K562 cells, and c-myb induced cell growth was blocked by IGFBP-3 overexpression in a dose-dependent manner. The growth-promoting effects of c-myb were mediated through two major intracellular signaling pathways, Akt and Erk. Activation of Akt and Erk by c-myb was completely blocked by IGF-IR and IGFBP-3 antibodies. These findings suggest that c-myb stimulates cell growth, in part, by regulating expression of the components of IGF-IGFBP axis in K562 cells. In addition, disruption of c-myb function by DN-myb may provide a useful strategy for treatment of leukemia.

  7. c-myb stimulates cell growth by regulation of insulin-like growth factor (IGF) and IGF-binding protein-3 in K562 leukemia cells

    International Nuclear Information System (INIS)

    c-myb plays an important role in the regulation of cell growth and differentiation, and is highly expressed in immature hematopoietic cells. The human chronic myelogenous leukemia cell K562, highly expresses IGF-I, IGF-II, IGF-IR, and IGF-induced cellular proliferation is mediated by IGF-IR. To characterize the impact of c-myb on the IGF-IGFBP-3 axis in leukemia cells, we overexpressed c-myb using an adenovirus gene transfer system in K562 cells. The overexpression of c-myb induced cell proliferation, compared to control, and c-myb induced cell growth was inhibited by anti-IGF-IR antibodies. c-myb overexpression resulted in a significant increase in the expression of IGF-I, IGF-II, and IGF-IR, and a decrease in IGFBP-3 expression. By contrast, disruption of c-myb function by DN-myb overexpression resulted in significant reduction of IGF-I, IGF-II, IGF-IR, and elevation of IGFBP-3 expression. In addition, exogenous IGFBP-3 inhibited the proliferation of K562 cells, and c-myb induced cell growth was blocked by IGFBP-3 overexpression in a dose-dependent manner. The growth-promoting effects of c-myb were mediated through two major intracellular signaling pathways, Akt and Erk. Activation of Akt and Erk by c-myb was completely blocked by IGF-IR and IGFBP-3 antibodies. These findings suggest that c-myb stimulates cell growth, in part, by regulating expression of the components of IGF-IGFBP axis in K562 cells. In addition, disruption of c-myb function by DN-myb may provide a useful strategy for treatment of leukemia.

  8. PIAS1 binds p300 and behaves as a coactivator or corepressor of the transcription factor c-Myb dependent on SUMO-status.

    Science.gov (United States)

    Ledsaak, Marit; Bengtsen, Mads; Molværsmyr, Ann-Kristin; Fuglerud, Bettina Maria; Matre, Vilborg; Eskeland, Ragnhild; Gabrielsen, Odd Stokke

    2016-05-01

    The PIAS proteins (Protein Inhibitor of Activated STATs) constitute a family of multifunctional nuclear proteins operating as SUMO E3 ligases and being involved in a multitude of interactions. They participate in a range of biological processes, also beyond their well-established role in the immune system and cytokine signalling. They act both as transcriptional corepressors and coactivators depending on the context. In the present work, we investigated mechanisms by which PIAS1 causes activation or repression of c-Myb dependent target genes. Analysis of global expression data shows that c-Myb and PIAS1 knockdowns affect a subset of common targets, but with a dual outcome consistent with a role of PIAS1 as either a corepressor or coactivator. Our mechanistic studies show that PIAS1 engages in a novel interaction with the acetyltransferase and coactivator p300. Interaction and ChIP analysis suggest a bridging function where PIAS1 enhances p300 recruitment to c-Myb-bound sites through interaction with both proteins. In addition, the E3 activity of PIAS1 enhances further its coactivation. Remarkably, the SUMO status of c-Myb had a decisive role, indicating a SUMO-dependent switch in the way PIAS1 affects c-Myb, either as a coactivator or corepressor. Removal of the two major SUMO-conjugation sites in c-Myb (2KR mutant), which enhances its activity significantly, turned PIAS1 into a corepressor. Also, p300 was less efficiently recruited to chromatin by c-Myb-2KR. We propose that PIAS1 acts as a "protein inhibitor of activated c-Myb" in the absence of SUMOylation while, in its presence, PIAS behaves as a "protein activator of repressed c-Myb". PMID:27032383

  9. Fbxw5 suppresses nuclear c-Myb activity via DDB1-Cul4-Rbx1 ligase-mediated sumoylation

    Energy Technology Data Exchange (ETDEWEB)

    Kanei-Ishii, Chie; Nomura, Teruaki; Egoh, Ayako [Laboratory of Molecular Genetics, RIKEN Tsukuba Institute, 3-1-1 Koyadai, Tsukuba, Ibaraki 305-0074 (Japan); Ishii, Shunsuke, E-mail: sishii@rtc.riken.jp [Laboratory of Molecular Genetics, RIKEN Tsukuba Institute, 3-1-1 Koyadai, Tsukuba, Ibaraki 305-0074 (Japan)

    2012-09-14

    Highlights: Black-Right-Pointing-Pointer Fbxw5 enhances sumoylation of c-Myb. Black-Right-Pointing-Pointer The DDB1-Cul4A-Rbx1 complex mediates c-Myb sumoylation. Black-Right-Pointing-Pointer The Fbxw5-DDB1-Cul4A-Rdx1 complex is a dual SUMO/ubiquitin ligase. Black-Right-Pointing-Pointer Fbxw5 suppresses the c-Myb trans-activating capacity. -- Abstract: The c-myb proto-oncogene product (c-Myb) is degraded in response to Wnt-1 signaling. In this process, Fbxw7{alpha}, the F-box protein of the SCF complex, binds to c-Myb via its C-terminal WD40 domain, and induces the ubiquitination of c-Myb. Here, we report that Fbxw5, another F-box protein, enhances sumoylation of nuclear c-Myb. Fbxw5 enhanced c-Myb sumoylation via the DDB1-Cul4A-Rbx1 complex. Since the Fbxw5-DDB1-Cul4A-Rbx1 complex was shown to act as a ubiquitin ligase for tumor suppressor TSC2, our results suggest that this complex can function as a dual SUMO/ubiquitin ligase. Fbxw5, which is localized to both nucleus and cytosol, enhanced sumoylation of nuclear c-Myb and induced the localization of c-Myb to nuclear dot-like domains. Co-expression of Fbxw5 suppressed the trans-activation of c-myc promoter by wild-type c-Myb, but not by v-Myb, which lacks the sumoylation sites. These results suggest that multiple E3 ligases suppress c-Myb activity through sumoylation or ubiquitination, and that v-Myb is no longer subject to these negative regulations.

  10. The same pocket in menin binds both MLL and JUND but has opposite effects on transcription

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    Huang, Jing; Gurung, Buddha; Wan, Bingbing; Matkar, Smita; Veniaminova, Natalia A.; Wan, Ke; Merchant, Juanita L.; Hua, Xianxin; Lei, Ming (Michigan); (Michigan-Med); (UPENN-MED)

    2013-04-08

    Menin is a tumour suppressor protein whose loss or inactivation causes multiple endocrine neoplasia 1 (MEN1), a hereditary autosomal dominant tumour syndrome that is characterized by tumorigenesis in multiple endocrine organs. Menin interacts with many proteins and is involved in a variety of cellular processes. Menin binds the JUN family transcription factor JUND and inhibits its transcriptional activity. Several MEN1 missense mutations disrupt the menin-JUND interaction, suggesting a correlation between the tumour-suppressor function of menin and its suppression of JUND-activated transcription. Menin also interacts with mixed lineage leukaemia protein 1 (MLL1), a histone H3 lysine 4 methyltransferase, and functions as an oncogenic cofactor to upregulate gene transcription and promote MLL1-fusion-protein-induced leukaemogenesis. A recent report on the tethering of MLL1 to chromatin binding factor lens epithelium-derived growth factor (LEDGF) by menin indicates that menin is a molecular adaptor coordinating the functions of multiple proteins. Despite its importance, how menin interacts with many distinct partners and regulates their functions remains poorly understood. Here we present the crystal structures of human menin in its free form and in complexes with MLL1 or with JUND, or with an MLL1-LEDGF heterodimer. These structures show that menin contains a deep pocket that binds short peptides of MLL1 or JUND in the same manner, but that it can have opposite effects on transcription. The menin-JUND interaction blocks JUN N-terminal kinase (JNK)-mediated JUND phosphorylation and suppresses JUND-induced transcription. In contrast, menin promotes gene transcription by binding the transcription activator MLL1 through the peptide pocket while still interacting with the chromatin-anchoring protein LEDGF at a distinct surface formed by both menin and MLL1.

  11. Crystal Structure of Menin Reveals Binding Site for Mixed Lineage Leukemia (MLL) Protein

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    Murai, Marcelo J.; Chruszcz, Maksymilian; Reddy, Gireesh; Grembecka, Jolanta; Cierpicki, Tomasz (Michigan); (UV)

    2014-10-02

    Menin is a tumor suppressor protein that is encoded by the MEN1 (multiple endocrine neoplasia 1) gene and controls cell growth in endocrine tissues. Importantly, menin also serves as a critical oncogenic cofactor of MLL (mixed lineage leukemia) fusion proteins in acute leukemias. Direct association of menin with MLL fusion proteins is required for MLL fusion protein-mediated leukemogenesis in vivo, and this interaction has been validated as a new potential therapeutic target for development of novel anti-leukemia agents. Here, we report the first crystal structure of menin homolog from Nematostella vectensis. Due to a very high sequence similarity, the Nematostella menin is a close homolog of human menin, and these two proteins likely have very similar structures. Menin is predominantly an {alpha}-helical protein with the protein core comprising three tetratricopeptide motifs that are flanked by two {alpha}-helical bundles and covered by a {beta}-sheet motif. A very interesting feature of menin structure is the presence of a large central cavity that is highly conserved between Nematostella and human menin. By employing site-directed mutagenesis, we have demonstrated that this cavity constitutes the binding site for MLL. Our data provide a structural basis for understanding the role of menin as a tumor suppressor protein and as an oncogenic co-factor of MLL fusion proteins. It also provides essential structural information for development of inhibitors targeting the menin-MLL interaction as a novel therapeutic strategy in MLL-related leukemias.

  12. Role of c-Myb in chondrogenesis.

    Science.gov (United States)

    Oralová, V; Matalová, E; Janečková, E; Drobná Krejčí, E; Knopfová, L; Šnajdr, P; Tucker, A S; Veselá, I; Šmarda, J; Buchtová, M

    2015-07-01

    The Myb locus encodes the c-Myb transcription factor involved in controlling a broad variety of cellular processes. Recently, it has been shown that c-Myb may play a specific role in hard tissue formation; however, all of these results were gathered from an analysis of intramembranous ossification. To investigate a possible role of c-Myb in endochondral ossification, we carried out our study on the long bones of mouse limbs during embryonic development. Firstly, the c-myb expression pattern was analyzed by in situ hybridization during endochondral ossification of long bones. c-myb positive areas were found in proliferating as well as hypertrophic zones of the growth plate. At early embryonic stages, localized expression was also observed in the perichondrium and interdigital areas. The c-Myb protein was found in proliferating chondrocytes and in the perichondrium of the forelimb bones (E14.5-E17.5). Furthermore, protein was detected in pre-hypertrophic as well as hypertrophic chondrocytes. Gain-of-function and loss-of-function approaches were used to test the effect of altered c-myb expression on chondrogenesis in micromass cultures established from forelimb buds of mouse embryos. A loss-of-function approach using c-myb specific siRNA decreased nodule formation, as well as downregulated the level of Sox9 expression, a major marker of chondrogenesis. Transient c-myb overexpression markedly increased the formation of cartilage nodules and the production of extracellular matrix as detected by intense staining with Alcian blue. Moreover, the expression of early chondrogenic genes such as Sox9, Col2a1 and activity of a Col2-LUC reporter were increased in the cells overexpressing c-myb while late chondrogenic markers such as Col10a1 and Mmp13 were not significantly changed or were downregulated. Taken together, the results of this study demonstrate that the c-Myb transcription factor is involved in the regulation and promotion of endochondral bone formation. PMID

  13. HIPK1 interacts with c-Myb and modulates its activity through phosphorylation

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    Matre, Vilborg; Nordgard, Oddmund; Alm-Kristiansen, Anne Hege; Ledsaak, Marit [Department of Molecular Biosciences, University of Oslo, N-0316 Oslo (Norway); Gabrielsen, Odd Stokke, E-mail: o.s.gabrielsen@imbv.uio.no [Department of Molecular Biosciences, University of Oslo, N-0316 Oslo (Norway)

    2009-10-09

    The transcription factor v-Myb is a potent inducer of myeloid leukaemias, and its cellular homologue c-Myb plays a crucial role in the regulation of haematopoiesis. In a yeast two-hybrid (Y2H) screening we identified the nuclear kinase HIPK1 as an interaction partner for human c-Myb. The interaction involves a C-terminal region of HIPK1, while a bipartite interaction surface was identified in c-Myb, including regions in its N-terminal DNA-binding domain as well as in its C-terminal region. HIPK1 and c-Myb co-localize in distinct nuclear foci upon co-transfection. c-Myb appears to be phosphorylated by HIPK1 in its negative regulatory domain as supported by both in vivo and in vitro data. A functional assay revealed that HIPK1 repressed the ability of c-Myb to activate a chromatin embedded target gene, mim-1, in haematopoetic cells. Our findings point to a novel link between an important kinase and a key regulator of haematopoiesis.

  14. c-Myb inhibits myoblast fusion

    Czech Academy of Sciences Publication Activity Database

    Kašpar, Petr; Ilenčíková, Kristina; Zíková, Martina; Horvath, Ondrej; Čermák, Vladimír; Bartůněk, Petr; Strnad, Hynek

    2013-01-01

    Roč. 8, č. 10 (2013), e76742. E-ISSN 1932-6203 R&D Projects: GA ČR GAP305/10/2133 Institutional support: RVO:68378050 Keywords : c-Myb * muscle cell differentiation * satellite cells Subject RIV: EB - Genetics ; Molecular Biology Impact factor: 3.534, year: 2013

  15. Expression and characterization of c-Myb in prenatal odontogenesis

    OpenAIRE

    Matalová, E.; Buchtová, M. (Marcela); Tucker, A.S.; Bender, T P; Janečková, E. (Eva); Lungová, V.; Balková, S. (Simona); Šmarda, J.

    2011-01-01

    The transcription factor c-Myb is involved in the control of cell proliferation, survival and differentiation. As these processes accompany the morphogenesis of developing teeth, this work investigates the possible role of c-Myb during odontogenesis. Analysis of the expression of c-Myb in the monophyodont mouse was followed by similar analysis in a diphyodont species, the pig, which has a dentition more closely resembling that of the human. The distribution of c-Myb was correlated with the pa...

  16. Experimental and molecular dynamics studies showed that CBP KIX mutation affects the stability of CBP:c-Myb complex.

    Science.gov (United States)

    Odoux, Anne; Jindal, Darren; Tamas, Tamara C; Lim, Benjamin W H; Pollard, Drake; Xu, Wu

    2016-06-01

    The coactivators CBP (CREBBP) and its paralog p300 (EP300), two conserved multi-domain proteins in eukaryotic organisms, regulate gene expression in part by binding DNA-binding transcription factors. It was previously reported that the CBP/p300 KIX domain mutant (Y650A, A654Q, and Y658A) altered both c-Myb-dependent gene activation and repression, and that mice with these three point mutations had reduced numbers of platelets, B cells, T cells, and red blood cells. Here, our transient transfection assays demonstrated that mouse embryonic fibroblast cells containing the same mutations in the KIX domain and without a wild-type allele of either CBP or p300, showed decreased c-Myb-mediated transcription. Dr. Wright's group solved a 3-D structure of the mouse CBP:c-Myb complex using NMR. To take advantage of the experimental structure and function data and improved theoretical calculation methods, we performed MD simulations of CBP KIX, CBP KIX with the mutations, and c-Myb, as well as binding energy analysis for both the wild-type and mutant complexes. The binding between CBP and c-Myb is mainly mediated by a shallow hydrophobic groove in the center where the side-chain of Leu302 of c-Myb plays an essential role and two salt bridges at the two ends. We found that the KIX mutations slightly decreased stability of the CBP:c-Myb complex as demonstrated by higher binding energy calculated using either MM/PBSA or MM/GBSA methods. More specifically, the KIX mutations affected the two salt bridges between CBP and c-Myb (CBP-R646 and c-Myb-E306; CBP-E665 and c-Myb-R294). Our studies also revealed differing dynamics of the hydrogen bonds between CBP-R646 and c-Myb-E306 and between CBP-E665 and c-Myb-R294 caused by the CBP KIX mutations. In the wild-type CBP:c-Myb complex, both of the hydrogen bonds stayed relatively stable. In contrast, in the mutant CBP:c-Myb complex, hydrogen bonds between R646 and E306 showed an increasing trend followed by a decreasing trend, and hydrogen

  17. Distinct regulation of c-myb gene expression by HoxA9, Meis1 and Pbx proteins in normal hematopoietic progenitors and transformed myeloid cells

    International Nuclear Information System (INIS)

    The proto-oncogenic protein c-Myb is an essential regulator of hematopoiesis and is frequently deregulated in hematological diseases such as lymphoma and leukemia. To gain insight into the mechanisms underlying the aberrant expression of c-Myb in myeloid leukemia, we analyzed and compared c-myb gene transcriptional regulation using two cell lines modeling normal hematopoietic progenitor cells (HPCs) and transformed myelomonocytic blasts. We report that the transcription factors HoxA9, Meis1, Pbx1 and Pbx2 bind in vivo to the c-myb locus and maintain its expression through different mechanisms in HPCs and leukemic cells. Our analysis also points to a critical role for Pbx2 in deregulating c-myb expression in murine myeloid cells cotransformed by the cooperative activity of HoxA9 and Meis1. This effect is associated with an intronic positioning of epigenetic marks and RNA polymerase II binding in the orthologous region of a previously described alternative promoter for c-myb. Taken together, our results could provide a first hint to explain the abnormal expression of c-myb in leukemic cells

  18. Identification and Regulation of c-Myb Target Genes in MCF-7 Cells

    International Nuclear Information System (INIS)

    The c-Myb transcription factor regulates differentiation and proliferation in hematopoietic cells, stem cells and epithelial cells. Although oncogenic versions of c-Myb were first associated with leukemias, over expression or rearrangement of the c-myb gene is common in several types of solid tumors, including breast cancers. Expression of the c-myb gene in human breast cancer cells is dependent on estrogen stimulation, but little is known about the activities of the c-Myb protein or what genes it regulates in estrogen-stimulated cells. We used chromatin immunoprecipitation coupled with whole genome promoter tiling microarrays to identify endogenous c-Myb target genes in human MCF-7 breast cancer cells and characterized the activity of c-Myb at a panel of target genes during different stages of estrogen deprivation and stimulation. By using different antibodies and different growth conditions, the c-Myb protein was found associated with over 10,000 promoters in MCF-7 cells, including many genes that encode cell cycle regulators or transcription factors and more than 60 genes that encode microRNAs. Several previously identified c-Myb target genes were identified, including CCNB1, MYC and CXCR4 and novel targets such as JUN, KLF4, NANOG and SND1. By studying a panel of these targets to validate the results, we found that estradiol stimulation triggered the association of c-Myb with promoters and that association correlated with increased target gene expression. We studied one target gene, CXCR4, in detail, showing that c-Myb associated with the CXCR4 gene promoter and activated a CXCR4 reporter gene in transfection assays. Our results show that c-Myb associates with a surprisingly large number of promoters in human cells. The results also suggest that estradiol stimulation leads to large-scale, genome-wide changes in c-Myb activity and subsequent changes in gene expression in human breast cancer cells

  19. c-Myb regulates NOX1/p38 to control survival of colorectal carcinoma cells.

    Science.gov (United States)

    Pekarčíková, Lucie; Knopfová, Lucia; Beneš, Petr; Šmarda, Jan

    2016-08-01

    The c-Myb transcription factor is important for maintenance of immature cells of many tissues including colon epithelium. Overexpression of c-Myb occurring in colorectal carcinomas (CRC) as well as in other cancers often marks poor prognosis. However, the molecular mechanism explaining how c-Myb contributes to progression of CRC has not been fully elucidated. To address this point, we investigated the way how c-Myb affects sensitivity of CRC cells to anticancer drugs. Using CRC cell lines expressing exogenous c-myb we show that c-Myb protects CRC cells from the cisplatin-, oxaliplatin-, and doxorubicin-induced apoptosis, elevates reactive oxygen species via up-regulation of NOX1, and sustains the pro-survival p38 MAPK pathway. Using pharmacological inhibitors and gene silencing of p38 and NOX1 we found that these proteins are essential for the protective effect of c-Myb and that NOX1 acts upstream of p38 activation. In addition, our result suggests that transcription of NOX1 is directly controlled by c-Myb and these genes are strongly co-expressed in human tumor tissue of CRC patients. The novel c-Myb/NOX1/p38 signaling axis that protects CRC cells from chemotherapy described in this study could provide a new base for design of future therapies of CRC. PMID:27107996

  20. The c-Myb target gene neuromedin U functions as a novel cofactor during the early stages of erythropoiesis

    OpenAIRE

    Gambone, Julia E; Dusaban, Stephanie S.; Loperena, Roxana; Nakata, Yuji; Shetzline, Susan E.

    2011-01-01

    The requirement of c-Myb during erythropoiesis spurred an interest in identifying c-Myb target genes that are important for erythroid development. Here, we determined that the neuropeptide neuromedin U (NmU) is a c-Myb target gene. Silencing NmU, c-myb, or NmU's cognate receptor NMUR1 expression in human CD34+ cells impaired burst-forming unit-erythroid (BFU-E) and colony-forming unit-erythroid (CFU-E) formation compared with control. Exogenous addition of NmU peptide to NmU or c-myb siRNA-tr...

  1. Two modes of c-myb activation in virus-induced mouse myeloid tumors.

    OpenAIRE

    1986-01-01

    Two modes of disruption of the protooncogene c-myb by viral insertional mutagenesis in mouse myeloid tumor cells are described. The first mode was found in six tumors in which a Moloney murine leukemia virus component had inserted in the same transcriptional orientation upstream of the 5'-most exon with v-myb homology (vE1). cDNA sequence data indicate the presence of a truncated c-myb mRNA that is initiated in the upstream 5' long terminal repeat of the integrated provirus and processed via ...

  2. Localization of c-MYB in differentiated cells during postnatal molar and alveolar bone development

    Czech Academy of Sciences Publication Activity Database

    Lungová, Vlasta; Buchtová, Marcela; Janečková, Eva; Tucker, A.S.; Knopfová, L.; Šmarda, J.; Matalová, Eva

    2012-01-01

    Roč. 120, č. 6 (2012), 495-504. ISSN 0909-8836 R&D Projects: GA ČR GCP302/12/J059 Institutional research plan: CEZ:AV0Z50450515 Keywords : c-myb * tooth * postnatal Subject RIV: FF - HEENT, Dentistry Impact factor: 1.420, year: 2012

  3. Effect of ischemic preconditioning on the expression of c-myb in the CA1 region of the gerbil hippocampus after ischemia/reperfusion injury

    Directory of Open Access Journals (Sweden)

    Hui Young Lee

    2016-06-01

    Conclusion: Our results show that a lethal transient ischemia significantly decreased c-myb immunoreactivity in the SP of the CA1 region and that IPC well preserved c-myb immunoreactivity in the SP of the CA1 region. We suggest that the maintenance of c-myb might be related with IPC-mediated neuroprotection after a lethal ischemic insult.

  4. Differentiation of mouse erythroleukemia cells is blocked by late up-regulation of a c-myb transgene.

    OpenAIRE

    McClinton, D; Stafford, J; Brents, L; Bender, T. P.; Kuehl, W M

    1990-01-01

    During chemically induced differentiation of Friend virus-infected mouse erythroleukemia (MEL) cell lines, there is a biphasic down-regulation of the c-myb proto-oncogene. A plasmid containing a murine c-myb cDNA controlled by a mouse metallothionein I promoter was transfected into the C19 MEL cell line. For six transfected clones, it was found that expression of the exogenous c-myb mRNA could be up-regulated by the addition of 120 microM ZnCl2 and that the N,N'-hexamethylenebisacetamide-indu...

  5. The Expression of c-Myb Correlates with the Levels of Rhabdomyosarcoma-specific Marker Myogenin

    Czech Academy of Sciences Publication Activity Database

    Kašpar, Petr; Zíková, Martina; Bartůněk, Petr; Štěrba, J.; Strnad, Hynek; Křen, L.; Sedláček, Radislav

    2015-01-01

    Roč. 5, Oct 14 (2015). ISSN 2045-2322 R&D Projects: GA ČR GAP305/10/2133; GA ČR GAP301/12/1478; GA MŠk(CZ) ED1.1.00/02.0109 Institutional support: RVO:68378050 Keywords : c-Myb * Rhabdomyosarcomas * C2C12 myoblast cell line * myogenin Subject RIV: EB - Genetics ; Molecular Biology Impact factor: 5.578, year: 2014

  6. c-Myb in incisor stem cell niches and in differentiation of odontogenic cells

    Czech Academy of Sciences Publication Activity Database

    Matalová, Eva; Lungová, Vlasta; Buchtová, Marcela; Švandová, Eva; Šmarda, J.

    Warwick: BSCB, 2012. 93-94. [British Society for Developmental Biology, Bristish Society for Cell Biology and Japanese Society of Developmental Biologists Joint Spring Meeting. 15.04.2012-18.04.2012, Warwick] R&D Projects: GA ČR GCP302/12/J059; GA ČR GAP304/11/1418 Institutional support: RVO:67985904 Keywords : c-Myb Subject RIV: EA - Cell Biology

  7. In vivo treatment of human leukemia in a scid mouse model with c-myb antisense oligodeoxynucleotides.

    OpenAIRE

    Ratajczak, M Z; Kant, J A; Luger, S M; Hijiya, N.; Zhang, J; Zon, G; Gewirtz, A. M.

    1992-01-01

    The c-myb protooncogene encodes proteins that are critical for hematopoietic cell proliferation and development. Disrupting c-myb function might, therefore, prove an effective therapeutic strategy for controlling leukemic cell growth. Antisense oligodeoxynucleotides have been utilized for this purpose in vitro, but their in vivo efficacy has not been reported. We therefore established human leukemia-scid mouse chimeras with K562 cells and treated diseased animals with phosphorothioate-modifie...

  8. c-Myb regulates matrix metalloproteinases 1/9, and cathepsin D: implications for matrix-dependent breast cancer cell invasion and metastasis

    Directory of Open Access Journals (Sweden)

    Knopfová Lucia

    2012-03-01

    Full Text Available Abstract Background The c-Myb transcription factor is essential for the maintenance of stem-progenitor cells in bone marrow, colon epithelia, and neurogenic niches. c-Myb malfunction contributes to several types of malignancies including breast cancer. However, the function of c-Myb in the metastatic spread of breast tumors remains unexplored. In this study, we report a novel role of c-Myb in the control of specific proteases that regulate the matrix-dependent invasion of breast cancer cells. Results Ectopically expressed c-Myb enhanced migration and ability of human MDA-MB-231 and mouse 4T1 mammary cancer cells to invade Matrigel but not the collagen I matrix in vitro. c-Myb strongly increased the expression/activity of cathepsin D and matrix metalloproteinase (MMP 9 and significantly downregulated MMP1. The gene coding for cathepsin D was suggested as the c-Myb-responsive gene and downstream effector of the migration-promoting function of c-Myb. Finally, we demonstrated that c-Myb delayed the growth of mammary tumors in BALB/c mice and affected the metastatic potential of breast cancer cells in an organ-specific manner. Conclusions This study identified c-Myb as a matrix-dependent regulator of invasive behavior of breast cancer cells.

  9. An oligomer complementary to c-myb-encoded mRNA inhibits proliferation of human myeloid leukemia cell lines.

    OpenAIRE

    Anfossi, G; Gewirtz, A M; Calabretta, B

    1989-01-01

    To study the role of the protooncogene c-myb in regulating myeloid leukemia cell proliferation and differentiation, we exposed cells of the human leukemia lines HL-60, ML-3, KG-1, and KG-1a to an oligodeoxynucleotide complementary to an 18-base-pair (bp) sequence of c-myb-encoded mRNA. This treatment resulted in a significant decrease in cell proliferation in all of the lines, which was most marked in HL-60 cells. After 5 days in culture, in several separate experiments with different oligome...

  10. Evidence for higher-order structure formation by the c-myb 18-mer phosphorothioate antisense (codons 2-7) oligodeoxynucleotide: potential relationship to antisense c-myb inhibition.

    Science.gov (United States)

    Vilenchik, M; Benimetsky, L; Kolbanovsky, A; Miller, P; Stein, C A

    2001-04-01

    We have demonstrated the formation of higher-order structures (presumably tetraplexes) by an 18-mer phosphorothioate antisense c-myb oligodeoxyribonucleotide that has been shown to have activity in the treatment of leukemia xenograft models. Although not observable by conventionally employed techniques, such as PAGE and dimethyl sulfate (DMS) protection, the formation of such higher-order structures by this oligonucleotide was revealed by several techniques. These included capillary gel electrophoresis (CGE), which demonstrated the presence of molecules with greatly increased retention time compared with the monomer; magnetic circular dichroism spectroscopy, which demonstrated a band at 290 nm, a characteristic of antiparallel tetraplexes; and fluorescence energy transfer measurements. For the last, the 18-mer phosphorothioate oligonucleotide was synthesized with a 5'-fluorescein group. Similar to the molecular beacon model, its fluorescence was quenched when combined in solution with tetraplex-forming oligomers that contained a 3'-Dabcyl moiety. 7-Deazaguanosine inhibits the formation of tetraplexes by eliminated Hoogsteen base pair interactions. The wild-type and 7-deazaguanosine-substituted antisense c-myb oligomers differentially downregulated the expression of the c-myb proto-oncogene in K562 and HL60 cells, with the wild-type oligomer being the least active. The 18-mer c-myb molecule can, therefore, form highly complex structures, whose analysis in solution cannot be limited to examination of slab gel electrophoresis results alone. PMID:11334144

  11. Effect of cisplatin and c-myb antisense phosphorothioate oligodeoxynucleotides combination on a human colon carcinoma cell line in vitro and in vivo.

    OpenAIRE

    Del Bufalo, D; Cucco, C.; C. Leonetti; Citro, G.; D'Agnano, I; M. Benassi; Geiser, T; Zon, G; Calabretta, B; Zupi, G.

    1996-01-01

    We investigated the effect of c-myb antisense phosphorothioate oligodeoxynucleotides [(S)ODNs] and cisplatin (CDDP) combination on the human colon carcinoma cell line LoVo Dx both in vitro and in nude mice bearing LoVo Dx solid tumour. We show that antisense (S)ODN treatment decreases c-myb mRNA and protein expression, induces growth arrest in the G1 phase of the cell cycle, and inhibits cell proliferation. In vivo treatment with c-myb antisense (S)ODNs results in a reduction in tumour growth...

  12. AML suppresses hematopoiesis by releasing exosomes that contain microRNAs targeting c-MYB.

    Science.gov (United States)

    Hornick, Noah I; Doron, Ben; Abdelhamed, Sherif; Huan, Jianya; Harrington, Christina A; Shen, Rongkun; Cambronne, Xiaolu A; Chakkaramakkil Verghese, Santhosh; Kurre, Peter

    2016-01-01

    Exosomes are paracrine regulators of the tumor microenvironment and contain complex cargo. We previously reported that exosomes released from acute myeloid leukemia (AML) cells can suppress residual hematopoietic stem and progenitor cell (HSPC) function indirectly through stromal reprogramming of niche retention factors. We found that the systemic loss of hematopoietic function is also in part a consequence of AML exosome-directed microRNA (miRNA) trafficking to HSPCs. Exosomes isolated from cultured AML or the plasma from mice bearing AML xenografts exhibited enrichment of miR-150 and miR-155. HSPCs cocultured with either of these exosomes exhibited impaired clonogenicity, through the miR-150- and miR-155-mediated suppression of the translation of transcripts encoding c-MYB, a transcription factor involved in HSPC differentiation and proliferation. To discover additional miRNA targets, we captured miR-155 and its target transcripts by coimmunoprecipitation with an attenuated RNA-induced silencing complex (RISC)-trap, followed by high-throughput sequencing. This approach identified known and previously unknown miR-155 target transcripts. Integration of the miR-155 targets with information from the protein interaction database STRING revealed proteins indirectly affected by AML exosome-derived miRNA. Our findings indicate a direct effect of AML exosomes on HSPCs that, through a stroma-independent mechanism, compromises hematopoiesis. Furthermore, combining miRNA target data with protein-protein interaction data may be a broadly applicable strategy to define the effects of exosome-mediated trafficking of regulatory molecules within the tumor microenvironment. PMID:27601730

  13. Fulvestrant up regulates UGT1A4 and MRPs through ERα and c-Myb pathways: a possible primary drug disposition mechanism.

    Science.gov (United States)

    Edavana, Vineetha K; Penney, Rosalind B; Yao-Borengasser, Aiwei; Williams, Suzanne; Rogers, Lora; Dhakal, Ishwori B; Kadlubar, Susan

    2013-01-01

    Fulvestrant (Faslodex™) is a pure antiestrogen that is effective in treating estrogen receptor-(ER) positive breast cancer tumors that are resistant to selective estrogen receptor modulators such as tamoxifen. Clinical trials investigating the utility of adding fulvestrant to other therapeutics have not been shown to affect cytochrome P450-mediated metabolism. Effects on phase II metabolism and drug resistance have not been explored. This study demonstrates that fulvestrant up regulates the expression of UDP glucuronosyltransferase 1A4 (UGT1A4) >2.5- and >3.5-fold in MCF7 and HepG2 cells, respectively. Up regulation occurred in a time- and concentration-dependent manner, and was inhibited by siRNA silencing of ERα. Fulvestrant also up regulates multidrug resistance-associated proteins (MRPs). There was an up regulation of MRP2 (1.5- and 3.5-fold), and MRP3 (5.5- and 4.5-fold) in MCF7 and HepG2 cell lines, respectively, and an up regulation of MRP1 (4-fold) in MCF7 cells. UGT1A4 mRNA up regulation was significantly correlated with UGT1A4 protein expression, anastrozole glucuronidation, ERα mRNA expression and MRP mRNA expression, but not with ERα protein expression. Genetic variants in the UGT1A4 promoter (-163A, -217G and -219T) reduced the basal activity of UGT1A4 by 40-60%. In silico analysis indicated that transcription factor c-Myb binding capacity may be affected by these variations. Luciferase activity assays demonstrate that silencing c-Myb abolished UGT1A4 up regulation by fulvestrant in promoters with the common genotype (-163G, -217 T and -219C) in MCF7 cells. These data indicate that fulvestrant can influence the disposition of other UGT1A4 substrates. These findings suggest a clinically significant role for UGT1A4 and MRPs in drug efficacy. PMID:24298433

  14. c-Maf Interacts with c-Myb To Regulate Transcription of an Early Myeloid Gene during Differentiation

    OpenAIRE

    Hegde, Shrikanth P.; Kumar, Alok; Kurschner, Cornelia; Shapiro, Linda H.

    1998-01-01

    The MafB transcriptional activator plays a pivotal role in regulating lineage-specific gene expression during hematopoiesis by repressing Ets-1-mediated transcription of key erythroid-specific genes in myeloid cells. To determine the effects of Maf family proteins on the transactivation of myeloid-specific genes in myeloid cells, we tested the ability of c-Maf to influence Ets-1- and c-Myb-dependent CD13/APN transcription. Expression of c-Maf in human immature myeloblastic cells inhibited CD1...

  15. RAV-1 insertional mutagenesis: disruption of the c-myb locus and development of avian B-cell lymphomas.

    OpenAIRE

    Pizer, E; Humphries, E H

    1989-01-01

    Infection of young chickens with RAV-1, a subgroup A isolate of avian leukosis virus, results in the development of lymphoid leukosis, a B-cell lymphoma characterized by provirus insertion into the c-myc locus. We report here that when 12- to 13-day-old embryos rather than 1-day-old chickens were infected with RAV-1, a novel B-cell lymphoma developed in which proviral insertions had activated expression of the c-myb gene. These tumors expressed elevated levels of a 4.5-kilobase myb-containing...

  16. MLL5 maintains spindle bipolarity by preventing aberrant cytosolic aggregation of PLK1.

    Science.gov (United States)

    Zhao, Wei; Liu, Jie; Zhang, Xiaoming; Deng, Lih-Wen

    2016-03-28

    Faithful chromosome segregation with bipolar spindle formation is critical for the maintenance of genomic stability. Perturbation of this process often leads to severe mitotic failure, contributing to tumorigenesis. MLL5 has been demonstrated to play vital roles in cell cycle progression and the maintenance of genomic stability. Here, we identify a novel interaction between MLL5 and PLK1 in the cytosol that is crucial for sustaining spindle bipolarity during mitosis. Knockdown of MLL5 caused aberrant PLK1 aggregation that led to acentrosomal microtubule-organizing center (aMTOC) formation and subsequent spindle multipolarity. Further molecular studies revealed that the polo-box domain (PBD) of PLK1 interacted with a binding motif on MLL5 (Thr887-Ser888-Thr889), and this interaction was essential for spindle bipolarity. Overexpression of wild-type MLL5 was able to rescue PLK1 mislocalization and aMTOC formation in MLL5-KD cells, whereas MLL5 mutants incapable of interacting with the PBD failed to do so. We thus propose that MLL5 preserves spindle bipolarity through maintaining cytosolic PLK1 in a nonaggregated form. PMID:27002166

  17. Molecular and Epigenetic Mechanisms of MLL in Human Leukemogenesis

    Energy Technology Data Exchange (ETDEWEB)

    Ballabio, Erica; Milne, Thomas A., E-mail: thomas.milne@imm.ox.ac.uk [MRC Molecular Haematology Unit, Weatherall Institute of Molecular Medicine, John Radcliffe Hospital Headington, Oxford OX3 9DS (United Kingdom)

    2012-09-10

    Epigenetics is often defined as the study of heritable changes in gene expression or chromosome stability that don’t alter the underlying DNA sequence. Epigenetic changes are established through multiple mechanisms that include DNA methylation, non-coding RNAs and the covalent modification of specific residues on histone proteins. It is becoming clear not only that aberrant epigenetic changes are common in many human diseases such as leukemia, but that these changes by their very nature are malleable, and thus are amenable to treatment. Epigenetic based therapies have so far focused on the use of histone deacetylase (HDAC) inhibitors and DNA methyltransferase inhibitors, which tend to have more general and widespread effects on gene regulation in the cell. However, if a unique molecular pathway can be identified, diseases caused by epigenetic mechanisms are excellent candidates for the development of more targeted therapies that focus on specific gene targets, individual binding domains, or specific enzymatic activities. Designing effective targeted therapies depends on a clear understanding of the role of epigenetic mutations during disease progression. The Mixed Lineage Leukemia (MLL) protein is an example of a developmentally important protein that controls the epigenetic activation of gene targets in part by methylating histone 3 on lysine 4. MLL is required for normal development, but is also mutated in a subset of aggressive human leukemias and thus provides a useful model for studying the link between epigenetic cell memory and human disease. The most common MLL mutations are chromosome translocations that fuse the MLL gene in frame with partner genes creating novel fusion proteins. In this review, we summarize recent work that argues MLL fusion proteins could function through a single molecular pathway, but we also highlight important data that suggests instead that multiple independent mechanisms underlie MLL mediated leukemogenesis.

  18. Molecular and Epigenetic Mechanisms of MLL in Human Leukemogenesis

    Directory of Open Access Journals (Sweden)

    Thomas A. Milne

    2012-09-01

    Full Text Available Epigenetics is often defined as the study of heritable changes in gene expression or chromosome stability that don’t alter the underlying DNA sequence. Epigenetic changes are established through multiple mechanisms that include DNA methylation, non-coding RNAs and the covalent modification of specific residues on histone proteins. It is becoming clear not only that aberrant epigenetic changes are common in many human diseases such as leukemia, but that these changes by their very nature are malleable, and thus are amenable to treatment. Epigenetic based therapies have so far focused on the use of histone deacetylase (HDAC inhibitors and DNA methyltransferase inhibitors, which tend to have more general and widespread effects on gene regulation in the cell. However, if a unique molecular pathway can be identified, diseases caused by epigenetic mechanisms are excellent candidates for the development of more targeted therapies that focus on specific gene targets, individual binding domains, or specific enzymatic activities. Designing effective targeted therapies depends on a clear understanding of the role of epigenetic mutations during disease progression. The Mixed Lineage Leukemia (MLL protein is an example of a developmentally important protein that controls the epigenetic activation of gene targets in part by methylating histone 3 on lysine 4. MLL is required for normal development, but is also mutated in a subset of aggressive human leukemias and thus provides a useful model for studying the link between epigenetic cell memory and human disease. The most common MLL mutations are chromosome translocations that fuse the MLL gene in frame with partner genes creating novel fusion proteins. In this review, we summarize recent work that argues MLL fusion proteins could function through a single molecular pathway, but we also highlight important data that suggests instead that multiple independent mechanisms underlie MLL mediated leukemogenesis.

  19. Molecular and Epigenetic Mechanisms of MLL in Human Leukemogenesis

    International Nuclear Information System (INIS)

    Epigenetics is often defined as the study of heritable changes in gene expression or chromosome stability that don’t alter the underlying DNA sequence. Epigenetic changes are established through multiple mechanisms that include DNA methylation, non-coding RNAs and the covalent modification of specific residues on histone proteins. It is becoming clear not only that aberrant epigenetic changes are common in many human diseases such as leukemia, but that these changes by their very nature are malleable, and thus are amenable to treatment. Epigenetic based therapies have so far focused on the use of histone deacetylase (HDAC) inhibitors and DNA methyltransferase inhibitors, which tend to have more general and widespread effects on gene regulation in the cell. However, if a unique molecular pathway can be identified, diseases caused by epigenetic mechanisms are excellent candidates for the development of more targeted therapies that focus on specific gene targets, individual binding domains, or specific enzymatic activities. Designing effective targeted therapies depends on a clear understanding of the role of epigenetic mutations during disease progression. The Mixed Lineage Leukemia (MLL) protein is an example of a developmentally important protein that controls the epigenetic activation of gene targets in part by methylating histone 3 on lysine 4. MLL is required for normal development, but is also mutated in a subset of aggressive human leukemias and thus provides a useful model for studying the link between epigenetic cell memory and human disease. The most common MLL mutations are chromosome translocations that fuse the MLL gene in frame with partner genes creating novel fusion proteins. In this review, we summarize recent work that argues MLL fusion proteins could function through a single molecular pathway, but we also highlight important data that suggests instead that multiple independent mechanisms underlie MLL mediated leukemogenesis

  20. c-Myb regulates matrix metalloproteinases 1/9, and cathepsin D: implications for matrix-dependent breast cancer cell invasion and metastasis

    Czech Academy of Sciences Publication Activity Database

    Knopfová, L.; Beneš, P.; Pekarčíková, L.; Hermanová, M.; Masařík, M.; Pernicová, Zuzana; Souček, Karel; Šmarda, J.

    2012-01-01

    Roč. 11, MAR 23 (2012), ID 15. ISSN 1476-4598 R&D Projects: GA MZd NS9600 Grant ostatní: GA AV ČR(CZ) IAA501630901 Institutional support: RVO:68081707 Keywords : c-Myb * Metastasis * Breast cancer Subject RIV: BO - Biophysics Impact factor: 5.134, year: 2012

  1. Pro Isomerization in MLL1 PHD3-Bromo Cassette Connects H3K4me Readout to CyP33 and HDAC-Mediated Repression

    Energy Technology Data Exchange (ETDEWEB)

    Wang, Zhanxin; Song, Jikui; Milne, Thomas A.; Wang, Gang G.; Li, Haitao; Allis, C. David; Patel, Dinshaw J. (MSKCC); (Rockefeller)

    2010-09-13

    The MLL1 gene is a frequent target for recurrent chromosomal translocations, resulting in transformation of hematopoietic precursors into leukemia stem cells. Here, we report on structure-function studies that elucidate molecular events in MLL1 binding of histone H3K4me3/2 marks and recruitment of the cyclophilin CyP33. CyP33 contains a PPIase and a RRM domain and regulates MLL1 function through HDAC recruitment. We find that the PPIase domain of CyP33 regulates the conformation of MLL1 through proline isomerization within the PHD3-Bromo linker, thereby disrupting the PHD3-Bromo interface and facilitating binding of the MLL1-PHD3 domain to the CyP33-RRM domain. H3K4me3/2 and CyP33-RRM target different surfaces of MLL1-PHD3 and can bind simultaneously to form a ternary complex. Furthermore, the MLL1-CyP33 interaction is required for repression of HOXA9 and HOXC8 genes in vivo. Our results highlight the role of PHD3-Bromo cassette as a regulatory platform, orchestrating MLL1 binding of H3K4me3/2 marks and cyclophilin-mediated repression through HDAC recruitment.

  2. Expression of p89c-Mybex9b, an alternatively spliced form of c-Myb, is required for proliferation and survival of p210BCR/ABL-expressing cells

    International Nuclear Information System (INIS)

    The c-Myb gene encodes the p75c-Myb isoform and less-abundant proteins generated by alternatively spliced transcripts. Among these, the best known is pc-Mybex9b, which contains 121 additional amino acids between exon 9 and 10, in a domain involved in protein–protein interactions and negative regulation. In hematopoietic cells, expression of pc-Mybex9b accounts for 10–15% of total c-Myb; these levels may be biologically relevant because modest changes in c-Myb expression affects proliferation and survival of leukemic cells and lineage choice and frequency of normal hematopoietic progenitors. In this study, we assessed biochemical activities of pc-Mybex9b and the consequences of perturbing its expression in K562 and primary chronic myeloid leukemia (CML) progenitor cells. Compared with p75c-Myb, pc-Mybex9b is more stable and more effective in transactivating Myb-regulated promoters. Ectopic expression of pc-Mybex9b enhanced proliferation and colony formation and reduced imatinib (IM) sensitivity of K562 cells; conversely, specific downregulation of pc-Mybex9b reduced proliferation and colony formation, enhanced IM sensitivity of K562 cells and markedly suppressed colony formation of CML CD34+ cells, without affecting the levels of p75c-Myb. Together, these studies indicate that expression of the low-abundance pc-Mybex9b isoform has an important role for the overall biological effects of c-Myb in BCR/ABL-transformed cells

  3. The formation and characteristics of the i-motif structure within the promoter of the c-myb proto-oncogene.

    Science.gov (United States)

    Li, Huihui; Hai, Jinhui; Zhou, Jiang; Yuan, Gu

    2016-09-01

    C-myb proto-oncogene is a potential therapeutic target for some human solid tumors and leukemias. A long cytosine-rich sequence, which locates the downstream of the transcription initiation site, is demonstrated to fold into an intramolecular i-motif DNA using electrospray ionization mass spectrometry (ESI-MS) and circular dichroism (CD) spectroscopy. Effects of factors, including the pH value, the number of C:C(+) dimers, the concentration of buffer, the molecular crowding condition, and the coexistence of the complementary DNA, on the formation and the structural stability of the i-motif DNA are systematically studied. We have demonstrated that the i-motif folding in the c-myb promoter could be accelerated upon synergistic physiological stimuli including intracellular molecular crowding and low pH values, as well as the large number of the i-motif C:C(+) dimers. Meanwhile, various inputs, such as acids/bases and metal ions, have exhibited their abilities in controlling the conformational switch of the c-myb GC-rich DNA. Acidic pH values and the presence of K(+) ions can induce the dissociation of the double helix. Our present strategy can greatly extend the potential usages of i-motif DNA molecules with specific sequences as conformational switch-controlled devices. Moreover, this work demonstrates the superiority of CD spectroscopy associated with ESI-MS as a rapid, more cost-effective and sensitive structural change responsive method in the research of DNA conformational switching. PMID:27487467

  4. Impact of the MLL1 morphemes on codon utilization and preservation in CpG islands.

    Science.gov (United States)

    Bina, Minou; Wyss, Phillip

    2015-09-01

    Previous studies have shown that Mixed Lineage Leukemia 1 (MLL1 or MLL) binds a group of CpG-rich motifs known as morphemes. To examine whether occurrences of MLL1 morphemes in genomic DNA may influence codon utilization, we analyzed the frequency of various 9-mers in human cDNAs and in total human genomic DNA. We uncovered preferential utilization of GGC for Gly, GCG for Ala, CCG for Pro, and TCG for Ser, in coding sequences (CDSs) that included MLL1 morphemes. We also examined weighted occurrences of CDS 9-mers in a 30-base window that moved along each human chromosome. In plots, we observed peaks with fluctuating intensities. High intensity peaks appeared within promoter and exons localized in CpG islands, encompassing sequences that included MLL1 morphemes. High intensity peaks included CCG/GGC repeats, whose expansion may cause neurological disorders and congenital malformations. Such repeats are generated from overlap of a morpheme (CGCCG/CGGCG), which depending on reading frame and orientation would produce runs of Ala, Gly, or Pro in proteins. Overall, our results point to a role for morpheme occurrences on synonymous codon utilization in human genomic DNA and indicate that regulatory instructions are dispersed not only in promoters but also in exons of human genes. PMID:25991579

  5. Molecular Insights in MLL Rearranged Acute Leukemia

    NARCIS (Netherlands)

    R.W. Stam (Ronald)

    2006-01-01

    textabstractAcute lymphoblastic leukemia (ALL) in infants (<1 year of age) is characterized by a high incidence (~80%) of rearrangements of the MLL gene, resistance to several important chemotherapeutic drugs, and a poor treatment outcome. With overall survival rates for infant ALL not exceeding 50%

  6. Both SEPT2 and MLL are down-regulated in MLL-SEPT2 therapy-related myeloid neoplasia

    International Nuclear Information System (INIS)

    A relevant role of septins in leukemogenesis has been uncovered by their involvement as fusion partners in MLL-related leukemia. Recently, we have established the MLL-SEPT2 gene fusion as the molecular abnormality subjacent to the translocation t(2;11)(q37;q23) in therapy-related acute myeloid leukemia. In this work we quantified MLL and SEPT2 gene expression in 58 acute myeloid leukemia patients selected to represent the major AML genetic subgroups, as well as in all three cases of MLL-SEPT2-associated myeloid neoplasms so far described in the literature. Cytogenetics, fluorescence in situ hybridization (FISH) and molecular studies (RT-PCR, qRT-PCR and qMSP) were used to characterize 58 acute myeloid leukemia patients (AML) at diagnosis selected to represent the major AML genetic subgroups: CBFB-MYH11 (n = 13), PML-RARA (n = 12); RUNX1-RUNX1T1 (n = 12), normal karyotype (n = 11), and MLL gene fusions other than MLL-SEPT2 (n = 10). We also studied all three MLL-SEPT2 myeloid neoplasia cases reported in the literature, namely two AML patients and a t-MDS patient. When compared with normal controls, we found a 12.8-fold reduction of wild-type SEPT2 and MLL-SEPT2 combined expression in cases with the MLL-SEPT2 gene fusion (p = 0.007), which is accompanied by a 12.4-fold down-regulation of wild-type MLL and MLL-SEPT2 combined expression (p = 0.028). The down-regulation of SEPT2 in MLL-SEPT2 myeloid neoplasias was statistically significant when compared with all other leukemia genetic subgroups (including those with other MLL gene fusions). In addition, MLL expression was also down-regulated in the group of MLL fusions other than MLL-SEPT2, when compared with the normal control group (p = 0.023) We found a significant down-regulation of both SEPT2 and MLL in MLL-SEPT2 myeloid neoplasias. In addition, we also found that MLL is under-expressed in AML patients with MLL fusions other than MLL-SEPT2

  7. Activation of c-myb by 5' retrovirus promoter insertion in myeloid neoplasms is dependent upon an intact alternative splice donor site (SD') in gag

    International Nuclear Information System (INIS)

    Alternative splicing in Mo-MuLV recruits a splice donor site, SD', within the gag that is required for optimal replication in vitro. Remarkably, this SD' site was also found to be utilized for production of oncogenic gag-myb fusion RNA in 100% of murine-induced myeloid leukemia (MML) in pristane-treated BALB/c mice. Therefore, we investigated the influence of silent mutations of SD' in this model. Although there was no decrease in the overall incidence of disease, there was a decrease in the incidence of myeloid leukemia with a concomitant increase in lymphoid leukemia. Importantly, there was a complete lack of myeloid tumors associated with 5' insertional mutagenic activation of c-myb, suggesting the specific requirement of the SD' site in this mechanism

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

    Directory of Open Access Journals (Sweden)

    Chen Carolyn

    2008-10-01

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

  9. Both SEPT2 and MLL are down-regulated in MLL-SEPT2 therapy-related myeloid neoplasia

    NARCIS (Netherlands)

    Cerveira, Nuno; Santos, Joana; Bizarro, Susana; Costa, Vera; Ribeiro, Franclim R.; Lisboa, Susana; Correia, Cecilia; Torres, Lurdes; Vieira, Joana; Snijder, Simone; Mariz, Jose M.; Norton, Lucilia; Mellink, Clemens H.; Buijs, Arjan; Teixeira, Manuel R.

    2009-01-01

    Background: A relevant role of septins in leukemogenesis has been uncovered by their involvement as fusion partners in MLL-related leukemia. Recently, we have established the MLL-SEPT2 gene fusion as the molecular abnormality subjacent to the translocation t(2;11)(q37;q23) in therapy-related acute m

  10. Lifelong persistence of AML associated MLL partial tandem duplications (MLL-PTD) in healthy adults.

    OpenAIRE

    Basecke, Jorg; Podleschny, Martina; Clemens, Robert; Schnittger, Susanne; Viereck, Volker; Trumper, Lorenz; Griesinger, Frank

    2006-01-01

    KEYWORDS CLASSIFICATION: Adult;Antigens,CD;blood;B-Lymphocytes;Female;Fetal Blood;genetics;Gene Duplication;Germany;Hematopoietic Stem Cells;Humans;Infant,Newborn;Leukemia;Leukemia,Myelocytic,Acute;Lymphoma,Mixed-Cell;metabolism;Male;mechanisms of carcinogenesis;Myeloid-Lymphoid Leukemia Protein;pathology;Research;T-Lymphocytes. AML-associated MLL-PTD contribute to leukemogenesis by a gain of function and confer an unfavorable prognosis. Like other leukemia associated aberrations they are ...

  11. The landscape of somatic mutations in infant MLL-rearranged acute lymphoblastic leukemias

    DEFF Research Database (Denmark)

    Andersson, Anna K; Ma, Jing; Wang, Jianmin;

    2015-01-01

    Infant acute lymphoblastic leukemia (ALL) with MLL rearrangements (MLL-R) represents a distinct leukemia with a poor prognosis. To define its mutational landscape, we performed whole-genome, exome, RNA and targeted DNA sequencing on 65 infants (47 MLL-R and 18 non-MLL-R cases) and 20 older childr...

  12. MLL1/WDR5 complex in leukemogenesis and epigenetic regulation

    Institute of Scientific and Technical Information of China (English)

    Min Wu; Hong-Bing Shu

    2011-01-01

    MLL1 is a histone H3Lys4 methyltransferase and forms a complex with WDR5 and other components. It plays important roles in developmental events, transcriptional regulation, and leukemogenesis. MLL1-fusion proteins resulting from chromosomal translocations are molecular hallmarks of a special type of leukemia, which occurs in over 70% infant leukemia patients and often accompanies poor prognosis. Investigations in the past years on leukemogenesis and the MLL1-WDR5 histone H3Lys4 methyltransferase complex demonstrate that epigenetic regulation is one of the key steps in development and human diseases.

  13. The MLL recombinome of acute leukemias in 2013

    DEFF Research Database (Denmark)

    Meyer, C; Hofmann, Julian; Burmeister, T;

    2013-01-01

    Chromosomal rearrangements of the human MLL (mixed lineage leukemia) gene are associated with high-risk infant, pediatric, adult and therapy-induced acute leukemias. We used long-distance inverse-polymerase chain reaction to characterize the chromosomal rearrangement of individual acute leukemia...... patients. We present data of the molecular characterization of 1590 MLL-rearranged biopsy samples obtained from acute leukemia patients. The precise localization of genomic breakpoints within the MLL gene and the involved translocation partner genes (TPGs) were determined and novel TPGs identified. All...... patients were classified according to their gender (852 females and 745 males), age at diagnosis (558 infant, 416 pediatric and 616 adult leukemia patients) and other clinical criteria. Combined data of our study and recently published data revealed a total of 121 different MLL rearrangements, of which 79...

  14. All-trans retinoic acid combined with 5-Aza-2 Prime -deoxycitidine induces C/EBP{alpha} expression and growth inhibition in MLL-AF9-positive leukemic cells

    Energy Technology Data Exchange (ETDEWEB)

    Fujiki, Atsushi [Department of Pediatrics, Graduate School of Medical Science, Kyoto Prefectural University of Medicine, Kyoto (Japan); Imamura, Toshihiko, E-mail: imamura@koto.kpu-m.ac.jp [Department of Pediatrics, Graduate School of Medical Science, Kyoto Prefectural University of Medicine, Kyoto (Japan); Sakamoto, Kenichi; Kawashima, Sachiko; Yoshida, Hideki; Hirashima, Yoshifumi; Miyachi, Mitsuru; Yagyu, Shigeki; Nakatani, Takuya [Department of Pediatrics, Graduate School of Medical Science, Kyoto Prefectural University of Medicine, Kyoto (Japan); Sugita, Kanji [Department of Pediatrics, University of Yamanashi, Yamanashi (Japan); Hosoi, Hajime [Department of Pediatrics, Graduate School of Medical Science, Kyoto Prefectural University of Medicine, Kyoto (Japan)

    2012-11-16

    Highlights: Black-Right-Pointing-Pointer We tested whether ATRA and 5-Aza affect AML cell differentiation and growth. Black-Right-Pointing-Pointer Cell differentiation and growth arrest were induced in MLL-AF9-expressing cells. Black-Right-Pointing-Pointer Increased expression of C/EBP{alpha}, C/EBP{epsilon}, and PU.1 were also observed. Black-Right-Pointing-Pointer MLL-AF4/AF5q31-expressing cells are less sensitive to ATRA and 5-Aza. Black-Right-Pointing-Pointer Different MLL fusion has distinct epigenetic properties related to RA pathway. -- Abstract: The present study tested whether all-trans retinoic acid (ATRA) and 5-Aza-2 Prime -deoxycitidine (5-Aza) affect AML cell differentiation and growth in vitro by acting on the CCAAT/enhancer binding protein {alpha} (C/EBP{alpha}) and c-Myc axis. After exposure to a combination of these agents, cell differentiation and growth arrest were significantly higher in human and murine MLL-AF9-expressing cells than in MLL-AF4/AF5q31-expressing cells, which were partly associated with increased expression of C/EBP{alpha}, C/EBP{epsilon}, and PU.1, and decreased expression of c-Myc. These findings indicate that MLL-AF9-expressing cells are more sensitive to ATRA and 5-Aza, indicating that different MLL fusion proteins possess different epigenetic properties associated with retinoic acid pathway inactivation.

  15. Simultaneous localization of MLL, AF4 and ENL genes in interphase nuclei by 3D-FISH: MLL translocation revisited

    Directory of Open Access Journals (Sweden)

    Sun Jian-Sheng

    2006-01-01

    Full Text Available Abstract Background Haematological cancer is characterised by chromosomal translocation (e.g. MLL translocation in acute leukaemia and two models have been proposed to explain the origins of recurrent reciprocal translocation. The first, established from pairs of translocated genes (such as BCR and ABL, considers the spatial proximity of loci in interphase nuclei (static "contact first" model. The second model is based on the dynamics of double strand break ends during repair processes (dynamic "breakage first" model. Since the MLL gene involved in 11q23 translocation has more than 40 partners, the study of the relative positions of the MLL gene with both the most frequent partner gene (AF4 and a less frequent partner gene (ENL, should elucidate the MLL translocation mechanism. Methods Using triple labeling 3D FISH experiments, we have determined the relative positions of MLL, AF4 and ENL genes, in two lymphoblastic and two myeloid human cell lines. Results In all cell lines, the ENL gene is significantly closer to the MLL gene than the AF4 gene (with P value loci would indicate a greater probability of the occurrence of t(11;19(q23;p13.3 compared to t(4;11(q21;q23. However this is in contradiction to the epidemiology of 11q23 translocation. Conclusion The simultaneous multi-probe hybridization in 3D-FISH is a new approach in addressing the correlation between spatial proximity and occurrence of translocation. Our observations are not consistent with the static "contact first" model of translocation. The recently proposed dynamic "breakage first" model offers an attractive alternative explanation.

  16. Requirement for CDK6 in MLL-rearranged acute myeloid leukemia.

    Science.gov (United States)

    Placke, Theresa; Faber, Katrin; Nonami, Atsushi; Putwain, Sarah L; Salih, Helmut R; Heidel, Florian H; Krämer, Alwin; Root, David E; Barbie, David A; Krivtsov, Andrei V; Armstrong, Scott A; Hahn, William C; Huntly, Brian J; Sykes, Stephen M; Milsom, Michael D; Scholl, Claudia; Fröhling, Stefan

    2014-07-01

    Chromosomal rearrangements involving the H3K4 methyltransferase mixed-lineage leukemia (MLL) trigger aberrant gene expression in hematopoietic progenitors and give rise to an aggressive subtype of acute myeloid leukemia (AML). Insights into MLL fusion-mediated leukemogenesis have not yet translated into better therapies because MLL is difficult to target directly, and the identity of the genes downstream of MLL whose altered transcription mediates leukemic transformation are poorly annotated. We used a functional genetic approach to uncover that AML cells driven by MLL-AF9 are exceptionally reliant on the cell-cycle regulator CDK6, but not its functional homolog CDK4, and that the preferential growth inhibition induced by CDK6 depletion is mediated through enhanced myeloid differentiation. CDK6 essentiality is also evident in AML cells harboring alternate MLL fusions and a mouse model of MLL-AF9-driven leukemia and can be ascribed to transcriptional activation of CDK6 by mutant MLL. Importantly, the context-dependent effects of lowering CDK6 expression are closely phenocopied by a small-molecule CDK6 inhibitor currently in clinical development. These data identify CDK6 as critical effector of MLL fusions in leukemogenesis that might be targeted to overcome the differentiation block associated with MLL-rearranged AML, and underscore that cell-cycle regulators may have distinct, noncanonical, and nonredundant functions in different contexts. PMID:24764564

  17. Zinc Finger Nuclease induced DNA double stranded breaks and rearrangements in MLL

    Energy Technology Data Exchange (ETDEWEB)

    Do, To Uyen [Graduate Group in Immunology, University of California Davis, Davis, CA 95616 (United States); Department of Radiation Oncology, University of California Davis, Sacramento CA 95817 (United States); Ho, Bay; Shih, Shyh-Jen [Department of Radiation Oncology, University of California Davis, Sacramento CA 95817 (United States); Vaughan, Andrew, E-mail: Andrew.vaughan@ucdmc.ucdavis.edu [Graduate Group in Immunology, University of California Davis, Davis, CA 95616 (United States); Department of Radiation Oncology, University of California Davis, Sacramento CA 95817 (United States)

    2012-12-15

    Highlights: ► A Zinc Finger Nuclease (ZFN) targeting a leukemogenic hot spot for rearrangement in MLL is created. ► The novel ZFN efficiently cleaves MLL exon 13. ► Despite MLL cleavage and evidence of mis-repair, no leukemogenic translocations were produced. ► MLL cleavage alone is insufficient to generate leukemogenic translocations. - Abstract: Radiation treatment or chemotherapy has been linked with a higher risk of secondary cancers such as therapy related Acute Myeloid Leukemia (tAML). Several of these cancers have been shown to be correlated to the introduction of double stranded breaks (DSB) and rearrangements within the Mixed Lineage Leukemia (MLL) gene. We used Zinc Finger Nucleases (ZFNs) to introduce precise cuts within MLL to examine how a single DNA DSB might lead to chromosomal rearrangements. A ZFN targeting exon 13 within the Breakpoint Cluster Region of MLL was transiently expressed in a human lymphoblast cell line originating from a CML patient. Although FISH analysis showed ZFN DSB at this region increased the rate of MLL fragmentation, we were unable to detect leukemogenic rearrangements or translocations via inverse PCR. Interestingly, gene fragmentation as well as small interstitial deletions, insertions and base substitutions increased with the inhibition of DNA-PK, suggesting repair of this particular DSB is linked to non-homologous end joining (NHEJ). Although mis-repair of DSBs may be necessary for the initiation of leukemogenic translocations, a MLL targeted DNA break alone is insufficient.

  18. LAF4, an AF4-related gene, is fused to MLL in infant acute lymphoblastic leukemia

    NARCIS (Netherlands)

    von Bergh, ARM; Beverloo, HB; Rombout, P; van Wering, ER; van Weel, MH; Beverstock, GC; Kluin, PM; Slater, RM; Schuuring, E

    2002-01-01

    Infant acute lymphoblastic leukemia (ALL) with MLL gene rearrangements is characterized by a proB phenotype and a poor clinical outcome. We analyzed an infant proB ALL with t(2; 11)(p 15;p 14) and an MLL rearrangement on Southern blot analysis, Rapid amplification of cDNA ends-polymerase chain react

  19. HGF-MET signals via the MLL-ETS2 complex in hepatocellular carcinoma.

    Science.gov (United States)

    Takeda, Shugaku; Liu, Han; Sasagawa, Satoru; Dong, Yiyu; Trainor, Paul A; Cheng, Emily H; Hsieh, James J

    2013-07-01

    HGF signals through its cognate receptor, MET, to orchestrate diverse biological processes, including cell proliferation, cell fate specification, organogenesis, and epithelial-mesenchymal transition. Mixed-lineage leukemia (MLL), an epigenetic regulator, plays critical roles in cell fate, stem cell, and cell cycle decisions. Here, we describe a role for MLL in the HGF-MET signaling pathway. We found a shared phenotype among Mll(-/-), Hgf(-/-), and Met(-/-) mice with common cranial nerve XII (CNXII) outgrowth and myoblast migration defects. Phenotypic analysis demonstrated that MLL was required for HGF-induced invasion and metastatic growth of hepatocellular carcinoma cell lines. HGF-MET signaling resulted in the accumulation of ETS2, which interacted with MLL to transactivate MMP1 and MMP3. ChIP assays demonstrated that activation of the HGF-MET pathway resulted in increased occupancy of the MLL-ETS2 complex on MMP1 and MMP3 promoters, where MLL trimethylated histone H3 lysine 4 (H3K4), activating transcription. Our results present an epigenetic link between MLL and the HGF-MET signaling pathway, which may suggest new strategies for therapeutic intervention. PMID:23934123

  20. The Histone Methyltransferase Activity of MLL1 Is Dispensable for Hematopoiesis and Leukemogenesis

    Directory of Open Access Journals (Sweden)

    Bibhu P. Mishra

    2014-05-01

    Full Text Available Despite correlations between histone methyltransferase (HMT activity and gene regulation, direct evidence that HMT activity is responsible for gene activation is sparse. We address the role of the HMT activity for MLL1, a histone H3 lysine 4 (H3K4 methyltransferase critical for maintaining hematopoietic stem cells (HSCs. Here, we show that the SET domain, and thus HMT activity of MLL1, is dispensable for maintaining HSCs and supporting leukemogenesis driven by the MLL-AF9 fusion oncoprotein. Upon Mll1 deletion, histone H4 lysine 16 (H4K16 acetylation is selectively depleted at MLL1 target genes in conjunction with reduced transcription. Surprisingly, inhibition of SIRT1 is sufficient to prevent the loss of H4K16 acetylation and the reduction in MLL1 target gene expression. Thus, recruited MOF activity, and not the intrinsic HMT activity of MLL1, is central for the maintenance of HSC target genes. In addition, this work reveals a role for SIRT1 in opposing MLL1 function.

  1. Identification of leukemia-associated genes by MLL-EEN fusion protein through dysregulation of histone modification and DNA methylation

    OpenAIRE

    Lui, Wing-chi; 呂穎芝

    2012-01-01

    Mixed lineage leukemia (MLL) gene undergoes chromosomal translocation with over 60 different fusion partner genes in human leukemias. The resultant MLL-fusion oncoproteins are profoundly implicated in leukemias with poor prognosis. Epigenetic dysregulations have been frequently reported in MLL-rearranged leukemogenesis. Our study aims to investigate the correlations between epigenetic alterations, including both histone modification and DNA methylation, and gene dysregulation in MLL-rearrange...

  2. A PTIP-PA1 subcomplex promotes transcription for IgH class switching independently from the associated MLL3/MLL4 methyltransferase complex.

    Science.gov (United States)

    Starnes, Linda M; Su, Dan; Pikkupeura, Laura M; Weinert, Brian T; Santos, Margarida A; Mund, Andreas; Soria, Rebeca; Cho, Young-Wook; Pozdnyakova, Irina; Kubec Højfeldt, Martina; Vala, Andrea; Yang, Wenjing; López-Méndez, Blanca; Lee, Ji-Eun; Peng, Weiqun; Yuan, Joan; Ge, Kai; Montoya, Guillermo; Nussenzweig, André; Choudhary, Chunaram; Daniel, Jeremy A

    2016-01-15

    Class switch recombination (CSR) diversifies antibodies for productive immune responses while maintaining stability of the B-cell genome. Transcription at the immunoglobulin heavy chain (Igh) locus targets CSR-associated DNA damage and is promoted by the BRCT domain-containing PTIP (Pax transactivation domain-interacting protein). Although PTIP is a unique component of the mixed-lineage leukemia 3 (MLL3)/MLL4 chromatin-modifying complex, the mechanisms for how PTIP promotes transcription remain unclear. Here we dissected the minimal structural requirements of PTIP and its different protein complexes using quantitative proteomics in primary lymphocytes. We found that PTIP functions in transcription and CSR separately from its association with the MLL3/MLL4 complex and from its localization to sites of DNA damage. We identified a tandem BRCT domain of PTIP that is sufficient for CSR and identified PA1 as its main functional protein partner. Collectively, we provide genetic and biochemical evidence that a PTIP-PA1 subcomplex functions independently from the MLL3/MLL4 complex to mediate transcription during CSR. These results further our understanding of how multifunctional chromatin-modifying complexes are organized by subcomplexes that harbor unique and distinct activities. PMID:26744420

  3. A PTIP–PA1 subcomplex promotes transcription for IgH class switching independently from the associated MLL3/MLL4 methyltransferase complex

    Science.gov (United States)

    Starnes, Linda M.; Su, Dan; Pikkupeura, Laura M.; Weinert, Brian T.; Santos, Margarida A.; Mund, Andreas; Soria, Rebeca; Cho, Young-Wook; Pozdnyakova, Irina; Kubec Højfeldt, Martina; Vala, Andrea; Yang, Wenjing; López-Méndez, Blanca; Lee, Ji-Eun; Peng, Weiqun; Yuan, Joan; Ge, Kai; Montoya, Guillermo; Nussenzweig, André; Choudhary, Chunaram; Daniel, Jeremy A.

    2016-01-01

    Class switch recombination (CSR) diversifies antibodies for productive immune responses while maintaining stability of the B-cell genome. Transcription at the immunoglobulin heavy chain (Igh) locus targets CSR-associated DNA damage and is promoted by the BRCT domain-containing PTIP (Pax transactivation domain-interacting protein). Although PTIP is a unique component of the mixed-lineage leukemia 3 (MLL3)/MLL4 chromatin-modifying complex, the mechanisms for how PTIP promotes transcription remain unclear. Here we dissected the minimal structural requirements of PTIP and its different protein complexes using quantitative proteomics in primary lymphocytes. We found that PTIP functions in transcription and CSR separately from its association with the MLL3/MLL4 complex and from its localization to sites of DNA damage. We identified a tandem BRCT domain of PTIP that is sufficient for CSR and identified PA1 as its main functional protein partner. Collectively, we provide genetic and biochemical evidence that a PTIP–PA1 subcomplex functions independently from the MLL3/MLL4 complex to mediate transcription during CSR. These results further our understanding of how multifunctional chromatin-modifying complexes are organized by subcomplexes that harbor unique and distinct activities. PMID:26744420

  4. HoxBlinc RNA Recruits Set1/MLL Complexes to Activate Hox Gene Expression Patterns and Mesoderm Lineage Development

    Directory of Open Access Journals (Sweden)

    Changwang Deng

    2016-01-01

    Full Text Available Trithorax proteins and long-intergenic noncoding RNAs are critical regulators of embryonic stem cell pluripotency; however, how they cooperatively regulate germ layer mesoderm specification remains elusive. We report here that HoxBlinc RNA first specifies Flk1+ mesoderm and then promotes hematopoietic differentiation through regulation of hoxb pathways. HoxBlinc binds to the hoxb genes, recruits Setd1a/MLL1 complexes, and mediates long-range chromatin interactions to activate transcription of the hoxb genes. Depletion of HoxBlinc by shRNA-mediated knockdown or CRISPR-Cas9-mediated genetic deletion inhibits expression of hoxb genes and other factors regulating cardiac/hematopoietic differentiation. Reduced hoxb expression is accompanied by decreased recruitment of Set1/MLL1 and H3K4me3 modification, as well as by reduced chromatin loop formation. Re-expression of hoxb2–b4 genes in HoxBlinc-depleted embryoid bodies rescues Flk1+ precursors that undergo hematopoietic differentiation. Thus, HoxBlinc plays an important role in controlling hoxb transcription networks that mediate specification of mesoderm-derived Flk1+ precursors and differentiation of Flk1+ cells into hematopoietic lineages.

  5. Nucleoporin Nup98 Associates with Trx/MLL and NSL Histone-Modifying Complexes and Regulates Hox Gene Expression

    Directory of Open Access Journals (Sweden)

    Pau Pascual-Garcia

    2014-10-01

    Full Text Available The nuclear pore complex is a transport channel embedded in the nuclear envelope and made up of 30 different components termed nucleoporins (Nups. In addition to their classical role in transport, a subset of Nups has a conserved role in the regulation of transcription via direct binding to chromatin. The molecular details of this function remain obscure, and it is unknown how metazoan Nups are recruited to their chromatin locations or what transcription steps they regulate. Here, we demonstrate genome-wide and physical association between Nup98 and histone-modifying complexes MBR-R2/NSL and Trx/MLL. Importantly, we identify a requirement for MBD-R2 in recruitment of Nup98 to many of its genomic target sites. Consistent with its interaction with the Trx/MLL complex, Nup98 is shown to be necessary for Hox gene expression in developing fly tissues. These findings introduce roles of Nup98 in epigenetic regulation that may underlie the basis of oncogenicity of Nup98 fusions in leukemia.

  6. The potential of clofarabine in MLL-rearranged infant acute lymphoblastic leukaemia.

    Science.gov (United States)

    Stumpel, Dominique J P M; Schneider, Pauline; Pieters, Rob; Stam, Ronald W

    2015-09-01

    MLL-rearranged acute lymphoblastic leukaemia (ALL) in infants is the most difficult-to-treat type of childhood ALL, displaying a chemotherapy-resistant phenotype, and unique histone modifications, gene expression signatures and DNA methylation patterns. MLL-rearranged infant ALL responds remarkably well to nucleoside analogue drugs in vitro, such as cytarabine and cladribine, and to the demethylating agents decitabine and zebularine as measured by cytotoxicity assays. These observations led to the inclusion of cytarabine into the treatment regimens currently used for infants with ALL. However, survival chances for infants with MLL-rearranged ALL do still not exceed 30-40%. Here we explored the in vitro potential of the novel nucleoside analogue clofarabine for MLL-rearranged infant ALL. Therefore we used both cell line models as well as primary patient cells. Compared with other nucleoside analogues, clofarabine effectively targeted primary MLL-rearranged infant ALL cells at the lowest concentrations, with median LC50 values of ∼25 nM. Interestingly, clofarabine displayed synergistic cytotoxic effects in combination with cytarabine. Furthermore, at concentrations of 5-10nM clofarabine induced demethylation of the promoter region of the tumour suppressor gene FHIT (Fragile Histidine Triad), a gene typically hypermethylated in MLL-rearranged ALL. Demethylation of the FHIT promoter region was accompanied by subtle re-expression of this gene both at the mRNA and protein level. We conclude that clofarabine is an interesting candidate for further studies in MLL-rearranged ALL in infants. PMID:26188848

  7. Spectrum of MLL2 (ALR) mutations in 110 cases of Kabuki syndrome

    Science.gov (United States)

    Hannibal, Mark C.; Buckingham, Kati J.; Ng, Sarah B.; Ming, Jeffrey E.; Beck, Anita E.; McMillin, Margaret J.; Gildersleeve, Heidi I.; Bigham, Abigail W.; Tabor, Holly K.; Mefford, Heather C.; Cook, Joseph; Yoshiura, Koh-ichiro; Matsumoto, Tadashi; Matsumoto, Naomichi; Miyake, Noriko; Tonoki, Hidefumi; Naritomi, Kenji; Kaname, Tadashi; Nagai, Toshiro; Ohashi, Hirofumi; Kurosawa, Kenji; Hou, Jia-Wei; Ohta, Tohru; Liang, Deshung; Sudo, Akira; Morris, Colleen A.; Banka, Siddharth; Black, Graeme C.; Clayton-Smith, Jill; Nickerson, Deborah A.; Zackai, Elaine H.; Shaikh, Tamim H.; Donnai, Dian; Niikawa, Norio; Shendure, Jay; Bamshad, Michael J.

    2011-01-01

    Kabuki syndrome is a rare, multiple malformation disorder characterized by a distinctive facial appearance, cardiac anomalies, skeletal abnormalities, and mild to moderate intellectual disability. Simplex cases make up the vast majority of the reported cases with Kabuki syndrome, but parent-to-child transmission in more than a half-dozen instances indicates that it is an autosomal dominant disorder. We recently reported that Kabuki syndrome is caused by mutations in MLL2, a gene that encodes a Trithorax-group histone methyltransferase, a protein important in the epigenetic control of active chromatin states. Here, we report on the screening of 110 families with Kabuki syndrome. MLL2 mutations were found in 81/110 (74%) of families. In simplex cases for which DNA was available from both parents, 25 mutations were confirmed to be de novo, while a transmitted MLL2 mutation was found in two of three familial cases. The majority of variants found to cause Kabuki syndrome were novel nonsense or frameshift mutations that are predicted to result in haploinsufficiency. The clinical characteristics of MLL2 mutation-positive cases did not differ significantly from MLL2 mutation-negative cases with the exception that renal anomalies were more common in MLL2 mutation-positive cases. These results are important for understanding the phenotypic consequences of MLL2 mutations for individuals and their families as well as for providing a basis for the identification of additional genes for Kabuki syndrome. PMID:21671394

  8. Role of c-Myb in chondrogenesis

    Czech Academy of Sciences Publication Activity Database

    Oralová, Veronika; Matalová, Eva; Janečková, Eva; Drobná Krejčí, E.; Knopfová, L.; Šnajdr, P.; Tucker, A. S.; Veselá, I.; Šmarda, J.; Buchtová, Marcela

    2015-01-01

    Roč. 76, č. 1 (2015), s. 97-106. ISSN 8756-3282 R&D Projects: GA ČR GCP302/12/J059; GA ČR GB14-37368G Institutional support: RVO:67985904 Keywords : micromass cultures * mouse limbs * endochondral bone Subject RIV: EA - Cell Biology Impact factor: 3.973, year: 2014

  9. Structure-based design and synthesis of small molecular inhibitors disturbing the interaction of MLL1-WDR5.

    Science.gov (United States)

    Li, Dong-Dong; Chen, Wei-Lin; Xu, Xiao-Li; Jiang, Fen; Wang, Lei; Xie, Yi-Yue; Zhang, Xiao-Jin; Guo, Xiao-Ke; You, Qi-Dong; Sun, Hao-Peng

    2016-08-01

    MLL1 complex catalyzes the methylation of H3K4, and plays important roles in the development of acute leukemia harboring MLL fusion proteins. Targeting MLL1-WDR5 protein-protein interaction (PPI) to inhibit the activity of histone methyltransferase of MLL1 complex is a novel strategy for treating of acute leukemia. WDR5-47 (IC50 = 0.3 μM) was defined as a potent small molecule to disturb the interaction of MLL1-WDR5. Here, we described structure-based design and synthesis of small molecular inhibitors to block MLL1-WDR5 PPI. Especially, compound 23 (IC50 = 104 nM) was the most potent small molecular, and about 3-times more potent than WDR5-47. We also discussed the SAR of these series of compounds with docking study, which may stimulate more potent compounds. PMID:27116709

  10. Validation and Structural Characterization of the LEDGF/p75-MLL Interface as a New Target for the Treatment of MLL-Dependent Leukemia

    Czech Academy of Sciences Publication Activity Database

    Čermáková, K.; Těšina, Petr; Demeulemeester, J.; El Ashkar, S.; Méreau, H.; Schwaller, J.; Řezáčová, Pavlína; Veverka, Václav; De Rijck, J.

    2014-01-01

    Roč. 74, č. 18 (2014), s. 5139-5151. ISSN 0008-5472 R&D Projects: GA MŠk(CZ) LK11205; GA MŠk(CZ) 7E08066; GA MŠk LO1302 Institutional support: RVO:61388963 Keywords : MLL * leukemia * LEDGF/p75 Subject RIV: CE - Biochemistry Impact factor: 9.329, year: 2014

  11. Histone H3K4 trimethylation by MLL3 as part of ASCOM complex is critical for NR activation of bile acid transporter genes and is downregulated in cholestasis

    OpenAIRE

    Ananthanarayanan, M.; Li, Yanfeng; Surapureddi, S.; Balasubramaniyan, N; Ahn, Jaeyong; Goldstein, J. A.; Suchy, Frederick J.

    2010-01-01

    The nuclear receptor Farnesoid x receptor (FXR) is a critical regulator of multiple genes involved in bile acid homeostasis. The coactivators attracted to promoters of FXR target genes and epigenetic modifications that occur after ligand binding to FXR have not been completely defined, and it is unknown whether these processes are disrupted during cholestasis. Using a microarray, we identified decreased expression of mixed lineage leukemia 3 (MLL3), a histone H3 lysine 4 (H3K4) lysine methyl ...

  12. Mutation spectrum of MLL2 in a cohort of kabuki syndrome patients

    Directory of Open Access Journals (Sweden)

    Renieri Alessandra

    2011-06-01

    Full Text Available Abstract Background Kabuki syndrome (Niikawa-Kuroki syndrome is a rare, multiple congenital anomalies/mental retardation syndrome characterized by a peculiar face, short stature, skeletal, visceral and dermatoglyphic abnormalities, cardiac anomalies, and immunological defects. Recently mutations in the histone methyl transferase MLL2 gene have been identified as its underlying cause. Methods Genomic DNAs were extracted from 62 index patients clinically diagnosed as affected by Kabuki syndrome. Sanger sequencing was performed to analyze the whole coding region of the MLL2 gene including intron-exon junctions. The putative causal and possible functional effect of each nucleotide variant identified was estimated by in silico prediction tools. Results We identified 45 patients with MLL2 nucleotide variants. 38 out of the 42 variants were never described before. Consistently with previous reports, the majority are nonsense or frameshift mutations predicted to generate a truncated polypeptide. We also identified 3 indel, 7 missense and 3 splice site. Conclusions This study emphasizes the relevance of mutational screening of the MLL2 gene among patients diagnosed with Kabuki syndrome. The identification of a large spectrum of MLL2 mutations possibly offers the opportunity to improve the actual knowledge on the clinical basis of this multiple congenital anomalies/mental retardation syndrome, design functional studies to understand the molecular mechanisms underlying this disease, establish genotype-phenotype correlations and improve clinical management.

  13. A human ESC model for MLL-AF4 leukemic fusion gene reveals an impaired early hematopoietic-endothelial specification

    Institute of Scientific and Technical Information of China (English)

    Clara Bueno; Agustin F Femández; Mario F Fraga; Inmaculada Moreno-Gimeno; Deborah Burks; Maria del Carmen Plaza-Calonge; Juan C Rodríguez-Manzaneque; Pablo Menendez; Rosa Montes; Gustavo J Melen; Verónica Ramos-Mejia; Pedro J Real; Verónica Ayllón; Laura Sanchez; Gertrudis Ligero; Iván Gutierrez-Aranda

    2012-01-01

    The MLL-AF4 fusion gene is a hallmark genomic aberration in high-risk acute lymphoblastic leukemia in inants.Although it is well established that MLL-AF4 arises prenatally during human development,its effects on hematopoieric development in utero remain unexplored.We have created a human-specific cellular system to study early hemato-endothelial development in MLL-AF4-expressing human embryonic stem cells (hESCs).Functional studies,clonal analysis and gene expression profiling reveal that expression of MLL-AF4 in hESCs has a phenotypic,functional and gene expression impact.MLL-AF4 acts as a global transcriptional activator and a positive regulator of homeobox gene expression in hESCs.Functionally,MLL-AF4 enhances the specification of hemogenic precursors from hESCs but strongly impairs further hematopoietic commitment in favor of an endothelial cell fate.MLL-AF4 hESCs are transcriptionally primed to differentiate towards hemogenic precursors prone to endothelial maturation,as reflected by the marked upregulation of master genes associated to vascular-endothelial functions and early hematopoiesis.Furthermore,we report that MLL-AF4 expression is not sufficient to transform hESC-derived hematopoietic cells.This work illustrates how hESCs may provide unique insights into human development and further our understanding of how leukemic fusion genes,known to arise prenatally,regulate human embryonic hematopoietic specification.

  14. MLL becomes functional through intra-molecular interaction not by proteolytic processing.

    Directory of Open Access Journals (Sweden)

    Akihiko Yokoyama

    Full Text Available The mixed lineage leukemia (MLL protein is an epigenetic transcriptional regulator that controls proliferative expansion of immature hematopoietic progenitors, whose aberrant activation triggers leukemogenesis. A mature MLL protein is produced by formation of an intra-molecular complex and proteolytic cleavage. However the biological significance of these two post-transcriptional events remains unclear. To address their in vivo roles, mouse mutant alleles were created that exclusively express either a variant protein incapable of intra-molecular interaction (designated de or an uncleavable mutant protein (designated uc. The de homozygous mice died during midgestation and manifested devastating failure in embryonic development and reduced numbers of hematopoietic progenitors, whereas uc homozygous mice displayed no apparent defects. Expression of MLL target genes was severely impaired in de homozygous fibroblasts but unaffected in uc homozygous fibroblasts. These results unequivocally demonstrate that intra-molecular complex formation is a crucial maturation step whereas proteolytic cleavage is dispensable for MLL-dependent gene activation and proliferation in vivo.

  15. Novel variants in MLL confer to bladder cancer recurrence identified by whole-exome sequencing

    Science.gov (United States)

    Wang, Yongqiang; Huang, Yi; Liu, Huan; Li, Feida; He, Luyun; Sun, Da; Yu, Yuan; Li, Qiaoling; Huang, Peide; Zhang, Meng; Zhao, Xin; Bi, Tengteng; Zhuang, Xuehan; Zhang, Liyan; Lu, Jingxiao; Sun, Xiaojuan; Zhou, Fangjian; Liu, Chunxiao; Yang, Guosheng; Hou, Yong; Fan, Zusen; Cai, Zhiming

    2016-01-01

    Bladder cancer (BC) is distinguished by high rate of recurrence after surgery, but the underlying mechanisms remain poorly understood. Here we performed the whole-exome sequencing of 37 BC individuals including 20 primary and 17 recurrent samples in which the primary and recurrent samples were not from the same patient. We uncovered that MLL, EP400, PRDM2, ANK3 and CHD5 exclusively altered in recurrent BCs. Specifically, the recurrent BCs and bladder cancer cells with MLL mutation displayed increased histone H3 tri-methyl K4 (H3K4me3) modification in tissue and cell levels and showed enhanced expression of GATA4 and ETS1 downstream. What's more, MLL mutated bladder cancer cells obtained with CRISPR/Cas9 showed increased ability of drug-resistance to epirubicin (a chemotherapy drug for bladder cancer) than wild type cells. Additionally, the BC patients with high expression of GATA4 and ETS1 significantly displayed shorter lifespan than patients with low expression. Our study provided an overview of the genetic basis of recrudescent bladder cancer and discovered that genetic alterations of MLL were involved in BC relapse. The increased modification of H3K4me3 and expression of GATA4 and ETS1 would be the promising targets for the diagnosis and therapy of relapsed bladder cancer. PMID:26625313

  16. MLL-AF9-mediated immortalization of human hematopoietic cells along different lineages changes during ontogeny

    NARCIS (Netherlands)

    Horton, S J; Jaques, J; Woolthuis, C; van Dijk, J; Mesuraca, M; Huls, G; Morrone, G; Vellenga, E; Schuringa, J J

    2013-01-01

    The MLL-AF9 fusion gene is associated with aggressive leukemias of both the myeloid and lymphoid lineage in infants, whereas in adults, this translocation is mainly associated with acute myeloid leukemia. These observations suggest that differences exist between fetal and adult tissues in terms of t

  17. MLL-AF9-mediated immortalization of human hematopoietic cells along different lineages changes during ontogeny.

    NARCIS (Netherlands)

    Horton, S.J.; Jaques, J.; Woolthuis, C.; Dijk, J. van; Mesuraca, M.; Huls, G.A.; Morrone, G.; Vellenga, E.; Schuringa, J.J.

    2013-01-01

    The MLL-AF9 fusion gene is associated with aggressive leukemias of both the myeloid and lymphoid lineage in infants, whereas in adults, this translocation is mainly associated with acute myeloid leukemia. These observations suggest that differences exist between fetal and adult tissues in terms of t

  18. A novel spliced fusion of MLL with CT45A2 in a pediatric biphenotypic acute leukemia

    International Nuclear Information System (INIS)

    Abnormalities of 11q23 involving the MLL gene are found in approximately 10% of human leukemias. To date, nearly 100 different chromosome bands have been described in rearrangements involving 11q23 and 64 fusion genes have been cloned and characterized at the molecular level. In this work we present the identification of a novel MLL fusion partner in a pediatric patient with de novo biphenotypic acute leukemia. Cytogenetics, fluorescence in situ hybridization (FISH), molecular studies (RT-PCR and LDI-PCR), and bioinformatic sequence analysis were used to characterize the CT45A2 gene as novel MLL fusion partner in pediatric acute leukemia. Fluorescence in situ hybridization of bone marrow G-banded metaphases demonstrated a cryptic insertion of 11q23 in Xq26.3 involving the MLL gene. Breakpoint fusion analysis revealed that a DNA fragment of 653 kb from 11q23, containing MLL exons 1-9 in addition to 16 other 11q23 genes, was inserted into the upstream region of the CT45A2 gene located at Xq26.3. In addition, a deletion at Xq26.3 encompassing the 3' region of the DDX26B gene (exons 9-16) and the entire CT45A1 gene was identified. RNA analysis revealed the presence of a novel MLL-CT45A2 fusion transcript in which the first 9 exons of the MLL gene were fused in-frame to exon 2 of the CT45A2 gene, resulting in a spliced MLL fusion transcript with an intact open reading frame. The resulting chimeric transcript predicts a fusion protein where the N-terminus of MLL is fused to the entire open reading frame of CT45A2. Finally, we demonstrate that all breakpoint regions are rich in long repetitive motifs, namely LINE/L1 and SINE/Alu sequences, but all breakpoints were exclusively identified outside these repetitive DNA sequences. We have identified CT45A2 as a novel spliced MLL fusion partner in a pediatric patient with de novo biphenotypic acute leukemia, as a result of a cryptic insertion of 11q23 in Xq26.3. Since CT45A2 is the first Cancer/Testis antigen family gene

  19. AF4 uses the SL1 components of RNAP1 machinery to initiate MLL fusion- and AEP-dependent transcription

    OpenAIRE

    Okuda, Hiroshi; Kanai, Akinori; Ito, Shinji; Matsui, Hirotaka; Yokoyama, Akihiko

    2015-01-01

    Gene rearrangements generate MLL fusion genes, which can lead to aggressive leukemia. In most cases, MLL fuses with a gene encoding a component of the AEP (AF4 family/ENL family/P-TEFb) coactivator complex. MLL–AEP fusion proteins constitutively activate their target genes to immortalize haematopoietic progenitors. Here we show that AEP and MLL–AEP fusion proteins activate transcription through selectivity factor 1 (SL1), a core component of the pre-initiation complex (PIC) of RNA polymerase ...

  20. TGIF1 is a negative regulator of MLL-rearranged acute myeloid leukemia

    DEFF Research Database (Denmark)

    Willer, Anton; Jakobsen, Janus Schou; Ohlsson, E;

    2015-01-01

    orchestrates a transcriptional program required for the maintenance of MLL-rearranged acute myeloid leukemia (AML). TGIF1/TGIF2 are relatively uncharacterized TALE transcription factors, which, in contrast to the remaining family, have been shown to act as transcriptional repressors. Given the general......-AF9-transformed cells promoted differentiation and cell cycle exit in vitro, and delayed leukemic onset in vivo. Mechanistically, we show that TGIF1 interferes with a MEIS1-dependent transcriptional program by associating with MEIS1-bound regions in a competitive manner and that the MEIS1:TGIF1 ratio...... influence the clinical outcome. Collectively, these findings demonstrate that TALE family members can act both positively and negatively on transcriptional programs responsible for leukemic maintenance and provide novel insights into the regulatory gene expression circuitries in MLL-rearranged AML...

  1. Spectrum of MLL2 (ALR) mutations in 110 cases of Kabuki syndrome

    OpenAIRE

    Hannibal, Mark C.; Buckingham, Kati J.; Ng, Sarah B.; Ming, Jeffrey E.; Beck, Anita E.; McMillin, Margaret J.; Gildersleeve, Heidi I.; Bigham, Abigail W.; Tabor, Holly K.; Mefford, Heather C.; Cook, Joseph; Yoshiura, Koh-ichiro; Matsumoto, Tadashi; Matsumoto, Naomichi; Miyake, Noriko

    2011-01-01

    Kabuki syndrome is a rare, multiple malformation disorder characterized by a distinctive facial appearance, cardiac anomalies, skeletal abnormalities, and mild to moderate intellectual disability. Simplex cases make up the vast majority of the reported cases with Kabuki syndrome, but parent-to-child transmission in more than a half-dozen instances indicates that it is an autosomal dominant disorder. We recently reported that Kabuki syndrome is caused by mutations in MLL2, a gene that encodes ...

  2. Infrequent Manifestations of Kabuki Syndrome in a Patient with Novel MLL2 Mutation

    OpenAIRE

    Zarate, Y.A.; Zhan, H.; Jones, J R

    2012-01-01

    We present a case of a 9-month-old Hispanic female with Kabuki syndrome with some infrequent manifestations including a single umbilical artery, butterfly vertebrae, a small larynx, a preauricular pit, microtia with internal ear abnormalities, abnormal calcium metabolism, premature thelarche, neonatal/persistent hypoglycemia and eventration of the diaphragm. She was found to have a previously unreported nonsense MLL2 mutation. This is the first case that includes all such findings occurring s...

  3. Roles for common MLL/COMPASS subunits and the 19S proteasome in regulating CIITA pIV and MHC class II gene expression and promoter methylation

    Directory of Open Access Journals (Sweden)

    Koues Olivia I

    2010-02-01

    Full Text Available Abstract Background Studies indicate that the 19S proteasome contributes to chromatin reorganization, independent of the role the proteasome plays in protein degradation. We have previously shown that components of the 19S proteasome are crucial for regulating inducible histone activation events in mammalian cells. The 19S ATPase Sug1 binds to histone-remodeling enzymes, and in the absence of Sug1, a subset of activating epigenetic modifications including histone H3 acetylation, H3 lysine 4 trimethylation and H3 arginine 17 dimethylation are inhibited at cytokine-inducible major histocompatibilty complex (MHC-II and class II transactivator (CIITA promoters, implicating Sug1 in events required to initiate mammalian transcription. Results Our previous studies indicate that H3 lysine 4 trimethylation at cytokine-inducible MHC-II and CIITA promoters is dependent on proteolytic-independent functions of 19S ATPases. In this report, we show that multiple common subunits of the mixed lineage leukemia (MLL/complex of proteins associated with Set I (COMPASS complexes bind to the inducible MHC-II and CIITA promoters; that overexpressing a single common MLL/COMPASS subunit significantly enhances promoter activity and MHC-II HLA-DRA expression; and that these common subunits are important for H3 lysine 4 trimethylation at MHC-II and CIITA promoters. In addition, we show that H3 lysine 27 trimethylation, which is inversely correlated with H3 lysine 4 trimethylation, is significantly elevated in the presence of diminished 19S ATPase Sug1. Conclusion Taken together, these experiments suggest that the 19S proteasome plays a crucial role in the initial reorganization of events enabling the relaxation of the repressive chromatin structure surrounding inducible promoters.

  4. MLL-rearranged acute lymphoblastic leukaemia stem cell interactions with bone marrow stroma promote survival and therapeutic resistance that can be overcome with CXCR4 antagonism

    OpenAIRE

    Sison, Edward Allan R; Rau, Rachel E.; McIntyre, Emily; LI Li; Small, Donald; Brown, Patrick

    2013-01-01

    Infants with MLL-rearranged (MLL-R) acute lymphoblastic leukaemia (ALL) have a dismal prognosis. While most patients achieve remission, approximately half of patients recur with a short latency to relapse. This suggests that chemotherapy-resistant leukaemia stem cells (LSCs) survive and can recapitulate the leukaemia. We hypothesized that interactions between LSCs and the bone marrow microenvironment mediate survival and chemotherapy resistance in MLL-R ALL. Using primary samples of infant ML...

  5. A complex MLL rearrangement identified five years after initial MDS diagnosis results in out-of-frame fusions without progression to acute leukemia.

    Science.gov (United States)

    Meyer, Claus; Kowarz, Eric; Yip, Sze-Fai; Wan, Thomas Shek-Kong; Chan, Tai-Kwong; Dingermann, Theo; Chan, Li-Chong; Marschalek, Rolf

    2011-10-01

    Chromosomal rearrangements of the MLL gene are uncommon in myelodysplastic syndromes (MDSs), and few studies of their molecular structures and oncogenic mechanisms exist. Here, we present a case of de novo MDS with a normal karyotype at initial diagnosis and a mild clinical course. Five years after the initial diagnosis, investigators identified a complex rearrangement of the MLL gene without progression to acute leukemia. The 5' part of the MLL gene is fused out of frame with the LOC100131626 gene, and the 3' part of the MLL gene out of frame with the TCF12 gene. Rapid amplification of complementary DNA 3' ends yielded two main fusion transcripts, which is in concordance with the two described isoforms of the LOC100131626 gene. For both isoform-fusion transcripts, the open reading frame terminates shortly after the breakpoint that is predicted to form two de facto truncated MLL proteins and disrupts the open reading frame of the LOC100131626, TCF12, and UBE4A genes. Neither dimerization nor a transcriptional activation domain, each of which is causally linked to MLL protein-mediated transformation, is present. This and other unusual MLL rearrangements probably represent a subclass of MLL gene abnormalities that have intrinsically no ability or only a weak ability to transform hematopoeitic cells and are identified only in the context of other hematopoetic malignancies. PMID:22137486

  6. EVI1 is critical for the pathogenesis of a subset of MLL-AF9–rearranged AMLs

    OpenAIRE

    Bindels, Eric M. J.; Havermans, Marije; Lugthart, Sanne; Erpelinck, Claudia; Wocjtowicz, Elizabeth; Krivtsov, Andrei V.; Rombouts, Elwin; Armstrong, Scott A; Taskesen, Erdogan; Haanstra, Jurgen R.; Beverloo, H. Berna; Döhner, Hartmut; Hudson, Wendy A.; Kersey, John H.; Delwel, Ruud

    2012-01-01

    The proto-oncogene EVI1 (ecotropic viral integration site-1), located on chromosome band 3q26, is aberrantly expressed in human acute myeloid leukemia (AML) with 3q26 rearrangements. In the current study, we showed, in a large AML cohort carrying 11q23 translocations, that ∼ 43% of all mixed lineage leukemia (MLL)–rearranged leukemias are EVI1pos. High EVI1 expression occurs in AMLs expressing the MLL-AF6, -AF9, -AF10, -ENL, or -ELL fusion genes. In addition, we present evidence that EVI1pos ...

  7. RUNX1 Is a Key Target in t(4;11 Leukemias that Contributes to Gene Activation through an AF4-MLL Complex Interaction

    Directory of Open Access Journals (Sweden)

    Adam C. Wilkinson

    2013-01-01

    Full Text Available The Mixed Lineage Leukemia (MLL protein is an important epigenetic regulator required for the maintenance of gene activation during development. MLL chromosomal translocations produce novel fusion proteins that cause aggressive leukemias in humans. Individual MLL fusion proteins have distinct leukemic phenotypes even when expressed in the same cell type, but how this distinction is delineated on a molecular level is poorly understood. Here, we highlight a unique molecular mechanism whereby the RUNX1 gene is directly activated by MLL-AF4 and the RUNX1 protein interacts with the product of the reciprocal AF4-MLL translocation. These results support a mechanism of transformation whereby two oncogenic fusion proteins cooperate by activating a target gene and then modulating the function of its downstream product.

  8. [Refractory primary myeloid sarcoma of the breast with MLL-AF9 rearrangement].

    Science.gov (United States)

    Uchida, Emi; Watanabe, Ken; Oshikawa, Gaku; Sakashita, Chizuko; Kurosu, Tetsuya; Fukuda, Tetsuya; Arai, Ayako; Murakami, Naomi; Miura, Osamu; Yamamoto, Masahide

    2016-01-01

    A 28-year-old woman presented with a right breast mass and axillary lymphadenopathy. Biopsy of the breast mass revealed myeloid sarcoma (MS) staining positive for CD4, CD13, CD33, and CD68/KP-1. Bone marrow aspiration revealed leukemic cell infiltration (9%). Leukemic cells possessed cytogenetic abnormalities of +8 and t(9;11)(p22;q23) with +22 (lymph node only), and molecular analyses confirmed the MLL-AF9 fusion gene. After induction chemotherapy and 2(nd) consolidation therapy, complete remission was maintained. However, during consolidation radiotherapy for the breast mass, the disease progressed in both the breast and bone marrow. She received re-induction therapy and proceeded to allogeneic stem cell transplantation. However, the disease relapsed in the breast soon after transplantation, and she died from disease progression. Trisomy 8 and the MLL-AF9 fusion gene have been reported in cases with MS in the breast. Trisomy 22 found additionally and exclusively in the extramedullary lesion implies extramedullary progression of MS from the medullary site of origin and may have been associated with the distinctive therapy resistance of these lesions in our case. PMID:26861104

  9. The broken MLL gene is frequently located outside the inherent chromosome territory in human lymphoid cells treated with DNA topoisomerase II poison etoposide.

    Directory of Open Access Journals (Sweden)

    Sergey I Glukhov

    Full Text Available The mixed lineage leukaemia (MLL gene is frequently rearranged in secondary leukaemias, in which it could fuse to a variety of different partners. Breakage in the MLL gene preferentially occurs within a ~8 kb region that possesses a strong DNA topoisomerase II cleavage site. It has been proposed that DNA topoisomerase II-mediated DNA cleavage within this and other regions triggers translocations that occur due to incorrect joining of broken DNA ends. To further clarify a possible mechanism for MLL rearrangements, we analysed the frequency of MLL cleavage in cells exposed to etoposide, a DNA topoisomerase II poison commonly used as an anticancer drug, and positioning of the broken 3'-end of the MLL gene in respect to inherent chromosomal territories. It was demonstrated that exposure of human Jurkat cells to etoposide resulted in frequent cleavage of MLL genes. Using MLL-specific break-apart probes we visualised cleaved MLL genes in ~17% of nuclei. Using confocal microscopy and 3D modelling, we demonstrated that in cells treated with etoposide and cultivated for 1 h under normal conditions, ~9% of the broken MLL alleles were present outside the chromosome 11 territory, whereas in both control cells and cells inspected immediately after etoposide treatment, virtually all MLL alleles were present within the chromosomal territory. The data are discussed in the framework of the "breakage first" model of juxtaposing translocation partners. We propose that in the course of repairing DNA topoisomerase II-mediated DNA lesions (removal of stalled DNA topoisomerase II complexes and non-homologous end joining, DNA ends acquire additional mobility, which allows the meeting and incorrect joining of translocation partners.

  10. Expression pattern of the septin gene family in acute myeloid leukemias with and without MLL-SEPT fusion genes

    NARCIS (Netherlands)

    Santos, Joana; Cerveira, Nuno; Bizarro, Susana; Ribeiro, Franclim R.; Correia, Cecilia; Torres, Lurdes; Lisboa, Susana; Vieira, Joana; Mariz, Jose M.; Norton, Lucilia; Snijder, Simone; Mellink, Clemens H.; Buijs, Arjan; Shih, Lee-Yung; Strehl, Sabine; Micci, Francesca; Heim, Sverre; Teixeira, Manuel R.

    2010-01-01

    Septins are proteins associated with crucial steps in cell division and cellular integrity. In humans, 14 septin genes have been identified, of which five (SEPT2, SEPT5, SEPT6, SEPT9, and SEPT11) are known to participate in reciprocal translocations with the MLL gene in myeloid neoplasias. We have r

  11. LEDGF/p75 as a target for treatment of MLL-dependent leukemia and HIV infection

    Czech Academy of Sciences Publication Activity Database

    Čermáková, K.; Těšina, Petr; Demeulemeester, J.; El Ashkar, S.; Méreau, H.; Schwaller, J.; Christ, F.; Debyser, Z.; Řezáčová, Pavlína; Veverka, Václav; De Rijck, J.

    2015-01-01

    Roč. 22, č. 1 (2015), s. 30. ISSN 1211-5894. [Discussions in Structural Molecular Biology . Annual Meeting of the Czech Society for Structural Biology /13./. 19.03.2015-21.03.2015, Nové Hrady] Institutional support: RVO:61388963 Keywords : LEDGF/p75 * leukemia * MLL Subject RIV: CE - Biochemistry

  12. High-Affinity Small-Molecule Inhibitors of the Menin-Mixed Lineage Leukemia (MLL) Interaction Closely Mimic a Natural Protein-Protein Interaction

    Energy Technology Data Exchange (ETDEWEB)

    He, Shihan; Senter, Timothy J.; Pollock, Jonathan; Han, Changho; Upadhyay, Sunil Kumar; Purohit, Trupta; Gogliotti, Rocco D.; Lindsley, Craig W.; Cierpicki, Tomasz; Stauffer, Shaun R.; Grembecka, Jolanta [Michigan; (Vanderbilt); (Vanderbilt-MED)

    2014-10-02

    The protein–protein interaction (PPI) between menin and mixed lineage leukemia (MLL) plays a critical role in acute leukemias, and inhibition of this interaction represents a new potential therapeutic strategy for MLL leukemias. We report development of a novel class of small-molecule inhibitors of the menin–MLL interaction, the hydroxy- and aminomethylpiperidine compounds, which originated from HTS of ~288000 small molecules. We determined menin–inhibitor co-crystal structures and found that these compounds closely mimic all key interactions of MLL with menin. Extensive crystallography studies combined with structure-based design were applied for optimization of these compounds, resulting in MIV-6R, which inhibits the menin–MLL interaction with IC50 = 56 nM. Treatment with MIV-6 demonstrated strong and selective effects in MLL leukemia cells, validating specific mechanism of action. Our studies provide novel and attractive scaffold as a new potential therapeutic approach for MLL leukemias and demonstrate an example of PPI amenable to inhibition by small molecules.

  13. Reflektiivisen työotteen hyödyntäminen : MLL:n lapsiperheiden ryhmänohjaajakoulutuksen käyneiden kokemuksia

    OpenAIRE

    Koikkalainen, Hilma; Hyvärinen, Sini

    2014-01-01

    Opinnäytetyön tarkoituksena oli kuvata Mannerheimin Lastensuojeluliiton (MLL) Järvi-Suomen piirin alueen Vahvuutta vanhemmuuteen -perheryhmien ohjaajakoulutuksen käyneiden kokemuksia koulutuksen vaikutuksista sekä oppien sovellettavuudesta heidän työssään lapsiperheiden parissa. Koulutus on osa MLL:n valtakunnallista Vahvuutta vanhemmuuteen -hanketta (toteuttamisaika 2010–2014). Opinnäytetyön tavoitteena oli tuottaa tietoa reflektiivisen työotteen käytöstä ja sovellettavuudesta. MLL voi hyödy...

  14. Expression and characterization of c-Myb in prenatal odontogenesis

    Czech Academy of Sciences Publication Activity Database

    Matalová, Eva; Buchtová, Marcela; Tucker, A. S.; Bender, T. P.; Janečková, Eva; Lungová, V.; Balková, Simona; Šmarda, J.

    2011-01-01

    Roč. 53, č. 6 (2011), s. 793-803. ISSN 0012-1592 R&D Projects: GA AV ČR KJB500450802; GA ČR GAP304/11/1418; GA ČR(CZ) GP304/08/P289; GA ČR GC524/08/J032 Institutional research plan: CEZ:AV0Z50450515 Keywords : morphogenesis * mouse * Myb Subject RIV: FF - HEENT, Dentistry Impact factor: 2.210, year: 2011

  15. [Effect of down-regulating mll-af9 gene expression on proliferation of acute monocytic leukemia cell line THP-1].

    Science.gov (United States)

    Li, Lei; Zhang, Ai-Hua; Liu, Ling-Bo; Bi, Lan; Wang, Li; Zhao, Ya-Jie; Zou, Ping

    2008-04-01

    This study was aimed to investigate the effect of small interfering RNA (siRNA) on the expression of mll-af9 oncogene and the proliferation of human acute monocytic leukemia cell line THP-1. One group of siRNA was designed targeting mll-af9 mRNA and finally obtained by chemosynthesis. Then the obtained siRNA was transfected into cultured human acute monocytic leukemia cell line THP-1 by lipofectamine. Flow cytometry was used to detect siRNA transfection efficiency. The level of mll-af9 mRNA expression was analyzed by reverse transcription polymerase chain reaction (RT-PCR). The cell proliferation rate was assayed by MTT. The change of cell cycles and apoptosis rate was detected by flow cytometry. The results showed that the siRNA transfection efficiency was 69.1%+/-1.8%. The level of mll-af9 mRNA expression was significantly inhibited in siRNA-transfected cells as compared with the controls. mll-af9-targeted siRNA inhibited the proliferation of THP-1 cells and induced cell apoptosis effectively after transfection. The percentage of G0/G1 phase cells significantly increased in siRNA-transfected cells in comparion with the control cells, but the percentage of S phase cells significantly decreased. It is concluded that the mll-af9-targeted siRNA can effectively inhibit the proliferation of human acute monocytic leukemia cell line THP-1. PMID:18426643

  16. Mll-AF4 Confers Enhanced Self-Renewal and Lymphoid Potential during a Restricted Window in Development

    Directory of Open Access Journals (Sweden)

    Neil A. Barrett

    2016-07-01

    Full Text Available MLL-AF4+ infant B cell acute lymphoblastic leukemia is characterized by an early onset and dismal survival. It initiates before birth, and very little is known about the early stages of the disease’s development. Using a conditional Mll-AF4-expressing mouse model in which fusion expression is targeted to the earliest definitive hematopoietic cells generated in the mouse embryo, we demonstrate that Mll-AF4 imparts enhanced B lymphoid potential and increases repopulation and self-renewal capacity during a putative pre-leukemic state. This occurs between embryonic days 12 and 14 and manifests itself most strongly in the lymphoid-primed multipotent progenitor population, thus pointing to a window of opportunity and a potential cell of origin. However, this state alone is insufficient to generate disease, with the mice succumbing to B cell lymphomas only after a long latency. Future analysis of the molecular details of this pre-leukemic state will shed light on additional events required for progression to acute leukemia.

  17. Mll-AF4 Confers Enhanced Self-Renewal and Lymphoid Potential during a Restricted Window in Development.

    Science.gov (United States)

    Barrett, Neil A; Malouf, Camille; Kapeni, Chrysa; Bacon, Wendi A; Giotopoulos, George; Jacobsen, Sten Eirik W; Huntly, Brian J; Ottersbach, Katrin

    2016-07-26

    MLL-AF4+ infant B cell acute lymphoblastic leukemia is characterized by an early onset and dismal survival. It initiates before birth, and very little is known about the early stages of the disease's development. Using a conditional Mll-AF4-expressing mouse model in which fusion expression is targeted to the earliest definitive hematopoietic cells generated in the mouse embryo, we demonstrate that Mll-AF4 imparts enhanced B lymphoid potential and increases repopulation and self-renewal capacity during a putative pre-leukemic state. This occurs between embryonic days 12 and 14 and manifests itself most strongly in the lymphoid-primed multipotent progenitor population, thus pointing to a window of opportunity and a potential cell of origin. However, this state alone is insufficient to generate disease, with the mice succumbing to B cell lymphomas only after a long latency. Future analysis of the molecular details of this pre-leukemic state will shed light on additional events required for progression to acute leukemia. PMID:27396339

  18. Dual DNA binding specificity of a petal epidermis-specific MYB transcription factor (MYB.Ph3) from Petunia hybrida.

    Science.gov (United States)

    Solano, R; Nieto, C; Avila, J; Cañas, L; Diaz, I; Paz-Ares, J

    1995-04-18

    The MYB.Ph3 protein recognized two DNA sequences that resemble the two known types of MYB DNA binding site: consensus I (MBSI), aaaAaaC(G/C)-GTTA, and consensus II (MBSII), aaaAGTTAGTTA. Optimal MBSI was recognized by animal c-MYB and not by Am305 from Antirrhinum, whereas MBSII showed the reverse behaviour. Different constraints on MYB.Ph3 binding to the two classes of sequences were demonstrated. DNA binding studies with mutated MBSI and MBSII and hydroxyl radical footprinting analysis, pointed to the N-terminal MYB repeat (R2) as the most involved in determining the dual DNA binding specificity of MYB.Ph3 and supported the idea that binding to MBSI and MBSII does not involve alternative orientations of the two repeats of MYB.Ph3. Minimal promoters containing either MBSI and MBSII were activated to the same extent by MYB.Ph3 in yeast, indicating that both types of binding site can be functionally equivalent. MYB.Ph3 binding sites are present in the promoter of flavonoid biosynthetic genes, such as the Petunia chsJ gene, which was transcriptionally activated by MYB.Ph3 in tobacco protoplasts. MYB.Ph3 was immunolocalized in the epidermal cell layer of petals, where flavonoid biosynthetic genes are actively expressed. This strongly suggests a role for MYB.Ph3 in the regulation of flavonoid biosynthesis. PMID:7737128

  19. The Superiority of Allogeneic Hematopoietic Stem Cell Transplantation Over Chemotherapy Alone in the Treatment of Acute Myeloid Leukemia Patients with Mixed Lineage Leukemia (MLL) Rearrangements

    Science.gov (United States)

    Yang, Hua; Huang, Sai; Zhu, Cheng-Ying; Gao, Li; Zhu, Hai-Yan; Lv, Na; Jing, Yu; Yu, Li

    2016-01-01

    Background Acute myeloid leukemia (AML) patients with mixed lineage leukemia (MLL) gene rearrangements always had a very poor prognosis. In this study, we report the incidence of MLL rearrangements in AML patients using gene analysis, as well as the clinical significance and prognostic features of these rearrangements. Material/Methods This retrospective study took place from April 2008 to November 2011 in the People’s Liberation Army General Hospital. A total 433 AML patients were screened by multiple nested reverse transcription polymerase chain reaction (RT-PCR) to determine the incidence of the 11 MLL gene rearrangements. There were 68 cases of MLL gene rearrangements, for a positive rate of 15.7%. A total of 24 patients underwent allogeneic hematopoietic stem cell transplantation (Allo-HSCT), and 34 patients received at least 4 cycles of chemotherapy. Ten patients were lost to follow-up. Results The median follow-up was 29 months. The complete remission (CR) rate was 85.4%. The overall survival (OS) was 57.4±5.9 months for the Allo-HSCT group and 21.0±2.1 months for the chemotherapy group. The Allo-HSCT group had superior survival compared with the chemotherapy group (5-year OS: 59±17% vs. 13±8%, P0.05). Multivariate analysis showed that transplantation, platelets >50×109/L at onset, and CR are associated with a better OS in MLL rearranged AML patients. Patients with thrombocytopenia and extramedullary involvement were prone to relapse. Conclusions Our results suggest that Allo-HSCT is superior to chemotherapy alone for treating MLL rearranged AML patients. Patients treated with Allo-HSCT have a better prognosis and a longer survival. CR is an independent prognostic factor for OS, and extramedullary involvement is an independent prognostic factor for DFS. MLL rearranged AML patients with thrombocytopenia at onset <50×109 had very bad OS and DFS. PMID:27373985

  20. A novel RT-qPCR assay for quantification of the MLL-MLLT3 fusion transcript in acute myeloid leukaemia

    DEFF Research Database (Denmark)

    Abildgaard, Lotte; Ommen, Hans Beier; Lausen, Birgitte Frederiksen;

    2013-01-01

    heterogeneity of translocation break points, the MLL-MLLT3 fusion gene is a challenging target. We hypothesised that MRD monitoring using MLL-MLLT3 as a RT-qPCR marker is feasible in the majority of patients with t(9;11)-positive AML. METHODS: Using a locked nucleic acid probe, we developed a sensitive RT......-qPCR assay for quantification of the most common break point region of the MLL-MLLT3 fusion gene. Five paediatric patients with t(9;11)-positive AML were monitored using the MLL-MLLT3 assay. RESULTS: A total of 43 bone marrow (BM) and 52 Peripheral blood (PB) samples were collected from diagnosis until......OBJECTIVES: Patients with acute myeloid leukaemia (AML) of the monocytic lineage often lack molecular markers for minimal residual disease (MRD) monitoring. The MLL-MLLT3 fusion transcript found in patients with AML harbouring t(9;11) is amenable to RT-qPCR quantification but because of the...

  1. Atg5-dependent autophagy contributes to the development of acute myeloid leukemia in an MLL-AF9-driven mouse model.

    Science.gov (United States)

    Liu, Qiang; Chen, Longgui; Atkinson, Jennifer M; Claxton, David F; Wang, Hong-Gang

    2016-01-01

    Acute myeloid leukemia (AML) is a hierarchical hematopoietic malignancy originating from leukemic stem cells (LSCs). Autophagy is a lysosomal degradation pathway that is hypothesized to be important for the maintenance of AML as well as contribute to chemotherapy response. Here we employ a mouse model of AML expressing the fusion oncogene MLL-AF9 and explore the effects of Atg5 deletion, a key autophagy protein, on the malignant transformation and progression of AML. Consistent with a transient decrease in colony-forming potential in vitro, the in vivo deletion of Atg5 in MLL-AF9-transduced bone marrow cells during primary transplantation prolonged the survival of recipient mice, suggesting that autophagy has a role in MLL-AF9-driven leukemia initiation. In contrast, deletion of Atg5 in malignant AML cells during secondary transplantation did not influence the survival or chemotherapeutic response of leukemic mice. Interestingly, autophagy was found to be involved in the survival of differentiated myeloid cells originating from MLL-AF9-driven LSCs. Taken together, our data suggest that Atg5-dependent autophagy may contribute to the development but not chemotherapy sensitivity of murine AML induced by MLL-AF9. PMID:27607576

  2. SWI/SNF Subunits SMARCA4, SMARCD2 and DPF2 Collaborate in MLL-Rearranged Leukaemia Maintenance

    DEFF Research Database (Denmark)

    Cruickshank, V Adam; Sroczynska, Patrycja; Sankar, Aditya;

    2015-01-01

    tumour-suppressor function in many solid tumours; recently however, it has been reported to sustain leukaemogenic transformation in MLL-rearranged leukaemia in mice. Here we further explore the role of SMARCA4 and the two SWI/SNF subunits SMARCD2/BAF60B and DPF2/BAF45D in leukaemia. We observed the...... selective requirement for these proteins for leukaemic cell expansion and self-renewal in-vitro as well as in leukaemia. Gene expression profiling in human cells of each of these three factors suggests that they have overlapping functions in leukaemia. The gene expression changes induced by loss of the...

  3. Favorable outcome in non-infant children with MLL-AF4-positive acute lymphoblastic leukemia: a report from the Tokyo Children's Cancer Study Group.

    Science.gov (United States)

    Tomizawa, Daisuke; Kato, Motohiro; Takahashi, Hiroyuki; Fujimura, Junya; Inukai, Takeshi; Fukushima, Takashi; Kiyokawa, Nobutaka; Koh, Katsuyoshi; Manabe, Atsushi; Ohara, Akira

    2015-11-01

    Unlike acute lymphoblastic leukemia (ALL) in infants, MLL gene rearrangement (MLL-r) is rare in ALL children (≥1 year old). The outcome and optimal treatment options for MLL-r ALL remain controversial. Among the 1827 children enrolled in the Tokyo Children's Cancer Study Group ALL studies L95-14, L99-15, L99-1502, L04-16, and L07-1602 (1995-2009), 25 MLL-r ALL patients (1.3 %) were identified. Their median age and leukocyte count at diagnosis was 2 years old (range 1-15 years) and 27,690/μL (range 1800-1,113,000/μL), respectively. All but one patient achieved complete remission (CR) after induction therapy, and 19 underwent allogeneic hematopoietic stem cell transplantation (HSCT) in first CR according to the protocol. The 5-year event-free survival (EFS) and overall survival (OS) rate were 60.0 % [standard error (SE), 9.7 %] and 64.0 % (SE 9.6 %), respectively. Notably, 9/12 cases with MLL-AF4-positive ALL are alive in continuous CR with a 75.0 % (SE 12.5 %) EFS rate. The causes of treatment failure were as follows: one induction failure, five relapses, and five transplant-related deaths. With intensive chemotherapy and allogeneic HSCT, favorable outcome of children (≥1 year old) with MLL-AF4-positive ALL was observed. However, considering the risk of acute and late toxicities associated with HSCT, its indication should be restricted. PMID:26410102

  4. MLL-Rearranged Acute Lymphoblastic Leukemias Activate BCL-2 through H3K79 Methylation and Are Sensitive to the BCL-2-Specific Antagonist ABT-199.

    Science.gov (United States)

    Benito, Juliana M; Godfrey, Laura; Kojima, Kensuke; Hogdal, Leah; Wunderlich, Mark; Geng, Huimin; Marzo, Isabel; Harutyunyan, Karine G; Golfman, Leonard; North, Phillip; Kerry, Jon; Ballabio, Erica; Chonghaile, Triona Ní; Gonzalo, Oscar; Qiu, Yihua; Jeremias, Irmela; Debose, LaKiesha; O'Brien, Eric; Ma, Helen; Zhou, Ping; Jacamo, Rodrigo; Park, Eugene; Coombes, Kevin R; Zhang, Nianxiang; Thomas, Deborah A; O'Brien, Susan; Kantarjian, Hagop M; Leverson, Joel D; Kornblau, Steven M; Andreeff, Michael; Müschen, Markus; Zweidler-McKay, Patrick A; Mulloy, James C; Letai, Anthony; Milne, Thomas A; Konopleva, Marina

    2015-12-29

    Targeted therapies designed to exploit specific molecular pathways in aggressive cancers are an exciting area of current research. Mixed Lineage Leukemia (MLL) mutations such as the t(4;11) translocation cause aggressive leukemias that are refractory to conventional treatment. The t(4;11) translocation produces an MLL/AF4 fusion protein that activates key target genes through both epigenetic and transcriptional elongation mechanisms. In this study, we show that t(4;11) patient cells express high levels of BCL-2 and are highly sensitive to treatment with the BCL-2-specific BH3 mimetic ABT-199. We demonstrate that MLL/AF4 specifically upregulates the BCL-2 gene but not other BCL-2 family members via DOT1L-mediated H3K79me2/3. We use this information to show that a t(4;11) cell line is sensitive to a combination of ABT-199 and DOT1L inhibitors. In addition, ABT-199 synergizes with standard induction-type therapy in a xenotransplant model, advocating for the introduction of ABT-199 into therapeutic regimens for MLL-rearranged leukemias. PMID:26711339

  5. MLL-Rearranged Acute Lymphoblastic Leukemias Activate BCL-2 through H3K79 Methylation and Are Sensitive to the BCL-2-Specific Antagonist ABT-199

    Directory of Open Access Journals (Sweden)

    Juliana M. Benito

    2015-12-01

    Full Text Available Targeted therapies designed to exploit specific molecular pathways in aggressive cancers are an exciting area of current research. Mixed Lineage Leukemia (MLL mutations such as the t(4;11 translocation cause aggressive leukemias that are refractory to conventional treatment. The t(4;11 translocation produces an MLL/AF4 fusion protein that activates key target genes through both epigenetic and transcriptional elongation mechanisms. In this study, we show that t(4;11 patient cells express high levels of BCL-2 and are highly sensitive to treatment with the BCL-2-specific BH3 mimetic ABT-199. We demonstrate that MLL/AF4 specifically upregulates the BCL-2 gene but not other BCL-2 family members via DOT1L-mediated H3K79me2/3. We use this information to show that a t(4;11 cell line is sensitive to a combination of ABT-199 and DOT1L inhibitors. In addition, ABT-199 synergizes with standard induction-type therapy in a xenotransplant model, advocating for the introduction of ABT-199 into therapeutic regimens for MLL-rearranged leukemias.

  6. B-cell acute lymphoblastic leukemia with mature phenotype and MLL rearrangement: report of five new cases and review of the literature.

    Science.gov (United States)

    Sajaroff, Elisa Olga; Mansini, Adrian; Rubio, Patricia; Alonso, Cristina Noemí; Gallego, Marta S; Coccé, Mariela C; Eandi-Eberle, Silvia; Bernasconi, Andrea Raquel; Ampatzidou, Maria; Paterakis, George; Papadhimitriou, Stefanos I; Petrikkos, Loizos; Papadakis, Vassilios; Polychronopoulou, Sophia; Rossi, Jorge G; Felice, Maria Sara

    2016-10-01

    The association between mature-B phenotype and MLL abnormalities in acute lymphoblastic leukemia (ALL) is a very unusual finding; only 14 pediatric cases have been reported so far. We describe the clinical and biological characteristics and outcome of five pediatric cases of newly diagnosed B lineage ALL with MLL abnormalities and mature immunophenotype based on light chain restriction and surface Ig expression. Blasts showed variable expression of CD10/CD34/TdT. MLL abnormalities with no MYC involvement were detected in all patients by G-banding, FISH, and/or RT-PCR. Three patients were treated according to Interfant protocol, one to ALLIC-09, and one received B-NHL-BFM-2004. All patients achieved complete remission and three of them relapsed. Despite the small cohort size, it could be postulated that B lineage ALL with MLL abnormalities and mature phenotype is a distinct entity that differs both from the typical Pro B ALL observed in infants and mature B-ALL with high MYC expression. PMID:26857438

  7. Spontaneous remission of acute myeloid leukemia relapse after hematopoietic cell transplantation in a high-risk patient with 11q23/MLL abnormality.

    Science.gov (United States)

    Hudecek, Michael; Bartsch, Kristina; Jäkel, Nadja; Heyn, Simone; Pfannes, Roald; Al-Ali, Haifa Kathrin; Cross, Michael; Pönisch, Wolfram; Gerecke, Ulrich; Edelmann, Jeanett; Ittel, Thomas; Niederwieser, Dietger

    2008-01-01

    A 35-year-old female patient was diagnosed with acute myeloid leukemia with multiple genetic aberrations [48 XX, del(3)(q21), +6, t(11;15)(q23;q15), +21] including an 11q23/MLL abnormality. The patient achieved a complete remission after one induction chemotherapy cycle. After three courses of consolidation, a matched unrelated hematopoietic cell transplantation (HCT) was performed. Following an upper respiratory tract infection 7 years after transplant, her blood counts declined to leukocytes of 1 x 10(9)/l, platelets of 51 x 10(9)/l and hemoglobin of 7.5 g/dl. A bone marrow aspirate revealed 55% leukemic blasts carrying the unfavorable genetic aberrations seen at initial diagnosis (11q23/MLL). In the absence of any disease-specific treatment, the leukemic blasts cleared from the bone marrow within 6 days after diagnosis of relapse and peripheral blood counts returned to normal. Molecular analysis of the 11q23/MLL rearrangement was used to evaluate minimal residual disease, which became undetectable in repetitive FISH analyses. This is the first report of spontaneous remission in a patient with initially a multiaberrant leukemic cell clone and a proven 11q23/MLL abnormality at relapse after HCT. PMID:18367831

  8. Sensitivity of MLL-rearranged AML cells to all-trans retinoic acid is associated with the level of H3K4me2 in the RARα promoter region

    International Nuclear Information System (INIS)

    All-trans retinoic acid (ATRA) is well established as differentiation therapy for acute promyelocytic leukemia (APL) in which the PML–RARα (promyelocytic leukemia-retinoic acid receptor α) fusion protein causes blockade of the retinoic acid (RA) pathway; however, in types of acute myeloid leukemia (AML) other than APL, the mechanism of RA pathway inactivation is not fully understood. This study revealed the potential mechanism of high ATRA sensitivity of mixed-lineage leukemia (MLL)-AF9-positive AML compared with MLL-AF4/5q31-positive AML. Treatment with ATRA induced significant myeloid differentiation accompanied by upregulation of RARα, C/EBPα, C/EBPε and PU.1 in MLL-AF9-positive but not in MLL-AF4/5q31-positive cells. Combining ATRA with cytarabine had a synergistic antileukemic effect in MLL-AF9-positive cells in vitro. The level of dimethyl histone H3 lysine 4 (H3K4me2) in the RARα gene-promoter region, PU.1 upstream regulatory region (URE) and RUNX1+24/+25 intronic enhancer was higher in MLL-AF9-positive cells than in MLL-AF4-positive cells, and inhibiting lysine-specific demethylase 1, which acts as a histone demethylase inhibitor, reactivated ATRA sensitivity in MLL-AF4-positive cells. These findings suggest that the level of H3K4me2 in the RARα gene-promoter region, PU.1 URE and RUNX1 intronic enhancer is determined by the MLL-fusion partner. Our findings provide insight into the mechanisms of ATRA sensitivity in AML and novel treatment strategies for ATRA-resistant AML

  9. EEN encodes for a member of a new family of proteins containing an Src homology 3 domain and is the third gene located on chromosome 19p13 that fuses to MLL in human leukemia

    OpenAIRE

    So, Chi Wai; Caldas, Carlos; Liu, Meng-Min; Chen, Sai-Juan; Huang, Qiu-Hua; Gu, Long-Jun; Sham, Mai Har; Wiedemann, Leanne Marie; Chan, Li Chong

    1997-01-01

    The MLL gene, the closest human homologue to the Drosophila trithorax gene, undergoes chromosomal translocation with a large number of different partner genes in both acute lymphoid and acute myeloid leukemias. We have identified a new partner gene, EEN, fused to MLL in a case of acute myeloid leukemia. The gene is located on chromosome 19p13, where two other MLL partner genes, ENL and ELL/MEN have also been identified. The deduced protein of 368 aa contains a central α-helical region and a C...

  10. Acquisition of a CD19-negative myeloid phenotype allows immune escape of MLL-rearranged B-ALL from CD19 CAR-T-cell therapy.

    Science.gov (United States)

    Gardner, Rebecca; Wu, David; Cherian, Sindhu; Fang, Min; Hanafi, Laïla-Aïcha; Finney, Olivia; Smithers, Hannah; Jensen, Michael C; Riddell, Stanley R; Maloney, David G; Turtle, Cameron J

    2016-05-19

    Administration of lymphodepletion chemotherapy followed by CD19-specific chimeric antigen receptor (CAR)-modified T cells is a remarkably effective approach to treating patients with relapsed and refractory CD19(+) B-cell malignancies. We treated 7 patients with B-cell acute lymphoblastic leukemia (B-ALL) harboring rearrangement of the mixed lineage leukemia (MLL) gene with CD19 CAR-T cells. All patients achieved complete remission (CR) in the bone marrow by flow cytometry after CD19 CAR-T-cell therapy; however, within 1 month of CAR-T-cell infusion, 2 of the patients developed acute myeloid leukemia (AML) that was clonally related to their B-ALL, a novel mechanism of CD19-negative immune escape. These reports have implications for the management of patients with relapsed and refractory MLL-B-ALL who receive CD19 CAR-T-cell therapy. PMID:26907630

  11. Transient potential receptor melastatin-2 (Trpm2) does not influence murine MLL-AF9-driven AML leukemogenesis or in vitro response to chemotherapy.

    Science.gov (United States)

    Haladyna, Jessica N; Pastuer, Taylor; Riedel, Simone S; Perraud, Anne-Laure; Bernt, Kathrin M

    2016-07-01

    Transient potential receptor melastatin-2 (TRPM2) is a nonselective cationic, Ca(2+)-permeable transmembrane pore that is preferentially expressed in cells of the myeloid lineage and modulates signaling pathways converging into NF-kB. This is of potential interest for acute myeloid leukemia (AML) therapy, as NF-κB signaling is emerging as a key pathway, mediating drug resistance and leukemia-initiating cell survival in AML. Inhibition of NF-κB signaling has been found to be synergistic with chemotherapy. TRPM2 is overexpressed in AML compared with normal bone marrow, with the highest levels in the FAB M3-6 subtypes. To determine the effect of TRPM2 depletions in a defined genetic model, we established MLL-AF9-driven AML on a Trpm2(-/-) genetic background. Trpm2(-/-) MLL-AF9 leukemias displayed reduced NF-κB phosphorylation as well as nuclear translocation. In vivo, primary and secondary recipients of Trpm2(-/-) MLL-AF9 leukemias exhibit increased latency compared with recipients of wild-type leukemia cells. However, the difference in latency was small and was lost in tertiary transplants. The effect of loss of Trpm2 in a BCR-ABL/NUP98-HOXA9 fusion model was even smaller. Given reports that loss or inhibition of TRPM2 enhanced killing by DNA-damaging agents in neuroblastoma, breast cancer, and prostate cancer cell lines, we exposed Trpm2(-/-) and Trpm2(wt) primary MLL-AF9 leukemias to doxorubicin, cytarabine, and etoposide, but found no difference in IC50 values. The in vitro response to decitabine was also unaffected. In summary, Trpm2 does not seem to play a major role in myeloid leukemogenesis. Additionally, loss of Trpm2 does not augment the cytotoxicity of standard AML chemotherapeutic agents. PMID:27033163

  12. An evolutionarily conserved interaction of tumor suppressor protein Pdcd4 with the poly(A)-binding protein contributes to translation suppression by Pdcd4.

    Science.gov (United States)

    Fehler, Olesja; Singh, Priyanka; Haas, Astrid; Ulrich, Diana; Müller, Jan P; Ohnheiser, Johanna; Klempnauer, Karl-Heinz

    2014-01-01

    The tumor suppressor protein programmed cell death 4 (Pdcd4) has been implicated in the translational regulation of specific mRNAs, however, the identities of the natural Pdcd4 target mRNAs and the mechanisms by which Pdcd4 affects their translation are not well understood. Pdcd4 binds to the eukaryotic translation initiation factor eIF4A and inhibits its helicase activity, which has suggested that Pdcd4 suppresses translation initiation of mRNAs containing structured 5'-untranslated regions. Recent work has revealed a second inhibitory mechanism, which is eIF4A-independent and involves direct RNA-binding of Pdcd4 to the target mRNAs. We have now identified the poly(A)-binding protein (PABP) as a novel direct interaction partner of Pdcd4. The ability to interact with PABP is shared between human and Drosophila Pdcd4, indicating that it has been highly conserved during evolution. Mutants of Pdcd4 that have lost the ability to interact with PABP fail to stably associate with ribosomal complexes in sucrose density gradients and to suppress translation, as exemplified by c-myb mRNA. Overall, our work identifies PABP as a novel functionally relevant Pdcd4 interaction partner that contributes to the regulation of translation by Pdcd4. PMID:25190455

  13. Hypothesis: Paralog Formation from Progenitor Proteins and Paralog Mutagenesis Spur the Rapid Evolution of Telomere Binding Proteins.

    Science.gov (United States)

    Lustig, Arthur J

    2016-01-01

    Through elegant studies in fungal cells and complex organisms, we propose a unifying paradigm for the rapid evolution of telomere binding proteins (TBPs) that associate with either (or both) telomeric DNA and telomeric proteins. TBPs protect and regulate telomere structure and function. Four critical factors are involved. First, TBPs that commonly bind to telomeric DNA include the c-Myb binding proteins, OB-fold single-stranded binding proteins, and G-G base paired Hoogsteen structure (G4) binding proteins. Each contributes independently or, in some cases, cooperatively, to provide a minimum level of telomere function. As a result of these minimal requirements and the great abundance of homologs of these motifs in the proteome, DNA telomere-binding activity may be generated more easily than expected. Second, telomere dysfunction gives rise to genome instability, through the elevation of recombination rates, genome ploidy, and the frequency of gene mutations. The formation of paralogs that diverge from their progenitor proteins ultimately can form a high frequency of altered TBPs with altered functions. Third, TBPs that assemble into complexes (e.g., mammalian shelterin) derive benefits from the novel emergent functions. Fourth, a limiting factor in the evolution of TBP complexes is the formation of mutually compatible interaction surfaces amongst the TBPs. These factors may have different degrees of importance in the evolution of different phyla, illustrated by the apparently simpler telomeres in complex plants. Selective pressures that can utilize the mechanisms of paralog formation and mutagenesis to drive TBP evolution along routes dependent on the requisite physiologic changes. PMID:26904098

  14. Mouse Af9 Is a Controller of Embryo Patterning, Like Mll, Whose Human Homologue Fuses with AF9 after Chromosomal Translocation in Leukemia

    OpenAIRE

    Collins, Emma C.; Appert, Alexandre; Ariza-McNaughton, Linda; Pannell, Richard; Yamada, Yoshihiro; Rabbitts, Terence H.

    2002-01-01

    Chromosomal translocation t(9;11)(p22;q23) in acute myeloid leukemia fuses the MLL and AF9 genes. We have inactivated the murine homologue of AF9 to elucidate its normal role. No effect on hematopoiesis was observed in mice with a null mutation of Af9. However, an Af9 null mutation caused perinatal lethality, and homozygous mice exhibited anomalies of the axial skeleton. Both the cervical and thoracic regions were affected by anterior homeotic transformation. Strikingly, mice lacking function...

  15. A Case of Therapy-related Acute Lymphoblastic Leukemia with t(11;19)(q23;p13.3) and MLL/MLLT1 Gene Rearrangement

    OpenAIRE

    Yoo, Byong-Joon; Nam, Myung-Hyun; Sung, Hwa-Jung; Lim, Chae-Seung; Lee, Chang-Kyu; Cho, Yun-Jung; Lee, Kap-No; Yoon, Soo-Young

    2011-01-01

    Therapy-related ALL (t-ALL) is a rare secondary leukemia that develops after chemotherapy and/or radiotherapy for primary malignancies. Chromosomal 11q23 abnormalities are the most common karyotypic alterations in t-ALL. The t(11;19)(q23;p13) aberration is extremely rare and has not been confirmed at the molecular genetic level. Here, we report a case of t-ALL with t(11;19)(q23;p13.3) and MLL-MLLT1 (alias ENL) gene rearrangement confirmed by cytogenetic analysis, multiplex reverse transcripti...

  16. FISH联合multiplex RT-PCR检测MLL基因重排的价值探讨%Diagnostic value of FISH combined with multiplex RT-PCR for detection of MLL rearrangement

    Institute of Scientific and Technical Information of China (English)

    何易; 李旭东; 王东宁; 肖若芝; 王文文; 胡元; 林东军

    2012-01-01

    AIM: To analyze the diagnostic value of fluorescence in situ hybridization (FISH) combined with reverse transcriplion — multiplex nested PCR ( multiplex RT — PCR) for the detection of mixed — lineage leukemia ( MLL) gene rearrangement from bone marrow aspirate in the patients with acute leukemia. METHODS: The bone marrow samples were obtained from 201 newly diagnosed acute leukemic patients in our hospital. MLL gene rearrangement was detected by both FISH and multiplex RT — PCR methods. FISH with the MLL dual color "break — apart" probe was performed following the procedures recommended by the manufacturer. Eleven common fusion transcripts of MLL were also determined by multiplex RT — PCR assay. Conventional cytogenetic analysis ( CCA) was performed on all specimens to observe the abnormality of Hq23. RESULTS; MLL gene rearrangement was observed in 19 patients, including 13 cases (10. 2% ) of acute mye-loid leukemia (AML) and 6 cases (8.2% ) of acute lymphoid leukemia (ALL). Among the MLL positive specimens, the rate of MLL rearrangement detected by FISH combined with multiplex RT — PCR was 9.45% , but 5. 47% by CCA merely. One case of MLL inversion and 3 cases of MLL amplification were found in 5 patients with normal karyotype and 3 patients with cytogenetic abnormalities uninvolving chromosome 11. Multiplex RT — PCR revealed 7 cases of dup (MLL) which failed to show in FISH and CCA. CONCLUSION: The combination of FISH and multiplex RT - PCR increases the sensitivity for detection of MLL rearrangement.%目的:采用荧光原位杂交(FISH)与逆转录多重巢式聚合酶链反应(multiplex RT-PCR)技术检测急性白血病中MLL基因重排的情况,分析两者联合应用的诊断价值.方法:对2008年1月~2011年5月在我院诊断为急性白血病的201例患者采用MLL双色断裂分离重排探针进行FISH检测,同时用multiplex RT-PCR技术检测11种较常见的MLL融合基因,观察MLL基因异常的检出率.所有患者均进

  17. Acute myocardial infarction during induction chemotherapy for acute MLL t(4;11 leukemia with lineage switch and extreme leukocytosis

    Directory of Open Access Journals (Sweden)

    Čolović Nataša

    2015-01-01

    Full Text Available Introduction. In patients with acute leukemias hemorrhage is the most frequent problem. Vein thrombotic events may appear rarely but arterial thromboses are exceptionally rare. We present a patient with acute leukemia and bilateral deep leg vein thrombosis who developed an acute myocardial infarction (AMI during induction chemotherapy. The etiology and treatment of AMI in patients with acute leukemia, which is a rare occurrence, is discussed. Case Outline. In April of 2012 a 37-year-old male presented with bilateral deep leg vein thrombosis and malaise. Laboratory data were as follows: Hb 118 g/L, WBC 354x109/L (with 91% blasts in differential leukocyte count, platelets 60Ч109/L. Bone marrow aspirate and immunophenotype revealed the presence of acute lymphoblastic leukemia. Cytogenetic analysis was as follows: 46,XY,t(4;11(q21:q23 [2]/62-82,XY,t(4;11[18]. Molecular analysis showed MLL-AF4 rearrangement. The patient was on low molecular weight heparin and combined chemotherapy according to protocol HyperCVAD. On day 10 after chemotherapy he got chest pain. Three days later AMI was diagnosed (creatine kinase 66 U/L, CK-MB 13U/L, troponin 1.19 μg/L. Electrocardiogram showed the ST elevation in leads D1, D2, aVL, V5 and V6 and “micro q” in D1. On echocardiography, hypokinesia of the left ventricle and ejection fraction of 39% was found. After recovering from AMI and restoring left ventricle ejection fraction to 59%, second course of HyperCVAD was given. The control bone marrow aspirate showed 88% of blasts but with monoblastic appearance. Flow cytometry confirmed a lineage switch from lymphoblasts to monoblasts. In further course of the disease he was treated with a variety of chemotherapeutic combinations without achieving remission. Eventually, palliative chemotherapy was administered to reduce the bulk of blasts. He died five months after the initial diagnosis. Conclusion. AMI in young adults with acute leukemia is a very rare complication

  18. Improved outcome with hematopoietic stem cell transplantation in a poor prognostic subgroup of infants with mixed-lineage-leukemia (MLL)-rearranged acute lymphoblastic leukemia: results from the Interfant-99 Study

    DEFF Research Database (Denmark)

    Mann, Georg; Attarbaschi, Andishe; Schrappe, Martin;

    2010-01-01

    To define a role for hematopoietic stem cell transplantation (HSCT) in infants with acute lymphoblastic leukemia and rearrangements of the mixed-lineage-leukemia gene (MLL(+)), we compared the outcome of MLL(+) patients from trial Interfant-99 who either received chemotherapy only or HSCT. Of 376...... such high-risk criteria, with 87 achieving CR. In this group, HSCT was associated with a 64% reduction in the risk of failure resulting from relapse or death in CR (hazard ratio = 0.36, 95% confidence interval, 0.15-0.86). In the remaining patients, there was no advantage for HSCT over chemotherapy only...

  19. Determination of the extraction efficiency for $^{233}$U source $\\alpha$-recoil ions from the MLL buffer-gas stopping cell

    CERN Document Server

    von der Wense, Lars; Laatiaoui, Mustapha; Thirolf, Peter G

    2016-01-01

    Following the $\\alpha$ decay of $^{233}$U, $^{229}$Th recoil ions are shown to be extracted in a significant amount from the MLL buffer-gas stopping cell. The produced recoil ions and subsequent daughter nuclei are mass purified with the help of a customized quadrupole mass spectrometer. The combined extraction and mass-purification efficiency for $^{229}$Th$^{3+}$ is determined via MCP-based measurements and via the direct detection of the $^{229}$Th $\\alpha$ decay. A large value of $(10\\pm2)$\\% for the combined extraction and mass-purification efficiency of $^{229}$Th$^{3+}$ is obtained at a mass resolution of about 1 u/e. In addition to $^{229}$Th, also other $\\alpha$-recoil ions of the $^{233,232}$U decay chains are addressed.

  20. Double minute chromosomes in acute myeloid leukemia, myelodysplastic syndromes, and chronic myelomonocytic leukemia are associated with micronuclei, MYC or MLL amplification, and complex karyotype.

    Science.gov (United States)

    Huh, Yang O; Tang, Guilin; Talwalkar, Sameer S; Khoury, Joseph D; Ohanian, Maro; Bueso-Ramos, Carlos E; Abruzzo, Lynne V

    2016-01-01

    Double minute chromosomes (dmin) are small, paired chromatin bodies that lack a centromere and represent a form of extrachromosomal gene amplification. Dmin are rare in myeloid neoplasms and are generally associated with a poor prognosis. Most studies of dmin in myeloid neoplasms are case reports or small series. In the current study, we present the clinicopathologic and cytogenetic features of 22 patients with myeloid neoplasms harboring dmin. These neoplasms included acute myeloid leukemia (AML) (n = 18), myelodysplastic syndrome (MDS) (n = 3), and chronic myelomonocytic leukemia (CMML) (n = 1). The AML cases consisted of AML with myelodysplasia-related changes (n = 13) and therapy-related AML (n = 5). Dmin were detected in initial pre-therapy samples in 14 patients with AML or CMML; they were acquired during the disease course in 8 patients who had AML or MDS. The presence of dmin was associated with micronuclei (18/18; 100%), complex karyotype (17/22; 77.3%), and amplification of MYC (12/16; 75%) or MLL (4/16; 25%). Immunohistochemical staining for MYC performed on bone marrow core biopsy or clot sections revealed increased MYC protein in all 19 cases tested. Except for one patient, most patients failed to respond to risk-adapted chemotherapies. At last follow up, all patients had died of disease after a median of 5 months following dmin detection. In conclusion, dmin in myeloid neoplasms commonly harbor MYC or MLL gene amplification and manifest as micronuclei within leukemic blasts. Dmin are often associated with myelodysplasia or therapy-related disease, and complex karyotypes. PMID:27318442

  1. Binding Procurement

    Science.gov (United States)

    Rao, Gopalakrishna M.; Vaidyanathan, Hari

    2007-01-01

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

  2. Activated KRAS Cooperates with MLL-AF4 to Promote Extramedullary Engraftment and Migration of Cord Blood CD34+ HSPC But Is Insufficient to Initiate Leukemia.

    Science.gov (United States)

    Prieto, Cristina; Stam, Ronald W; Agraz-Doblas, Antonio; Ballerini, Paola; Camos, Mireia; Castaño, Julio; Marschalek, Rolf; Bursen, Aldeheid; Varela, Ignacio; Bueno, Clara; Menendez, Pablo

    2016-04-15

    The MLL-AF4 (MA4) fusion gene is the genetic hallmark of an aggressive infant pro-B-acute lymphoblastic leukemia (B-ALL). Our understanding of MA4-mediated transformation is very limited. Whole-genome sequencing studies revealed a silent mutational landscape, which contradicts the aggressive clinical outcome of this hematologic malignancy. Only RAS mutations were recurrently detected in patients and found to be associated with poorer outcome. The absence of MA4-driven B-ALL models further questions whether MA4 acts as a single oncogenic driver or requires cooperating mutations to manifest a malignant phenotype. We explored whether KRAS activation cooperates with MA4 to initiate leukemia in cord blood-derived CD34(+) hematopoietic stem/progenitor cells (HSPC). Clonogenic and differentiation/proliferation assays demonstrated that KRAS activation does not cooperate with MA4 to immortalize CD34(+) HSPCs. Intrabone marrow transplantation into immunodeficient mice further showed that MA4 and KRAS(G12V) alone or in combination enhanced hematopoietic repopulation without impairing myeloid-lymphoid differentiation, and that mutated KRAS did not cooperate with MA4 to initiate leukemia. However, KRAS activation enhanced extramedullary hematopoiesis of MA4-expressing cell lines and CD34(+) HSPCs that was associated with leukocytosis and central nervous system infiltration, both hallmarks of infant t(4;11)(+) B-ALL. Transcriptional profiling of MA4-expressing patients supported a cell migration gene signature underlying the mutant KRAS-mediated phenotype. Collectively, our findings demonstrate that KRAS affects the homeostasis of MA4-expressing HSPCs, suggesting that KRAS activation in MA4(+) B-ALL is important for tumor maintenance rather than initiation. Cancer Res; 76(8); 2478-89. ©2016 AACR. PMID:26837759

  3. Molecular Recognition by Templated Folding of an Intrinsically Disordered Protein

    Science.gov (United States)

    Toto, Angelo; Camilloni, Carlo; Giri, Rajanish; Brunori, Maurizio; Vendruscolo, Michele; Gianni, Stefano

    2016-02-01

    Intrinsically disordered proteins often become structured upon interacting with their partners. The mechanism of this ‘folding upon binding’ process, however, has not been fully characterised yet. Here we present a study of the folding of the intrinsically disordered transactivation domain of c-Myb (c-Myb) upon binding its partner KIX. By determining the structure of the folding transition state for the binding of wild-type and three mutational variants of KIX, we found a remarkable plasticity of the folding pathway of c-Myb. To explain this phenomenon, we show that the folding of c-Myb is templated by the structure of KIX. This adaptive folding behaviour, which occurs by heterogeneous nucleation, differs from the robust homogeneous nucleation typically observed for globular proteins. We suggest that this templated folding mechanism may enable intrinsically disordered proteins to achieve specific and reliable binding with multiple partners while avoiding aberrant interactions.

  4. ”Kuinka tärkee määkin oon ollu monille perheille ja toisaalta kuinka tärkee toi kokemus on ollu mulle” : MLL:n lastenhoitotoiminta nuoren hoitajan näkökulmasta

    OpenAIRE

    Stankowski, Tarja

    2014-01-01

    Opinnäytetyöni tehtävänä on selvittää mitä merkityksiä Mannerheimin Lastensuojeluliiton (MLL) nuoret hoitajat (alle 25 v) antavat lastenhoitotoiminnassa mukana olemiselle ja mitä kehittämisehdotuksia nuorilla on lastenhoitotoimintaan. Hyvinvointivaltion kivijalka on kansalaisyhteiskunta, jossa järjestöjen rooli on ollut keskeinen. Nuorten kannustaminen aktiiviseen kansalaisuuteen on esillä niin kansallisesti kuin kansainvälisestikin. Mitä on aktiivinen kansalaisuus? Millaisia aktiivisia ...

  5. Systematic in-vitro evaluation of the NCI/NIH Developmental Therapeutics Program Approved Oncology Drug Set for the identification of a candidate drug repertoire for MLL-rearranged leukemia

    Directory of Open Access Journals (Sweden)

    Hoeksema KA

    2011-09-01

    Full Text Available Kimberley A Hoeksema1, Aarthi Jayanthan1, Todd Cooper2, Lia Gore3, Tanya Trippett4, Jessica Boklan6, Robert J Arceci5, Aru Narendran11Division of Pediatric Oncology, Alberta Children's Hospital, Calgary, AB, Canada; 2Aflac Cancer Center and Blood Disorders Service, Children's Healthcare of Atlanta, Emory University, Atlanta, GA, USA; 3Center for Cancer and Blood Disorders, Children's Hospital, University of Colorado Denver, Aurora, CO, USA; 4Memorial Sloan-Kettering Cancer Center, New York, NY, USA; 5Sidney Kimmel Comprehensive Cancer Center, Johns Hopkins University, Baltimore, MD, USA; 6Center for Cancer and Blood Disorders, Phoenix Children's Hospital, Phoenix, AZ, USAAbstract: Despite significant progress made in the overall cure rate, the prognosis for relapsed and refractory malignancies in children remains extremely poor. Hence, there is an urgent need for studies that enable the timely selection of appropriate agents for Phase I clinical studies. The Pediatric Oncology Experimental Therapeutics Investigators' Consortium (POETIC is systematically evaluating libraries of known and novel compounds for activity against subsets of high-risk pediatric malignancies with defined molecular aberrations for future clinical development. In this report, we describe the in-vitro activity of a diverse panel of approved oncology drugs against MLL-rearranged pediatric leukemia cell lines. Agents in the Approved Oncology Drug Set II (National Cancer Institute/National Institutes of Health Developmental Therapeutics Program were evaluated by in-vitro cytotoxicity assays in pediatric acute lymphoblastic leukemia and acute myeloid leukemia cell lines with MLL gene rearrangements. Validation studies were carried out with patient leukemia cells in culture. Comparative analysis for toxicity against nonmalignant cells was evaluated in normal bone marrow stromal cells and normal human lymphocytes. Results from this study show that 42 of the 89 agents tested have

  6. Higher expression levels of the HOXA9 gene, closely associated with MLL-PTD and EZH2 mutations, predict inferior outcome in acute myeloid leukemia

    Directory of Open Access Journals (Sweden)

    Gao L

    2016-02-01

    Full Text Available Li Gao,1,* Junzhong Sun,2,* Fang Liu,2,3,* Hui Zhang,1 Yigai Ma1 1Department of Hematology, China–Japan Friendship Hospital, 2Department of Hematology and Oncology, The First Affiliated Hospital of Chinese PLA General Hospital, 3Department of Oncology, Chinese PLA General Hospital, Beijing, People’s Republic of China *These authors contributed equally to this work Background: Although the biological insight of acute myeloid leukemia (AML has increased in the past few years, the discovery of novel discriminative biomarkers remains of utmost value for improving outcome predictions. Systematical studies concerning the clinical implications and genetic correlations of HOXA9 aberrations in patients with AML are relatively promising.Materials and methods: Here, we investigated mutational status and the mRNA levels of the HOXA9 gene in 258 patients with AML. Furthermore, hematological characteristics, chromosome abnormalities, and genetic mutations associated with AML were analyzed, followed by the assessment of clinical survival. Besides, the expression level and mutational status of MEIS1, a cofactor of HOXA9, were also detected in patients with AML with the aim of a deeper understanding about the homeodomain-containing transcription factors associated with hematological characteristics.Results: HOXA9 and MEIS1 mutations were detected in 4.26% and 3.49% AML cases, respectively. No correlations were detected between mutation status and clinical characteristics, cytogenetic and genetic aberrations, and clinical survival. Higher HOXA9 expression levels were correlated with white blood cell count and closely associated with unfavorable karyotype as well as MLL-PTD and EZH2 mutations, whereas, there was an inverse correlation with the French–American–British M3 subtype. Compared with patients with lower HOXA9 expression levels, those with higher HOXA9 expression levels had a lower complete remission rate and inferior survivals in both AML and

  7. Complex three-way translocation involving MLL, ELL, RREB1, and CMAHP genes in an infant with acute myeloid leukemia and t(6;19;11)(p22.2;p13.1;q23.3)

    DEFF Research Database (Denmark)

    Tuborgh, A; Meyer, C; Marschalek, R;

    2013-01-01

    Rearrangements affecting the MLL gene in hematological malignancies are associated with poor prognosis. Most often they are reciprocal translocations and more rarely complex forms involving at least 3 chromosomes. We describe an unusual case with cutaneous leukemic infiltrates that waxed and waned...... until progression to acute myeloid leukemia, AML-M5. The leukemic cells harbored a novel apparent 3-way translocation t(6;19;11)(p22.2;p13.1;q23.3). We utilized advanced molecular cytogenetic methods including 24-color karyotyping, high-resolution array comparative genomic hybridization (aCGH) and DNA...... initial stages of disease before clear morphological signs of bone marrow involvement. The patient responded well to therapy and remains in remission>6 years from diagnosis. This apparent 3-way translocation is remarkable because of its rarity and presentation with myeloid sarcoma, and may, as more cases...

  8. RT-PCR ANALYSIS OF E2A-PBX1, TEL-AML1, BCR-ABL AND MLL-AF4 FUSION GENE TRANSCRIPTS IN B-LINEAGE ACUTE LYMPHOBLASTIC LEUKEMIA

    Directory of Open Access Journals (Sweden)

    Iuliu-Cristian Ivanov

    2013-11-01

    Full Text Available Acute lymphoblastic leukemia represents a heterogeneous group of hematological malignancies, defined by clonal proliferation of lymphoid cells. Immunophenotyping by flow cytometry and molecular analysis for the detection of genetic anomalies are clinical standard procedures for diagnosis, sub-classification and post-therapeutic evaluation. Samples from 105 patients diagnosed with acute lymphoblastic leukemia were immunophenotyped at diagnosis and were investigated by molecular analysis in order to identify the occurrence of four fusion genes: MLL-AF4, TEL-AML-1, BCR-ABL-p190, E2A-PBX-1. There were no associations found between the immunophenotype and the presence of any fusion genes evaluated. Both methods in combination remain a prerequisite for an improved subclassification of hematological malignancies, therapeutic decision, and evaluation of treatment response.

  9. 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 ...

  10. Plant Hormone Binding Sites

    OpenAIRE

    Napier, Richard

    2004-01-01

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

  11. Python bindings for libcloudph++

    OpenAIRE

    Jarecka, Dorota; Arabas, Sylwester; Del Vento, Davide

    2015-01-01

    This technical note introduces the Python bindings for libcloudph++. The libcloudph++ is a C++ library of algorithms for representing atmospheric cloud microphysics in numerical models. The bindings expose the complete functionality of the library to the Python users. The bindings are implemented using the Boost.Python C++ library and use NumPy arrays. This note includes listings with Python scripts exemplifying the use of selected library components. An example solution for using the Python ...

  12. DNS & Bind Cookbook

    CERN Document Server

    Liu, Cricket

    2011-01-01

    The DNS & BIND Cookbook presents solutions to the many problems faced by network administrators responsible for a name server. Following O'Reilly's popular problem-and-solution cookbook format, this title is an indispensable companion to DNS & BIND, 4th Edition, the definitive guide to the critical task of name server administration. The cookbook contains dozens of code recipes showing solutions to everyday problems, ranging from simple questions, like, "How do I get BIND?" to more advanced topics like providing name service for IPv6 addresses. It's full of BIND configuration files that yo

  13. Python bindings for libcloudph++

    CERN Document Server

    Jarecka, Dorota; Del Vento, Davide

    2015-01-01

    This technical note introduces the Python bindings for libcloudph++. The libcloudph++ is a C++ library of algorithms for representing atmospheric cloud microphysics in numerical models. The bindings expose the complete functionality of the library to the Python users. The bindings are implemented using the Boost.Python C++ library and use NumPy arrays. This note includes listings with Python scripts exemplifying the use of selected library components. An example solution for using the Python bindings to access libcloudph++ from Fortran is presented.

  14. Successful Administration of Recombinant Human Soluble Thrombomodulin α (Recomodulin for Disseminated Intravascular Coagulation during Induction Chemotherapy in an Elderly Patient with Acute Monoblastic Leukemia Involving the t(9;11(p22;q23 MLL/AF9 Translocation

    Directory of Open Access Journals (Sweden)

    Kazutaka Takagi

    2011-01-01

    Full Text Available Patients with acute myelogenous leukemia complicate with disseminated intravascular coagulation (DIC, not only at the time of the initially leukemia diagnosis, but also during induction chemotherapy. In Japan, recently, a recombinant human soluble thrombomodulin alpha (Recomodulin has been introduced as a new type of anti-DIC agent for clinical use in patients with hematological cancer or infectious disease. We describe a 67-year-old female case in which 25,600 units of Recomodulin for 6 days were successfully administered for both initially complicating and therapy-induced DIC without any troubles of bleeding in an acute monoblastic leukemia (AML-M5a patient with the MLL gene translocation. Furthermore, the levels of DIC biomarkers recovered rapidly after the Recomodulin treatment. Our case suggests that DIC control using Recomodulin is one of the crucial support-therapies during remission induction chemotherapy in patients with acute leukemia of which type tends to complicate extramedullary or extranodal infiltration having potential to onset DIC.

  15. Melanin-binding radiopharmaceuticals

    Energy Technology Data Exchange (ETDEWEB)

    Packer, S; Fairchild, R G; Watts, K P; Greenberg, D; Hannon, S J

    1980-01-01

    The scope of this paper is limited to an analysis of the factors that are important to the relationship of radiopharmaceuticals to melanin. While the authors do not attempt to deal with differences between melanin-binding vs. melanoma-binding, a notable variance is assumed. (PSB)

  16. Melanin-binding radiopharmaceuticals

    International Nuclear Information System (INIS)

    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

  17. DNS BIND Server Configuration

    Directory of Open Access Journals (Sweden)

    Radu MARSANU

    2011-01-01

    Full Text Available After a brief presentation of the DNS and BIND standard for Unix platforms, the paper presents an application which has a principal objective, the configuring of the DNS BIND 9 server. The general objectives of the application are presented, follow by the description of the details of designing the program.

  18. SHBG (Sex Hormone Binding Globulin)

    Science.gov (United States)

    ... as: Testosterone-estrogen Binding Globulin; TeBG Formal name: Sex Hormone Binding Globulin Related tests: Testosterone , Free Testosterone, ... I should know? How is it used? The sex hormone binding globulin (SHBG) test may be used ...

  19. Inhibition of selectin binding

    Energy Technology Data Exchange (ETDEWEB)

    Nagy, Jon O. (Rodeo, CA); Spevak, Wayne R. (Albany, CA); Dasgupta, Falguni (New Delhi, IN); Bertozzi, Caroline (Albany, CA)

    2001-10-09

    This invention provides compositions for inhibiting the binding between two cells, one expressing P- or L-selectin on the surface and the other expressing the corresponding ligand. A covalently crosslinked lipid composition is prepared having saccharides and acidic group on separate lipids. The composition is then interposed between the cells so as to inhibit binding. Inhibition can be achieved at an effective oligosaccharide concentration as low as 10.sup.6 fold below that of the free saccharide. Since selectins are involved in recruiting cells to sites of injury, these composition scan be used to palliate certain inflammatory and immunological conditions.

  20. Inhibition of selectin binding

    Energy Technology Data Exchange (ETDEWEB)

    Nagy, J.O.; Spevak, W.R.; Dasgupta, F.; Bertozzi, C.

    1999-10-05

    This invention provides a system for inhibiting the binding between two cells, one expressing P- or L-selectin on the surface and the other expressing the corresponding ligand. A covalently crosslinked lipid composition is prepared having saccharides and acidic group on separate lipids. The composition is then interposed between the cells so as to inhibit binding. Inhibition can be achieved at an effective oligosaccharide concentration as low as 10{sup 6} fold below that of the free saccharide. Since selectins are involved in recruiting cells to sites of injury, this system can be used to palliate certain inflammatory and immunological conditions.

  1. Inhibition of selectin binding

    Energy Technology Data Exchange (ETDEWEB)

    Nagy, Jon O. (Rodeo, CA); Spevak, Wayne R. (Albany, CA); Dasgupta, Falguni (New Delhi, IN); Bertozzi, Caroline (Albany, CA)

    1999-01-01

    This invention provides compositions for inhibiting the binding between two cells, one expressing P- or L-selectin on the surface and the other expressing the corresponding ligand. A covalently crosslinked lipid composition is prepared having saccharides and acidic group on separate lipids. The composition is then interposed between the cells so as to inhibit binding. Inhibition can be achieved at an effective oligosaccharide concentration as low as 10.sup.6 fold below that of the free saccharide. Since selectins are involved in recruiting cells to sites of injury, these composition scan be used to palliate certain inflammatory and immunological conditions.

  2. Inhibition of selectin binding

    Energy Technology Data Exchange (ETDEWEB)

    Nagy, J.O.; Spevak, W.R.; Dasgupta, F.; Bertozzi, C.

    1999-11-16

    This invention provides compositions for inhibiting the binding between two cells, one expressing P- or L-selectin on the surface and the other expressing the corresponding ligand. A covalently crosslinked lipid composition is prepared having saccharides and acidic group on separate lipids. The composition is then interposed between the cells so as to inhibit binding. Inhibition can be achieved at an effective oligosaccharide concentration as low as 10{sup 6} fold below that of the free saccharide. Since selectins are involved in recruiting cells to sites of injury, these composition scan be used to palliate certain inflammatory and immunological conditions.

  3. Inhibition of selectin binding

    Energy Technology Data Exchange (ETDEWEB)

    Nagy, Jon O. (Rodeo, CA); Spevak, Wayne R. (Albany, CA); Dasgupta, Falguni (New Delhi, IN); Bertozzi, Carolyn (Albany, CA)

    1999-10-05

    This invention provides a system for inhibiting the binding between two cells, one expressing P- or L-selectin on the surface and the other expressing the corresponding ligand. A covalently crosslinked lipid composition is prepared having saccharides and acidic group on separate lipids. The composition is then interposed between the cells so as to inhibit binding. Inhibition can be achieved at an effective oligosaccharide concentration as low as 10.sup.6 fold below that of the free saccharide. Since selectins are involved in recruiting cells to sites of injury, this system can be used to palliate certain inflammatory and immunological conditions.

  4. 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.

  5. DNA-binding residues and binding mode prediction with binding-mechanism concerned models

    OpenAIRE

    Oyang Yen-Jen; Liu Yu-Cheng; Huang Chun-Chin; Huang Yu-Feng; Huang Chien-Kang

    2009-01-01

    Abstract Background Protein-DNA interactions are essential for fundamental biological activities including DNA transcription, replication, packaging, repair and rearrangement. Proteins interacting with DNA can be classified into two categories of binding mechanisms - sequence-specific and non-specific binding. Protein-DNA specific binding provides a mechanism to recognize correct nucleotide base pairs for sequence-specific identification. Protein-DNA non-specific binding shows sequence indepe...

  6. Carboplatin binding to histidine

    International Nuclear Information System (INIS)

    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

  7. 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.

  8. Collagen binding to Staphylococcus aureus

    International Nuclear Information System (INIS)

    Staphylococcus aureus can bind soluble collagen in a specific, saturable manner. We have previously shown that some variability exists in the degree of collagen binding between different strains of heat-killed, formaldehyde-fixed S. aureus which are commercially available as immunologic reagents. The present study demonstrates that live S. aureus of the Cowan 1 strain binds amounts of collagen per organism equivalent to those demonstrated previously in heat-killed, formaldehyde-fixed bacteria but has an affinity over 100 times greater, with Kd values of 9.7 X 10(-11) M and 4.3 X 10(-8) M for live and heat-killed organisms, respectively. Studies were also carried out with S. aureus killed by ionizing radiation, since this method of killing the organism seemed less likely to alter the binding moieties on the surface than did heat killing. Bacteria killed by exposure to gamma radiation bound collagen in a manner essentially indistinguishable from that of live organisms. Binding of collagen to irradiated cells of the Cowan 1 strain was rapid, with equilibrium reached by 30 min at 22 degrees C, and was fully reversible. The binding was not inhibited by fibronectin, fibrinogen, C1q, or immunoglobulin G, suggesting a binding site for collagen distinct from those for these proteins. Collagen binding was virtually eliminated in trypsin-treated organisms, indicating that the binding site has a protein component. Of four strains examined, Cowan 1 and S. aureus ATCC 25923 showed saturable, specific binding, while strains Woods and S4 showed a complete lack of binding. These results suggest that some strains of S. aureus contain high-affinity binding sites for collagen. While the number of binding sites per bacterium varied sixfold in the two collagen-binding strains, the apparent affinity was similar

  9. Melanin binding radiopharmaceuticals

    International Nuclear Information System (INIS)

    We have determined the biodistribution an uptake by the Greene melanoma in the Syrian golden hamster with 21 radiopharmaceuticals. Maximum % uptake and the time at which this occurred are listed. It is essential to know maximum tumor to background ration and the time after injection that this occurs to determine suitability for tumor scanning. The importance of species variation deserves mention. Detection of eye melanoma in humans was quite variable whereas in hamsters it was quite easy to obtain a positive scan with a single pinhole. We then looked at brain uptake in man and found it (the brain scan) to be significant. In addition, we found a high uptake by the lung, something not found in hamsters but not entirely unsuspected of a amine, such as 123I-4,3DMQ. Finally, our clinical experience has shown us some of the vagaries of melanoma-seeking radiopharmaceuticals. This reflects the complexity of melanin and melanin-binding and points out the necessity for a more detailed analysis of the mechanisms involved in melanin binding radionuclides

  10. c-MYB in the mouse incisor and hair follicle stem cell niches and surrounding tissues - correlation with proliferation and apoptosis

    Czech Academy of Sciences Publication Activity Database

    Veselá, Barbora; Švandová, Eva; Šmarda, J.; Hampl, A.; Matalová, Eva

    Lipsko : German Society for Stem Cell Research, 2012. 113-114. [Annual Congress of theGerman Society for Stem Cell Research /7./. 29.11.2012-30.11.2012, Lipsko] R&D Projects: GA ČR GAP304/11/1418 Institutional support: RVO:67985904 Keywords : apoptosis Subject RIV: EA - Cell Biology

  11. Heteronuclear two-and three-dimensional NMR studies on the R1-R2-R3 domain of Drosophila melanogaster c-myb protein: spin system identifications

    International Nuclear Information System (INIS)

    Advantages of heteronuclear two- and three-dimensional NMR experiments in obtaining better dispersion of peaks in spectra of large protein molecules have been described. The basic experimental techniques have been qualitatively presented and their application to a protein of 160 amino acid residues has been described. Several residue-type specific signals have been identified. The analysis of three-dimensional 13C resolved 1H-1H TOCSY (total correlated spectroscopy) spectra for spin system identifications has been described in some detail. (author). 42 refs., 9 figs

  12. Quarkonium Binding and Entropic Force

    CERN Document Server

    Satz, Helmut

    2015-01-01

    A Q-Qbar bound state represents a balance between repulsive kinetic and attractive potential energy. In a hot quark-gluon plasma, the interaction potential experiences medium effects. Color screening modifies the attractive binding force between the quarks, while the increase of entropy with Q-Qbar separation gives rise to a growing repulsion. We study the role of these phenomena for in-medium Q-Qbar binding and dissociation. It is found that the relevant potential for Q-Qbar binding is the free energy F; with increasing Q-Qbar separation, further binding through the internal energy U is compensated by repulsive entropic effects.

  13. A conserved patch of hydrophobic amino acids modulates Myb activity by mediating protein-protein interactions.

    Science.gov (United States)

    Dukare, Sandeep; Klempnauer, Karl-Heinz

    2016-07-01

    The transcription factor c-Myb plays a key role in the control of proliferation and differentiation in hematopoietic progenitor cells and has been implicated in the development of leukemia and certain non-hematopoietic tumors. c-Myb activity is highly dependent on the interaction with the coactivator p300 which is mediated by the transactivation domain of c-Myb and the KIX domain of p300. We have previously observed that conservative valine-to-isoleucine amino acid substitutions in a conserved stretch of hydrophobic amino acids have a profound effect on Myb activity. Here, we have explored the function of the hydrophobic region as a mediator of protein-protein interactions. We show that the hydrophobic region facilitates Myb self-interaction and binding of the histone acetyl transferase Tip60, a previously identified Myb interacting protein. We show that these interactions are affected by the valine-to-isoleucine amino acid substitutions and suppress Myb activity by interfering with the interaction of Myb and the KIX domain of p300. Taken together, our work identifies the hydrophobic region in the Myb transactivation domain as a binding site for homo- and heteromeric protein interactions and leads to a picture of the c-Myb transactivation domain as a composite protein binding region that facilitates interdependent protein-protein interactions of Myb with regulatory proteins. PMID:27080133

  14. Alcohol Binding to the Odorant Binding Protein LUSH: Multiple Factors Affecting Binding Affinities

    OpenAIRE

    Ader, Lauren; Jones, David N. M.; Lin, Hai

    2010-01-01

    Density function theory (DFT) calculations have been carried out to investigate the binding of alcohols to the odorant binding protein LUSH from Drosophila melanogaster. LUSH is one of the few proteins known to bind to ethanol at physiologically relevant concentrations and where high-resolution structural information is available for the protein bound to alcohol at these concentrations. The structures of the LUSH–alcohol complexes identify a set of specific hydrogen-bonding interactions as cr...

  15. [3]tetrahydrotrazodone binding. Association with serotonin binding sites

    International Nuclear Information System (INIS)

    High (17 nM) and low (603 nM) affinity binding sites for [3]tetrahydrotrazodone ([3] THT), a biologically active analogue of trazodone, have been identified in rat brain membranes. The substrate specificity, concentration, and subcellular and regional distributions of these sites suggest that they may represent a component of the serotonin transmitter system. Pharmacological analysis of [3]THT binding, coupled with brain lesion and drug treatment experiments, revealed that, unlike other antidepressants, [3] THT does not attach to either a biogenic amine transporter or serotonin binding sites. Rather, it would appear that [3]THT may be an antagonist ligand for the serotonin binding site. This probe may prove of value in defining the mechanism of action of trazodone and in further characterizing serotonin receptors

  16. Windows Presentation Foundation & Data Binding

    OpenAIRE

    JANDA, Vilém

    2010-01-01

    The aim of this work is a course in the form of e-learning study materials for the interpretation of technology Data Binding in Windows Presentation Foundation (WPF). In the first, mostly theoretical part will be done a description and interpretation of the elements of technology, focusing on WPF Data Binding. In the second part, is available methodology and training course with their own interpretive audio-visual files for self-study. The lectures are supplemented by solved examples, and exa...

  17. Water binding in legume seeds

    Science.gov (United States)

    Vertucci, C. W.; Leopold, A. C.

    1987-01-01

    The physical status of water in seeds has a pivotal role in determining the physiological reactions that can take place in the dry state. Using water sorption isotherms from cotyledon and axis tissue of five leguminous seeds, the strength of water binding and the numbers of binding sites have been estimated using van't Hoff analyses and the D'Arcy/Watt equation. These parameters of water sorption are calculated for each of the three regions of water binding and for a range of temperatures. Water sorption characteristics are reflective of the chemical composition of the biological materials as well as the temperature at which hydration takes place. Changes in the sorption characteristics with temperature and hydration level may suggest hydration-induced structural changes in cellular components.

  18. Megalin binds and mediates cellular internalization of folate binding protein

    DEFF Research Database (Denmark)

    Birn, Henrik; Zhai, Xiaoyue; Holm, Jan;

    2005-01-01

    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...

  19. Computational Prediction of RNA-Binding Proteins and Binding Sites

    Directory of Open Access Journals (Sweden)

    Jingna Si

    2015-11-01

    Full Text Available Proteins and RNA interaction have vital roles in many cellular processes such as protein synthesis, sequence encoding, RNA transfer, and gene regulation at the transcriptional and post-transcriptional levels. Approximately 6%–8% of all proteins are RNA-binding proteins (RBPs. Distinguishing these RBPs or their binding residues is a major aim of structural biology. Previously, a number of experimental methods were developed for the determination of protein–RNA interactions. However, these experimental methods are expensive, time-consuming, and labor-intensive. Alternatively, researchers have developed many computational approaches to predict RBPs and protein–RNA binding sites, by combining various machine learning methods and abundant sequence and/or structural features. There are three kinds of computational approaches, which are prediction from protein sequence, prediction from protein structure, and protein-RNA docking. In this paper, we review all existing studies of predictions of RNA-binding sites and RBPs and complexes, including data sets used in different approaches, sequence and structural features used in several predictors, prediction method classifications, performance comparisons, evaluation methods, and future directions.

  20. 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.

  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. BINDING ISOTHERMS SURFACTANT-PROTEINS

    OpenAIRE

    Elena Irina Moater; Cristiana Radulescu; Ionica Ionita

    2011-01-01

    The interactions between surfactants and proteins shows some similarities with interactions between surfactants and polymers, but the hydrophobic amphoteric nature of proteins and their secondary and tertiary structure components make them different from conventional polymer systems. Many studies from the past about surfactant - proteins bonding used the dialysis techniques. Other techniques used to determine the binding isotherm, included ultrafiltration, ultracentrifugation, potentiometry, ...

  3. Positive Emotion Facilitates Audiovisual Binding.

    Science.gov (United States)

    Kitamura, Miho S; Watanabe, Katsumi; Kitagawa, Norimichi

    2015-01-01

    It has been shown that positive emotions can facilitate integrative and associative information processing in cognitive functions. The present study examined whether emotions in observers can also enhance perceptual integrative processes. We tested 125 participants in total for revealing the effects of emotional states and traits in observers on the multisensory binding between auditory and visual signals. Participants in Experiment 1 observed two identical visual disks moving toward each other, coinciding, and moving away, presented with a brief sound. We found that for participants with lower depressive tendency, induced happy moods increased the width of the temporal binding window of the sound-induced bounce percept in the stream/bounce display, while no effect was found for the participants with higher depressive tendency. In contrast, no effect of mood was observed for a simple audiovisual simultaneity discrimination task in Experiment 2. These results provide the first empirical evidence of a dependency of multisensory binding upon emotional states and traits, revealing that positive emotions can facilitate the multisensory binding processes at a perceptual level. PMID:26834585

  4. Radioligand Binding at Muscarinic Receptors

    Czech Academy of Sciences Publication Activity Database

    El-Fakahany, E. E.; Jakubík, Jan

    New York: Springer, 2016 - (Mysliveček, J.; Jakubík, J.), s. 37-68. (Neuromethods. 107). ISBN 978-1-4939-2857-6 R&D Projects: GA ČR(CZ) GBP304/12/G069 Institutional support: RVO:67985823 Keywords : muscarinic acetylcholine receptors * radioligand binding Subject RIV: ED - Physiology

  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...

  6. Antimicrobial Peptide-Lipid Binding Interactions and Binding Selectivity

    OpenAIRE

    Lad, Mitaben D.; Birembaut, Fabrice; Clifton, Luke A.; Frazier, Richard A.; Webster, John R. P.; Green, Rebecca J.

    2007-01-01

    Surface pressure measurements, external reflection-Fourier transform infrared spectroscopy, and neutron reflectivity have been used to investigate the lipid-binding behavior of three antimicrobial peptides: melittin, magainin II, and cecropin P1. As expected, all three cationic peptides were shown to interact more strongly with the anionic lipid, 1,2 dihexadecanoyl-sn-glycerol-3-(phosphor-rac-(1-glycerol)) (DPPG), compared to the zwitterionic lipid, 1,2 dihexadecanoyl-sn-glycerol-3-phosphocho...

  7. Characteristics of human erythrocyte insulin binding sites.

    OpenAIRE

    Okada, Yoshio

    1981-01-01

    Insulin and human erythrocyte cell membrane interactions were studied with respect to binding and dissociation. The per cent of specific binding of 125I-labeled insulin to erythrocytes was directly proportional to the cell concentration. The optimum pH for binding was 8.1. The initial binding rate was directly proportional to, and the steady state insulin binding was reversely proportional to, the incubation temperature. The per cent of specific binding of 125I-labeled insulin was 12.10 +/- 1...

  8. Dissection of the Critical Binding Determinants of Cellular Retinoic Acid Binding Protein II by Mutagenesis and Fluorescence Binding Assay

    OpenAIRE

    Vasileiou, Chrysoula; Lee, Kin Sing Stephen; Crist, Rachael M.; Vaezeslami, Soheila; Goins, Sarah M.; Geiger, James H.; Borhan, Babak

    2009-01-01

    The binding of retinoic acid to mutants of Cellular Retinoic Acid Binding Protein II (CRABPII) was evaluated to better understand the importance of the direct protein/ligand interactions. The important role of Arg111 for the correct structure and function of the protein was verified and other residues that directly affect retinoic acid binding have been identified. Furthermore, retinoic acid binding to CRABPII mutants that lack all previously identified interacting amino acids was rescued by ...

  9. Probing protein phosphatase substrate binding

    DEFF Research Database (Denmark)

    Højlys-Larsen, Kim B.; Sørensen, Kasper Kildegaard; Jensen, Knud Jørgen; Gammeltoft, Steen

    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...

  10. Optical binding of unlike particles

    Czech Academy of Sciences Publication Activity Database

    Karásek, Vítězslav; Zemánek, Pavel

    Bellingham : SPIE, 2012, 86970T: 1-6. ISBN 978-0-8194-9481-8. [CPS 2012. Czech-Polish-Slovak Optical Conference on Wave and Quantum Aspects of Contemporary Optics /18./. Ostravice (CZ), 03.09.2012-07.09.2012] R&D Projects: GA ČR GPP205/12/P868 Institutional support: RVO:68081731 Keywords : Optical binding * Optical tweezers * self-arrangement * colloids Subject RIV: BH - Optics, Masers, Lasers

  11. Calcium binding by dietary fibre

    International Nuclear Information System (INIS)

    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. Positive Emotion Facilitates Audiovisual Binding

    OpenAIRE

    Kitamura, Miho S.; Watanabe, Katsumi; Kitagawa, Norimichi

    2016-01-01

    It has been shown that positive emotions can facilitate integrative and associative information processing in cognitive functions. The present study examined whether emotions in observers can also enhance perceptual integrative processes. We tested 125 participants in total for revealing the effects of emotional states and traits in observers on the multisensory binding between auditory and visual signals. Participants in Experiment 1 observed two identical visual disks moving toward each oth...

  13. Binding effects and nuclear shadowing

    OpenAIRE

    Indumathi, D.; Wei ZHU

    1996-01-01

    The effects of nuclear binding on nuclear structure functions have so far been studied mainly at fixed target experiments, and there is currently much interest in obtaining a clearer understanding of this phenomenon. We use an existing dynamical model of nuclear structure functions, that gives good agreement with current data, to study this effect in a kinematical regime (low $x$, high $Q^2$) that can possibly be probed by an upgrade of {\\sc hera} at {\\sc desy} into a nuclear accelerator.

  14. 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.

  15. 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.

  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. Erythropoietin binding protein from mammalian serum

    Energy Technology Data Exchange (ETDEWEB)

    Clemons, G.K.

    1997-04-29

    Purified mammalian erythropoietin binding-protein is disclosed, and its isolation, identification, characterization, purification, and immunoassay are described. The erythropoietin binding protein can be used for regulation of erythropoiesis by regulating levels and half-life of erythropoietin. A diagnostic kit for determination of level of erythropoietin binding protein is also described. 11 figs.

  18. Erythropoietin binding protein from mammalian serum

    Energy Technology Data Exchange (ETDEWEB)

    Clemons, Gisela K. (Berkeley, CA)

    1997-01-01

    Purified mammalian erythropoietin binding-protein is disclosed, and its isolation, identification, characterization, purification, and immunoassay are described. The erythropoietin binding protein can be used for regulation of erythropoiesis by regulating levels and half-life of erythropoietin. A diagnostic kit for determination of level of erythropoietin binding protein is also described.

  19. Solute-vacancy binding in aluminum

    International Nuclear Information System (INIS)

    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

  20. Binding of quasi two-dimensional biexcitons

    DEFF Research Database (Denmark)

    Birkedal, Dan; Singh, J; Vadim, Lyssenko; Hvam, Jørn Märcher

    Summary form only given. In this presentation we report on a determination of the biexciton binding energies in GaAs-AlGaAs quantum wells of different widths and the results of a theoretical calculation of the ratio of the biexciton binding energy to that of the exciton. We determine the binding ...

  1. Glucocorticoid receptor transformation and DNA binding

    International Nuclear Information System (INIS)

    The overall goal is to probe the mechanism whereby glucocorticoid receptors are transformed from a non-DNA-binding form to their active DNA-binding form. The author has examined the effect of an endogenous inhibitor purified from rat liver cytosol on receptor binding to DNA. The inhibitor binds to transformed receptors in whole cytosol and prevent their binding to DNA. He also examined the role of sulfhydryl groups in determining the DNA binding activity of the transformed receptor and in determining the transformation process. Treatment of rat liver cytosol containing temperature-transformed, [3H]dexamethasone-bound receptors at 00C with the sulfhydryl modifying reagent methyl methanethiosulfonate inhibits the DNA-binding activity of the receptor, and DNA-binding activity is restored after addition of dithiothreitol. In addition, he has examined the relationship between receptor phosphorylation and DNA binding. Untransformed receptor complexes purified from cytosol prepared from mouse L cells grown in medium containing [32P]orthophosphate contain two components, a 100 k-Da and a 90-kDa subunit, both of which are phosphoproteins. On transformation, the receptor dissociates from the 90-kDa protein. Transformation of the complex under cell free conditions does not result in a dephosphorylation of the 100-kDa steroid-binding protein. Transformed receptor that has been bound to DNA and purified by monoclonal antibody is still in a phosphorylated form. These results suggest that dephosphorylation is not required for receptor binding to DNA

  2. 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 per albumin molecule, and binding of the first sulfamethizole molecule is possibly reduced as well. Diazepam binds with equal affinity to the fetal and adult proteins. Among the two main albumin drug-binding functions, for warfarin and diazepam, the former is thus compromised in the newborn...

  3. Insulin binding to individual rat skeletal muscles

    International Nuclear Information System (INIS)

    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

  4. Erythropoietin binding sites in human foetal tissues

    Energy Technology Data Exchange (ETDEWEB)

    Pekonen, F.; Rosenloef, K.; Rutanen, E.-M.

    1987-01-01

    Using /sup 125/I labelled recombinant DNA human erythropoietin (EP), we have explored the presence and properties of EP binding sites in foetal human tissues. The EP binding site is present in the foetal liver already during the first trimester of pregnancy. The binding site has a equilibrium association constant of 4.1-6.2 x 10/sup 9/l/mol and is specific for EP. The cross-reactivities of FSH, TSH, hCG, insulin and renin substrate were less than 0.01%. The EP binding capacity of foetal liver was 5.4-16 fmol/mg membrane protein. In foetal lung tissue, a slight EP binding activity was observed, whereas foetal spleen, muscle, brain, thyroid and placental tissues were virtually devoid of EP binding capacity. The same level of binding was reached at 37 deg. C in 1 h and at 4 deg. C in 24 h. The binding was pH-dependent with maximal specific binding at pH 7.7. SDS-PAGE gel electrophoresis analysis of covalently cross-linked /sup 125/I-EP to foetal liver membranes suggested that the EP binding site was composed of two subunits with an apparent mol wt of 41000 and 86000 dalton, respectively.

  5. Binding characteristics of swine erythrocyte insulin receptors

    International Nuclear Information System (INIS)

    Crossbred gilts had 8.8 +/- 1.1% maximum binding of [125I]insulin to insulin receptors on erythrocytes. The number of insulin-binding sites per cell was 137 +/- 19, with a binding affinity ranging from 7.4 X 10(7)M-1 to 11.2 X 10(7)M-1 and mean of 8.8 X 10(7)M-1. Pregnant sows had a significant increase in maximum binding due to an increase in number of receptor sites per cell. Lactating sows fed a high-fiber diet and a low-fiber diet did not develop a significant difference in maximum binding of insulin. Sows fed the low-fiber diet had a significantly higher number of binding sites and a significantly lower binding affinity than did sows fed a high-fiber diet. Receptor-binding affinity was lower in the low-fiber diet group than in cycling gilts, whereas data from sows fed the high-fiber diet did not differ from data for cycling gilts. Data from this study indicated that insulin receptors of swine erythrocytes have binding characteristics similar to those in other species. Pregnancy and diet will alter insulin receptor binding in swine

  6. Erythropoietin binding sites in human foetal tissues

    International Nuclear Information System (INIS)

    Using 125I labelled recombinant DNA human erythropoietin (EP), we have explored the presence and properties of EP binding sites in foetal human tissues. The EP binding site is present in the foetal liver already during the first trimester of pregnancy. The binding site has a equilibrium association constant of 4.1-6.2 x 109l/mol and is specific for EP. The cross-reactivities of FSH, TSH, hCG, insulin and renin substrate were less than 0.01%. The EP binding capacity of foetal liver was 5.4-16 fmol/mg membrane protein. In foetal lung tissue, a slight EP binding activity was observed, whereas foetal spleen, muscle, brain, thyroid and placental tissues were virtually devoid of EP binding capacity. The same level of binding was reached at 37 deg. C in 1 h and at 4 deg. C in 24 h. The binding was pH-dependent with maximal specific binding at pH 7.7. SDS-PAGE gel electrophoresis analysis of covalently cross-linked 125I-EP to foetal liver membranes suggested that the EP binding site was composed of two subunits with an apparent mol wt of 41000 and 86000 dalton, respectively. (author)

  7. 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

  8. Methods for Improving Aptamer Binding Affinity

    OpenAIRE

    Hijiri Hasegawa; Nasa Savory; Koichi Abe; Kazunori Ikebukuro

    2016-01-01

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

  9. Predicted metal binding sites for phytoremediation

    OpenAIRE

    Sharma, Ashok; Roy, Sudeep; Tripathi, Kumar Parijat; Roy, Pratibha; Mishra, Manoj; Khan, Feroz; Meena, Abha

    2009-01-01

    Metal ion binding domains are found in proteins that mediate transport, buffering or detoxification of metal ions. The objective of the study is to design and analyze metal binding motifs against the genes involved in phytoremediation. This is being done on the basis of certain pre-requisite amino-acid residues known to bind metal ions/metal complexes in medicinal and aromatic plants (MAP's). Earlier work on MAP's have shown that heavy metals accumulated by aromatic and medicinal plants do no...

  10. RNA Binding Specificity of Drosophila Muscleblind†

    OpenAIRE

    Goers, Emily S.; Voelker, Rodger B.; Gates, Devika P.; Berglund, J. Andrew

    2008-01-01

    Members of the muscleblind family of RNA binding proteins found in Drosophila and mammals are key players in both the human disease myotonic dystrophy and the regulation of alternative splicing. Recently, the mammalian muscleblind-like protein, MBNL1, has been shown to have interesting RNA binding properties with both endogenous and disease-related RNA targets. Here we report the characterization of RNA binding properties of the Drosophila muscleblind protein Mbl. Mutagenesis of double-strand...

  11. Exciton Binding Energy of Monolayer WS2

    OpenAIRE

    Bairen Zhu; Xi Chen; Xiaodong Cui

    2015-01-01

    The optical properties of monolayer transition metal dichalcogenides (TMDC) feature prominent excitonic natures. Here we report an experimental approach toward measuring the exciton binding energy of monolayer WS2 with linear differential transmission spectroscopy and two-photon photoluminescence excitation spectroscopy (TP-PLE). TP-PLE measurements show the exciton binding energy of 0.71eV around K valley in the Brillouin zone. The trion binding energy of 34meV, two-photon absorption cross s...

  12. A computational model for feature binding

    Institute of Scientific and Technical Information of China (English)

    SHI ZhiWei; SHI ZhongZhi; LIU Xi; SHI ZhiPing

    2008-01-01

    The "Binding Problem" is an important problem across many disciplines, including psychology, neuroscience, computational modeling, and even philosophy. In this work, we proposed a novel computational model, Bayesian Linking Field Model, for feature binding in visual perception, by combining the idea of noisy neuron model, Bayesian method, Linking Field Network and competitive mechanism.Simulation Experiments demonstrated that our model perfectly fulfilled the task of feature binding in visual perception and provided us some enlightening idea for future research.

  13. Binding of cryptococcal polysaccharide to Cryptococcus neoformans.

    OpenAIRE

    Kozel, T R; Hermerath, C A

    1984-01-01

    Radioiodinated cryptococcal polysaccharide was used to study binding of the soluble polysaccharide to encapsulated and non-encapsulated cryptoccoci. Binding of polysaccharide to non-encapsulated cryptococci occurred rapidly over a 30-min period and was largely complete after 2 h. Bound, labeled polysaccharide was slowly eluted from Cryptococcus neoformans after the addition of unlabeled polysaccharide, indicating reversibility of binding. Non-encapsulated cryptococci bound polysaccharide in t...

  14. A computational model for feature binding

    Institute of Scientific and Technical Information of China (English)

    2008-01-01

    The "Binding Problem" is an important problem across many disciplines, including psychology, neuroscience, computational modeling, and even philosophy. In this work, we proposed a novel computational model, Bayesian Linking Field Model, for feature binding in visual perception, by combining the idea of noisy neuron model, Bayesian method, Linking Field Network and competitive mechanism. Simulation Experiments demonstrated that our model perfectly fulfilled the task of feature binding in visual perception and provided us some enlightening idea for future research.

  15. Copper(II) binding properties of hepcidin

    OpenAIRE

    Kulprachakarn, Kanokwan; Chen, Yu-Lin; Kong, Xiaole; Arno, Maria Chiara; Hider, Robert Charles; Srichairatanakool, Somdet; Bansal, Sukhvinder

    2016-01-01

    Hepcidin is a peptide hormone that regulates the homeostasis of iron metabolism. The N-terminal domain of hepcidin is conserved amongst a range of species and is capable of binding CuII and NiII through the amino terminal copper–nickel binding motif (ATCUN). It has been suggested that the binding of copper to hepcidin may have biological relevance. In this study we have investigated the binding of CuII with model peptides containing the ATCUN motif, fluorescently labelled hepcidin and hepcidi...

  16. Advances on Plant Pathogenic Mycotoxin Binding Proteins

    Institute of Scientific and Technical Information of China (English)

    WANG Chao-hua; DONG Jin-gao

    2002-01-01

    Toxin-binding protein is one of the key subjects in plant pathogenic mycotoxin research. In this paper, new advances in toxin-binding proteins of 10 kinds of plant pathogenic mycotoxins belonging to Helminthosporium ,Alternaria ,Fusicoccum ,Verticillium were reviewed, especially the techniques and methods of toxin-binding proteins of HS-toxin, HV-toxin, HMT-toxin, HC-toxin. It was proposed that the isotope-labeling technique and immunological chemistry technique should be combined together in research of toxin-binding protein, which will be significant to study the molecular recognition mechanism between host and pathogenic fungus.

  17. 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.

  18. Thermodynamics of ligand binding to acyl-coenzyme A binding protein studied by titration calorimetry

    DEFF Research Database (Denmark)

    Færgeman, Nils J.; Sigurskjold, B W; Kragelund, B B;

    1996-01-01

    Ligand binding to recombinant bovine acyl-CoA binding protein (ACBP) was examined using isothermal microcalorimetry. Microcalorimetric measurements confirm that the binding affinity of acyl-CoA esters for ACBP is strongly dependent on the length of the acyl chain with a clear preference for acyl-...

  19. Molecularly Responsive Binding through Co-occupation of Binding Space: A Lock-Key Story.

    Science.gov (United States)

    Awino, Joseph K; Hu, Lan; Zhao, Yan

    2016-04-01

    When two guest molecules co-occupy a binding pocket of a water-soluble host, the first guest could be used as a signal molecule to turn on the binding of the second. This type of molecularly responsive binding strongly depends on the size of the two guests and the location of the signal molecule. PMID:27001464

  20. CTCF Binding Polarity Determines Chromatin Looping

    NARCIS (Netherlands)

    de Wit, Elzo; Vos, Erica S M; Holwerda, Sjoerd J B; Valdes-Quezada, Christian; Verstegen, Marjon J A M; Teunissen, Hans; Splinter, Erik; Wijchers, Patrick J; Krijger, Peter H L; de Laat, Wouter

    2015-01-01

    CCCTC-binding factor (CTCF) is an architectural protein involved in the three-dimensional (3D) organization of chromatin. In this study, we assayed the 3D genomic contact profiles of a large number of CTCF binding sites with high-resolution 4C-seq. As recently reported, our data also suggest that ch

  1. Localization-enhanced biexciton binding in semiconductors

    DEFF Research Database (Denmark)

    Langbein, Wolfgang Werner; Hvam, Jørn Märcher

    1999-01-01

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

  2. Gravitational Binding Energy in Charged Cylindrical Symmetry

    CERN Document Server

    Sharif, M

    2014-01-01

    We consider static cylindrically symmetric charged gravitating object with perfect fluid and investigate the gravitational binding energy. It is found that only the localized part of the mass function provides the gravitational binding energy, whereas the non-localized part generated by the electric coupling does not contribute for such energy.

  3. (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.

  4. [3H] diazepam binding to human granulocytes

    International Nuclear Information System (INIS)

    [3H]-diazepam binds to sites on human granulocyte membranes, with little or no binding to platelets or lymphocytes. These [3H]-diazepam binding sites are of the peripheral type, being strongly inhibited by R05-4864 (Ki=6.23nM) but only weakly by clonazepam (Ki=14μM). Binding of [3H] diazepam at 00 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

  5. Bacterial periplasmic sialic acid-binding proteins exhibit a conserved binding site

    Energy Technology Data Exchange (ETDEWEB)

    Gangi Setty, Thanuja [Institute for Stem Cell Biology and Regenerative Medicine, NCBS Campus, GKVK Post, Bangalore, Karnataka 560 065 (India); Cho, Christine [Carver College of Medicine, University of Iowa, Iowa City, IA 52242-1109 (United States); Govindappa, Sowmya [Institute for Stem Cell Biology and Regenerative Medicine, NCBS Campus, GKVK Post, Bangalore, Karnataka 560 065 (India); Apicella, Michael A. [Carver College of Medicine, University of Iowa, Iowa City, IA 52242-1109 (United States); Ramaswamy, S., E-mail: ramas@instem.res.in [Institute for Stem Cell Biology and Regenerative Medicine, NCBS Campus, GKVK Post, Bangalore, Karnataka 560 065 (India)

    2014-07-01

    Structure–function studies of sialic acid-binding proteins from F. nucleatum, P. multocida, V. cholerae and H. influenzae reveal a conserved network of hydrogen bonds involved in conformational change on ligand binding. Sialic acids are a family of related nine-carbon sugar acids that play important roles in both eukaryotes and prokaryotes. These sialic acids are incorporated/decorated onto lipooligosaccharides as terminal sugars in multiple bacteria to evade the host immune system. Many pathogenic bacteria scavenge sialic acids from their host and use them for molecular mimicry. The first step of this process is the transport of sialic acid to the cytoplasm, which often takes place using a tripartite ATP-independent transport system consisting of a periplasmic binding protein and a membrane transporter. In this paper, the structural characterization of periplasmic binding proteins from the pathogenic bacteria Fusobacterium nucleatum, Pasteurella multocida and Vibrio cholerae and their thermodynamic characterization are reported. The binding affinities of several mutations in the Neu5Ac binding site of the Haemophilus influenzae protein are also reported. The structure and the thermodynamics of the binding of sugars suggest that all of these proteins have a very well conserved binding pocket and similar binding affinities. A significant conformational change occurs when these proteins bind the sugar. While the C1 carboxylate has been identified as the primary binding site, a second conserved hydrogen-bonding network is involved in the initiation and stabilization of the conformational states.

  6. Bacterial periplasmic sialic acid-binding proteins exhibit a conserved binding site

    International Nuclear Information System (INIS)

    Structure–function studies of sialic acid-binding proteins from F. nucleatum, P. multocida, V. cholerae and H. influenzae reveal a conserved network of hydrogen bonds involved in conformational change on ligand binding. Sialic acids are a family of related nine-carbon sugar acids that play important roles in both eukaryotes and prokaryotes. These sialic acids are incorporated/decorated onto lipooligosaccharides as terminal sugars in multiple bacteria to evade the host immune system. Many pathogenic bacteria scavenge sialic acids from their host and use them for molecular mimicry. The first step of this process is the transport of sialic acid to the cytoplasm, which often takes place using a tripartite ATP-independent transport system consisting of a periplasmic binding protein and a membrane transporter. In this paper, the structural characterization of periplasmic binding proteins from the pathogenic bacteria Fusobacterium nucleatum, Pasteurella multocida and Vibrio cholerae and their thermodynamic characterization are reported. The binding affinities of several mutations in the Neu5Ac binding site of the Haemophilus influenzae protein are also reported. The structure and the thermodynamics of the binding of sugars suggest that all of these proteins have a very well conserved binding pocket and similar binding affinities. A significant conformational change occurs when these proteins bind the sugar. While the C1 carboxylate has been identified as the primary binding site, a second conserved hydrogen-bonding network is involved in the initiation and stabilization of the conformational states

  7. Specific insulin binding in bovine chromaffin cells; demonstration of preferential binding to adrenalin-storing cells

    Energy Technology Data Exchange (ETDEWEB)

    Serck-Hanssen, G.; Soevik, O.

    1987-12-28

    Insulin binding was studied in subpopulations of bovine chromaffin cells enriched in adrenalin-producing cells (A-cells) or noradrenalin-producing cells (NA-cells). Binding of /sup 125/I-insulin was carried out at 15/sup 0/C for 3 hrs in the absence or presence of excess unlabeled hormone. Four fractions of cells were obtained by centrifugation on a stepwise bovine serum albumin gradient. The four fractions were all shown to bind insulin in a specific manner and the highest binding was measured in the cell layers of higher densities, containing mainly A-cells. The difference in binding of insulin to the four subpopulations of chromaffin cells seemed to be related to differences in numbers of receptors as opposed to receptor affinities. The authors conclude that bovine chromaffin cells possess high affinity binding sites for insulin and that these binding sites are mainly confined to A-cells. 24 references, 2 figures, 1 table.

  8. DNA Triplexes That Bind Several Cofactor Molecules.

    Science.gov (United States)

    Vollmer, Sven; Richert, Clemens

    2015-12-14

    Cofactors are critical for energy-consuming processes in the cell. Harnessing such processes for practical applications requires control over the concentration of cofactors. We have recently shown that DNA triplex motifs with a designed binding site can be used to capture and release nucleotides with low micromolar dissociation constants. In order to increase the storage capacity of such triplex motifs, we have explored the limits of ligand binding through designed cavities in the oligopurine tract. Oligonucleotides with up to six non-nucleotide bridges between purines were synthesized and their ability to bind ATP, cAMP or FAD was measured. Triplex motifs with several single-nucleotide binding sites were found to bind purines more tightly than triplexes with one large binding site. The optimized triplex consists of 59 residues and four C3-bridges. It can bind up to four equivalents of ligand with apparent Kd values of 52 µM for ATP, 9 µM for FAD, and 2 µM for cAMP. An immobilized version fuels bioluminescence via release of ATP at body temperature. These results show that motifs for high-density capture, storage and release of energy-rich biomolecules can be constructed from synthetic DNA. PMID:26561335

  9. Copper(II) binding properties of hepcidin.

    Science.gov (United States)

    Kulprachakarn, Kanokwan; Chen, Yu-Lin; Kong, Xiaole; Arno, Maria C; Hider, Robert C; Srichairatanakool, Somdet; Bansal, Sukhvinder S

    2016-06-01

    Hepcidin is a peptide hormone that regulates the homeostasis of iron metabolism. The N-terminal domain of hepcidin is conserved amongst a range of species and is capable of binding Cu(II) and Ni(II) through the amino terminal copper-nickel binding motif (ATCUN). It has been suggested that the binding of copper to hepcidin may have biological relevance. In this study we have investigated the binding of Cu(II) with model peptides containing the ATCUN motif, fluorescently labelled hepcidin and hepcidin using MALDI-TOF mass spectrometry. As with albumin, it was found that tetrapeptide models of hepcidin possessed a higher affinity for Cu(II) than that of native hepcidin. The log K 1 value of hepcidin for Cu(II) was determined as 7.7. Cu(II) binds to albumin more tightly than hepcidin (log K 1 = 12) and in view of the serum concentration difference of albumin and hepcidin, the bulk of kinetically labile Cu(II) present in blood will be bound to albumin. It is estimated that the concentration of Cu(II)-hepcidin will be less than one femtomolar in normal serum and thus the binding of copper to hepcidin is unlikely to play a role in iron homeostasis. As with albumin, small tri and tetra peptides are poor models for the metal binding properties of hepcidin. PMID:26883683

  10. Calmodulin Binding Proteins and Alzheimer's Disease.

    Science.gov (United States)

    O'Day, Danton H; Eshak, Kristeen; Myre, Michael A

    2015-01-01

    The small, calcium-sensor protein, calmodulin, is ubiquitously expressed and central to cell function in all cell types. Here the literature linking calmodulin to Alzheimer's disease is reviewed. Several experimentally-verified calmodulin-binding proteins are involved in the formation of amyloid-β plaques including amyloid-β protein precursor, β-secretase, presenilin-1, and ADAM10. Many others possess potential calmodulin-binding domains that remain to be verified. Three calmodulin binding proteins are associated with the formation of neurofibrillary tangles: two kinases (CaMKII, CDK5) and one protein phosphatase (PP2B or calcineurin). Many of the genes recently identified by genome wide association studies and other studies encode proteins that contain putative calmodulin-binding domains but only a couple (e.g., APOE, BIN1) have been experimentally confirmed as calmodulin binding proteins. At least two receptors involved in calcium metabolism and linked to Alzheimer's disease (mAchR; NMDAR) have also been identified as calmodulin-binding proteins. In addition to this, many proteins that are involved in other cellular events intimately associated with Alzheimer's disease including calcium channel function, cholesterol metabolism, neuroinflammation, endocytosis, cell cycle events, and apoptosis have been tentatively or experimentally verified as calmodulin binding proteins. The use of calmodulin as a potential biomarker and as a therapeutic target is discussed. PMID:25812852

  11. Glycolipid binding preferences of Shiga toxin variants.

    Directory of Open Access Journals (Sweden)

    Sayali S Karve

    Full Text Available The major virulence factor of Shiga toxin producing E. coli, is Shiga toxin (Stx, an AB5 toxin that consists of a ribosomal RNA-cleaving A-subunit surrounded by a pentamer of receptor-binding B subunits. The two major isoforms, Stx1 and Stx2, and Stx2 variants (Stx2a-h significantly differ in toxicity. The exact reason for this toxicity difference is unknown, however different receptor binding preferences are speculated to play a role. Previous studies used enzyme linked immunosorbent assay (ELISA to study binding of Stx1 and Stx2a toxoids to glycolipid receptors. Here, we studied binding of holotoxin and B-subunits of Stx1, Stx2a, Stx2b, Stx2c and Stx2d to glycolipid receptors globotriaosylceramide (Gb3 and globotetraosylceramide (Gb4 in the presence of cell membrane components such as phosphatidylcholine (PC, cholesterol (Ch and other neutral glycolipids. In the absence of PC and Ch, holotoxins of Stx2 variants bound to mixtures of Gb3 with other glycolipids but not to Gb3 or Gb4 alone. Binding of all Stx holotoxins significantly increased in the presence of PC and Ch. Previously, Stx2a has been shown to form a less stable B-pentamer compared to Stx1. However, its effect on glycolipid receptor binding is unknown. In this study, we showed that even in the absence of the A-subunit, the B-subunits of both Stx1 and Stx2a were able to bind to the glycolipids and the more stable B-pentamer formed by Stx1 bound better than the less stable pentamer of Stx2a. B-subunit mutant of Stx1 L41Q, which shows similar stability as Stx2a B-subunits, lacked glycolipid binding, suggesting that pentamerization is more critical for binding of Stx1 than Stx2a.

  12. Binding of uranyl by humic acid

    International Nuclear Information System (INIS)

    The binding of tracer level UO2+2 to a soil humic acid was measured by a solvent extraction technique. The binding is interpreted as involving only the carboxylate groups of the humate and both 1:1 and 1:2 UO2+2:CO2-binding is observed. Estimates based on these values indicate that uranyl complexing by humic and/or fulvic materials is not significant in sea water but may play a role in fresh water systems. Retention of uranyl from ground water by soil humics would be strong. (author)

  13. Measuring Binding Affinity of Protein-Ligand Interaction Using Spectrophotometry: Binding of Neutral Red to Riboflavin-Binding Protein

    Science.gov (United States)

    Chenprakhon, Pirom; Sucharitakul, Jeerus; Panijpan, Bhinyo; Chaiyen, Pimchai

    2010-01-01

    The dissociation constant, K[subscript d], of the binding of riboflavin-binding protein (RP) with neutral red (NR) can be determined by titrating RP to a fixed concentration of NR. Upon adding RP to the NR solution, the maximum absorption peak of NR shifts to 545 nm from 450 nm for the free NR. The change of the absorption can be used to determine…

  14. Genetics Home Reference: mannose-binding lectin deficiency

    Science.gov (United States)

    ... Health Conditions mannose-binding lectin deficiency mannose-binding lectin deficiency Enable Javascript to view the expand/collapse ... PDF Open All Close All Description Mannose-binding lectin deficiency is a condition that affects the immune ...

  15. Peptide binding specificity of the chaperone calreticulin

    DEFF Research Database (Denmark)

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

    2007-01-01

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

  16. Hardware device binding and mutual authentication

    Energy Technology Data Exchange (ETDEWEB)

    Hamlet, Jason R; Pierson, Lyndon G

    2014-03-04

    Detection and deterrence of device tampering and subversion by substitution may be achieved by including a cryptographic unit within a computing device for binding multiple hardware devices and mutually authenticating the devices. The cryptographic unit includes a physically unclonable function ("PUF") circuit disposed in or on the hardware device, which generates a binding PUF value. The cryptographic unit uses the binding PUF value during an enrollment phase and subsequent authentication phases. During a subsequent authentication phase, the cryptographic unit uses the binding PUF values of the multiple hardware devices to generate a challenge to send to the other device, and to verify a challenge received from the other device to mutually authenticate the hardware devices.

  17. Hydrogen binding in vacancy clusters in platinum

    International Nuclear Information System (INIS)

    The binding of hydrogen in different vacancy complexes in platinum metal was investigated with atomic-scale sensitivity using perturbed angular correlations of gamma rays (PAC). Hydrogen was introduced by cathodic charging. Detrapping was monitored microscopically during desorption at 294 K by changes in site fractions of hydrogen-decorated and undecorated complexes. Analysis of desorption includes effects of retrapping of hydrogen at other sites. Assuming a trap concentration of 10-3, binding enthalpies of 0.23(2), 0.28(1), 0.24(1) and >0.20 eV are obtained for hydrogen atoms in 1V to 4V complexes, respectively. The small differences between the binding enthalpies demonstrate that hydrogen binding is insensitive to the detailed geometrical structure of small vacancy complexes. However, the magnitudes found here are a factor of two smaller than in the literature. (orig.)

  18. System Support for Managing Invalid Bindings

    CERN Document Server

    Das, Lachhman; Shah, Azhar; Khoumbati, Khalil; 10.5121/iju.2011.2303

    2011-01-01

    Context-aware adaptation is a central aspect of pervasive computing applications, enabling them to adapt and perform tasks based on contextual information. One of the aspects of context-aware adaptation is reconfiguration in which bindings are created between application component and remote services in order to realize new behaviour in response to contextual information. Various research efforts provide reconfiguration support and allow the development of adaptive context-aware applications from high-level specifications, but don't consider failure conditions that might arise during execution of such applications, making bindings between application and remote services invalid. To this end, we propose and implement our design approach to reconfiguration to manage invalid bindings. The development and modification of adaptive context-aware applications is a complex task, and an issue of an invalidity of bindings further complicates development efforts. To reduce the development efforts, our approach provides ...

  19. Binding of heparan sulfate to Staphylococcus aureus.

    OpenAIRE

    Liang, O D; Ascencio, F; Fransson, L A; Wadström, T

    1992-01-01

    Heparan sulfate binds to proteins present on the surface of Staphylococcus aureus cells. Binding of 125I-heparan sulfate to S. aureus was time dependent, saturable, and influenced by pH and ionic strength, and cell-bound 125I-heparan sulfate was displaced by unlabelled heparan sulfate or heparin. Other glycosaminoglycans of comparable size (chondroitin sulfate and dermatan sulfate), highly glycosylated glycoprotein (hog gastric mucin), and some anionic polysaccharides (dextran sulfate and RNA...

  20. DNA-Aptamers Binding Aminoglycoside Antibiotics

    OpenAIRE

    Nadia Nikolaus; Beate Strehlitz

    2014-01-01

    Aptamers are short, single stranded DNA or RNA oligonucleotides that are able to bind specifically and with high affinity to their non-nucleic acid target molecules. This binding reaction enables their application as biorecognition elements in biosensors and assays. As antibiotic residues pose a problem contributing to the emergence of antibiotic-resistant pathogens and thereby reducing the effectiveness of the drug to fight human infections, we selected aptamers targeted against the aminog...

  1. Penicillin-Binding Protein Imaging Probes

    OpenAIRE

    Kocaoglu, Ozden; Carlson, Erin E.

    2013-01-01

    Penicillin-binding proteins (PBPs) are membrane-associated proteins involved in the biosynthesis of peptidoglycan (PG), the main component of bacterial cell walls. These proteins were discovered and named for their affinity to bind the β-lactam antibiotic penicillin. The importance of the PBPs has long been appreciated; however, the apparent functional redundancy of the ~5–15 proteins that most bacteria possess makes determination of their individual roles difficult. Existing techniques to st...

  2. Photonic Binding in Silicon-Colloid Microcavities

    OpenAIRE

    Xifré-Pérez, E.; García de Abajo, Francisco Javier; Fenollosa Esteve, Roberto; Meseguer, Francisco

    2009-01-01

    Photonic binding between two identical silicon-colloid-based microcavities is studied by using a generalized multipolar expansion. In contrast with previous works, we focus on low-order cavity modes that resemble low-energy electronic orbitals. For conservative light intensities, the interaction between cavity modes with moderate Q factors produces extremely large particle acceleration values. Optical forces dominate over vanderWaals, gravity, and Brownian motion, and they show a binding-anti...

  3. Liver Fatty Acid Binding Protein and Obesity

    OpenAIRE

    Atshaves, B.P.; Martin, G G; Hostetler, H.A.; McIntosh, A.L.; Kier, A B; Schroeder, F.

    2010-01-01

    While low levels of unesterified long chain fatty acids (LCFAs) are normal metabolic intermediates of dietary and endogenous fat, LCFAs are also potent regulators of key receptors/enzymes, and at high levels become toxic detergents within the cell. Elevated levels of LCFAs are associated with diabetes, obesity, and metabolic syndrome. Consequently, mammals evolved fatty acid binding proteins (FABPs) that bind/sequester these potentially toxic free fatty acids in the cytosol and present them f...

  4. Radiation damage to DNA-binding proteins

    International Nuclear Information System (INIS)

    The DNA-binding properties of proteins are strongly affected upon irradiation. The tetrameric lactose repressor (a dimer of dimers) losses its ability to bind operator DNA as soon as at least two damages per protomer of each dimer occur. The monomeric MC1 protein losses its ability to bind DNA in two steps : i) at low doses only the specific binding is abolished, whereas the non-specific one is still possible; ii) at high doses all binding vanishes. Moreover, the DNA bending induced by MC1 binding is less pronounced for a protein that underwent the low dose irradiation. When the entire DNA-protein complexes are irradiated, the observed disruption of the complexes is mainly due to the damage of the proteins and not to that of DNA. The doses necessary for complex disruption are higher than those inactivating the free protein. This difference, larger for MC1 than for lactose repressor, is due to the protection of the protein by the bound DNA. The oxidation of the protein side chains that are accessible to the radiation-induced hydroxyl radicals seems to represent the inactivating damage

  5. Impact of receptor clustering on ligand binding

    Directory of Open Access Journals (Sweden)

    Caré Bertrand R

    2011-03-01

    Full Text Available Abstract Background Cellular response to changes in the concentration of different chemical species in the extracellular medium is induced by ligand binding to dedicated transmembrane receptors. Receptor density, distribution, and clustering may be key spatial features that influence effective and proper physical and biochemical cellular responses to many regulatory signals. Classical equations describing this kind of binding kinetics assume the distributions of interacting species to be homogeneous, neglecting by doing so the impact of clustering. As there is experimental evidence that receptors tend to group in clusters inside membrane domains, we investigated the effects of receptor clustering on cellular receptor ligand binding. Results We implemented a model of receptor binding using a Monte-Carlo algorithm to simulate ligand diffusion and binding. In some simple cases, analytic solutions for binding equilibrium of ligand on clusters of receptors are provided, and supported by simulation results. Our simulations show that the so-called "apparent" affinity of the ligand for the receptor decreases with clustering although the microscopic affinity remains constant. Conclusions Changing membrane receptors clustering could be a simple mechanism that allows cells to change and adapt its affinity/sensitivity toward a given stimulus.

  6. The readiness potential reflects intentional binding

    Directory of Open Access Journals (Sweden)

    Han-Gue eJo

    2014-06-01

    Full Text Available When a voluntary action is causally linked with a sensory outcome, the action and its consequent effect are perceived as being closer together in time. This effect is called intentional binding. Although many experiments were conducted on this phenomenon, the underlying neural mechanisms are not well understood. While intentional binding is specific to voluntary action, we presumed that preconscious brain activity (the readiness potential, RP, which occurs before an action is made, might play an important role in this binding effect. In this study, the brain dynamics were recorded with electroencephalography (EEG and analyzed in single-trials in order to estimate whether intentional binding is correlated with the early neural processes. Moreover, we were interested in different behavioral performance between meditators and non-meditators since meditators are expected to be able to keep attention more consistently on a task. Thus, we performed the intentional binding paradigm with twenty mindfulness meditators and compared them to matched controls. Although, we did not observe a group effect on either behavioral data or EEG recordings, we found that self-initiated movements following ongoing negative deflections of slow cortical potentials (SCPs result in a stronger binding effect compared to positive potentials, especially regarding the perceived time of the consequent effect. Our results provide the first direct evidence that the early neural activity within the range of SCPs affects perceived time of a sensory outcome that is caused by intentional action.

  7. DNA-Aptamers Binding Aminoglycoside Antibiotics

    Directory of Open Access Journals (Sweden)

    Nadia Nikolaus

    2014-02-01

    Full Text Available Aptamers are short, single stranded DNA or RNA oligonucleotides that are able to bind specifically and with high affinity to their non-nucleic acid target molecules. This binding reaction enables their application as biorecognition elements in biosensors and assays. As antibiotic residues pose a problem contributing to the emergence of antibiotic-resistant pathogens and thereby reducing the effectiveness of the drug to fight human infections, we selected aptamers targeted against the aminoglycoside antibiotic kanamycin A with the aim of constructing a robust and functional assay that can be used for water analysis. With this work we show that aptamers that were derived from a Capture-SELEX procedure targeting against kanamycin A also display binding to related aminoglycoside antibiotics. The binding patterns differ among all tested aptamers so that there are highly substance specific aptamers and more group specific aptamers binding to a different variety of aminoglycoside antibiotics. Also the region of the aminoglycoside antibiotics responsible for aptamer binding can be estimated. Affinities of the different aptamers for their target substance, kanamycin A, are measured with different approaches and are in the micromolar range. Finally, the proof of principle of an assay for detection of kanamycin A in a real water sample is given.

  8. Protein Dynamics in an RNA Binding Protein

    Science.gov (United States)

    Hall, Kathleen

    2006-03-01

    Using ^15N NMR relaxation measurements, analyzed with the Lipari-Szabo formalism, we have found that the human U1A RNA binding protein has ps-ns motions in those loops that make contact with RNA. Specific mutations can alter the extent and pattern of motions, and those proteins inevitably lose RNA binding affinity. Proteins with enhanced mobility of loops and termini presumably lose affinity due to increased conformational sampling by those parts of the protein that interact directly with RNA. There is an entropic penalty associated with locking down those elements upon RNA binding, in addition to a loss of binding efficiency caused by the increased number of conformations adopted by the protein. However, in addition to local conformational heterogeneity, analysis of molecular dynamics trajectories by Reorientational Eigenmode Dynamics reveals that loops of the wild type protein undergo correlated motions that link distal sites across the binding surface. Mutations that disrupt correlated motions result in weaker RNA binding, implying that there is a network of interactions across the surface of the protein. (KBH was a Postdoctoral Fellow with Al Redfield from 1985-1990). This work was supported by the NIH (to KBH) and NSF (SAS).

  9. The readiness potential reflects intentional binding

    Science.gov (United States)

    Jo, Han-Gue; Wittmann, Marc; Hinterberger, Thilo; Schmidt, Stefan

    2014-01-01

    When a voluntary action is causally linked with a sensory outcome, the action and its consequent effect are perceived as being closer together in time. This effect is called intentional binding. Although many experiments were conducted on this phenomenon, the underlying neural mechanisms are not well understood. While intentional binding is specific to voluntary action, we presumed that preconscious brain activity (the readiness potential, RP), which occurs before an action is made, might play an important role in this binding effect. In this study, the brain dynamics were recorded with electroencephalography (EEG) and analyzed in single-trials in order to estimate whether intentional binding is correlated with the early neural processes. Moreover, we were interested in different behavioral performance between meditators and non-meditators since meditators are expected to be able to keep attention more consistently on a task. Thus, we performed the intentional binding paradigm with 20 mindfulness meditators and compared them to matched controls. Although, we did not observe a group effect on either behavioral data or EEG recordings, we found that self-initiated movements following ongoing negative deflections of slow cortical potentials (SCPs) result in a stronger binding effect compared to positive potentials, especially regarding the perceived time of the consequent effect. Our results provide the first direct evidence that the early neural activity within the range of SCPs affects perceived time of a sensory outcome that is caused by intentional action. PMID:24959135

  10. Theoretical studies of binding of mannose-binding protein to monosaccharides

    Science.gov (United States)

    Aida-Hyugaji, Sachiko; Takano, Keiko; Takada, Toshikazu; Hosoya, Haruo; Kojima, Naoya; Mizuochi, Tsuguo; Inoue, Yasushi

    2004-11-01

    Binding properties of mannose-binding protein (MBP) to monosaccharides are discussed based on ab initio molecular orbital calculations for cluster models constructed. The calculated binding energies indicate that MBP has an affinity for N-acetyl- D-glucosamine, D-mannose, L-fucose, and D-glucose rather than D-galactose and N-acetyl- D-galactosamine, which is consistent with the biochemical experimental results. Electrostatic potential surfaces at the binding site of four monosaccharides having binding properties matched well with that of MBP. A vacant frontier orbital was found to be localized around the binding site of MBP, suggesting that MBP-monosaccharide interaction may occur through electrostatic and orbital interactions.

  11. To Bind or not to Bind: It’s in the Contract

    DEFF Research Database (Denmark)

    Tvarnø, Christina D.

    2016-01-01

    This article discusses the formalization of collaboration through partnering contracts in the construction industry in the USA, Great Britain and Denmark. The article compares the different types of collaborative partnering contracts in the three countries, and provides a conclusion on whether the...... collaborative partnering contract should be binding or non-binding, based on the three empirical contracts analyzed in this article. The partnering contracts in Great Britain and Denmark are legally binding, while in the USA the partnering agreements are non-binding charters or letters of intent. This article...... discusses, in a theoretical perspective, the legal reasoning behind the different partnering approaches, both from a historical and contract law perspective, and furthermore applies a game theoretical approach in evaluating binding versus non-binding partnering contracts. The analysis focuses on private...

  12. Thermodynamic parameters of the binding of retinol to binding proteins and to membranes

    International Nuclear Information System (INIS)

    Retinol (vitamin A alcohol) is a hydrophobic compound and distributes in vivo mainly between binding proteins and cellular membranes. To better clarify the nature of the interactions of retinol with these phases which have a high affinity for it, the thermodynamic parameters of these interactions were studied. The temperature-dependence profiles of the binding of retinol to bovine retinol binding protein, bovine serum albumin, unilamellar vesicles of dioleoylphosphatidylcholine, and plasma membranes from rat liver were determined. It was found that binding of retinol to retinol binding protein is characterized by a large increase in entropy and no change in enthalpy. Binding to albumin is driven by enthalpy and is accompanied by a decrease in entropy. Partitioning of retinal into unilamellar vesicles and into plasma membranes is stabilized both by enthalpic and by entropic components. The implications of these finding are discussed

  13. Fucose-binding Lotus tetragonolobus lectin binds to human polymorphonuclear leukocytes and induces a chemotactic response.

    Science.gov (United States)

    VanEpps, D E; Tung, K S

    1977-09-01

    Fucose-binding L. tetragonolobus lectin to the surface of human polymorphonuclear leukocytes (PMN) and induces a chemotactic response. Both surface binding and chemotaxis are inhibited by free fucose but not by fructose, mannose, or galactose. The lectin-binding sites on PMN are unrelated to the A, B, or O blood group antigen. Utilization of this lectin should be a useful tool in isolating PMN membrane components and in analyzing the mechanism of neutrophil chemotaxis. PMID:330752

  14. Effect of solid surface charge on the binding behaviour of a metal-binding peptide

    OpenAIRE

    Donatan, Senem; Sarikaya, Mehmet; TAMERLER, Candan; Urgen, Mustafa

    2012-01-01

    Over the last decade, solid-binding peptides have been increasingly used as molecular building blocks coupling bio- and nanotechnology. Despite considerable research being invested in this field, the effects of many surface-related parameters that define the binding of peptide to solids are still unknown. In the quest to control biological molecules at solid interfaces and, thereby, tailoring the binding characteristics of the peptides, the use of surface charge of the solid surface may proba...

  15. Solution Structure and Backbone Dynamics of Human Liver Fatty Acid Binding Protein: Fatty Acid Binding Revisited

    OpenAIRE

    Cai, Jun; Lücke, Christian; Chen, Zhongjing; Qiao, Ye; Klimtchuk, Elena; Hamilton, James A.

    2012-01-01

    Liver fatty acid binding protein (L-FABP), a cytosolic protein most abundant in liver, is associated with intracellular transport of fatty acids, nuclear signaling, and regulation of intracellular lipolysis. Among the members of the intracellular lipid binding protein family, L-FABP is of particular interest as it can i), bind two fatty acid molecules simultaneously and ii), accommodate a variety of bulkier physiological ligands such as bilirubin and fatty acyl CoA. To better understand the p...

  16. Hemoglobin binding activity and hemoglobin-binding protein of prevotella nigrescens

    OpenAIRE

    Miyashita M; Oishi S; Kiso A; Kikuchi Y; Ueda O; Hirai K; Shibata Y; Fujimura S

    2010-01-01

    Abstract Prevotella nigrescens, lacking siderophores was found to bind to the hemoproteins. The binding was observed also in the envelope which was prepared by sonication of the cell. The binding occurred in the pH-dependent manner; the binding was observed below neutral pHs of the incubation mixtures but only slightly observed in the neutral and alkaline pHs. Furthermore, hemoglobin bound to the envelope was dissociated at high pHs buffers. Maximum amounts of hemoglobin bound to 1 mg envelop...

  17. Crystal Structure of the Botulinum Neurotoxin Type G Binding Domain: Insight into Cell Surface Binding

    Energy Technology Data Exchange (ETDEWEB)

    Stenmark, Pål; Dong, Min; Dupuy, Jérôme; Chapman, Edwin R.; Stevens, Raymond C. (Scripps); (UW)

    2011-11-02

    Botulinum neurotoxins (BoNTs) typically bind the neuronal cell surface via dual interactions with both protein receptors and gangliosides. We present here the 1.9-{angstrom} X-ray structure of the BoNT serotype G (BoNT/G) receptor binding domain (residues 868-1297) and a detailed view of protein receptor and ganglioside binding regions. The ganglioside binding motif (SxWY) has a conserved structure compared to the corresponding regions in BoNT serotype A and BoNT serotype B (BoNT/B), but several features of interactions with the hydrophilic face of the ganglioside are absent at the opposite side of the motif in the BoNT/G ganglioside binding cleft. This may significantly reduce the affinity between BoNT/G and gangliosides. BoNT/G and BoNT/B share the protein receptor synaptotagmin (Syt) I/II. The Syt binding site has a conserved hydrophobic plateau located centrally in the proposed protein receptor binding interface (Tyr1189, Phe1202, Ala1204, Pro1205, and Phe1212). Interestingly, only 5 of 14 residues that are important for binding between Syt-II and BoNT/B are conserved in BoNT/G, suggesting that the means by which BoNT/G and BoNT/B bind Syt diverges more than previously appreciated. Indeed, substitution of Syt-II Phe47 and Phe55 with alanine residues had little effect on the binding of BoNT/G, but strongly reduced the binding of BoNT/B. Furthermore, an extended solvent-exposed hydrophobic loop, located between the Syt binding site and the ganglioside binding cleft, may serve as a third membrane association and binding element to contribute to high-affinity binding to the neuronal membrane. While BoNT/G and BoNT/B are homologous to each other and both utilize Syt-I/Syt-II as their protein receptor, the precise means by which these two toxin serotypes bind to Syt appears surprisingly divergent.

  18. Promoter-distal RNA polymerase II binding discriminates active from inactive CCAAT/ enhancer-binding protein beta binding sites

    Science.gov (United States)

    Savic, Daniel; Roberts, Brian S.; Carleton, Julia B.; Partridge, E. Christopher; White, Michael A.; Cohen, Barak A.; Cooper, Gregory M.; Gertz, Jason; Myers, Richard M.

    2015-01-01

    Transcription factors (TFs) bind to thousands of DNA sequences in mammalian genomes, but most of these binding events appear to have no direct effect on gene expression. It is unclear why only a subset of TF bound sites are actively involved in transcriptional regulation. Moreover, the key genomic features that accurately discriminate between active and inactive TF binding events remain ambiguous. Recent studies have identified promoter-distal RNA polymerase II (RNAP2) binding at enhancer elements, suggesting that these interactions may serve as a marker for active regulatory sequences. Despite these correlative analyses, a thorough functional validation of these genomic co-occupancies is still lacking. To characterize the gene regulatory activity of DNA sequences underlying promoter-distal TF binding events that co-occur with RNAP2 and TF sites devoid of RNAP2 occupancy using a functional reporter assay, we performed cis-regulatory element sequencing (CRE-seq). We tested more than 1000 promoter-distal CCAAT/enhancer-binding protein beta (CEBPB)-bound sites in HepG2 and K562 cells, and found that CEBPB-bound sites co-occurring with RNAP2 were more likely to exhibit enhancer activity. CEBPB-bound sites further maintained substantial cell-type specificity, indicating that local DNA sequence can accurately convey cell-type–specific regulatory information. By comparing our CRE-seq results to a comprehensive set of genome annotations, we identified a variety of genomic features that are strong predictors of regulatory element activity and cell-type–specific activity. Collectively, our functional assay results indicate that RNAP2 occupancy can be used as a key genomic marker that can distinguish active from inactive TF bound sites. PMID:26486725

  19. Binding of Fidarestat Stereoisomers with Aldose Reductase

    Directory of Open Access Journals (Sweden)

    Dae-Sil Lee

    2006-11-01

    Full Text Available The stereospecificity in binding to aldose reductase (ALR2 of two fidarestat {6-fluoro-2',5'-dioxospiro[chroman-4,4'-imidazolidine]-2-carboxamide} stereoisomers [(2S,4Sand (2R,4S] has been investigated by means of molecular dynamics simulations using freeenergy integration techniques. The difference in the free energy of binding was found to be2.0 ± 1.7 kJ/mol in favour of the (2S,4S-form, in agreement with the experimentalinhibition data. The relative mobilities of the fidarestats complexed with ALR2 indicate alarger entropic penalty for hydrophobic binding of (2R,4S-fidarestat compared to (2S,4S-fidarestat, partially explaining its lower binding affinity. The two stereoisomers differmainly in the orientation of the carbamoyl moiety with respect to the active site and rotationof the bond joining the carbamoyl substituent to the ring. The detailed structural andenergetic insights obtained from out simulations allow for a better understanding of thefactors determining stereospecific inhibitor-ALR2 binding in the EPF charges model.

  20. Donkey anaphora is in-scope binding

    Directory of Open Access Journals (Sweden)

    Chris Barker

    2008-05-01

    Full Text Available We propose that the antecedent of a donkey pronoun takes scope over and binds the donkey pronoun, just like any other quantificational antecedent would bind a pronoun. We flesh out this idea in a grammar that compositionally derives the truth conditions of donkey sentences containing conditionals and relative clauses, including those involving modals and proportional quantifiers. For example, an indefinite in the antecedent of a conditional can bind a donkey pronoun in the consequent by taking scope over the entire conditional. Our grammar manages continuations using three independently motivated type-shifters, Lift, Lower, and Bind. Empirical support comes from donkey weak crossover (*He beats it if a farmer owns a donkey: in our system, a quantificational binder need not c-command a pronoun that it binds, but must be evaluated before it, so that donkey weak crossover is just a special case of weak crossover. We compare our approach to situation-based E-type pronoun analyses, as well as to dynamic accounts such as Dynamic Predicate Logic. A new 'tower' notation makes derivations considerably easier to follow and manipulate than some previous grammars based on continuations. http://dx.doi.org/10.3765/sp.1.1 BibTeX info See also the interactive tutorial about the system in this paper

  1. Conformational heterogeneity of the calmodulin binding interface

    Science.gov (United States)

    Shukla, Diwakar; Peck, Ariana; Pande, Vijay S.

    2016-04-01

    Calmodulin (CaM) is a ubiquitous Ca2+ sensor and a crucial signalling hub in many pathways aberrantly activated in disease. However, the mechanistic basis of its ability to bind diverse signalling molecules including G-protein-coupled receptors, ion channels and kinases remains poorly understood. Here we harness the high resolution of molecular dynamics simulations and the analytical power of Markov state models to dissect the molecular underpinnings of CaM binding diversity. Our computational model indicates that in the absence of Ca2+, sub-states in the folded ensemble of CaM's C-terminal domain present chemically and sterically distinct topologies that may facilitate conformational selection. Furthermore, we find that local unfolding is off-pathway for the exchange process relevant for peptide binding, in contrast to prior hypotheses that unfolding might account for binding diversity. Finally, our model predicts a novel binding interface that is well-populated in the Ca2+-bound regime and, thus, a candidate for pharmacological intervention.

  2. 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.

  3. Binding of anandamide to bovine serum albumin

    DEFF Research Database (Denmark)

    Bojesen, I.N.; Hansen, Harald S.

    2003-01-01

    The endocannabinoid anandamide is of lipid nature and may thus bind to albumin in the vascular system, as do fatty acids. The knowledge of the free water-phase concentration of anandamide is essential for the investigations of its transfer from the binding protein to cellular membranes, because a...... water-phase shuttle of monomers mediates such transfers. We have used our method based upon the use of albumin-filled red cell ghosts as a dispersed biological "reference binder" to measure the water-phase concentrations of anandamide. These concentrations were measured in buffer (pH 7.3) in equilibrium...... data suggest that BSA has one high-affinity binding site for anandamide at all four temperatures. The free energy of anandamide binding (¿G) is calculated to -43.05 kJ mol with a large enthalpy (¿H ) contribution of -42.09 kJ mol. Anandamide has vasodilator activity, and the binding to albumin may...

  4. Characterization of DNA Binding and Retinoic Acid Binding Properties of Retinoic Acid Receptor

    Science.gov (United States)

    Yang, Na; Schule, Roland; Mangelsdorf, David J.; Evans, Ronald M.

    1991-05-01

    High-level expression of the full-length human retinoic acid receptor (RAR) α and the DNA binding domain of the RAR in Escherichia coli was achieved by using a T7 RNA polymerase-directed expression system. After induction, full-length RAR protein was produced at an estimated level of 20% of the total bacterial proteins. Both intact RAR molecules and the DNA binding domain bind to the cognate DNA response element with high specificity in the absence of retinoic acid. However, this binding is enhanced to a great extent upon the addition of eukaryotic cell extracts. The factor responsible for this enhancement is heat-sensitive and forms a complex with RAR that binds to DNA and exhibits a distinct migration pattern in the gel-mobility-shift assay. The interaction site of the factor with RAR is localized in the 70-amino acid DNA binding region of RAR. The hormone binding ability of the RARα protein was assayed by a charcoal absorption assay and the RAR protein was found to bind to retinoic acid with a K_d of 2.1 x 10-10 M.

  5. Estradiol Binds to Insulin and Insulin Receptor Decreasing Insulin Binding in vitro

    OpenAIRE

    RobertRoot-Bernstein

    2014-01-01

    Rationale: Insulin resistance associated with hyperestrogenemias occurs in gestational diabetes mellitus, polycystic ovary syndrome, ovarian hyperstimulation syndrome, estrogen therapies, metabolic syndrome and obesity. The mechanism by which insulin and estrogen interact is unknown. We hypothesize that estrogen binds directly to insulin and the insulin receptor producing insulin resistance. Objectives: To determine the binding constants of steroid hormones to insulin, the insulin recepto...

  6. The interrelationship between ligand binding and self-association of the folate binding protein

    DEFF Research Database (Denmark)

    Holm, Jan; Schou, Christian; Babol, Linnea N.;

    2011-01-01

    The folate binding protein (FBP) regulates homeostasis and intracellular trafficking of folic acid, a vitamin of decisive importance in cell division and growth. We analyzed whether interrelationship between ligand binding and self-association of FBP plays a significant role in the physiology of...

  7. Natural ligand binding and transfer from liver fatty acid binding protein (LFABP) to membranes.

    Science.gov (United States)

    De Gerónimo, Eduardo; Hagan, Robert M; Wilton, David C; Córsico, Betina

    2010-09-01

    Liver fatty acid-binding protein (LFABP) is distinctive among fatty acid-binding proteins because it binds more than one molecule of long-chain fatty acid and a variety of diverse ligands. Also, the transfer of fluorescent fatty acid analogues to model membranes under physiological ionic strength follows a different mechanism compared to most of the members of this family of intracellular lipid binding proteins. Tryptophan insertion mutants sensitive to ligand binding have allowed us to directly measure the binding affinity, ligand partitioning and transfer to model membranes of natural ligands. Binding of fatty acids shows a cooperative mechanism, while acyl-CoAs binding presents a hyperbolic behavior. Saturated fatty acids seem to have a stronger partition to protein vs. membranes, compared to unsaturated fatty acids. Natural ligand transfer rates are more than 200-fold higher compared to fluorescently-labeled analogues. Interestingly, oleoyl-CoA presents a markedly different transfer behavior compared to the rest of the ligands tested, probably indicating the possibility of specific targeting of ligands to different metabolic fates. PMID:20541621

  8. Asporin competes with decorin for collagen binding, binds calcium and promotes osteoblast collagen mineralization

    DEFF Research Database (Denmark)

    Kalamajski, Sebastian; Aspberg, Anders; Lindblom, Karin;

    2009-01-01

    The interactions of the ECM (extracellular matrix) protein asporin with ECM components have previously not been investigated. Here, we show that asporin binds collagen type I. This binding is inhibited by recombinant asporin fragment LRR (leucine-rich repeat) 10-12 and by full-length decorin, but...... not by biglycan. We demonstrate that the polyaspartate domain binds calcium and regulates hydroxyapatite formation in vitro. In the presence of asporin, the number of collagen nodules, and mRNA of osteoblastic markers Osterix and Runx2, were increased. Moreover, decorin or the collagen-binding asporin...... fragment LRR 10-12 inhibited the pro-osteoblastic activity of full-length asporin. Our results suggest that asporin and decorin compete for binding to collagen and that the polyaspartate in asporin directly regulates collagen mineralization. Therefore asporin has a role in osteoblast-driven collagen...

  9. The receptor binding domain of botulinum neurotoxin serotype C binds phosphoinositides.

    Science.gov (United States)

    Zhang, Yanfeng; Varnum, Susan M

    2012-03-01

    Botulinum neurotoxins (BoNTs) are the most toxic proteins known for humans and animals with an extremely low LD(50) of ∼1 ng/kg. BoNTs generally require a protein and a ganglioside on the cell membrane surface for binding, which is known as a "dual receptor" mechanism for host intoxication. Recent studies have suggested that in addition to gangliosides, other membrane lipids such as phosphoinositides may be involved in the interactions with the receptor binding domain (HCR) of BoNTs for better membrane penetration. Using two independent lipid-binding assays, we tested the interactions of BoNT/C-HCR with lipids in vitro domain. BoNT/C-HCR was found to bind negatively charged phospholipids, preferentially phosphoinositides in both assays. Interactions with phosphoinositides may facilitate tighter binding between neuronal membranes and BoNT/C. PMID:22120109

  10. Conformation-controlled binding kinetics of antibodies

    Science.gov (United States)

    Galanti, Marta; Fanelli, Duccio; Piazza, Francesco

    2016-01-01

    Antibodies are large, extremely flexible molecules, whose internal dynamics is certainly key to their astounding ability to bind antigens of all sizes, from small hormones to giant viruses. In this paper, we build a shape-based coarse-grained model of IgG molecules and show that it can be used to generate 3D conformations in agreement with single-molecule Cryo-Electron Tomography data. Furthermore, we elaborate a theoretical model that can be solved exactly to compute the binding rate constant of a small antigen to an IgG in a prescribed 3D conformation. Our model shows that the antigen binding process is tightly related to the internal dynamics of the IgG. Our findings pave the way for further investigation of the subtle connection between the dynamics and the function of large, flexible multi-valent molecular machines.

  11. Predicting binding free energies in solution

    CERN Document Server

    Jensen, Jan H

    2015-01-01

    Recent predictions of absolute binding free energies of host-guest complexes in aqueous solution using electronic structure theory have been encouraging for some systems, while other systems remain problematic for others. In paper I summarize some of the many factors that could easily contribute 1-3 kcal/mol errors at 298 K: three-body dispersion effects, molecular symmetry, anharmonicity, spurious imaginary frequencies, insufficient conformational sampling, wrong or changing ionization states, errors in the solvation free energy of ions, and explicit solvent (and ion) effects that are not well-represented by continuum models. While the paper is primarily a synthesis of previously published work there are two new results: the adaptation of Legendre transformed free energies to electronic structure theory and a use of water clusters that maximizes error cancellation in binding free energies computed using explicit solvent molecules. While I focus on binding free energies in aqueous solution the approach also a...

  12. Binding of calcium and carbonate to polyacrylates.

    Science.gov (United States)

    Tribello, Gareth A; Liew, CheeChin; Parrinello, Michele

    2009-05-21

    Polyacrylate molecules can be used to slow the growth of calcium carbonate. However, little is known about the mechanism by which the molecules impede the growth rate. A recent computational study (Bulo et al. Macromolecules 2007, 40, 3437) used metadynamics to investigate the binding of calcium to polyacrylate chains and has thrown some light on the coiling and precipitation of these polymers. We extend these simulations to examine the binding of calcium and carbonate to polyacrylate chains. We show that calcium complexed with both carbonate and polyacrylate is a very stable species. The free energies of calcium-carbonate-polyacrylate complexes, with different polymer configurations, are calculated, and differences in the free energy of the binding of carbonate are shown to be due to differences in the amount of steric hindrance about the calcium, which prevents the approach of the carbonate ion. PMID:19400592

  13. Mercury-binding proteins of Mytilus edulis

    Energy Technology Data Exchange (ETDEWEB)

    Roesijadi, G.; Morris, J. E.; Calabrese, A.

    1981-11-01

    Mytilus edulis possesses low molecular weight, mercury-binding proteins. The predominant protein isolated from gill tissue is enriched in cysteinyl residues (8%) and possesses an amino acid composition similar to cadmium-binding proteins of mussels and oysters. Continuous exposure of mussels to 5 ..mu..g/l mercury results in spillover of mercury from these proteins to high molecular weight proteins. Antibodies to these proteins have been isolated, and development of immunoassays is presently underway. Preliminary studies to determine whether exposure of adult mussels to mercury will result in induction of mercury-binding proteins in offspring suggest that such proteins occur in larvae although additional studies are indicated for a conclusive demonstration.

  14. Binding in short-term visual memory.

    Science.gov (United States)

    Wheeler, Mary E; Treisman, Anne M

    2002-03-01

    The integration of complex information in working memory, and its effect on capacity, shape the limits of conscious cognition. The literature conflicts on whether short-term visual memory represents information as integrated objects. A change-detection paradigm using objects defined by color with location or shape was used to investigate binding in short-term visual memory. Results showed that features from the same dimension compete for capacity, whereas features from different dimensions can be stored in parallel. Binding between these features can occur, but focused attention is required to create and maintain the binding over time, and this integrated format is vulnerable to interference. In the proposed model, working memory capacity is limited both by the independent capacity of simple feature stores and by demands on attention networks that integrate this distributed information into complex but unified thought objects. PMID:11900102

  15. [Water binding of adsorptive immobilized lipases].

    Science.gov (United States)

    Loose, S; Meusel, D; Muschter, A; Ruthe, B

    1990-01-01

    It is supposed that not only the total water content of lipase preparations but more their state of water binding is of technological importance in enzymatic interesterification reactions in systems nearly free from water. The isotherms at 65 degrees C of two microbial lipases immobilized on various adsorbents as well as different adsorbents themselves are shown. The water binding capacity in the range of water content of technological interest decreases from the anion exchange resin Amberlyst A 21 via nonpolar adsorbent Amberlite XAD-2 to kieselguhr Celite 545. It is demonstrated that water binding by lipases is depending on temperature but is also affected by adsorptive immobilization. Adsorptive immobilized lipases show hysteresis, which is very important for preparing a definite water content of the enzyme preparations. PMID:2325750

  16. Receptor binding studies of the living heart

    International Nuclear Information System (INIS)

    Receptors form a class of intrinsic membrane proteins (or glycoproteins) defined by the high affinity and specificity with which they bind ligands. Many receptors are associated directly or indirectly with membrane ion channels that open or close after a conformational change of the receptor induced by the binding of the neurotransmitter. Changes in number and/or affinity of cardiac neurotransmitter receptors have been associated with myocardial ischemia and infarction, congestive heart failure, and cardiomyopathy as well as diabetes or thyroid-induced heart muscle disease. These alterations of cardiac receptors have been demonstrated in vitro on membrane homogenates from samples collected mainly during surgery or postmortem. The disadvantage of these in vitro binding techniques is that receptors lose their natural environment and their relationships with the other components of the tissue

  17. Dendrimers bind antioxidant polyphenols and cisplatin drug.

    Directory of Open Access Journals (Sweden)

    Amine Abderrezak

    Full Text Available Synthetic polymers of a specific shape and size play major role in drug delivery systems. Dendrimers are unique synthetic macromolecules of nanometer dimensions with a highly branched structure and globular shape with potential applications in gene and drug delivery. We examine the interaction of several dendrimers of different compositions mPEG-PAMAM (G3, mPEG-PAMAM (G4 and PAMAM (G4 with hydrophilic and hydrophobic drugs cisplatin, resveratrol, genistein and curcumin at physiological conditions. FTIR and UV-visible spectroscopic methods as well as molecular modeling were used to analyse drug binding mode, the binding constant and the effects of drug complexation on dendrimer stability and conformation. Structural analysis showed that cisplatin binds dendrimers in hydrophilic mode via Pt cation and polymer terminal NH(2 groups, while curcumin, genistein and resveratrol are located mainly in the cavities binding through both hydrophobic and hydrophilic contacts. The overall binding constants of durg-dendrimers are ranging from 10(2 M(-1 to 10(3 M(-1. The affinity of dendrimer binding was PAMAM-G4>mPEG-PAMAM-G4>mPEG-PAMAM-G3, while the order of drug-polymer stability was curcumin>cisplatin>genistein>resveratrol. Molecular modeling showed larger stability for genisten-PAMAM-G4 (ΔG = -4.75 kcal/mol than curcumin-PAMAM-G4 ((ΔG = -4.53 kcal/mol and resveratrol-PAMAM-G4 ((ΔG = -4.39 kcal/mol. Dendrimers might act as carriers to transport hydrophobic and hydrophilic drugs.

  18. Nickel binding sites in histone proteins

    OpenAIRE

    Zoroddu, Maria Antonietta; Peana, Massimiliano Francesco; Solinas, Costantino; Medici, Serenella

    2012-01-01

    Nickel compounds are well known as human carcinogens, though the molecular events that are responsible for this are not well understood. It has been proposed that a crucial element in the mechanism of carcinogenesis is the binding of Ni(II) ions within the cell nucleus. It is known that DNA polymer binds Ni(II) only weakly, leaving the proteins of the cell nucleus as the likely Ni(II) targets. Being histone proteins the most abundant among them, they can be considered the primary sites fo...

  19. Ice-Binding Proteins and Their Function.

    Science.gov (United States)

    Bar Dolev, Maya; Braslavsky, Ido; Davies, Peter L

    2016-06-01

    Ice-binding proteins (IBPs) are a diverse class of proteins that assist organism survival in the presence of ice in cold climates. They have different origins in many organisms, including bacteria, fungi, algae, diatoms, plants, insects, and fish. This review covers the gamut of IBP structures and functions and the common features they use to bind ice. We discuss mechanisms by which IBPs adsorb to ice and interfere with its growth, evidence for their irreversible association with ice, and methods for enhancing the activity of IBPs. The applications of IBPs in the food industry, in cryopreservation, and in other technologies are vast, and we chart out some possibilities. PMID:27145844

  20. On Feature Binding in Space and Time

    OpenAIRE

    Chennu, Srivas

    2008-01-01

    When presented with a yellow Volkswagen and a red Ferrari, how does the brain �gure out which color goes with which car? The binding problem refers to how the visual system pre-consciously combines visual features of objects in the physical world to create coherent mental equivalents in our consciousness. I discuss why feature binding is a problem for our brains despite its seemingly e�ortless resolution in every-day life. Drawing from experimental cognitive psychology, I demonstrate how i...

  1. Binding Energy Distribution Analysis Method: Hamiltonian Replica Exchange with Torsional Flattening for Binding Mode Prediction and Binding Free Energy Estimation.

    Science.gov (United States)

    Mentes, Ahmet; Deng, Nan-Jie; Vijayan, R S K; Xia, Junchao; Gallicchio, Emilio; Levy, Ronald M

    2016-05-10

    Molecular dynamics modeling of complex biological systems is limited by finite simulation time. The simulations are often trapped close to local energy minima separated by high energy barriers. Here, we introduce Hamiltonian replica exchange (H-REMD) with torsional flattening in the Binding Energy Distribution Analysis Method (BEDAM), to reduce energy barriers along torsional degrees of freedom and accelerate sampling of intramolecular degrees of freedom relevant to protein-ligand binding. The method is tested on a standard benchmark (T4 Lysozyme/L99A/p-xylene complex) and on a library of HIV-1 integrase complexes derived from the SAMPL4 blind challenge. We applied the torsional flattening strategy to 26 of the 53 known binders to the HIV Integrase LEDGF site found to have a binding energy landscape funneled toward the crystal structure. We show that our approach samples the conformational space more efficiently than the original method without flattening when starting from a poorly docked pose with incorrect ligand dihedral angle conformations. In these unfavorable cases convergence to a binding pose within 2-3 Å from the crystallographic pose is obtained within a few nanoseconds of the Hamiltonian replica exchange simulation. We found that torsional flattening is insufficient in cases where trapping is due to factors other than torsional energy, such as the formation of incorrect intramolecular hydrogen bonds and stacking. Work is in progress to generalize the approach to handle these cases and thereby make it more widely applicable. PMID:27070865

  2. Studies on folate binding and a radioassay for serum and whole blood folate using goat milk as binding agent

    International Nuclear Information System (INIS)

    Preparations of cow, goat, buffalo, and human milk in addition to pig plasma were tested for folate binding properties. Of these, only pig plasma and goat milk showed sufficient binding to enable use as binding agents in a radioassay for serum and whole blood folate. The binding of folate by cow mild preparations in particular was found to be very poor. (orig.)

  3. Estradiol Binds to Insulin and Insulin Receptor Decreasing Insulin Binding in vitro

    Directory of Open Access Journals (Sweden)

    RobertRoot-Bernstein

    2014-07-01

    Methods: Ultraviolet spectroscopy, capillary electrophoresis and NMR demonstrated estrogen binding to insulin and its receptor. Horse-radish peroxidase-linked insulin was used in an ELISA-like procedure to measure the effect of estradiol on binding of insulin to its receptor. Measurements: Binding constants for estrogens to insulin and the insulin receptor were determined by concentration-dependent spectral shifts. The effect of estradiol on insulin-HRP binding to its receptor was determined by shifts in the insulin binding curve. Main Results: Estradiol bound to insulin with a Kd of 12 x 10-9 M and to the insulin receptor with a Kd of 24 x 10-9 M, while other hormones had significantly less affinity. 200 nM estradiol shifted the binding curve of insulin to its receptor 0.8 log units to the right. Conclusions: Estradiol concentrations in many hyperestrogenemic syndromes are sufficient to interfere with insulin binding to its receptor producing significant insulin resistance.

  4. Cross-Modal Binding in Developmental Dyslexia

    Science.gov (United States)

    Jones, Manon W.; Branigan, Holly P.; Parra, Mario A.; Logie, Robert H.

    2013-01-01

    The ability to learn visual-phonological associations is a unique predictor of word reading, and individuals with developmental dyslexia show impaired ability in learning these associations. In this study, we compared developmentally dyslexic and nondyslexic adults on their ability to form cross-modal associations (or "bindings") based…

  5. Tension-induced binding of semiflexible biopolymers

    CERN Document Server

    Benetatos, Panayotis; Zippelius, Annette

    2014-01-01

    We investigate theoretically the effect of polymer tension on the collective behavior of reversibly binding cross-links. For this purpose, we employ a model of two weakly bending wormlike chains aligned in parallel by a tensile force, with a sequence of inter-chain binding sites regularly spaced along the contours. Reversible cross-links attach and detach at the sites with an affinity controlled by a chemical potential. In a mean-field approach, we calculate the free energy of the system and find the emergence of a free-energy barrier which controls the reversible (un)binding. The tension affects the conformational entropy of the chains which competes with the binding energy of the cross-links. This competition gives rise to a sudden increase in the fraction of bound sites as the tension increases. We show that this transition is related to the cross-over between weak and strong localization of a directed polymer in a pinning potential. The cross-over to the strongly bound state can be interpreted as a mechan...

  6. The Double Bind: The next Generation

    Science.gov (United States)

    Malcom, Lindsey E.; Malcom, Shirley M.

    2011-01-01

    In this foreword, Shirley Malcom and Lindsey Malcom speak to the history and current status of women of color in science, technology, engineering, and mathematics (STEM) fields. As the author of the seminal report "The Double Bind: The Price of Being a Minority Woman in Science", Shirley Malcom is uniquely poised to give us an insightful…

  7. Binding properties of Treponema denticola lipooligosaccharide

    Directory of Open Access Journals (Sweden)

    Daniel Grenier

    2013-09-01

    Full Text Available Background and objective: The cell-surface lipooligosaccharide (LOS of Treponema denticola possesses several biological properties. The aim of this study was to investigate the binding properties of T. denticola LOS to extracellular matrix (ECM proteins, mucosal cells, and oral bacteria. Design: LOS was isolated from T. denticola and labeled with tritium. Tritium-labeled LOS was placed in ECM protein-, epithelial cell-, fibroblast-, or bacterium-coated wells of a 96-well microplate. Following incubation, unattached LOS was removed by extensive washing, and the amount of bound LOS was determined by measuring the radioactivity in the wells. Peptostreptococcus micros coated with LOS was used to stimulate fibroblasts, and the secretion of interleukin-6 (IL-6 and interleukin-8 (IL-8 by the fibroblasts was determined by ELISA. Results: T. denticola LOS had a high affinity for laminin. It also bound to gingival epithelial cells and fibroblasts. Soluble CD14 significantly increased the binding of LOS to fibroblasts. More LOS bound to P. micros than the other oral bacterial species tested. Stimulating fibroblasts with LOS-coated P. micros induced the secretion of IL-6 and IL-8. Conclusions: Our study provided evidence that T. denticola LOS possesses the capacity to bind to ECM proteins, mucosal cells, and oral bacteria. In addition, LOS binding to bacteria may increase their pro-inflammatory potential.

  8. The Case against Binding Interest Arbitration.

    Science.gov (United States)

    Ecker, Charles I.

    1984-01-01

    The author contends that districts should reject binding interest arbitration as a means of resolving an impasse in contract negotiations, charging that it hampers good faith bargaining, adversely affects fiscal and operational management of the school system, and diminishes the governing role of the board of education. (MJL)

  9. Oxytocin binding sites in bovine mammary tissue

    Energy Technology Data Exchange (ETDEWEB)

    Zhao, Xin.

    1989-01-01

    Oxytocin binding sites were identified and characterized in bovine mammary tissue. ({sup 3}H)-oxytocin binding reached equilibrium by 50 min at 20{degree}C and by 8 hr at 4{degree}C. The half-time of displacement at 20{degree}C was approximately 1 hr. Thyrotropin releasing hormone, adrenocorticotropin, angiotensin I, angiotensin II, pentagastrin, bradykinin, xenopsin and L-valyl-histidyl-L-leucyl-L-threonyl-L-prolyl-L-valyl-L-glutamyl-L-lysine were not competitive. In the presence of 10 nM LiCl, addition of oxytocin to dispersed bovine mammary cells, in which phosphatidylinositol was pre-labelled, caused a time and dose-dependent increase in radioactive inositiol monophosphate incorporation. The possibility that there are distinct vasopressin receptors in bovine mammary tissue was investigated. ({sup 3}H)-vasopressin binding reached equilibrium by 40 min at 20{degree}. The half-time of displacement at 20{degree}C was approximately 1 hr. The ability of the peptides to inhibit ({sup 3}H)-vasopressin binding was: (Thr{sup 4},Gly{sup 7})-oxytocin > Arg{sup 8}-vasopressin > (lys{sup 8})-vasopressin > (Deamino{sup 1},D-arg{sup 8})-vasopressin > oxytocin > d (CH{sub 2}){sub 5}Tyr(Me)AVP.

  10. Non-binding relationship between visual features

    Directory of Open Access Journals (Sweden)

    Dragan Rangelov

    2014-10-01

    Full Text Available The answer as to how visual attributes processed in different brain loci at different speeds are bound together to give us our unitary experience of the visual world remains unknown. In this study we investigated whether bound representations arise, as commonly assumed, through physiological interactions between cells in the visual areas. In a focal attentional task in which correct responses from either bound or unbound representations were possible, participants discriminated the colour or orientation of briefly presented single bars. On the assumption that representations of the two attributes are bound, the accuracy of reporting the colour and orientation should co-vary. By contrast, if the attributes are not mandatorily bound, the accuracy of reporting the two attributes should be independent. The results of our psychophysical studies reported here supported the latter, non-binding, relationship between visual features, suggesting that binding does not necessarily occur even under focal attention. We propose a task-contingent binding mechanism, postulating that binding occurs at late, post-perceptual, stages through the intervention of memory.

  11. ALG-2, a multifunctional calcium binding protein?

    DEFF Research Database (Denmark)

    Tarabykina, Svetlana; Mollerup, Jens; Winding Gojkovic, P.;

    2004-01-01

    ALG-2 was originally discovered as a pro-apoptotic protein in a genetic screen. Due to its ability to bind calcium with high affinity it was postulated to provide a link between the known effect of calcium in programmed cell death and the molecular death execution machinery. This review article...

  12. Treponema pallidum Fibronectin-Binding Proteins

    OpenAIRE

    Cameron, Caroline E.; Brown, Elizabeth L.; Kuroiwa, Janelle M. Y.; Schnapp, Lynn M.; Brouwer, Nathan L.

    2004-01-01

    Putative adhesins were predicted by computer analysis of the Treponema pallidum genome. Two treponemal proteins, Tp0155 and Tp0483, demonstrated specific attachment to fibronectin, blocked bacterial adherence to fibronectin-coated slides, and supported attachment of fibronectin-producing mammalian cells. These results suggest Tp0155 and Tp0483 are fibronectin-binding proteins mediating T. pallidum-host interactions.

  13. Inhibition of histone binding by supramolecular hosts

    Science.gov (United States)

    Allen, Hillary F.; Daze, Kevin D.; Shimbo, Takashi; Lai, Anne; Musselman, Catherine A.; Sims, Jennifer K.; Wade, Paul A.; Hof†, Fraser; Kutateladze, Tatiana G.

    2015-01-01

    The tandem PHD (plant homeodomain) fingers of the CHD4 (chromodomain helicase DNA-binding protein 4) ATPase are epigenetic readers that bind either unmodified histone H3 tails or H3K9me3 (histone H3 trimethylated at Lys9). This dual function is necessary for the transcriptional and chromatin remodelling activities of the NuRD (nucleosome remodelling and deacetylase) complex. In the present paper, we show that calixarene-based supramolecular hosts disrupt binding of the CHD4 PHD2 finger to H3K9me3, but do not affect the interaction of this protein with the H3K9me0 (unmodified histone H3) tail. A similar inhibitory effect, observed for the association of chromodomain of HP1γ (heterochromatin protein 1γ) with H3K9me3, points to a general mechanism of methyl-lysine caging by calixarenes and suggests a high potential for these compounds in biochemical applications. Immunofluorescence analysis reveals that the supramolecular agents induce changes in chromatin organization that are consistent with their binding to and disruption of H3K9me3 sites in living cells. The results of the present study suggest that the aromatic macrocyclic hosts can be used as a powerful new tool for characterizing methylation-driven epigenetic mechanisms. PMID:24576085

  14. Binding dynamics of single-stranded DNA binding proteins to fluctuating bubbles in breathing DNA

    International Nuclear Information System (INIS)

    We investigate the dynamics of a single local denaturation zone in a DNA molecule, a so-called DNA bubble, in the presence of single-stranded DNA binding proteins (SSBs). In particular, we develop a dynamical description of the process in terms of a two-dimensional master equation for the time evolution of the probability distribution of having a bubble of size m with n bound SSBs, for the case when m and n are the slowest variables in the system. We derive explicit expressions for the equilibrium statistical weights for a given m and n, which depend on the statistical weight u associated with breaking a base-pair interaction, the loop closure exponent c, the cooperativity parameter σ0, the SSB size λ, and binding strength κ. These statistical weights determine, through the detailed balance condition, the transfer coefficient in the master equation. For the case of slow and fast binding dynamics the problem can be reduced to one-dimensional master equations. In the latter case, we perform explicitly the adiabatic elimination of the fast variable n. Furthermore, we find that for the case that the loop closure is neglected and the binding dynamics is vanishing (but with arbitrary σ0) the eigenvalues and the eigenvectors of the master equation can be obtained analytically, using an orthogonal polynomial approach. We solve the general case numerically (i.e., including SSB binding and the loop closure) as a function of statistical weight u, binding protein size λ, and binding strength κ, and compare to the fast and slow binding limits. In particular, we find that the presence of SSBs in general increases the relaxation time, compared to the case when no binding proteins are present. By tuning the parameters, we can drive the system from regular bubble fluctuation in the absence of SSBs to full denaturation, reflecting experimental and in vivo situations

  15. Characterization of (3H)-nicotine binding in rodent brain and comparison with the binding of other labelled nicotinic ligands

    International Nuclear Information System (INIS)

    In an investigation of the receptor through which nicotine exerts its central actions, radioactively labelled nicotine was used in biochemical in vitro binding studies. Tritium-labelled nicotine (tritium-NIC) binding to mouse hippocampus was studied and the effect of temperature on the binding was analyzed by saturation-binding experiments. The specific tritium-NIC binding was found to be approximately four times higher at 4 C than at 25 C

  16. Yeast TATA-binding protein TFIID binds to TATA elements with both consensus and nonconsensus DNA sequences.

    OpenAIRE

    S. Hahn; Buratowski, S.; Sharp, P A; Guarente, L

    1989-01-01

    The DNA binding properties of the yeast TATA element-binding protein TFIID were investigated. The affinity (apparent equilibrium dissociation constant) of TFIID for the adenovirus major late promoter consensus TATA element is 2 x 10(-9) M, a value similar to the affinity of gene-specific regulatory proteins for their binding sites. TFIID binding is highly specific and recognizes nonspecific sites with approximately 10(5)-fold lower affinity. Despite this specificity, TFIID also binds with hig...

  17. Acyl-CoA-binding protein/diazepam-binding inhibitor gene and pseudogenes

    DEFF Research Database (Denmark)

    Mandrup, S; Hummel, R; Ravn, S;

    1992-01-01

    Acyl-CoA-binding protein (ACBP) is a 10 kDa protein isolated from bovine liver by virtue of its ability to bind and induce the synthesis of medium-chain acyl-CoA esters. Surprisingly, it turned out to be identical to a protein named diazepam-binding Inhibitor (DBI) claimed to be an endogenous...... remarkable correspondence between the structural modules of ACBP/DBI as determined by 1H nuclear magnetic resonance spectroscopy and the exon-intron architecture of the ACBP/DBI gene. Detailed analyses of transcription of the ACBP/DBI gene in brain and liver were performed to map transcription initiation...

  18. Automatic Binding Time Analysis for a Typed Lambda-Calculus

    DEFF Research Database (Denmark)

    Nielson, Hanne Riis; Nielson, Flemming

    1988-01-01

    A binding time analysis imposes a distinction between the computations to be performed early (e.g. at compile-time) and those to be performed late (e.g. at run-time). For the lambda-calculus this distinction is formalized by a two-level lambda-calculus. The authors present an algorithm for static...... analysis of the binding times of a typed lambda-calculus with products, sums, lists and general recursive types. Given partial information about the binding times of some of the subexpressions it will complete that information such that (i) early bindings may be turned into late bindings but not vice versa......, (ii) the resulting two-level lambda-expression reflects our intuition about binding times, e.g. that early bindings are performed before late bindings, and (iii) as few changes as possible have been made compared with the initial binding information. The results can be applied in the implementation...

  19. Effects of ATP on calcium binding to synaptic plasma membrane

    International Nuclear Information System (INIS)

    The release of labeled norepinephrine from preloaded synaptosomes requires the presence of potassium and calcium. ATP-dependent binding of calcium to synaptic plasma membranes (SPM) may provide a means of maintaining the cation in a readily available pool for the triggering of transmitter release. A high Ca-binding capacity was demonstrated in SPM. The Km for calcium is 5.5 X 10(-5) M. The dependence of the system on the gamma phosphate of ATP was demonstrated by an increase in Ca-binding with increasing ATP concentration and by competitive inhibition of binding by ADP and AMP. Magnesium is also required for ATP-dependent Ca-binding. The optimum pH for the Ca binding was 7.0. Pretreatment of SPM with phospholipase A2 lowered the binding capacity. Sulfhydryl groups are also critical for ATP-dependent Ca binding to occur. A model for ATP-dependent Ca-binding was proposed

  20. Protein-binding RNA aptamers affect molecular interactions distantly from their binding sites

    DEFF Research Database (Denmark)

    Dupont, Daniel Miotto; Thuesen, Cathrine K; Bøtkjær, Kenneth A;

    2015-01-01

    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...

  1. Oligomerization of mannan-binding lectin dictates binding properties and complement activation

    DEFF Research Database (Denmark)

    Kjaer, Troels R; Jensen, Lisbeth; Hansen, Annette;

    2016-01-01

    altered self-surfaces. Associated with the collagen-like stems of these PRMs are three mannan-binding lectin (MBL)-associated serine proteases (MASPs) and two MBL-associated proteins (MAps). The most studied of the PRMs, MBL, is present in serum mainly as trimeric and tetrameric oligomers of the...... structural subunit. We hypothesized that oligomerization of MBL may influence both the potential to bind to microorganisms and the interaction with the MASPs and MAps, thus influencing the ability to initiate complement activation. When testing binding at 37°C, we found higher binding of tetrameric MBL to...... Staphylococcus aureus (S. aureus) than trimeric and dimeric MBL. In serum, we found that tetrameric MBL was the main oligomeric form present in complexes with the MASPs and MAp44. Such preference was confirmed using purified forms of recombinant MBL (rMBL) oligomers, where tetrameric rMBL interacted stronger...

  2. 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...... by a simple two step protocol combining ion exchange chromatography and gel filtration. Dissociation constants for binding of oleic acid, arachidonic acid, oleoyl-CoA, lysophosphatidic acid and the peroxisomal proliferator bezafibrate to L-FABP have been determined by titration calorimetry. All ligands were...... bound in a 2:1 stoichiometry, the dissociation constants for the first ligand bound were all in the micro molar range. Oleic acid was bound with the highest affinity and a Kd of 0.26 microM. Furthermore, binding of cholesterol to L-FABP was investigated with the Lipidex assay, a liposome binding assay...

  3. The inhibition of anti-DNA binding to DNA by nucleic acid binding polymers.

    Directory of Open Access Journals (Sweden)

    Nancy A Stearns

    Full Text Available Antibodies to DNA (anti-DNA are the serological hallmark of systemic lupus erythematosus (SLE and can mediate disease pathogenesis by the formation of immune complexes. Since blocking immune complex formation can attenuate disease manifestations, the effects of nucleic acid binding polymers (NABPs on anti-DNA binding in vitro were investigated. The compounds tested included polyamidoamine dendrimer, 1,4-diaminobutane core, generation 3.0 (PAMAM-G3, hexadimethrine bromide, and a β-cylodextrin-containing polycation. As shown with plasma from patients with SLE, NABPs can inhibit anti-DNA antibody binding in ELISA assays. The inhibition was specific since the NABPs did not affect binding to tetanus toxoid or the Sm protein, another lupus autoantigen. Furthermore, the polymers could displace antibody from preformed complexes. Together, these results indicate that NABPs can inhibit the formation of immune complexes and may represent a new approach to treatment.

  4. A simple ligand-binding assay for thyroxine-binding globulin on reusable Sephadex columns

    International Nuclear Information System (INIS)

    A method for the assay of thyroxine-binding globulin on reusable Sephadex G-25 columns is described. It depends upon elution by diluted iodothyronine-free serum of protein-bound [125I]thyroxine from the columns under conditions where binding to thyroxine-binding prealbumin and albumin are abolished. It is simple, rapid and precise, and permits determinations inlarge numbers of samples. Values (mg/l; mean +- S.D.) were: normals 31.6+-5.4, hyperthyroid 28.3+-4.8, hypothyroid 40.6+-7.5, oral contraceptives 40.1+-6.8, pregnant 50.3+-5.4, cirrhotics 20.7+-4.3. Concentrations were reduced in serum heated at 56degC, while the uptake of [125I]triiodothyronine was increased. There was a significant negative correlation between thyroxine-binding globulin concentration and triiodothyronine uptake in the heated serum samples and in euthyroid subjects

  5. Relating the shape of protein binding sites to binding affinity profiles: is there an association?

    Directory of Open Access Journals (Sweden)

    Bitter István

    2010-10-01

    Full Text Available Abstract Background Various pattern-based methods exist that use in vitro or in silico affinity profiles for classification and functional examination of proteins. Nevertheless, the connection between the protein affinity profiles and the structural characteristics of the binding sites is still unclear. Our aim was to investigate the association between virtual drug screening results (calculated binding free energy values and the geometry of protein binding sites. Molecular Affinity Fingerprints (MAFs were determined for 154 proteins based on their molecular docking energy results for 1,255 FDA-approved drugs. Protein binding site geometries were characterized by 420 PocketPicker descriptors. The basic underlying component structure of MAFs and binding site geometries, respectively, were examined by principal component analysis; association between principal components extracted from these two sets of variables was then investigated by canonical correlation and redundancy analyses. Results PCA analysis of the MAF variables provided 30 factors which explained 71.4% of the total variance of the energy values while 13 factors were obtained from the PocketPicker descriptors which cumulatively explained 94.1% of the total variance. Canonical correlation analysis resulted in 3 statistically significant canonical factor pairs with correlation values of 0.87, 0.84 and 0.77, respectively. Redundancy analysis indicated that PocketPicker descriptor factors explain 6.9% of the variance of the MAF factor set while MAF factors explain 15.9% of the total variance of PocketPicker descriptor factors. Based on the salient structures of the factor pairs, we identified a clear-cut association between the shape and bulkiness of the drug molecules and the protein binding site descriptors. Conclusions This is the first study to investigate complex multivariate associations between affinity profiles and the geometric properties of protein binding sites. We found that

  6. Evidence for an intrinsic binding force between dodecaborate dianions and receptors with hydrophobic binding pockets.

    Science.gov (United States)

    Warneke, Jonas; Jenne, Carsten; Bernarding, Johannes; Azov, Vladimir A; Plaumann, Markus

    2016-05-01

    A gas phase binding study revealed strong intrinsic intermolecular interactions between dianionic halogenated closo-dodecaborates [B12X12](2-) and several neutral organic receptors. Oxidation of a tetrathiafulvalene host allowed switching between two host-guest binding modes in a supramolecular complex. Complexes of β-cyclodextrin with [B12F12](2-) show remarkable stability in the gas phase and were successfully tested as carriers for the delivery of boron clusters into cancer cells. PMID:27087168

  7. Binding of fluorescent lanthanides to rat liver mitochondrial membranes and calcium ion-binding proteins.

    Science.gov (United States)

    Mikkelsen, R B; Wallach, D F

    1976-05-21

    (1) Tb3+ binding to mitochondrial membranes can be monitored by enhanced ion fluorescence at 545 nm with excitation at 285 nm. At low protein concentrations (less than 30 mug/ml) no inner filter effects are observed. (2) This binding is localized at the external surface of the inner membrane and is unaffected by inhibitors of respiration or oxidative phosphorylation. (3) A soluble Ca2+ binding protein isolated according to Lehninger, A.L. ((1971) Biochem. Biophys. Res. Commun. 42, 312-317) also binds Tb3+ with enhanced ion fluorescence upon excitation at 285 nm. The excitation spectrum of the isolated protein and of the intact mitochondria are indicative of an aromatic amino acid at the cation binding site. (4) Further characterization of the Tb3+-protein interaction revealed that there is more than one binding site per protein molecule and that these sites are clustered (less than 20 A). Neuraminidase treatment or organic solvent extraction of the protein did not affect fluorescent Tb3+ binding. (5) pH dependency studies of Tb3+ binding to the isolated protein or intact mitochondria demonstrated the importance of an ionizable group of pK greater than 6. At pH less than 7.5 the amount of Tb3+ bound to the isolated protein decreased with increase in pH as monitored by Tb3+ fluorescence. With intact mitochondria the opposite occurred with a large increase in Tb3+ fluorescence at higher pH. This increase was not observed when the mitochondria were preincubated with antimycin A and rotenone. PMID:6061

  8. Sequence similarity between the erythrocyte binding domain of the Plasmodium vivax Duffy binding protein and the V3 loop of HIV-1 strain MN reveals a functional heparin binding motif involved in binding to the Duffy antigen receptor for chemokines

    Directory of Open Access Journals (Sweden)

    Bolton Michael J

    2011-11-01

    Full Text Available Abstract Background The HIV surface glycoprotein gp120 (SU, gp120 and the Plasmodium vivax Duffy binding protein (PvDBP bind to chemokine receptors during infection and have a site of amino acid sequence similarity in their binding domains that often includes a heparin binding motif (HBM. Infection by either pathogen has been found to be inhibited by polyanions. Results Specific polyanions that inhibit HIV infection and bind to the V3 loop of X4 strains also inhibited DBP-mediated infection of erythrocytes and DBP binding to the Duffy Antigen Receptor for Chemokines (DARC. A peptide including the HBM of PvDBP had similar affinity for heparin as RANTES and V3 loop peptides, and could be specifically inhibited from heparin binding by the same polyanions that inhibit DBP binding to DARC. However, some V3 peptides can competitively inhibit RANTES binding to heparin, but not the PvDBP HBM peptide. Three other members of the DBP family have an HBM sequence that is necessary for erythrocyte binding, however only the protein which binds to DARC, the P. knowlesi alpha protein, is inhibited by heparin from binding to erythrocytes. Heparitinase digestion does not affect the binding of DBP to erythrocytes. Conclusion The HBMs of DBPs that bind to DARC have similar heparin binding affinities as some V3 loop peptides and chemokines, are responsible for specific sulfated polysaccharide inhibition of parasite binding and invasion of red blood cells, and are more likely to bind to negative charges on the receptor than cell surface glycosaminoglycans.

  9. A thermodynamic signature for drug-DNA binding mode.

    Science.gov (United States)

    Chaires, Jonathan B

    2006-09-01

    A number of small molecules bind directly and selectively to DNA, acting as chemotherapeutic agents by inhibiting replication, transcription or topoisomerase activity. Two common binding modes for these small molecules are intercalation or groove-binding. Intercalation results from insertion of a planar aromatic substituent between DNA base pairs, with concomitant unwinding and lengthening of the DNA helix. Groove binding, in contrast, does not perturb the duplex structure to any great extent. Groove-binders are typically crescent-shaped, and fit snugly into the minor groove with little distortion of the DNA structure. Recent calorimetric studies have determined the enthalpic and entropic contributions to the DNA binding of representative DNA binding compounds. Analysis of such thermodynamic data culled from the literature reveals distinctive thermodynamic signatures for groove-binding and intercalating compounds. Plots of the binding enthalpy (DeltaH) against binding entropy (-TDeltaS) for 26 drug-DNA interactions reveal that groove-binding interactions are clustered in a region of the graph with favorable entropy contributions to the free energy, while intercalators are clustered in a region with unfavorable entropy but favorable enthalpy contributions. Groove-binding is predominantly entropically driven, while intercalation in enthalpically driven. The molecular basis of the contrasting thermodynamic signatures for the two binding modes is by no means clear, but the pattern should be of use in categorizing new DNA binding agents. PMID:16730635

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

    Science.gov (United States)

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

    2013-10-01

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

  11. Antibodies against the calcium-binding protein

    International Nuclear Information System (INIS)

    Plant microsomes contain a protein clearly related to a calcium-binding protein, calsequestrin, originally found in the sarcoplasmic reticulum of muscle cells, responsible for the rapid release and uptake of Ca2+ within the cells. The location and role of calsequestrin in plant cells is unknown. To generate monoclonal antibodies specific to plant calsequestrin, mice were immunized with a microsomal fraction from cultured cells of Streptanthus tortuosus (Brassicaceae). Two clones cross-reacted with one protein band with a molecular weight equal to that of calsequestrin (57 kilodaltons) by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and immunoblotting. This band is able to bind 45Ca2+ and can be recognized by a polyclonal antibody against the canine cardiac muscle calsequestrin. Rabbit skeletal muscle calsequestrin cross-reacted with the plant monoclonal antibodies. The plant monoclonal antibodies generated here are specific to calsequestrin protein

  12. tPA-binding RNA Aptamers

    DEFF Research Database (Denmark)

    Bjerregaard, Nils

    2015-01-01

    -density lipoprotein receptor Related Protein-1 (LRP-1). Here, we describe the selection and characterisation of structured RNA ligands (“RNA aptamers”) to tPA, K18 and K32. Both aptamers were truncated to minimal 32-nucleotide constructs (v2) with improved or unchanged activities, and were shown to bind tPA with low...... nanomolar affinities and efficiently inhibit tPA-LRP-1 binding and LRP-1 mediated cellular endocytosis. Both aptamers minimally affected the fibrinolytic properties of tPA despite efficiently inhibiting plasminogen activation stimulated by a soluble fibrin fragment. K18v2 additionally inhibited plasminogen......, and upon conjugation to serum albumin. K18v2 was able to inhibit tPA-induced fibrinogen depletion in vitro, which may provide additional benefits in stroke treatment. A conjugate of both aptamers separated by a linker encompassed the activities of both constituent sequences, and additionally possessed...

  13. Autologous antibodies that bind neuroblastoma cells.

    Science.gov (United States)

    Sun, Yujing; Sholler, Giselle S; Shukla, Girja S; Pero, Stephanie C; Carman, Chelsea L; Zhao, Ping; Krag, David N

    2015-11-01

    Antibody therapy of neuroblastoma is promising and our goal is to derive antibodies from patients with neuroblastoma for developing new therapeutic antibodies. The feasibility of using residual bone marrow obtained for clinical indications as a source of tumor cells and a source of antibodies was assessed. From marrow samples, neuroblastoma cells were recovered, grown in cell culture and also implanted into mice to create xenografts. Mononuclear cells from the marrow were used as a source to generate phage display antibody libraries and also hybridomas. Growth of neuroblastoma patient cells was possible both in vitro and as xenografts. Antibodies from the phage libraries and from the monoclonal hybridomas bound autologous neuroblastoma cells with some selectivity. It appears feasible to recover neuroblastoma cells from residual marrow specimens and to generate human antibodies that bind autologous neuroblastoma cells. Expansion of this approach is underway to collect more specimens, optimize methods to generate antibodies, and to evaluate the bioactivity of neuroblastoma-binding antibodies. PMID:26210205

  14. Insulin-induced lipid binding to hemoglobin

    Directory of Open Access Journals (Sweden)

    VESNA NIKETIC

    2003-01-01

    Full Text Available Under hypoglycemic conditions, concomitant hyperinsulinism causes an apparent modification of hemoglobin (Hb which is manifested by its aggregation (Niketi} et al., Clin. Chim. Acta 197 (1991 47. In the present work the causes and mechanisms underlying this Hb modification were studied. Hemoglobin isolated from normal erythrocytes incubated with insulin was analyzed by applying 31P-spectrometry and lipid extraction and analysis. To study the dynamics of the plasma membrane during hyperinsulinism, a fluorescent lipid-analog was applied. In the presence of insulin, phosphatidylserine (PS, phosphatidylethanolamine (PE and cholesterol were found to bind to Hb. Lipid binding resulted in Hb aggregation, a condition that can be reproduced when phospholipids are incubated with Hb in vitro. Using a fluorescent lipid-analog, it was also shown that exposing erythrocytes to supraphysiological concentrations of insulin in vitro resulted in the internalization of lipids. The results presented in this work may have relevance to cases of diabetes mellitus and hypoglycemia.

  15. Lectin binding in normal donkey eyeball

    Directory of Open Access Journals (Sweden)

    Khaled Aly

    2013-10-01

    Full Text Available In the present study, the distribution of various sugar residues in the eyeball tissues of sexually mature donkey was examined by employing fluorescein isothiocyanate-conjugated lectins. Our results revealed the presence of mannose (labeled by lectins ConA, galactose (labeled by PNA, GSAI, ECA, GalNAc (labeled by SBA, VVA, and GlcNAc (labeled by WGA residues in the donkey ocular tissues. The epithelium and stroma of the ocular tissues were labeled with mannose (ConA and GlcNAc (WGA binding lectins. Binding sites for WGA and PNA to the rod and cone cells of the retina were evident. The lectins Con A, WGA and GSAI are bound strongly to the endothelium of blood vessels and to smooth muscle cells of the iris. In conclusion, the findings of the present study clearly indicate that the donkey eyeball contains a wide range of glycoconjugates (bearing mannosyl, galactosyl and glucosly residues, and it lacks fucosyl residues.

  16. Odorant-binding proteins in insects.

    Science.gov (United States)

    Zhou, Jing-Jiang

    2010-01-01

    Our understanding of the molecular and biochemical mechanisms that mediate chemoreception in insects has been greatly improved after the discovery of olfactory and taste receptor proteins. However, after 50 years of the discovery of first insect sex pheromone from the silkmoth Bombyx mori, it is still unclear how hydrophobic compounds reach the dendrites of sensory neurons in vivo across aqueous space and interact with the sensory receptors. The presence of soluble polypeptides in high concentration in the lymph of chemosensilla still poses unanswered questions. More than two decades after their discovery and despite the wealth of structural and biochemical information available, the physiological function of odorant-binding proteins (OBPs) is not well understood. Here, I review the structural properties of different subclasses of insect OBPs and their binding to pheromones and other small ligands. Finally, I discuss current ideas and models on the role of such proteins in insect chemoreception. PMID:20831949

  17. Tight-binding treatment of conjugated polymers

    DEFF Research Database (Denmark)

    Lynge, Thomas Bastholm

    This PhD thesis concerns conjugated polymers which constitute a constantly growing research area. Today, among other things, conjugated polymers play a role in plastic based solar cells, photodetectors and light emitting diodes, and even today such plastic-based components constitute an alternative...... of tomorrow. This thesis specifically treats the three conjugated polymers trans-polyacetylene (tPA), poly(para-phenylene) (PPP) and poly(para-phe\\-nylene vinylene) (PPV). The present results, which are derived within the tight-binding model, are divided into two parts. In one part, analytic results...... are derived for the optical properties of the polymers expressed in terms of the optical susceptibility both in the presence and in the absence of a static electric field. In the other part, the cumputationally efficient Density Functional-based Tight-Binding (DFTB) model is applied to the description...

  18. Quantifying drug-protein binding in vivo

    International Nuclear Information System (INIS)

    Accelerator mass spectrometry (AMS) provides precise quantitation of isotope labeled compounds that are bound to biological macromolecules such as DNA or proteins. The sensitivity is high enough to allow for sub-pharmacological (''micro-'') dosing to determine macromolecular targets without inducing toxicities or altering the system under study, whether it is healthy or diseased. We demonstrated an application of AMS in quantifying the physiologic effects of one dosed chemical compound upon the binding level of another compound in vivo at sub-toxic doses [4].We are using tissues left from this study to develop protocols for quantifying specific binding to isolated and identified proteins. We also developed a new technique to quantify nanogram to milligram amounts of isolated protein at precisions that are comparable to those for quantifying the bound compound by AMS

  19. In vivo binding of retinol to chromatin

    International Nuclear Information System (INIS)

    The authors have previously shown that exposure of responding cells to vitamin A leads to profound modifications of chromatin structure as revealed by an increased susceptibility to DNase I digestion, modified patterns of histone acetylation, and impaired synthesis of a nonhistone chromosomal protein. The present results show that these effects are most probably due to the direct interaction between retinol and chromatin, and analysis of mononucleosomes and higher oligomers obtained from retinol-treated cells shows that retinol is indeed tightly bound to chromatin. Enzymatic digestions of vitamin A containing nucleosomes with proteinase K, phospholipase C, and phospholipase A2 support a model where the final binding of retinol to chromatin is mediated by a lipoprotein: the recognition of the binding sites on DNA being dictated by the proteic component while the hydrophobic retinol is solubilized in the fatty acid moiety

  20. Comparison of Transcription Factor Binding Site Models

    KAUST Repository

    Bhuyan, Sharifulislam

    2012-05-01

    Modeling of transcription factor binding sites (TFBSs) and TFBS prediction on genomic sequences are important steps to elucidate transcription regulatory mechanism. Dependency of transcription regulation on a great number of factors such as chemical specificity, molecular structure, genomic and epigenetic characteristics, long distance interaction, makes this a challenging problem. Different experimental procedures generate evidence that DNA-binding domains of transcription factors show considerable DNA sequence specificity. Probabilistic modeling of TFBSs has been moderately successful in identifying patterns from a family of sequences. In this study, we compare performances of different probabilistic models and try to estimate their efficacy over experimental TFBSs data. We build a pipeline to calculate sensitivity and specificity from aligned TFBS sequences for several probabilistic models, such as Markov chains, hidden Markov models, Bayesian networks. Our work, containing relevant statistics and evaluation for the models, can help researchers to choose the most appropriate model for the problem at hand.

  1. The Actin Binding Protein Adseverin Regulates Osteoclastogenesis

    OpenAIRE

    Hassanpour, Siavash; Jiang, Hongwei; Wang, Yongqiang; Kuiper, Johannes W. P.; Glogauer, Michael

    2014-01-01

    Adseverin (Ads), a member of the Gelsolin superfamily of actin binding proteins, regulates the actin cytoskeleton architecture by severing and capping existing filamentous actin (F-actin) strands and nucleating the assembly of new F-actin filaments. Ads has been implicated in cellular secretion, exocytosis and has also been shown to regulate chondrogenesis and megakaryoblastic leukemia cell differentiation. Here we report for the first time that Ads is involved in regulating osteoclastogenesi...

  2. Microtubule binding distinguishes dystrophin from utrophin

    OpenAIRE

    Belanto, Joseph J.; Mader, Tara L.; Eckhoff, Michael D.; Strandjord, Dana M.; Banks, Glen B.; Gardner, Melissa K.; Lowe, Dawn A.; Ervasti, James M.

    2014-01-01

    Our in vitro analyses reveal that dystrophin, the protein absent in Duchenne muscular dystrophy patients, binds microtubules with high affinity and pauses microtubule polymerization, whereas utrophin, the autosomal homologue of dystrophin thought to mirror many known functions of dystrophin, has no activity in either assay. We also report that transgenic utrophin overexpression does not correct subsarcolemmal microtubule lattice disorganization, physical inactivity after mild exercise, or los...

  3. Binding of episodic memories in the rat

    OpenAIRE

    Crystal, Jonathon D; Smith, Alexandra E.

    2014-01-01

    People remember an event as a coherent scene [1-4]. Memory of such an episode is thought to reflect binding of a fully integrated representation, rather than memory of unconnected features [4-7]. However, it is not known if rodents form bound representations. Here we show that rats remember episodes as bound representations. Rats were presented with multiple features of unique episodes at memory encoding: what (food flavor), where (maze location), source (self-generated food seeking–running t...

  4. Copper Binding in the Prion Protein†

    OpenAIRE

    Millhauser, Glenn L.

    2004-01-01

    A conformational change of the prion protein is responsible for a class of neurodegenerative diseases called the transmissible spongiform encephalopathies that include mad cow disease and the human afflictions kuru and Creutzfeldt–Jakob disease. Despite the attention given to these diseases, the normal function of the prion protein in healthy tissue is unknown. Research over the past few years, however, demonstrates that the prion protein is a copper binding protein with high selectivity for ...

  5. Optical sorting due to optical binding

    Czech Academy of Sciences Publication Activity Database

    Karásek, Vítězslav; Zemánek, Pavel

    Bellingham: SPIE, 2013, 881027:1-8. ISSN 0277-786X. [Optical Trapping and Optical Micromanipulation /10./. San Diego (US), 25.08.2013-29.08.2013] R&D Projects: GA ČR GPP205/12/P868 Institutional support: RVO:68081731 Keywords : optical binding * optical sorting * particles * optical trapping * bessel beam s * code division multiplexing * numerical simuklations Subject RIV: BH - Optics, Masers, Laser s

  6. Brain hyaluronan binding protein inhibits tumor growth

    Institute of Scientific and Technical Information of China (English)

    高锋; 曹曼林; 王蕾

    2004-01-01

    Background Great efforts have been made to search for the angiogenic inhibitors in avascular tissues. Several proteins isolated from cartilage have been proved to have anti-angiogenic or anti-tumour effects. Because cartilage contains a great amount of hyaluronic acid (HA) oligosaccharides and abundant HA binding proteins (HABP), therefore, we speculated that HABP might be one of the factors regulating vascularization in cartilage or anti-angiogenesis in tumours. The purpose of this research was to evaluale the effects of hyaluronan binding protein on inhibiting tumour growth both in vivo and vitro. Methods A unique protein termed human brain hyaluronan (HA) binding protein (b-HABP) was cloned from human brain cDNA library. MDA-435 human breast cancer cell line was chosen as a transfectant. The in vitro underlying mechanisms were investigated by determining the possibilities of MDA-435/b-HABP colony formation on soft agar, the effects of the transfectant on the proliferation of endothelial cells and the expression levels of caspase 3 and FasL from MDA-435/b-HABP. The in vivo study included tumour growth on the chorioallantoic membrane (CAM) of chicken embryos and nude mice. Results Colony formation assay revealed that the colonies formed by MDA-435/b-HABP were greatly reduced compared to mock transfectants. The conditioned media from MDA-435/b-HABP inhibited the growth of endothelial cells in culture. Caspase 3 and FasL expressions were induced by MDA-435/b-HABP. The size of tumours of MDA-435/b-HABP in both CAM and nude mice was much smaller than that of MDA-435 alone. Conclusions Human brain hyaluronan binding protein (b-HABP) may represent a new kind of naturally existing anti-tumour substance. This brain-derived glycoprotein may block tumour growth by inducing apoptosis of cancer cells or by decreasing angiogenesis in tumour tissue via inhibiting proliferation of endothelial cells.

  7. Binding of Actinobacillus pleuropneumoniae to Phosphatidylethanolamine

    OpenAIRE

    Jeannotte, Marie-Eve; Abul-Milh, Maan; Dubreuil, J. Daniel; Jacques, Mario

    2003-01-01

    The gram-negative bacterium Actinobacillus pleuropneumoniae is the causative agent of porcine fibrinohemorrhagic necrotizing pleuropneumonia, a disease that causes important economic losses to the swine industry worldwide. In general, the initial step of bacterial colonization is attachment to host cells. The purpose of the present study was to evaluate the binding of A. pleuropneumoniae serotype 1 to phospholipids, which are the major constituents of biological membranes. Phospholipids serve...

  8. Tight Binding Models in Cold Atoms Physics

    Science.gov (United States)

    Zakrzewski, J.

    2007-05-01

    Cold atomic gases placed in optical lattice potentials offer a unique tool to study simple tight binding models. Both the standard cases known from the condensed matter theory as well as novel situations may be addressed. Cold atoms setting allows for a precise control of parameters of the systems discussed, stimulating new questions and problems. The attempts to treat disorder in a controlled fashion are addressed in detail.

  9. Superconductivity in tight-binding approximation

    International Nuclear Information System (INIS)

    An interpretation of Barisic's relation for transition elements between the d-electron contribution to the cohesive energy and the local atomic parameter eta is presented. This relation is extended to a lattice with more than one atom per unit cell in the tight- binding approximation of rigid ions. It is conjectured that Barisic's relation is correct to first order approximation for transition metal alloys, provided the phonon induced d-d coupling is the dominant mechanism for superconductivity

  10. Alternative polyadenylation and RNA-binding proteins.

    Science.gov (United States)

    Erson-Bensan, Ayse Elif

    2016-08-01

    Our understanding of the extent of microRNA-based gene regulation has expanded in an impressive pace over the past decade. Now, we are beginning to better appreciate the role of 3'-UTR (untranslated region) cis-elements which harbor not only microRNA but also RNA-binding protein (RBP) binding sites that have significant effect on the stability and translational rate of mRNAs. To add further complexity, alternative polyadenylation (APA) emerges as a widespread mechanism to regulate gene expression by producing shorter or longer mRNA isoforms that differ in the length of their 3'-UTRs or even coding sequences. Resulting shorter mRNA isoforms generally lack cis-elements where trans-acting factors bind, and hence are differentially regulated compared with the longer isoforms. This review focuses on the RBPs involved in APA regulation and their action mechanisms on APA-generated isoforms. A better understanding of the complex interactions between APA and RBPs is promising for mechanistic and clinical implications including biomarker discovery and new therapeutic approaches. PMID:27208003

  11. SUMO-1 possesses DNA binding activity

    Directory of Open Access Journals (Sweden)

    Wieruszeski Jean-Michel

    2010-05-01

    Full Text Available Abstract Background Conjugation of small ubiquitin-related modifiers (SUMOs is a frequent post-translational modification of proteins. SUMOs can also temporally associate with protein-targets via SUMO binding motifs (SBMs. Protein sumoylation has been identified as an important regulatory mechanism especially in the regulation of transcription and the maintenance of genome stability. The precise molecular mechanisms by which SUMO conjugation and association act are, however, not understood. Findings Using NMR spectroscopy and protein-DNA cross-linking experiments, we demonstrate here that SUMO-1 can specifically interact with dsDNA in a sequence-independent fashion. We also show that SUMO-1 binding to DNA can compete with other protein-DNA interactions at the example of the regulatory domain of Thymine-DNA Glycosylase and, based on these competition studies, estimate the DNA binding constant of SUMO1 in the range 1 mM. Conclusion This finding provides an important insight into how SUMO-1 might exert its activity. SUMO-1 might play a general role in destabilizing DNA bound protein complexes thereby operating in a bottle-opener way of fashion, explaining its pivotal role in regulating the activity of many central transcription and DNA repair complexes.

  12. Binding Energy and Equilibrium of Compact Objects

    Directory of Open Access Journals (Sweden)

    Germano M.

    2014-04-01

    Full Text Available The theoretical analysis of the existence of a limit mass for compact astronomic ob- jects requires the solution of the Einstein’s equations of g eneral relativity together with an appropriate equation of state. Analytical solutions exi st in some special cases like the spherically symmetric static object without energy sou rces that is here considered. Solutions, i.e. the spacetime metrics, can have a singular m athematical form (the so called Schwarzschild metric due to Hilbert or a nonsingula r form (original work of Schwarzschild. The former predicts a limit mass and, conse quently, the existence of black holes above this limit. Here it is shown that, the origi nal Schwarzschild met- ric permits compact objects, without mass limit, having rea sonable values for central density and pressure. The lack of a limit mass is also demonst rated analytically just imposing reasonable conditions on the energy-matter densi ty, of positivity and decreas- ing with radius. Finally the ratio between proper mass and to tal mass tends to 2 for high values of mass so that the binding energy reaches the lim it m (total mass seen by a distant observer. As it is known the negative binding energ y reduces the gravitational mass of the object; the limit of m for the binding energy provides a mechanism for stable equilibrium of any amount of mass to contrast the gravitatio nal collapse.

  13. The aesthetic experience of 'contour binding'.

    Science.gov (United States)

    Casco, Clara; Guzzon, Daniela

    2008-01-01

    To find the diagnostic spatial frequency information in different painting styles (cubism, impressionism and realism), we have compared sensitivity (d') in distinguishing signal (subject of the painting) from noise with normal, high-pass and low-pass filtered images at long (150 ms) and short (30 ms) exposure. We found that for cubist-style images, d' increases with high-pass filtering compared with normal and low-pass filtered images, but decreases with low-pass filtering compared with normal images. These results indicate that channels with high spatial resolution provide the diagnostic information to solve the binding problem. Sensitivity for images in impressionist style was instead reduced by both low- and high-pass filtering. This indicates that both high and low spatial frequency channels play a role in solving the binding problem, suggesting the involvement of large collator units that group the response of small channels tuned to the same orientation. The difference between realism, which shows higher sensitivity for low-frequency filtering at short durations and cubism in which the binding problem is solved by high spatial frequency channels, has a corresponding difference in aesthetic judgment: the probability of judging a painting as 'intriguing' is larger with low-pass filtering than with high-pass filtering in realism, while the opposite is true for cubism. This suggests that the aesthetic experience is available during early processing of an image, and could preferentially influence high-level categorization of the subject of a painting. PMID:18534105

  14. STUDY OF ESTROGEN BINDING SITE ON HUMAN EJACULATED SPERMATOZOA

    Institute of Scientific and Technical Information of China (English)

    CHUJin-Shong; WANGYi-Fei

    1989-01-01

    The specific estrogen binding site for 17β-estradiol has been investigated on human spermatozoa by electron microscopec autoradiography. The results show that the binding sites were distributed over the surface of human spermatozoa: acrosomal cap, equatorial

  15. Streptococcal IgA-binding proteins bind in the Calpha 2-Calpha 3 interdomain region and inhibit binding of IgA to human CD89.

    Science.gov (United States)

    Pleass, R J; Areschoug, T; Lindahl, G; Woof, J M

    2001-03-16

    Certain pathogenic bacteria express surface proteins that bind to the Fc part of human IgA or IgG. These bacterial proteins are important as immunochemical tools and model systems, but their biological function is still unclear. Here, we describe studies of three streptococcal proteins that bind IgA: the Sir22 and Arp4 proteins of Streptococcus pyogenes and the unrelated beta protein of group B streptococcus. Analysis of IgA domain swap and point mutants indicated that two loops at the Calpha2/Calpha3 domain interface are critical for binding of the streptococcal proteins. This region is also used in binding the human IgA receptor CD89, an important mediator of IgA effector function. In agreement with this finding, the three IgA-binding proteins and a 50-residue IgA-binding peptide derived from Sir22 blocked the ability of IgA to bind CD89. Further, the Arp4 protein inhibited the ability of IgA to trigger a neutrophil respiratory burst via CD89. Thus, we have identified residues on IgA-Fc that play a key role in binding of different streptococcal IgA-binding proteins, and we have identified a mechanism by which a bacterial IgA-binding protein may interfere with IgA effector function. PMID:11096107

  16. Architecture of the sugar binding sites in carbohydrate binding proteins--a computer modeling study.

    Science.gov (United States)

    Rao, V S; Lam, K; Qasba, P K

    1998-11-01

    Different sugars, Gal, GalNAc and Man were docked at the monosaccharide binding sites of Erythrina corallodenron (EcorL), peanut lectin (PNA), Lathyrus ochrus (LOLI), and pea lectin (PSL). To study the lectin-carbohydrate interactions, in the complexes, the hydroxymethyl group in Man and Gal favors, gg and gt conformations respectively, and is the dominant recognition determination. The monosaccharide binding site in lectins that are specific to Gal/GalNAc is wider due to the additional amino acid residues in loop D as compared to that in lectins specific to Man/Glc, and affects the hydrogen bonds of the sugar involving residues from loop D, but not its orientation in the binding site. The invariant amino acid residues Asp from loop A, and Asn and an aromatic residue (Phe or Tyr) in loop C provides the basic architecture to recognize the common features in C4 epimers. The invariant Gly in loop B together with one or two residues in the variable region of loop D/A holds the sugar tightly at both ends. Loss of any one of these hydrogen bonds leads to weak interaction. While the subtle variations in the sequence and conformation of peptide fragment that resulted due to the size and location of gaps present in amino acid sequence in the neighborhood of the sugar binding site of loop D/A seems to discriminate the binding of sugars which differ at C4 atom (galacto and gluco configurations). The variations at loop B are important in discriminating Gal and GalNAc binding. The present study thus provides a structural basis for the observed specificities of legume lectins which uses the same four invariant residues for binding. These studies also bring out the information that is important for the design/engineering of proteins with the desired carbohydrate specificity. PMID:9849627

  17. The RNA binding domain of Pumilio antagonizes poly-adenosine binding protein and accelerates deadenylation.

    Science.gov (United States)

    Weidmann, Chase A; Raynard, Nathan A; Blewett, Nathan H; Van Etten, Jamie; Goldstrohm, Aaron C

    2014-08-01

    PUF proteins are potent repressors that serve important roles in stem cell maintenance, neurological processes, and embryonic development. These functions are driven by PUF protein recognition of specific binding sites within the 3' untranslated regions of target mRNAs. In this study, we investigated mechanisms of repression by the founding PUF, Drosophila Pumilio, and its human orthologs. Here, we evaluated a previously proposed model wherein the Pumilio RNA binding domain (RBD) binds Argonaute, which in turn blocks the translational activity of the eukaryotic elongation factor 1A. Surprisingly, we found that Argonautes are not necessary for repression elicited by Drosophila and human PUFs in vivo. A second model proposed that the RBD of Pumilio represses by recruiting deadenylases to shorten the mRNA's polyadenosine tail. Indeed, the RBD binds to the Pop2 deadenylase and accelerates deadenylation; however, this activity is not crucial for regulation. Rather, we determined that the poly(A) is necessary for repression by the RBD. Our results reveal that poly(A)-dependent repression by the RBD requires the poly(A) binding protein, pAbp. Furthermore, we show that repression by the human PUM2 RBD requires the pAbp ortholog, PABPC1. Pumilio associates with pAbp but does not disrupt binding of pAbp to the mRNA. Taken together, our data support a model wherein the Pumilio RBD antagonizes the ability of pAbp to promote translation. Thus, the conserved function of the PUF RBD is to bind specific mRNAs, antagonize pAbp function, and promote deadenylation. PMID:24942623

  18. AB-Bind: Antibody binding mutational database for computational affinity predictions.

    Science.gov (United States)

    Sirin, Sarah; Apgar, James R; Bennett, Eric M; Keating, Amy E

    2016-02-01

    Antibodies (Abs) are a crucial component of the immune system and are often used as diagnostic and therapeutic agents. The need for high-affinity and high-specificity antibodies in research and medicine is driving the development of computational tools for accelerating antibody design and discovery. We report a diverse set of antibody binding data with accompanying structures that can be used to evaluate methods for modeling antibody interactions. Our Antibody-Bind (AB-Bind) database includes 1101 mutants with experimentally determined changes in binding free energies (ΔΔG) across 32 complexes. Using the AB-Bind data set, we evaluated the performance of protein scoring potentials in their ability to predict changes in binding free energies upon mutagenesis. Numerical correlations between computed and observed ΔΔG values were low (r = 0.16-0.45), but the potentials exhibited predictive power for classifying variants as improved vs weakened binders. Performance was evaluated using the area under the curve (AUC) for receiver operator characteristic (ROC) curves; the highest AUC values for 527 mutants with |ΔΔG| > 1.0 kcal/mol were 0.81, 0.87, and 0.88 using STATIUM, FoldX, and Discovery Studio scoring potentials, respectively. Some methods could also enrich for variants with improved binding affinity; FoldX and Discovery Studio were able to correctly rank 42% and 30%, respectively, of the 80 most improved binders (those with ΔΔG < -1.0 kcal/mol) in the top 5% of the database. This modest predictive performance has value but demonstrates the continuing need to develop and improve protein energy functions for affinity prediction. PMID:26473627

  19. Cellular studies of binding, internalization and retention of a radiolabeled EGFR-binding affibody molecule

    Energy Technology Data Exchange (ETDEWEB)

    Nordberg, Erika [Rudbeck Laboratory, Department of Oncology, Radiology and Clinical Immunology, Biomedical Radiation Sciences, Uppsala University, SE-751 85 Uppsala (Sweden)], E-mail: erika.nordberg@bms.uu.se; Friedman, Mikaela [Department of Molecular Biotechnology, AlbaNova University Center, Kungl Tekniska Hoegskolan (KTH), SE-106 91 Stockholm (Sweden); Goestring, Lovisa [Rudbeck Laboratory, Department of Oncology, Radiology and Clinical Immunology, Biomedical Radiation Sciences, Uppsala University, SE-751 85 Uppsala (Sweden); Affibody AB, PO Box 20137, SE-161 02 Bromma (Sweden); Adams, Gregory P. [Department of Medical Oncology, Fox Chase Cancer Center, Philadelphia, PA 19111 (United States); Brismar, Hjalmar [Department of Cell Physics, AlbaNova University Center, Kungl Tekniska Hoegskolan (KTH), SE-106 91 Stockholm (Sweden); Nilsson, Fredrik Y. [Rudbeck Laboratory, Department of Oncology, Radiology and Clinical Immunology, Biomedical Radiation Sciences, Uppsala University, SE-751 85 Uppsala (Sweden); Affibody AB, PO Box 20137, SE-161 02 Bromma (Sweden); Stahl, Stefan [Department of Molecular Biotechnology, AlbaNova University Center, Kungl Tekniska Hoegskolan (KTH), SE-106 91 Stockholm (Sweden); Glimelius, Bengt [Rudbeck Laboratory, Oncology, Radiology and Clinical Immunology, Uppsala University, SE-751 85 Uppsala (Sweden); Carlsson, Joergen [Rudbeck Laboratory, Department of Oncology, Radiology and Clinical Immunology, Biomedical Radiation Sciences, Uppsala University, SE-751 85 Uppsala (Sweden)

    2007-08-15

    Introduction: The cellular binding and processing of an epidermal growth factor receptor (EGFR) targeting affibody molecule, (Z{sub EGFR:955}){sub 2}, was studied. This new and small molecule is aimed for applications in nuclear medicine. The natural ligand epidermal growth factor (EGF) and the antibody cetuximab were studied for comparison. Methods: All experiments were made with cultured A431 squamous carcinoma cells. Receptor specificity, binding time patterns, retention and preliminary receptor binding site localization studies were all made after {sup 125}I labeling. Internalization was studied using Oregon Green 488, Alexa Fluor 488 and CypHer5E markers. Results: [{sup 125}I](Z{sub EGFR:955}){sub 2} and [{sup 125}I]cetuximab gave a maximum cellular uptake of {sup 125}I within 4 to 8 h of incubation, while [{sup 125}I]EGF gave a maximum uptake already after 2 h. The retention studies showed that the cell-associated fraction of {sup 125}I after 48 h of incubation was {approx}20% when delivered as [{sup 125}I](Z{sub EGFR:955}){sub 2} and {approx}25% when delivered as [{sup 125}I]cetuximab. [{sup 125}I]EGF-mediated delivery gave a faster {sup 125}I release, where almost all cell-associated radioactivity had disappeared within 24 h. All three substances were internalized as demonstrated with confocal microscopy. Competitive binding studies showed that both EGF and cetuximab inhibited binding of (Z{sub EGFR:955}){sub 2} and indicated that the three substances competed for an overlapping binding site. Conclusion: The results gave information on cellular processing of radionuclides when delivered with (Z{sub EGFR:955}){sub 2} in comparison to delivery with EGF and cetuximab. Competition assays suggested that [{sup 125}I](Z{sub EGFR:955}){sub 2} bind to Domain III of EGFR. The affibody molecule (Z{sub EGFR:955}){sub 2} can be a candidate for EGFR imaging applications in nuclear medicine.

  20. Biophysical characterization of DNA binding from single molecule force measurements

    OpenAIRE

    Chaurasiya, Kathy R.; Paramanathan, Thayaparan; McCauley, Micah J.; Williams, Mark C.

    2010-01-01

    Single molecule force spectroscopy is a powerful method that uses the mechanical properties of DNA to explore DNA interactions. Here we describe how DNA stretching experiments quantitatively characterize the DNA binding of small molecules and proteins. Small molecules exhibit diverse DNA binding modes, including binding into the major and minor grooves and intercalation between base pairs of double-stranded DNA (dsDNA). Histones bind and package dsDNA, while other nuclear proteins such as hig...

  1. Allosteric, chiral-selective drug binding to DNA

    OpenAIRE

    Qu, Xiaogang; Trent, John O.; Fokt, Izabela; Priebe, Waldemar; Chaires, Jonathan B.

    2000-01-01

    The binding interactions of (−)-daunorubicin (WP900), a newly synthesized enantiomer of the anticancer drug (+)-daunorubicin, with right- and left-handed DNA, have been studied quantitatively by equilibrium dialysis, fluorescence spectroscopy, and circular dichroism. (+)-Daunorubicin binds selectively to right-handed DNA, whereas the enantiomeric WP900 ligand binds selectively to left-handed DNA. Further, binding of the enantiomeric pair to DNA is clearly chirally ...

  2. The hepcidin-binding site on ferroportin is evolutionarily conserved

    OpenAIRE

    De Domenico, Ivana; Nemeth, Elizabeta; Nelson, Jenifer M.; Phillips, John D.; Ajioka, Richard S.; Kay, Michael S.; Kushner, James P.; Ganz, Tomas; Ward, Diane M.; Kaplan, Jerry

    2008-01-01

    Mammalian iron homeostasis is regulated by the interaction of the liver-produced peptide hepcidin and its receptor, the iron transporter ferroportin. Hepcidin binds to ferroportin resulting in degradation of ferroportin and decreased cellular iron export. We identify the hepcidin-binding domain (HBD) on ferroportin and show that a synthetic 19 amino acid peptide corresponding to the HBD recapitulates the characteristics and specificity of hepcidin binding to cell surface ferroportin. The bind...

  3. A streptavidin mutant with altered ligand-binding specificity

    OpenAIRE

    Reznik, Gabriel O.; Vajda, Sandor; Sano, Takeshi; Cantor, Charles R.

    1998-01-01

    The biotin-binding site of streptavidin was modified to alter its ligand-binding specificity. In natural streptavidin, the side chains of N23 and S27 make two of the three hydrogen bonds with the ureido oxygen of biotin. These two residues were mutated to severely weaken biotin binding while attempting to maintain the affinity for two biotin analogs, 2-iminobiotin and diaminobiotin. Redesigning of the biotin-binding site used the difference in local electrostatic charge distribution between b...

  4. Specific receptor binding of staphylococcal enterotoxins by murine splenic lymphocytes.

    OpenAIRE

    Buxser, S; Bonventre, P F; Archer, D L

    1981-01-01

    We describe a reliable assay to measure the specific binding of 125I-labeled staphylococcal enterotoxin A (SEA) by murine spleen cells. Toxin binding by lymphocytes was specific in that it was inhibited by unlabeled SEA but not by unrelated proteins. The biological activity of SEA (T-lymphocyte mitogenesis) correlated with toxin binding to splenic lymphocytes. In the presence of high concentrations of [125I]SEA, specific binding increased rapidly and approached saturation after 2 h. Toxin bin...

  5. Biomimetic supramolecular metallohosts for binding and activation of dioxygen

    NARCIS (Netherlands)

    Sprakel, Vera Stefanie Irene

    2004-01-01

    Host-guest chemistry involves the binding of a specific substrate in a receptor via molecular recognition based on supramolecular interactions. Metal-containing derivatives of receptors for the selective supramolecular binding of dihydroxybenzene substrates, which receptors model oxygen binding enz

  6. Rapid determination of thyroxine binding proteins of human serum

    Directory of Open Access Journals (Sweden)

    Arima,Terukatsu

    1976-02-01

    Full Text Available A simple method is described for determing thyroxine binding proteins in human serum by electrophoresis at pH 8.6, using cellulose acetate membrane as the supporting medium. The procedure had high reliability in sera of normal subjects, pregnant women and patients with decreased thyroxine binding capacity of thyroxine binding globulin.

  7. A structural classification of substrate-binding proteins

    NARCIS (Netherlands)

    Berntsson, Ronnie P. -A.; Smits, Sander H. J.; Schmitt, Lutz; Slotboom, Dirk-Jan; Poolman, Bert; Rydström, Jan

    2010-01-01

    Substrate-binding proteins (SBP) are associated with a wide variety of protein complexes. The proteins are part of ATP-binding cassette transporters for substrate uptake, ion gradient driven transporters, DNA-binding proteins, as well as channels and receptors from both pro-and eukaryotes. A wealth

  8. Landscape of protein-small ligand binding modes.

    Science.gov (United States)

    Kasahara, Kota; Kinoshita, Kengo

    2016-09-01

    Elucidating the mechanisms of specific small-molecule (ligand) recognition by proteins is a long-standing conundrum. While the structures of these molecules, proteins and ligands, have been extensively studied, protein-ligand interactions, or binding modes, have not been comprehensively analyzed. Although methods for assessing similarities of binding site structures have been extensively developed, the methods for the computational treatment of binding modes have not been well established. Here, we developed a computational method for encoding the information about binding modes as graphs, and assessing their similarities. An all-against-all comparison of 20,040 protein-ligand complexes provided the landscape of the protein-ligand binding modes and its relationships with protein- and chemical spaces. While similar proteins in the same SCOP Family tend to bind relatively similar ligands with similar binding modes, the correlation between ligand and binding similarities was not very high (R(2)  = 0.443). We found many pairs with novel relationships, in which two evolutionally distant proteins recognize dissimilar ligands by similar binding modes (757,474 pairs out of 200,790,780 pairs were categorized into this relationship, in our dataset). In addition, there were an abundance of pairs of homologous proteins binding to similar ligands with different binding modes (68,217 pairs). Our results showed that many interesting relationships between protein-ligand complexes are still hidden in the structure database, and our new method for assessing binding mode similarities is effective to find them. PMID:27327045

  9. Thioredoxin binding site of phosphoribulokinase overlaps the catalytic site

    International Nuclear Information System (INIS)

    The ATP-regulatory binding site of phosphoribulokinase was studied using bromoacetylethanolamine phosphate (BrAcNHEtOP). BrAcNHEtOP binds to the active-regulatory binding site of the protein. Following trypsin degradation of the labeled protein, fragments were separated by HPLC and sequenced. (DT)

  10. Binding of Intrinsic and Extrinsic Features in Working Memory

    Science.gov (United States)

    Ecker, Ullrich K. H.; Maybery, Murray; Zimmer, Hubert D.

    2013-01-01

    There is ongoing debate concerning the mechanisms of feature binding in working memory. In particular, there is controversy regarding the extent to which these binding processes are automatic. The present article demonstrates that binding mechanisms differ depending on whether the to-be-integrated features are perceived as forming a coherent…

  11. Identification of Treponema pallidum penicillin-binding proteins.

    OpenAIRE

    Cunningham, T M; Miller, J N; Lovett, M A

    1987-01-01

    Penicillin-binding proteins of 180, 89, 80, 68, 61, 41, and 38 kilodaltons were identified in Treponema pallidum (Nichols) by their covalent binding of [35S]benzylpenicillin. Penicillin-binding proteins are localized in the plasma membranes of many bacterial species and may serve as useful markers for determining plasma membrane intactness in T. pallidum fractionation studies.

  12. Oncogenic conversion of Ets affects redox regulation in-vivo and in-vitro.

    OpenAIRE

    Wasylyk, C; Wasylyk, B.

    1993-01-01

    The avian acute leukemia virus E26 encodes a fusion protein between viral Gag and the cellular transcription factors cMyb and cEts1(p68). vEts on its own transforms more mature erythroid cells. We have compared the properties of vEts and cEts1(p68). vEts interacts preferentially with an antibody that recognizes the active conformation of the DNA-binding domain. The DNA-binding activity of vEts is particularly sensitive to incubation conditions for band-shift assays, phosphorylation and modifi...

  13. Modelling the binding affinity of steroids to zebrafish sex hormone-binding globulin.

    Science.gov (United States)

    Saxena, A K; Devillers, J; Pery, A R R; Beaudouin, R; Balaramnavar, V M; Ahmed, S

    2014-01-01

    The circulating endogenous steroids are transported in the bloodstream. These are bound to a highly specific sex hormone-binding globulin (SHBG) and in lower affinity to proteins such as the corticosteroid-binding protein and albumin in vertebrates, including fish. It is generally believed that the glycoprotein SHBG protects these steroids from rapid metabolic degradation and thus intervenes in its availability at the target tissues. Endocrine disrupters binding to SHBG affect the normal activity of natural steroids. Since xenobiotics are primarily released in the aquatic environment, there is a need to evaluate the binding affinity of xenosteroid mimics on fish SHBG, especially in zebrafish (Danio rerio), a small freshwater fish originating in India and widely employed in ecotoxicology, toxicology, and genetics. In this context, a zebrafish SHBG (zfSHBG) homology model was developed using the human SHBG (hSHBG) receptor structure as template. It was shown that interactions with amino acids Ser-36, Asp-59 and Thr-54 were important for binding affinity. A ligand-based pharmacophore model was also developed for both zfSHBG and hSHBG inhibitors that differentiated binders from non-binders, but also demonstrated structural requirements for zfSHBG and hSHBG ligands. The study provides insights into the mechanism of action of endocrine disruptors in zebrafish as well as providing a useful tool for identifying anthropogenic compounds inhibiting zfSHBG. PMID:24874994

  14. The Receptor Binding Domain of Botulinum Neurotoxin Stereotype C Binds Phosphoinositides

    Energy Technology Data Exchange (ETDEWEB)

    Zhang, Yanfeng; Varnum, Susan M.

    2012-03-01

    Botulinum neurotoxins (BoNTs) are the most toxic proteins known for humans and animals with an extremely low LD50 of {approx} 1 ng/kg. BoNTs generally require a protein and a ganglioside on the cell membrane surface for binding, which is known as a 'dual receptor' mechanism for host intoxication. Recent studies have suggested that in addition to gangliosides, other membrane lipids such as phosphoinositides may be involved in the interactions with the receptor binding domain (HCR) of BoNTs for better membrane penetration. Here, using two independent lipid-binding assays, we tested the interactions of BoNT/C-HCR with lipids in vitro. BoNT/C-HCR was found to bind negatively charged phospholipids, preferentially phosphoinositides. Additional interactions to phosphoinositides may help BoNT/C bind membrane more tightly and transduct signals for subsequent steps of intoxication. Our results provide new insights into the mechanisms of host cell membrane recognition by BoNTs.

  15. 14C-glucose binding assay of the glucose transporter binding sites in muscular cell membrane

    International Nuclear Information System (INIS)

    A method of determining the binding sites of glucose transporter in rat muscular cell membrane was introduced. The crude products of cell membrane form the skeletal muscle of control and insulin treated rats were prepared, and then fractionated in sucrose gradient. Both plasma membrane and microsome membrane were incubated with D-[U-14C] glucose respectively for the measurement of radioactivity and Scatchard plot analysis. It was found that the binding sites of glucose transporter in plasma membrane and intracellular membrane were 5.6 nmol 14C-glucose/mg protein and 8.7 nmol 14C-glucose-mg protein respectively at basic state. Insulin treatment in experimental groups caused approximately 146% increase in plasma membrane fraction and 88% decrease in intracellular membrane fraction. Moreover, the kinetic data of Scatchard plot curve were similar to those of the [3H]-cytochalasin B binding assay. D-[U-14C] glucose binding assay of glucose transporter binding sites in muscular cell membrane is simple, easy and practicable. The D-[U-14C] glucose is commercially available

  16. Tetrapyrrole binding affinity of the murine and human p22HBP heme-binding proteins.

    Science.gov (United States)

    Micaelo, Nuno M; Macedo, Anjos L; Goodfellow, Brian J; Félix, Vítor

    2010-11-01

    We present the first systematic molecular modeling study of the binding properties of murine (mHBP) and human (hHBP) p22HBP protein (heme-binding protein) with four tetrapyrrole ring systems belonging to the heme biosynthetic pathway: iron protoporphyrin IX (HEMIN), protoporphyrin IX (PPIX), coproporphyrin III (CPIII), coproporphyrin I (CPI). The relative binding affinities predicted by our computational study were found to be similar to those observed experimentally, providing a first rational structural analysis of the molecular recognition mechanism, by p22HBP, toward a number of different tetrapyrrole ligands. To probe the structure of these p22HBP protein complexes, docking, molecular dynamics and MM-PBSA methodologies supported by experimental NMR ring current shift data have been employed. The tetrapyrroles studied were found to bind murine p22HBP with the following binding affinity order: HEMIN> PPIX> CPIII> CPI, which ranged from -22.2 to -6.1 kcal/mol. In general, the protein-tetrapyrrole complexes are stabilized by non-bonded interactions between the tetrapyrrole propionate groups and basic residues of the protein, and by the preferential solvation of the complex compared to the unbound components. PMID:20800521

  17. Codeine-binding RNA aptamers and rapid determination of their binding constants using a direct coupling surface plasmon resonance assay

    OpenAIRE

    Win, Maung Nyan; Klein, Joshua S.; Smolke, Christina D.

    2006-01-01

    RNA aptamers that bind the opium alkaloid codeine were generated using an iterative in vitro selection process. The binding properties of these aptamers, including equilibrium and kinetic rate constants, were determined through a rapid, high-throughput approach using surface plasmon resonance (SPR) analysis to measure real-time binding. The approach involves direct coupling of the target small molecule onto a sensor chip without utilization of a carrier protein. Two highest binding aptamer se...

  18. Zooming into the binding groove of HLA molecules : which positions and which substitutions change peptide binding most?

    NARCIS (Netherlands)

    van Deutekom, Hanneke W M; Kesmir, C.

    2015-01-01

    Human leukocyte antigen (HLA) genes are the most polymorphic genes in the human genome. Almost all polymorphic residues are located in the peptide-binding groove, resulting in different peptide-binding preferences. Whether a single amino acid change can alter the peptide-binding repertoire of an HLA

  19. Coenzyme A Binding to the Aminoglycoside Acetyltransferase (3)-IIIb Increases Conformational Sampling of Antibiotic Binding Site

    Energy Technology Data Exchange (ETDEWEB)

    Hu, Xiaohu [ORNL; Norris, Adrianne [University of Tennessee, Knoxville (UTK); Baudry, Jerome Y [ORNL; Serpersu, Engin H [University of Tennessee, Knoxville (UTK)

    2011-01-01

    NMR spectroscopy experiments and molecular dynamics simulations were performed to describe the dynamic properties of the aminoglycoside acetyltransferase (3)-IIIb (AAC) in its apo and coenzyme A (CoASH) bound forms. The {sup 15}N-{sup 1}H HSQC spectra indicate a partial structural change and coupling of the CoASH binding site with another region in the protein upon the CoASH titration into the apo enzyme. Molecular dynamics simulations indicate a significant structural and dynamic variation of the long loop in the antibiotic binding domain in the form of a relatively slow (250 ns), concerted opening motion in the CoASH enzyme complex and that binding of the CoASH increases the structural flexibility of the loop, leading to an interchange between several similar equally populated conformations.

  20. Progesterone Binding and Inhibition of Growth in Trichophyton mentagrophytes

    OpenAIRE

    1986-01-01

    Specific binding of [3H]progesterone to cytosol of Trichophyton mentagrophytes was demonstrated. Scatchard analysis of [3H]progesterone binding showed a single class of binding sites with a dissociation constant of 9.5 X 10(-8) [corrected] +/- 2.4 X 10(-8) M (standard deviation) and a maximal binding capacity of 4,979 +/- 3,489 fmol/mg of cytosol protein. Deoxycorticosterone and dihydrotestosterone competitively inhibited binding by 50% at molar ratios of 10:1 and 20:1, respectively. Other st...

  1. Binding of collagen to Staphylococcus aureus Cowan 1.

    OpenAIRE

    Speziale, P; Raucci, G; Visai, L.; Switalski, L M; Timpl, R; Höök, M

    1986-01-01

    Collagen binds to a receptor protein present on the surfaces of Staphylococcus aureus cells. Binding of 125I-labeled type II collagen to its bacterial receptor is reversible, and Scatchard plot analysis indicates the presence of one class of receptor that occurs on an average of 3 X 10(4) copies per cell and binds type II collagen with a Kd of 10(-7) M. Studies on the specificity of collagen cell binding indicate that the receptor does not recognize noncollagenous proteins but binds all of th...

  2. Human pentraxin 3 binds to the complement regulator c4b-binding protein.

    Directory of Open Access Journals (Sweden)

    Anne Braunschweig

    Full Text Available The long pentraxin 3 (PTX3 is a soluble recognition molecule with multiple functions including innate immune defense against certain microbes and the clearance of apoptotic cells. PTX3 interacts with recognition molecules of the classical and lectin complement pathways and thus initiates complement activation. In addition, binding of PTX3 to the alternative complement pathway regulator factor H was shown. Here, we show that PTX3 binds to the classical and lectin pathway regulator C4b-binding protein (C4BP. A PTX3-binding site was identified within short consensus repeats 1-3 of the C4BP α-chain. PTX3 did not interfere with the cofactor activity of C4BP in the fluid phase and C4BP maintained its complement regulatory activity when bound to PTX3 on surfaces. While C4BP and factor H did not compete for PTX3 binding, the interaction of C4BP with PTX3 was inhibited by C1q and by L-ficolin. PTX3 bound to human fibroblast- and endothelial cell-derived extracellular matrices and recruited functionally active C4BP to these surfaces. Whereas PTX3 enhanced the activation of the classical/lectin pathway and caused enhanced C3 deposition on extracellular matrix, deposition of terminal pathway components and the generation of the inflammatory mediator C5a were not increased. Furthermore, PTX3 enhanced the binding of C4BP to late apoptotic cells, which resulted in an increased rate of inactivation of cell surface bound C4b and a reduction in the deposition of C5b-9. Thus, in addition to complement activators, PTX3 interacts with complement inhibitors including C4BP. This balanced interaction on extracellular matrix and on apoptotic cells may prevent excessive local complement activation that would otherwise lead to inflammation and host tissue damage.

  3. Glycan masking of Plasmodium vivax Duffy Binding Protein for probing protein binding function and vaccine development.

    Directory of Open Access Journals (Sweden)

    Sowmya Sampath

    Full Text Available Glycan masking is an emerging vaccine design strategy to focus antibody responses to specific epitopes, but it has mostly been evaluated on the already heavily glycosylated HIV gp120 envelope glycoprotein. Here this approach was used to investigate the binding interaction of Plasmodium vivax Duffy Binding Protein (PvDBP and the Duffy Antigen Receptor for Chemokines (DARC and to evaluate if glycan-masked PvDBPII immunogens would focus the antibody response on key interaction surfaces. Four variants of PVDBPII were generated and probed for function and immunogenicity. Whereas two PvDBPII glycosylation variants with increased glycan surface coverage distant from predicted interaction sites had equivalent binding activity to wild-type protein, one of them elicited slightly better DARC-binding-inhibitory activity than wild-type immunogen. Conversely, the addition of an N-glycosylation site adjacent to a predicted PvDBP interaction site both abolished its interaction with DARC and resulted in weaker inhibitory antibody responses. PvDBP is composed of three subdomains and is thought to function as a dimer; a meta-analysis of published PvDBP mutants and the new DBPII glycosylation variants indicates that critical DARC binding residues are concentrated at the dimer interface and along a relatively flat surface spanning portions of two subdomains. Our findings suggest that DARC-binding-inhibitory antibody epitope(s lie close to the predicted DARC interaction site, and that addition of N-glycan sites distant from this site may augment inhibitory antibodies. Thus, glycan resurfacing is an attractive and feasible tool to investigate protein structure-function, and glycan-masked PvDBPII immunogens might contribute to P. vivax vaccine development.

  4. 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.

  5. Liver fatty acid-binding protein binds monoacylglycerol in vitro and in mouse liver cytosol.

    Science.gov (United States)

    Lagakos, William S; Guan, Xudong; Ho, Shiu-Ying; Sawicki, Luciana Rodriguez; Corsico, Betina; Kodukula, Sarala; Murota, Kaeko; Stark, Ruth E; Storch, Judith

    2013-07-01

    Liver fatty acid-binding protein (LFABP; FABP1) is expressed both in liver and intestinal mucosa. Mice null for LFABP were recently shown to have altered metabolism of not only fatty acids but also monoacylglycerol, the two major products of dietary triacylglycerol hydrolysis (Lagakos, W. S., Gajda, A. M., Agellon, L., Binas, B., Choi, V., Mandap, B., Russnak, T., Zhou, Y. X., and Storch, J. (2011) Am. J. Physiol. Gastrointest. Liver Physiol. 300, G803-G814). Nevertheless, the binding and transport of monoacylglycerol (MG) by LFABP are uncertain, with conflicting reports in the literature as to whether this single chain amphiphile is in fact bound by LFABP. In the present studies, gel filtration chromatography of liver cytosol from LFABP(-/-) mice shows the absence of the low molecular weight peak of radiolabeled monoolein present in the fractions that contain LFABP in cytosol from wild type mice, indicating that LFABP binds sn-2 MG in vivo. Furthermore, solution-state NMR spectroscopy demonstrates two molecules of sn-2 monoolein bound in the LFABP binding pocket in positions similar to those found for oleate binding. Equilibrium binding affinities are ∼2-fold lower for MG compared with fatty acid. Finally, kinetic studies examining the transfer of a fluorescent MG analog show that the rate of transfer of MG is 7-fold faster from LFABP to phospholipid membranes than from membranes to membranes and occurs by an aqueous diffusion mechanism. These results provide strong support for monoacylglycerol as a physiological ligand for LFABP and further suggest that LFABP functions in the efficient intracellular transport of MG. PMID:23658011

  6. Liver Fatty Acid-binding Protein Binds Monoacylglycerol in Vitro and in Mouse Liver Cytosol*

    Science.gov (United States)

    Lagakos, William S.; Guan, Xudong; Ho, Shiu-Ying; Sawicki, Luciana Rodriguez; Corsico, Betina; Kodukula, Sarala; Murota, Kaeko; Stark, Ruth E.; Storch, Judith

    2013-01-01

    Liver fatty acid-binding protein (LFABP; FABP1) is expressed both in liver and intestinal mucosa. Mice null for LFABP were recently shown to have altered metabolism of not only fatty acids but also monoacylglycerol, the two major products of dietary triacylglycerol hydrolysis (Lagakos, W. S., Gajda, A. M., Agellon, L., Binas, B., Choi, V., Mandap, B., Russnak, T., Zhou, Y. X., and Storch, J. (2011) Am. J. Physiol. Gastrointest. Liver Physiol. 300, G803–G814). Nevertheless, the binding and transport of monoacylglycerol (MG) by LFABP are uncertain, with conflicting reports in the literature as to whether this single chain amphiphile is in fact bound by LFABP. In the present studies, gel filtration chromatography of liver cytosol from LFABP−/− mice shows the absence of the low molecular weight peak of radiolabeled monoolein present in the fractions that contain LFABP in cytosol from wild type mice, indicating that LFABP binds sn-2 MG in vivo. Furthermore, solution-state NMR spectroscopy demonstrates two molecules of sn-2 monoolein bound in the LFABP binding pocket in positions similar to those found for oleate binding. Equilibrium binding affinities are ∼2-fold lower for MG compared with fatty acid. Finally, kinetic studies examining the transfer of a fluorescent MG analog show that the rate of transfer of MG is 7-fold faster from LFABP to phospholipid membranes than from membranes to membranes and occurs by an aqueous diffusion mechanism. These results provide strong support for monoacylglycerol as a physiological ligand for LFABP and further suggest that LFABP functions in the efficient intracellular transport of MG. PMID:23658011

  7. Thyroxine binding to serum thyronine-binding globulin in thyroidectomized adult and normal neonatal rats

    International Nuclear Information System (INIS)

    The amount of tracer [125I]T4 bound to serum thyronine-binding globulin (TBG) was measured by polyacrylamide gel electrophoresis in adult thyroidectomized (TX) rats and normal 1-day to 4-week-old rat puts. Thyroidectomy was associated with the appearance of significant amounts of [125I]T4 binding to serum TBG in lean rats, but not in obese Zucker rats. Treatment of the TX rats in vivo with replacement doses of T4 prevented this increase in TBG binding, but enrichment of serum from TX rats with T4 did not. Significant amounts of tracer [125I]T4 binding to TBG was present in serum from 1- to 3-week-old normal rat pups, but not in 1-day- or 4-week-old pups. There were significantly higher levels of TBG binding of [125I]T4 in serum from 2-week-old rat pups raised in litters of 16 pups compared to those raised in litters of 4 pups. All manipulations that result in the appearance of TBG in rat serum also result in either weight loss or a slowing in the rate of growth, suggesting that the appearance of TBG in rat serum has a nutritional component. This possibility is further supported by the observations that increases in TBG binding of [125I]T4 are not found in obese Zucker rats fed a low protein-high carbohydrate diet for 14 days or fasted for 7 days, or after thyroidectomy, perhaps owing to the large stores of fuel in the obese rat

  8. Metal ion binding to iron oxides

    Science.gov (United States)

    Ponthieu, M.; Juillot, F.; Hiemstra, T.; van Riemsdijk, W. H.; Benedetti, M. F.

    2006-06-01

    The biogeochemistry of trace elements (TE) is largely dependent upon their interaction with heterogeneous ligands including metal oxides and hydrous oxides of iron. The modeling of TE interactions with iron oxides has been pursued using a variety of chemical models. The objective of this work is to show that it is possible to model the adsorption of protons and TE on a crystallized oxide (i.e., goethite) and on an amorphous oxide (HFO) in an identical way. Here, we use the CD-MUSIC approach in combination with valuable and reliable surface spectroscopy information about the nature of surface complexes of the TE. The other objective of this work is to obtain generic parameters to describe the binding of the following elements (Cd, Co, Cu, Ni, Pb, and Zn) onto both iron oxides for the CD-MUSIC approach. The results show that a consistent description of proton and metal ion binding is possible for goethite and HFO with the same set of model parameters. In general a good prediction of almost all the collected experimental data sets corresponding to metal ion binding to HFO is obtained. Moreover, dominant surface species are in agreement with the recently published surface complexes derived from X-ray absorption spectroscopy (XAS) data. Until more detailed information on the structure of the two iron oxides is available, the present option seems a reasonable approximation and can be used to describe complex geochemical systems. To improve our understanding and modeling of multi-component systems we need more data obtained at much lower metal ion to iron oxide ratios in order to be able to account eventually for sites that are not always characterized in spectroscopic studies.

  9. DNA and RNA Quadruplex-Binding Proteins

    Directory of Open Access Journals (Sweden)

    Václav Brázda

    2014-09-01

    Full Text Available Four-stranded DNA structures were structurally characterized in vitro by NMR, X-ray and Circular Dichroism spectroscopy in detail. Among the different types of quadruplexes (i-Motifs, minor groove quadruplexes, G-quadruplexes, etc., the best described are G-quadruplexes which are featured by Hoogsteen base-paring. Sequences with the potential to form quadruplexes are widely present in genome of all organisms. They are found often in repetitive sequences such as telomeric ones, and also in promoter regions and 5' non-coding sequences. Recently, many proteins with binding affinity to G-quadruplexes have been identified. One of the initially portrayed G-rich regions, the human telomeric sequence (TTAGGGn, is recognized by many proteins which can modulate telomerase activity. Sequences with the potential to form G-quadruplexes are often located in promoter regions of various oncogenes. The NHE III1 region of the c-MYC promoter has been shown to interact with nucleolin protein as well as other G-quadruplex-binding proteins. A number of G-rich sequences are also present in promoter region of estrogen receptor alpha. In addition to DNA quadruplexes, RNA quadruplexes, which are critical in translational regulation, have also been predicted and observed. For example, the RNA quadruplex formation in telomere-repeat-containing RNA is involved in interaction with TRF2 (telomere repeat binding factor 2 and plays key role in telomere regulation. All these fundamental examples suggest the importance of quadruplex structures in cell processes and their understanding may provide better insight into aging and disease development.

  10. Triazatriangulene as binding group for molecular electronics

    DEFF Research Database (Denmark)

    Wei, Zhongming; Wang, Xintai; Borges, Anders;

    2014-01-01

    The triazatriangulene (TATA) ring system was investigated as a binding group for tunnel junctions of molecular wires on gold surfaces. Self-assembled monolayers (SAMs) of TATA platforms with three different lengths of phenylene wires were fabricated, and their electrical conductance was recorded by...... platform displays a contact resistance only slightly larger than the thiols. This surprising finding has not been reported before and was analyzed by theoretical computations of the transmission functions of the TATA anchored molecular wires. The relatively low contact resistance of the TATA platform along...

  11. Defining Starch Binding by Glucan Phosphatases

    DEFF Research Database (Denmark)

    Auger, Kyle; Raththagala, Madushi; Wilkens, Casper;

    2015-01-01

    Starch is a vital energy molecule in plants that has a wide variety of uses in industry, such as feedstock for biomaterial processing and biofuel production. Plants employ a three enzyme cyclic process utilizing kinases, amylases, and phosphatases to degrade starch in a diurnal manner. Starch is...... comprised of the branched glucan amylopectin and the more linear glucan amylose. Our lab has determined the first structures of these glucan phosphatases and we have defined their enzymatic action. Despite this progress, we lacked a means to quickly and efficiently quantify starch binding to glucan...

  12. Systematic Calculations of Total Atomic Binding Energies

    International Nuclear Information System (INIS)

    We have calculated total atomic binding energies of 3- to 91-electron ions of all atoms with Z=3 to 118, in the Dirac-Fock model, for applications to atomic mass determination from highly-charged ions. In this process we have determined the ground-state configuration of many ions for which it was not known. We also provide total electronic correlation including Breit correlation for iso-electronic series of beryllium, neon, magnesium and argon, using the multiconfiguration Dirac-Fock approach.

  13. Particles in motion driven by optical binding

    Czech Academy of Sciences Publication Activity Database

    Karásek, Vítězslav; Zemánek, Pavel

    Bellingham : SPIE, 2014, 944103:1-6. ISBN 9781628415568. ISSN 0277-786X. [Polish-Slovak-Czech Optical Conference on Wave and Quantum Aspects of Contemporary Optics /19./. Jelenia Góra (PL), 08.09.2014-12.09.2014] R&D Projects: GA ČR GAP205/11/1687; GA MŠk(CZ) LO1212; GA MŠk ED0017/01/01 Institutional support: RVO:68081731 Keywords : optical binding * particles * scattering * optical micromanipulation * code division multiplexing * laser s Subject RIV: BH - Optics , Masers, Laser s

  14. Where metal ions bind in proteins.

    OpenAIRE

    Yamashita, M M; Wesson, L.; Eisenman, G.; Eisenberg, D.

    1990-01-01

    The environments of metal ions (Li+, Na+, K+, Ag+, Cs+, Mg2+, Ca2+, Mn2+, Cu2+, Zn2+) in proteins and other metal-host molecules have been examined. Regardless of the metal and its precise pattern of ligation to the protein, there is a common qualitative feature to the binding site: the metal is ligated by a shell of hydrophilic atomic groups (containing oxygen, nitrogen, or sulfur atoms) and this hydrophilic shell is embedded within a larger shell of hydrophobic atomic groups (containing car...

  15. Receptor binding studies of soft anticholinergic agents

    OpenAIRE

    Huang, Fenglei; Buchwald, Peter; Browne, Clinton E.; Farag, Hassan H.; Wu, Wnei-Mei; Ji, Fubao; Hochhaus, Guenther; Bodor, Nicholas

    2001-01-01

    Receptor binding studies were performed on 24 soft anticholinergic agents and 5 conventional anticholinergic agents using 4 cloned human muscarinic receptor subtypes. The measured pKi values of the soft anticholinergic agents ranged from 6.5 to 9.5, with the majority being in the range of 7.5 to 8.5. Strong correlation was observed between the pKis determined here and the pA2 values measured earlier in guinea pig ileum contraction assays. The corresponding correlation coefficients (r2) were 0...

  16. DNS and BIND on IPv6

    CERN Document Server

    Liu, Cricket

    2011-01-01

    If you're preparing to roll out IPv6 on your network, this concise book provides the essentials you need to support this protocol with DNS. You'll learn how DNS was extended to accommodate IPv6 addresses, and how you can configure a BIND name server to run on the network. This book also features methods for troubleshooting problems with IPv6 forward- and reverse-mapping, and techniques for helping islands of IPv6 clients communicate with IPv4 resources. Topics include: DNS and IPv6-Learn the structure and representation of IPv6 addresses, and the syntaxes of AAAA and PTR records in the ip6.a

  17. The metal binding properties of kraft lignin

    OpenAIRE

    Waltersson, Johanna

    2009-01-01

    There is a strong driving force to increase the competitiveness of the pulping industry by finding new business opportunities. In this context full utilisation of the wood raw material used in conventional pulping mills is of vital importance. One focus area is to increase the utilisation areas of lignin. LignoBoost is a new method to obtain kraft lignin of high purity. The aim of the project was to investigate and increase the ability of LignoBoost kraft lignins to bind metals in aqueous sol...

  18. Binding of Glutamate to the Umami Receptor

    OpenAIRE

    Lopez Cacales, J.; Oliviera Costa, S.; de Groot, B.; Walters, D

    2010-01-01

    Abstract The umami taste receptor is a heterodimer composed of two members of the T1R taste receptor family: T1R1 and T1R3. It detects glutamate in humans, and is a more general amino acid detector in other species. We have constructed homology models of the ligand binding domains of the human umami receptor (based on crystallographic structures of the metabotropic glutamate receptor of the central nervous system). We have carried out molecular dynamics simulations of the ligand bi...

  19. Binding of Actinomyces naeslundii to glycosphingolipids.

    OpenAIRE

    Brennan, M J; Joralmon, R A; Cisar, J O; Sandberg, A L

    1987-01-01

    The type 2 fimbrial lectin of Actinomyces naeslundii WVU45 mediated the binding of this bacterium to glycosphingolipids chromatographed on thin-layer silica gel plates. Radioiodinated bacteria attached to GM1, GD1b, and globoside. After chromatograms were treated with sialidase, the bacteria also bound to GD1a and GT1b. The actinomyces lectin apparently recognized the Gal beta 3GalNAc termini of these gangliosides and the GalNAc beta 3Gal terminus of globoside, suggesting that glycolipids con...

  20. The complex interplay between ligand binding and conformational structure of the folate binding protein (folate receptor)

    DEFF Research Database (Denmark)

    Holm, Jan; Bruun, Susanne Wrang; Hansen, Steen I.

    2015-01-01

    folate, probably due to shielding of binding sites between interacting hydrophobic patches. Titration with folate removes apo-monomers, favoring dissociation of self-associated apo-FBP into apo-monomers. Folate anchors to FBP through a network of hydrogen bonds and hydrophobic interactions, and the...... binding induces a conformational change with formation of hydrophilic and stable holo-FBP. Holo-FBP exhibits a ligand-mediated concentration-dependent self-association into multimers of great thermal and chemical stability due to strong intermolecular forces. Both ligand and FBP are thus protected against...

  1. Binding characterization, synthesis and biological evaluation of RXRα antagonists targeting the coactivator binding site.

    Science.gov (United States)

    Xu, Dingyu; Guo, Shangjie; Chen, Ziwen; Bao, Yuzhou; Huang, Fengyu; Xu, Dan; Zhang, Xindao; Zeng, Zhiping; Zhou, Hu; Zhang, Xiaokun; Su, Ying

    2016-08-15

    Previously we identified the first retinoid X receptor-alpha (RXRα) modulators that regulate the RXRα biological function via binding to the coregulator-binding site. Here we report the characterization of the interactions between the hit molecule and RXRα through computational modeling, mutagenesis, SAR and biological evaluation. In addition, we reported studies of additional new compounds and identified a molecule that mediated the NF-κB pathway by inhibiting the TNFα-induced IκBα degradation and p65 nuclear translocation. PMID:27450787

  2. The species-specific RNA polymerase I transcription factor SL-1 binds to upstream binding factor.

    OpenAIRE

    Hempel, W M; Cavanaugh, A H; Hannan, R D; Taylor, L.; Rothblum, L I

    1996-01-01

    Transcription of the 45S rRNA genes is carried out by RNA polymerase I and at least two trans-acting factors, upstream binding factor (UBF) and SL-1. We have examined the hypothesis that SL-1 and UBF interact. Coimmunoprecipitation studies using an antibody to UBF demonstrated that TATA-binding protein, a subunit of SL-1, associates with UBF in the absence of DNA. Inclusion of the detergents sodium dodecyl sulfate and deoxycholate disrupted this interaction. In addition, partially purified UB...

  3. A unique bivalent binding and inhibition mechanism by the yatapoxvirus interleukin 18 binding protein.

    Directory of Open Access Journals (Sweden)

    Brian Krumm

    Full Text Available Interleukin 18 (IL18 is a cytokine that plays an important role in inflammation as well as host defense against microbes. Mammals encode a soluble inhibitor of IL18 termed IL18 binding protein (IL18BP that modulates IL18 activity through a negative feedback mechanism. Many poxviruses encode homologous IL18BPs, which contribute to virulence. Previous structural and functional studies on IL18 and IL18BPs revealed an essential binding hot spot involving a lysine on IL18 and two aromatic residues on IL18BPs. The aromatic residues are conserved among the very diverse mammalian and poxviruses IL18BPs with the notable exception of yatapoxvirus IL18BPs, which lack a critical phenylalanine residue. To understand the mechanism by which yatapoxvirus IL18BPs neutralize IL18, we solved the crystal structure of the Yaba-Like Disease Virus (YLDV IL18BP and IL18 complex at 1.75 Å resolution. YLDV-IL18BP forms a disulfide bonded homo-dimer engaging IL18 in a 2∶2 stoichiometry, in contrast to the 1∶1 complex of ectromelia virus (ECTV IL18BP and IL18. Disruption of the dimer interface resulted in a functional monomer, however with a 3-fold decrease in binding affinity. The overall architecture of the YLDV-IL18BP:IL18 complex is similar to that observed in the ECTV-IL18BP:IL18 complex, despite lacking the critical lysine-phenylalanine interaction. Through structural and mutagenesis studies, contact residues that are unique to the YLDV-IL18BP:IL18 binding interface were identified, including Q67, P116 of YLDV-IL18BP and Y1, S105 and D110 of IL18. Overall, our studies show that YLDV-IL18BP is unique among the diverse family of mammalian and poxvirus IL-18BPs in that it uses a bivalent binding mode and a unique set of interacting residues for binding IL18. However, despite this extensive divergence, YLDV-IL18BP binds to the same surface of IL18 used by other IL18BPs, suggesting that all IL18BPs use a conserved inhibitory mechanism by blocking a putative receptor-binding

  4. Specific albumin binding to microvascular endothelium in culture

    International Nuclear Information System (INIS)

    The specific binding of rat serum albumin (RSA) to confluent microvascular endothelial cells in culture derived from the vasculature of the rat epididymal fat pad was studied at 4 degree C by radioassay and immunocytochemistry. Radioiodinated RSA (125I-RSA) binding to the cells reached equilibrium at ∼ 20 min incubation. Albumin binding was a slowly saturating function over concentrations ranging from 0.01 to 50 mg/ml. Specific RSA binding with a moderate apparent affinity constant of 1.0 mg/ml and with a maximum binding concentration of 90 ng/cm2 was immunolocalized with anti-RSA antibody to the outer (free) side of the enothelium. Scatchard analysis of the binding yielded a nonlinear binding curve with a concave-upward shape. Dissociation rate analysis supports negative cooperativity of albumin binding, but multiple binding sites may also be present. Albumin binding fulfilled many requirements for ligand specificity including saturability, reversibility, competibility, and dependence on both cell type and cell number. The results are discussed in terms of past in situ investigations on the localization of albumin binding to vascular endothelium and its effect on transendothelial molecular transport

  5. 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.

  6. Cooperative binding modes of Cu(II) in prion protein

    Science.gov (United States)

    Hodak, Miroslav; Chisnell, Robin; Lu, Wenchang; Bernholc, Jerry

    2007-03-01

    The misfolding of the prion protein, PrP, is responsible for a group of neurodegenerative diseases including mad cow disease and Creutzfeldt-Jakob disease. It is known that the PrP can efficiently bind copper ions; four high-affinity binding sites located in the octarepeat region of PrP are now well known. Recent experiments suggest that at low copper concentrations new binding modes, in which one copper ion is shared between two or more binding sites, are possible. Using our hybrid Thomas-Fermi/DFT computational scheme, which is well suited for simulations of biomolecules in solution, we investigate the geometries and energetics of two, three and four binding sites cooperatively binding one copper ion. These geometries are then used as inputs for classical molecular dynamics simulations. We find that copper binding affects the secondary structure of the PrP and that it stabilizes the unstructured (unfolded) part of the protein.

  7. A structure-based model for predicting serum albumin binding.

    Directory of Open Access Journals (Sweden)

    Katrina W Lexa

    Full Text Available One of the many factors involved in determining the distribution and metabolism of a compound is the strength of its binding to human serum albumin. While experimental and QSAR approaches for determining binding to albumin exist, various factors limit their ability to provide accurate binding affinity for novel compounds. Thus, to complement the existing tools, we have developed a structure-based model of serum albumin binding. Our approach for predicting binding incorporated the inherent flexibility and promiscuity known to exist for albumin. We found that a weighted combination of the predicted logP and docking score most accurately distinguished between binders and nonbinders. This model was successfully used to predict serum albumin binding in a large test set of therapeutics that had experimental binding data.

  8. Binding of Diphtheria Toxin to Phospholipids in Liposomes

    Science.gov (United States)

    Alving, Carl R.; Iglewski, Barbara H.; Urban, Katharine A.; Moss, Joel; Richards, Roberta L.; Sadoff, Jerald C.

    1980-04-01

    Diphtheria toxin bound to the phosphate portion of some, but not all, phospholipids in liposomes. Liposomes consisting of dimyristoyl phosphatidylcholine and cholesterol did not bind toxin. Addition of 20 mol% (compared to dimyristoyl phosphatidylcholine) of dipalmitoyl phosphatidic acid, dicetyl phosphate, phosphatidylinositol phosphate, cardiolipin, or phosphatidylserine in the liposomes resulted in substantial binding of toxin. Inclusion of phosphatidylinositol in dimyristol phosphatidylcholine / cholesterol liposomes did not result in toxin binding. The calcium salt of dipalmitoyl phosphatidic acid was more effective than the sodium salt, and the highest level of binding occurred with liposomes consisting only of dipalmitoyl phosphatidic acid (calcium salt) and cholesterol. Binding of toxin to liposomes was dependent on pH, and the pattern of pH dependence varied with liposomes having different compositions. Incubation of diphtheria toxin with liposomes containing dicetyl phosphate resulted in maximal binding at pH 3.6, whereas binding to liposomes containing phosphatidylinositol phosphate was maximal above pH 7. Toxin did not bind to liposomes containing 20 mol% of a free fatty acid (palmitic acid) or a sulfated lipid (3-sulfogalactosylceramide). Toxin binding to dicetyl phosphate or phosphatidylinositol phosphate was inhibited by UTP, ATP, phosphocholine, or p-nitrophenyl phosphate, but not by uracil. We conclude that (a) diphtheria toxin binds specifically to the phosphate portion of certain phospholipids, (b) binding to phospholipids in liposomes is dependent on pH, but is not due only to electrostatic interaction, and (c) binding may be strongly influenced by the composition of adjacent phospholipids that do not bind toxin. We propose that a minor membrane phospholipid (such as phosphatidylinositol phosphate or phosphatidic acid), or that some other phosphorylated membrane molecule (such as a phosphoprotein) may be important in the initial binding of

  9. An aprotinin binding site localized in the hormone binding domain of the estrogen receptor from calf uterus.

    Science.gov (United States)

    Nigro, V; Medici, N; Abbondanza, C; Minucci, S; Moncharmont, B; Molinari, A M; Puca, G A

    1990-07-31

    It has been proposed that the estrogen receptor bears proteolytic activity responsible for its own transformation. This activity was inhibited by aprotinin. Incubation of transformed ER with aprotinin modified the proteolytic digestion of the hormone binding subunit by proteinase K. The smallest hormone-binding fragment of the ER, obtained by tryptic digestion, was still able to bind to aprotinin. These results suggest that aprotinin interacts with ER and the hormone-binding domain of ER is endowed with a specific aprotinin-binding site. PMID:1696480

  10. Knowledge-based fragment binding prediction.

    Science.gov (United States)

    Tang, Grace W; Altman, Russ B

    2014-04-01

    Target-based drug discovery must assess many drug-like compounds for potential activity. Focusing on low-molecular-weight compounds (fragments) can dramatically reduce the chemical search space. However, approaches for determining protein-fragment interactions have limitations. Experimental assays are time-consuming, expensive, and not always applicable. At the same time, computational approaches using physics-based methods have limited accuracy. With increasing high-resolution structural data for protein-ligand complexes, there is now an opportunity for data-driven approaches to fragment binding prediction. We present FragFEATURE, a machine learning approach to predict small molecule fragments preferred by a target protein structure. We first create a knowledge base of protein structural environments annotated with the small molecule substructures they bind. These substructures have low-molecular weight and serve as a proxy for fragments. FragFEATURE then compares the structural environments within a target protein to those in the knowledge base to retrieve statistically preferred fragments. It merges information across diverse ligands with shared substructures to generate predictions. Our results demonstrate FragFEATURE's ability to rediscover fragments corresponding to the ligand bound with 74% precision and 82% recall on average. For many protein targets, it identifies high scoring fragments that are substructures of known inhibitors. FragFEATURE thus predicts fragments that can serve as inputs to fragment-based drug design or serve as refinement criteria for creating target-specific compound libraries for experimental or computational screening. PMID:24762971

  11. Xylanase inhibitors bind to nonstarch polysaccharides.

    Science.gov (United States)

    Fierens, Ellen; Gebruers, Kurt; Courtin, Christophe M; Delcour, Jan A

    2008-01-23

    This study is an in-depth investigation of the interaction between polysaccharides and the proteinaceous xylanase inhibitors, Triticum aestivum xylanase inhibitor (TAXI), xylanase inhibitor protein (XIP), and thaumatin-like xylanase inhibitor (TLXI). The binding affinities of all three known types of xylanase inhibitors from wheat are studied by measuring the residual xylanase inhibition activity after incubation of the inhibitors in the presence of different polysaccharides, such as beta-glucans and (arabino)xylans. The binding affinities of all three xylanase inhibitors for (arabino)xylans increased with a decreasing arabinose/xylose ratio (A/X ratio). This phenomenon was observed both with water-extractable and water-unextractable (arabino)xylans. The inhibitors also interacted with different soluble and insoluble beta-glucans. None of the inhibitors tested had the ability to hydrolyze the polysaccharides investigated. The present findings contribute to the unraveling of the function of xylanase inhibitors in nature and to the prediction of the effect of added xylanases in cereal-based biotechnological processes, such as bread making and gluten-starch separation. PMID:18092758

  12. Reflection-Based Python-C++ Bindings

    International Nuclear Information System (INIS)

    Python is a flexible, powerful, high-level language with excellent interactive and introspective capabilities and a very clean syntax. As such, it can be a very effective tool for driving physics analysis. Python is designed to be extensible in low-level C-like languages, and its use as a scientific steering language has become quite widespread. To this end, existing and custom-written C or C++ libraries are bound to the Python environment as so-called extension modules. A number of tools for easing the process of creating such bindings exist, such as SWIG and Boost. Python. Yet, the process still requires a considerable amount of effort and expertise. The C++ language has few built-in introspective capabilities, but tools such as LCGDict and CINT add this by providing so-called dictionaries: libraries that contain information about the names, entry points, argument types, etc. of other libraries. The reflection information from these dictionaries can be used for the creation of bindings and so the process can be fully automated, as dictionaries are already provided for many end-user libraries for other purposes, such as object persistency. PyLCGDict is a Python extension module that uses LCG dictionaries, as PyROOT uses CINT reflection information, to allow /cwPython users to access C++ libraries with essentially no preparation on the users' behalf. In addition, and in a similar way, PyROOT gives ROOT users access to Python libraries

  13. Specific binding assay technique; standardization of reagent

    International Nuclear Information System (INIS)

    The standardization of a labelled constituent, such as anti-IgE, for use in a specific binding assay method is disclosed. A labelled ligand, such as IgE, is standardized against a ligand reference substance, such as WHO standard IgE, to determine the weight of IgE protein represented by the labelled ligand. Anti-light chain antibodies are contacted with varying concentrations of the labelled ligand. The ligand is then contacted with the labelled constituent which is then quantitated in relation to the amount of ligand protein present. The preparation of 131I-labelled IgE is described. Also disclosed is an improved specific binding assay test method for determining the potency of an allergen extract in serum from an allergic individual. The improvement involved using a parallel model system of a second complex which consisted of anti-light chain antibodies, labelled ligand and the standardized labelled constituent (anti-IgE). The amount of standardized labelled constituent bound to the ligand in the first complex was determined, as described above, and the weight of ligand inhibited by addition of soluble allergen was then used as a measure of the potency of the allergen extract. (author)

  14. Grafting of protein-protein binding sites

    Institute of Scientific and Technical Information of China (English)

    2000-01-01

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

  15. Ligand binding studies in the mouse olfactory bulb: identification and characterisation of a L-[3H]carnosine binding site

    International Nuclear Information System (INIS)

    Binding sites for the dipeptide L-carnosine (β-alanyl-t-histidine) have been detected in membranes prepared from mouse olfactory bulbs. The binding of L-[3H]- carnosine was saturable, reversible and stereospecific and had a Ksub(d) of about 770 nM. The stereospecific binding of L-carnosine represented about 30% of the totoal binding at pH 6.8, and decreased markedly with increasing pH. Binding was stimulated by calcium, unaffected by zinc, magnesium or manganese and inhibted by sodium and potassium. Carnosine binding was sensitive to trypsin and phospholipases A and C, but not to neuraminidase. Nystatin and filipin, which interact with membrane lipids, also interfered with binding. Some peptide analogues of carnosine were potent inhibitors of binding, but a variety of drugs serving as potent inhibitors in other binding systems had no effect on carnosine binding. Carnosine binding to mouse olfactory bulb membranes was 15-fold higher than that seen in membranes prepared from cerebral hemispheres, 5-fold higher than in cerebellum membranes and 3-fold higher than in membranes from spinal medulla and the olfactory tubercle-lateral olfactory tract area. (Auth.)

  16. Characterization of nicotine binding in mouse brain and comparison with the binding of alpha-bungarotoxin and quinuclidinyl benzilate

    International Nuclear Information System (INIS)

    The binding of [3H]nicotine to mouse brain has been measured and subsequently compared with the binding of [125I]alpha-bungarotoxin (alpha-BTX) and L-[3H]quinuclidinyl benzilate (QNB). The binding of nicotine was saturable, reversible, and stereospecific. The average KD and Bmax were 59 nM and 88 fmoles/mg of protein, respectively. Although the rates of association and dissociation of nicotine were temperature-dependent, the incubation temperature had no effect on either KD or Bmax. When measured at 20 degrees or 37 degrees, nicotine appeared to bind to a single class of binding sites, but a second, very low-affinity, binding site was observed at 4 degrees. Nicotine binding was unaffected by the addition of NaCl, KCl, CaCl2, or MgSO4 to the incubation medium. Nicotinic cholinergic agonists were potent inhibitors of nicotine binding; however, nicotinic antagonists were poor inhibitors. The regional distribution of binding was not uniform: midbrain and striatum contained the highest number of receptors, whereas cerebellum had the fewest. Differences in site densities, regional distribution, inhibitor potencies, and thermal denaturation indicated that nicotine binding was not the same as either QNB or alpha-BTX binding, and therefore that receptors for nicotine may represent a unique population of cholinergic receptors

  17. Binding modes of thrombin binding aptamers investigated by simulations and experiments

    Science.gov (United States)

    Trapaidze, A.; Bancaud, A.; Brut, M.

    2015-01-01

    Thrombin binding aptamers HD1 and HD22 are the most studied aptamers, both for therapeutic and sensing purposes. Yet, there is still no commercialized aptamer-based sensor device for thrombin detection, suggesting that the binding modes of these aptamers remain to be precisely described. Here, we investigate thrombin-aptamer interactions with molecular dynamics simulations, and show that the different solved structures of HD1-thrombin complex are energetically similar and consequently possibly co-existing. Conversely, HD22 folding is much more stable, and its binding energy with thrombin is significantly larger than that of HD1 complexes. These results are confronted to experiments, which consist in monitoring aggregation of aptamer-functionalized gold nanoparticles triggered by thrombin. HD1 alone, but not HD22, can trigger aggregation, meaning that this aptamer has multiple sites of interactions with thrombin. Furthermore, pre-incubation of HD22 with thrombin impedes HD1 aggregation, suggesting that HD1 and HD22 have competing affinities for the same binding site. Altogether, this study shows that the characterization of aptamer-thrombin interactions by structural and kinetic experiments joined to simulations is necessary for the development of biosensors.

  18. The RNA binding domain of Pumilio antagonizes poly-adenosine binding protein and accelerates deadenylation

    OpenAIRE

    Weidmann, Chase A.; Raynard, Nathan A.; Blewett, Nathan H.; Van Etten, Jamie; Goldstrohm, Aaron C.

    2014-01-01

    This article analyzes the mechanism by which Pumilio represses the translation of its targets. The results show, rather surprisingly, that promotion of deadenylation is not required for expression. Instead, Pumilio interacts with poly(A) binding protein and somehow interferes with its activity.

  19. Structure of the RNA-Binding Domain of Telomerase: Implications For RNA Recognition and Binding

    Energy Technology Data Exchange (ETDEWEB)

    Rouda,S.; Skordalakes, E.

    2007-01-01

    Telomerase, a ribonucleoprotein complex, replicates the linear ends of eukaryotic chromosomes, thus taking care of the 'end of replication problem.' TERT contains an essential and universally conserved domain (TRBD) that makes extensive contacts with the RNA (TER) component of the holoenzyme, and this interaction is thought to facilitate TERT/TER assembly and repeat-addition processivity. Here, we present a high-resolution structure of TRBD from Tetrahymena thermophila. The nearly all-helical structure comprises a nucleic acid-binding fold suitable for TER binding. An extended pocket on the surface of the protein, formed by two conserved motifs (CP and T motifs) comprises TRBD's RNA-binding pocket. The width and the chemical nature of this pocket suggest that it binds both single- and double-stranded RNA, possibly stem I, and the template boundary element (TBE). Moreover, the structure provides clues into the role of this domain in TERT/TER stabilization and telomerase repeat-addition processivity.

  20. Cooperative binding of copper(I) to the metal binding domains in Menkes disease protein

    DEFF Research Database (Denmark)

    Jensen, P Y; Bonander, N; Møller, L B;

    1999-01-01

    We have optimised the overexpression and purification of the N-terminal end of the Menkes disease protein expressed in Escherichia coli, containing one, two and six metal binding domains (MBD), respectively. The domain(s) have been characterised using circular dichroism (CD) and fluorescence spec...

  1. The TRPV5/6 calcium channels contain multiple calmodulin binding sites with differential binding properties.

    NARCIS (Netherlands)

    Kovalevskaya, N.V.; Bokhovchuk, F.M.; Vuister, G.W.

    2012-01-01

    The epithelial Ca(2+) channels TRPV5/6 (transient receptor potential vanilloid 5/6) are thoroughly regulated in order to fine-tune the amount of Ca(2+) reabsorption. Calmodulin has been shown to be involved into calcium-dependent inactivation of TRPV5/6 channels by binding directly to the distal C-t

  2. Palmitate and stearate binding to human serum albumin. Determination of relative binding constants

    DEFF Research Database (Denmark)

    Vorum, H; Fisker, K; Honoré, B

    1997-01-01

    Multiple binding equilibria of two apparently insoluble ligands, palmitate and stearate, to defatted human serum albumin were studied in a 66 mM sodium phosphate buffer (pH 7.4) at 37 degrees C, by determination of dialytic exchange rates of ligands among identical equilibrium solutions. The expe...

  3. Distinct binding and immunogenic properties of the gonococcal homologue of meningococcal factor h binding protein.

    Directory of Open Access Journals (Sweden)

    Ilse Jongerius

    Full Text Available Neisseria meningitidis is a leading cause of sepsis and meningitis. The bacterium recruits factor H (fH, a negative regulator of the complement system, to its surface via fH binding protein (fHbp, providing a mechanism to avoid complement-mediated killing. fHbp is an important antigen that elicits protective immunity against the meningococcus and has been divided into three different variant groups, V1, V2 and V3, or families A and B. However, immunisation with fHbp V1 does not result in cross-protection against V2 and V3 and vice versa. Furthermore, high affinity binding of fH could impair immune responses against fHbp. Here, we investigate a homologue of fHbp in Neisseria gonorrhoeae, designated as Gonococcal homologue of fHbp (Ghfp which we show is a promising vaccine candidate for N. meningitidis. We demonstrate that Gfhp is not expressed on the surface of the gonococcus and, despite its high level of identity with fHbp, does not bind fH. Substitution of only two amino acids in Ghfp is sufficient to confer fH binding, while the corresponding residues in V3 fHbp are essential for high affinity fH binding. Furthermore, immune responses against Ghfp recognise V1, V2 and V3 fHbps expressed by a range of clinical isolates, and have serum bactericidal activity against N. meningitidis expressing fHbps from all variant groups.

  4. Distinct binding and immunogenic properties of the gonococcal homologue of meningococcal factor h binding protein.

    Science.gov (United States)

    Jongerius, Ilse; Lavender, Hayley; Tan, Lionel; Ruivo, Nicola; Exley, Rachel M; Caesar, Joseph J E; Lea, Susan M; Johnson, Steven; Tang, Christoph M

    2013-01-01

    Neisseria meningitidis is a leading cause of sepsis and meningitis. The bacterium recruits factor H (fH), a negative regulator of the complement system, to its surface via fH binding protein (fHbp), providing a mechanism to avoid complement-mediated killing. fHbp is an important antigen that elicits protective immunity against the meningococcus and has been divided into three different variant groups, V1, V2 and V3, or families A and B. However, immunisation with fHbp V1 does not result in cross-protection against V2 and V3 and vice versa. Furthermore, high affinity binding of fH could impair immune responses against fHbp. Here, we investigate a homologue of fHbp in Neisseria gonorrhoeae, designated as Gonococcal homologue of fHbp (Ghfp) which we show is a promising vaccine candidate for N. meningitidis. We demonstrate that Gfhp is not expressed on the surface of the gonococcus and, despite its high level of identity with fHbp, does not bind fH. Substitution of only two amino acids in Ghfp is sufficient to confer fH binding, while the corresponding residues in V3 fHbp are essential for high affinity fH binding. Furthermore, immune responses against Ghfp recognise V1, V2 and V3 fHbps expressed by a range of clinical isolates, and have serum bactericidal activity against N. meningitidis expressing fHbps from all variant groups. PMID:23935503

  5. Kinetics characterization of c-Src binding to lipid membranes: Switching from labile to persistent binding.

    Science.gov (United States)

    Le Roux, Anabel-Lise; Busquets, Maria Antònia; Sagués, Francesc; Pons, Miquel

    2016-02-01

    Cell signaling by the c-Src proto-oncogen requires the attachment of the protein to the inner side of the plasma membrane through the myristoylated N-terminal region, known as the SH4 domain. Additional binding regions of lower affinity are located in the neighbor intrinsically disordered Unique domain and the structured SH3 domain. Here we present a surface plasmon resonance study of the binding of a myristoylated protein including the SH4, Unique and SH3 domains of c-Src to immobilized liposomes. Two distinct binding processes were observed: a fast and a slow one. The second process lead to a persistently bound form (PB) with a slower binding and a much slower dissociation rate than the first one. The association and dissociation of the PB form could be detected using an anti-SH4 antibody. The kinetic analysis revealed that binding of the PB form follows a second order rate law suggesting that it involves the formation of c-Src dimers on the membrane surface. A kinetically equivalent PB form is observed in a myristoylated peptide containing only the SH4 domain but not in a construct including the three domains but with a 12-carbon lauroyl substituent instead of the 14-carbon myristoyl group. The PB form is observed with neutral lipids but its population increases when the immobilized liposomes contain negatively charged lipids. We suggest that the PB form may represent the active signaling form of c-Src while the labile form provides the capacity for fast 2D search of the target signaling site on the membrane surface. PMID:26638178

  6. Family 42 carbohydrate-binding modules display multiple arabinoxylan-binding interfaces presenting different ligand affinities.

    Science.gov (United States)

    Ribeiro, Teresa; Santos-Silva, Teresa; Alves, Victor D; Dias, Fernando M V; Luís, Ana S; Prates, José A M; Ferreira, Luís M A; Romão, Maria J; Fontes, Carlos M G A

    2010-10-01

    Enzymes that degrade plant cell wall polysaccharides display a modular architecture comprising a catalytic domain bound to one or more non-catalytic carbohydrate-binding modules (CBMs). CBMs display considerable variation in primary structure and are grouped into 59 sequence-based families organized in the Carbohydrate-Active enZYme (CAZy) database. Here we report the crystal structure of CtCBM42A together with the biochemical characterization of two other members of family 42 CBMs from Clostridium thermocellum. CtCBM42A, CtCBM42B and CtCBM42C bind specifically to the arabinose side-chains of arabinoxylans and arabinan, suggesting that various cellulosomal components are targeted to these regions of the plant cell wall. The structure of CtCBM42A displays a beta-trefoil fold, which comprises 3 sub-domains designated as alpha, beta and gamma. Each one of the three sub-domains presents a putative carbohydrate-binding pocket where an aspartate residue located in a central position dominates ligand recognition. Intriguingly, the gamma sub-domain of CtCBM42A is pivotal for arabinoxylan binding, while the concerted action of beta and gamma sub-domains of CtCBM42B and CtCBM42C is apparently required for ligand sequestration. Thus, this work reveals that the binding mechanism of CBM42 members is in contrast with that of homologous CBM13s where recognition of complex polysaccharides results from the cooperative action of three protein sub-domains presenting similar affinities. PMID:20637315

  7. Memory binding and white matter integrity in familial Alzheimer's disease.

    Science.gov (United States)

    Parra, Mario A; Saarimäki, Heini; Bastin, Mark E; Londoño, Ana C; Pettit, Lewis; Lopera, Francisco; Della Sala, Sergio; Abrahams, Sharon

    2015-05-01

    Binding information in short-term and long-term memory are functions sensitive to Alzheimer's disease. They have been found to be affected in patients who meet criteria for familial Alzheimer's disease due to the mutation E280A of the PSEN1 gene. However, only short-term memory binding has been found to be affected in asymptomatic carriers of this mutation. The neural correlates of this dissociation are poorly understood. The present study used diffusion tensor magnetic resonance imaging to investigate whether the integrity of white matter structures could offer an account. A sample of 19 patients with familial Alzheimer's disease, 18 asymptomatic carriers and 21 non-carrier controls underwent diffusion tensor magnetic resonance imaging, neuropsychological and memory binding assessment. The short-term memory binding task required participants to detect changes across two consecutive screens displaying arrays of shapes, colours, or shape-colour bindings. The long-term memory binding task was a Paired Associates Learning Test. Performance on these tasks were entered into regression models. Relative to controls, patients with familial Alzheimer's disease performed poorly on both memory binding tasks. Asymptomatic carriers differed from controls only in the short-term memory binding task. White matter integrity explained poor memory binding performance only in patients with familial Alzheimer's disease. White matter water diffusion metrics from the frontal lobe accounted for poor performance on both memory binding tasks. Dissociations were found in the genu of corpus callosum which accounted for short-term memory binding impairments and in the hippocampal part of cingulum bundle which accounted for long-term memory binding deficits. The results indicate that white matter structures in the frontal and temporal lobes are vulnerable to the early stages of familial Alzheimer's disease and their damage is associated with impairments in two memory binding functions known to

  8. An Electrostatic Funnel in the GABA-Binding Pathway

    Science.gov (United States)

    Lightstone, Felice C.

    2016-01-01

    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. PMID:27119953

  9. 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.

  10. Pulmonary surfactant protein A (SP-A) specifically binds dipalmitoylphosphatidylcholine

    International Nuclear Information System (INIS)

    Phospholipids are the major components of pulmonary surfactant. Dipalmitoylphosphatidylcholine is believed to be especially essential for the surfactant function of reducing the surface tension at the air-liquid interface. Surfactant protein A (SP-A) with a reduced denatured molecular mass of 26-38 kDa, characterized by a collagen-like structure and N-linked glycosylation, interacts strongly with a mixture of surfactant-like phospholipids. In the present study the direct binding of SP-A to phospholipids on a thin layer chromatogram was visualized using 125I-SP-A as a probe, so that the phospholipid specificities of SP-A binding and the structural requirements of SP-A and phospholipids for the binding could be examined. Although 125I-SP-A bound phosphatidylcholine and sphingomyeline, it was especially strong in binding dipalmitoylphosphatidylcholine, but failed to bind phosphatidylglycerol, phosphatidylinositol, phosphatidylethanolamine, and phosphatidylserine. Labeled SP-A also exhibited strong binding to distearoylphosphatidylcholine, but weak binding to dimyristoyl-, 1-palmitoyl-2-linoleoyl-, and dilinoleoylphosphatidylcholine. Unlabeled SP-A readily competed with labeled SP-A for phospholipid binding. SP-A strongly bound dipalmitoylglycerol produced by phospholipase C treatment of dipalmitoylphosphatidylcholine, but not palmitic acid. This protein also failed to bind lysophosphatidylcholine produced by phospholipase A2 treatment of dipalmitoylphosphatidylcholine. 125I-SP-A shows almost no binding to dipalmitoylphosphatidylglycerol and dipalmitoylphosphatidylethanolamine. The addition of 10 mM EGTA into the binding buffer reduced much of the 125I-SP-A binding to phospholipids. Excess deglycosylated SP-A competed with labeled SP-A for binding to dipalmitoylphosphatidylcholine, but the excess collagenase-resistant fragment of SP-A failed

  11. Binding biological motion and visual features in working memory.

    Science.gov (United States)

    Ding, Xiaowei; Zhao, Yangfan; Wu, Fan; Lu, Xiqian; Gao, Zaifeng; Shen, Mowei

    2015-06-01

    Working memory mechanisms for binding have been examined extensively in the last decade, yet few studies have explored bindings relating to human biological motion (BM). Human BM is the most salient and biologically significant kinetic information encountered in everyday life and is stored independently from other visual features (e.g., colors). The current study explored 3 critical issues of BM-related binding in working memory: (a) how many BM binding units can be retained in working memory, (b) whether involuntarily object-based binding occurs during BM binding, and (c) whether the maintenance of BM bindings in working memory requires attention above and beyond that needed to maintain the constituent dimensions. We isolated motion signals of human BM from non-BM sources by using point-light displays as to-be-memorized BM and presented the participants colored BM in a change detection task. We found that working memory capacity for BM-color bindings is rather low; only 1 or 2 BM-color bindings could be retained in working memory regardless of the presentation manners (Experiments 1-3). Furthermore, no object-based encoding took place for colored BM stimuli regardless of the processed dimensions (Experiments 4 and 5). Central executive attention contributes to the maintenance of BM-color bindings, yet maintaining BM bindings in working memory did not require more central attention than did maintaining the constituent dimensions in working memory (Experiment 6). Overall, these results suggest that keeping BM bindings in working memory is a fairly resource-demanding process, yet central executive attention does not play a special role in this cross-module binding. PMID:25893683

  12. Calculation of binding constants and concentration of binding sites in a reaction of a ligand with a heterogeneous system of binding sites

    International Nuclear Information System (INIS)

    A method is presented for the calculation of association constants and the concentration of binding sites in a reaction of a ligand with a heterogeneous system of binding sites. The Scatchard plot for such a system is curvelinear and the method employs previously established relationships between the parameters of the limiting slopes to such a curve and the above mentioned association constants and concentrations of binding sites. The special case of a system with two different and non-interacting groups of binding sites was solved. The expressions thus obtained were used to characterize the reaction of a polypeptide neurotoxin with its specific binding sites in a membranal preparation from insect central nervous system. Moreover it is evident from these expressions that the widely accepted method to analyze such system, by an intuitive generalization of the method applicable to homogeneous systems, is erroneous and should be avoided. (author)

  13. Variations of nuclear binding with quark masses

    CERN Document Server

    Carrillo-Serrano, M E; Tsushima, K; Thomas, A W; Afnan, I R

    2012-01-01

    We investigate the variation with light quark mass of the mass of the nucleon as well as the masses of the mesons commonly used in a one-boson-exchange model of the nucleon-nucleon force. Care is taken to evaluate the meson mass shifts at the kinematic point relevant to that problem. Using these results, the corresponding changes in the energy of the 1 S0 anti-bound state, the binding energies of the deuteron, triton and selected finite nuclei are evaluated using a one-boson exchange model. The results are discussed in the context of possible corrections to the standard scenario for big bang nucleosynthesis in the case where, as suggested by recent observations of quasar absorption spectra, the quark masses may have changed over the age of the Universe.

  14. DNA binding and aggregation by carbon nanoparticles

    International Nuclear Information System (INIS)

    Significant environmental and health risks due to the increasing applications of engineered nanoparticles in medical and industrial activities have been concerned by many communities. The interactions between nanomaterials and genomes have been poorly studied so far. This study examined interactions of DNA with carbon nanoparticles (CNP) using atomic force microscopy (AFM). We experimentally assessed how CNP affect DNA molecule and bacterial growth of Escherichia coli. We found that CNP were bound to the DNA molecules during the DNA replication in vivo. The results revealed that the interaction of DNA with CNP resulted in DNA molecule binding and aggregation both in vivo and in vitro in a dose-dependent manner, and consequently inhabiting the E. coli growth. While this was a preliminary study, our results showed that this nanoparticle may have a significant impact on genomic activities.

  15. Hafnium binding to rat serum transferrin

    Energy Technology Data Exchange (ETDEWEB)

    Then, G.; Appel, H.; Thies, W.G.; Duffield, J.; Taylor, D.M. (Kernforschungszentrum Karlsruhe G.m.b.H. (Germany, F.R.). Inst. fuer Genetik und Toxikologie von Spaltstoffen); Zell, I. (Karlsruhe Univ. (T.H.) (Germany, F.R.). Inst. fuer Experimentelle Kernphysik)

    1983-12-01

    Using the TDPAC-technique binding parameters for Hf were determined after in vivo uptake of /sup 181/Hf in rat plasma. As much as 98.5% of the metal ions proved to be bound to protein, essentially to transferrin. The main fraction of the /sup 181/Hf ions experiences a well defined electric quadrupole perturbation frequency (..gamma..sub(Q1) = (1516 +- 15)MHz, deltasub(1) = (5.3 +- 0.8)%) connected with a marked relaxation damping (lambda = (46 +- 8)MHz). The remaining Hf nuclei are subject to a fairly broad distribution of electric field gradients (..gamma..sub(Q2) = (1014 +- 37)MHz, deltasub(2) = (16 +- 3)%). The results are compared with data obtained with in vitro /sup 181/Hf-labeled human transferrin.

  16. Sugared biomaterial binding lectins: achievements and perspectives.

    Science.gov (United States)

    Bojarová, P; Křen, V

    2016-07-19

    Lectins, a distinct group of glycan-binding proteins, play a prominent role in the immune system ranging from pathogen recognition and tuning of inflammation to cell adhesion or cellular signalling. The possibilities of their detailed study expanded along with the rapid development of biomaterials in the last decade. The immense knowledge of all aspects of glycan-lectin interactions both in vitro and in vivo may be efficiently used in bioimaging, targeted drug delivery, diagnostic and analytic biological methods. Practically applicable examples comprise photoluminescence and optical biosensors, ingenious three-dimensional carbohydrate microarrays for high-throughput screening, matrices for magnetic resonance imaging, targeted hyperthermal treatment of cancer tissues, selective inhibitors of bacterial toxins and pathogen-recognising lectin receptors, and many others. This review aims to present an up-to-date systematic overview of glycan-decorated biomaterials promising for interactions with lectins, especially those applicable in biology, biotechnology or medicine. The lectins of interest include galectin-1, -3 and -7 participating in tumour progression, bacterial lectins from Pseudomonas aeruginosa (PA-IL), E. coli (Fim-H) and Clostridium botulinum (HA33) or DC-SIGN, receptors of macrophages and dendritic cells. The spectrum of lectin-binding biomaterials covered herein ranges from glycosylated organic structures, calixarene and fullerene cores over glycopeptides and glycoproteins, functionalised carbohydrate scaffolds of cyclodextrin or chitin to self-assembling glycopolymer clusters, gels, micelles and liposomes. Glyconanoparticles, glycan arrays, and other biomaterials with a solid core are described in detail, including inorganic matrices like hydroxyapatite or stainless steel for bioimplants. PMID:27075026

  17. Signal transduction by guanine nucleotide binding proteins.

    Science.gov (United States)

    Spiegel, A M

    1987-01-01

    High affinity binding of guanine nucleotides and the ability to hydrolyze bound GTP to GDP are characteristics of an extended family of intracellular proteins. Subsets of this family include cytosolic initiation and elongation factors involved in protein synthesis, and cytoskeletal proteins such as tubulin (Hughes, S.M. (1983) FEBS Lett. 164, 1-8). A distinct subset of guanine nucleotide binding proteins is membrane-associated; members of this subset include the ras gene products (Ellis, R.W. et al. (1981) Nature 292, 506-511) and the heterotrimeric G-proteins (also termed N-proteins) (Gilman, A.G. (1984) Cell 36, 577-579). Substantial evidence indicates that G-proteins act as signal transducers by coupling receptors (R) to effectors (E). A similar function has been suggested but not proven for the ras gene products. Known G-proteins include Gs and Gi, the G-proteins associated with stimulation and inhibition, respectively, of adenylate cyclase; transducin (TD), the G-protein coupling rhodopsin to cGMP phosphodiesterase in rod photoreceptors (Bitensky, M.W. et al. (1981) Curr. Top. Membr. Transp. 15, 237-271; Stryer, L. (1986) Annu. Rev. Neurosci. 9, 87-119), and Go, a G-protein of unknown function that is highly abundant in brain (Sternweis, P.C. and Robishaw, J.D. (1984) J. Biol. Chem. 259, 13806-13813; Neer, E.J. et al. (1984) J. Biol. Chem. 259, 14222-14229). G-proteins also participate in other signal transduction pathways, notably that involving phosphoinositide breakdown. In this review, I highlight recent progress in our understanding of the structure, function, and diversity of G-proteins. PMID:2435586

  18. Diazepam binding inhibitor and the endocrine pancreas.

    Science.gov (United States)

    Ostenson, C G; Ahrén, B; Johansson, O; Karlsson, S; Hilliges, M; Efendic, S

    1991-12-01

    Regulation of blood glucose homeostasis is complex. Its major hormonal regulators include insulin, glucagon and somatostatin from the endocrine pancreas. Secretion of these hormones is controlled predominantly by the supply of nutrients in the circulation but also by nerve signals and other peptides. Thus, it is likely that peptides, released from cells of the gut or endocrine pancreas or from peptidergic nerves, affect glucose homeostasis by modulating the secretion of insulin, glucagon and somatostatin. When searching for novel gut peptides with such effects, diazepam binding inhibitor (DBI) was isolated from the porcine small intestine. By immunocytochemistry, DBI has been demonstrated to occur not only in the gut but also in endocrine cells of the pancreatic islets, namely in the somatostatin-producing D-cells in pig and man, and in the glucagon-producing A-cells in rat. Porcine DBI (pDBI; 10(-8)-10(-7) M) has been shown to suppress glucose-stimulated release of insulin from both isolated islets and perfused pancreas of the rat. Furthermore, secretion of insulin stimulated by either the sulfonylurea glibenclamide or the phosphodiesterase inhibitor 3-isobutyl-1-methylxanthine (IBMX), was inhibited by the peptide. In contrast, arginine-induced release of insulin was unaffected by pDBI. Moreover, pDBI decreased arginine-induced release of glucagon from the perfused rat pancreas, whereas release of somatostatin was unchanged. Notably, rat DBI, structurally identical with rat acyl-CoA-binding protein, has also been demonstrated to inhibit glucose-stimulated release of insulin in the rat, both in vivo and in vitro. Long-term exposure of cultured fetal rat islets to pDBI (10(-8) M) significantly decreased the synthesis of DNA in islet cells.(ABSTRACT TRUNCATED AT 250 WORDS) PMID:1780037

  19. Quantitative radiommunoassay for DNA-binding antibodies

    International Nuclear Information System (INIS)

    A radioimmunoassay (RIA) is described for the measurement of serum immunoglobulins capable of binding to double-standard or single-standard DNA. DNA attached to Sephadex G-50 by ultraviolet radiation was used as a solid- phase immunoabsorbent for DNA-binding proteins from serum. Goat anti-human (GAH) IgG (125I-labeled) were used to detect the human immunoglobulins bound onto the washed DNA-Sephadex. The quantities of immunoglobulins bound were determined by comparison with a standard curve constructed by dilution of a plasma from an systemic lupus erythematosus (SLE) patient containing known amounts of bound, DNA-specific IgM and IgG. Another RIA was employed for measuring levels of IgG and IgM. In combination with measurements of the total serum IgM and IgG, the RIA allowed for the determination of the fraction of the total serum IgM or IgG that was specific for double- or single-standard DNA. For a pool of normal human sera the quantities were as follows: 0.04% of the total IgM and 0.001% of the total IgG bound double-standard DNA; 0.22% of the total IgM and 0.05% of the total IgG bound single-stranded DNA. This capability is important because information regarding the quantitative measurement of antibodies to DNA and their class determination may be of significance in monitoring the status of subjects with SLE

  20. Niobium Uptake and Release by Bacterial Ferric Ion Binding Protein

    Directory of Open Access Journals (Sweden)

    Yanbo Shi

    2010-01-01

    Full Text Available Ferric ion binding proteins (Fbps transport FeIII across the periplasm and are vital for the virulence of many Gram negative bacteria. Iron(III is tightly bound in a hinged binding cleft with octahedral coordination geometry involving binding to protein side chains (including tyrosinate residues together with a synergistic anion such as phosphate. Niobium compounds are of interest for their potential biological activity, which has been little explored. We have studied the binding of cyclopentadienyl and nitrilotriacetato NbV complexes to the Fbp from Neisseria gonorrhoeae by UV-vis spectroscopy, chromatography, ICP-OES, mass spectrometry, and Nb K-edge X-ray absorption spectroscopy. These data suggest that NbV binds strongly to Fbp and that a dinuclear NbV centre can be readily accommodated in the interdomain binding cleft. The possibility of designing niobium-based antibiotics which block iron uptake by pathogenic bacteria is discussed.

  1. Concentration-dependent Cu(II) binding to prion protein

    Science.gov (United States)

    Hodak, Miroslav; Lu, Wenchang; Bernholc, Jerry

    2008-03-01

    The prion protein plays a causative role in several neurodegenerative diseases, including mad cow disease in cattle and Creutzfeldt-Jakob disease in humans. The normal function of the prion protein is unknown, but it has been linked to its ability to bind copper ions. Experimental evidence suggests that copper can be bound in three distinct modes depending on its concentration, but only one of those binding modes has been fully characterized experimentally. Using a newly developed hybrid DFT/DFT method [1], which combines Kohn-Sham DFT with orbital-free DFT, we have examined all the binding modes and obtained their detailed binding geometries and copper ion binding energies. Our results also provide explanation for experiments, which have found that when the copper concentration increases the copper binding mode changes, surprisingly, from a stronger to a weaker one. Overall, our results indicate that prion protein can function as a copper buffer. 1. Hodak, Lu, Bernholc, JCP, in press.

  2. Hardware device to physical structure binding and authentication

    Energy Technology Data Exchange (ETDEWEB)

    Hamlet, Jason R.; Stein, David J.; Bauer, Todd M.

    2013-08-20

    Detection and deterrence of device tampering and subversion may be achieved by including a cryptographic fingerprint unit within a hardware device for authenticating a binding of the hardware device and a physical structure. The cryptographic fingerprint unit includes an internal physically unclonable function ("PUF") circuit disposed in or on the hardware device, which generate an internal PUF value. Binding logic is coupled to receive the internal PUF value, as well as an external PUF value associated with the physical structure, and generates a binding PUF value, which represents the binding of the hardware device and the physical structure. The cryptographic fingerprint unit also includes a cryptographic unit that uses the binding PUF value to allow a challenger to authenticate the binding.

  3. Aging neuromodulation impairs associative binding: a neurocomputational account.

    Science.gov (United States)

    Li, Shu-Chen; Naveh-Benjamin, Moshe; Lindenberger, Ulman

    2005-06-01

    Relative to young adults, older adults are particularly impaired in episodic memory tasks requiring associative binding of separate components into compound episodes, such as tasks requiring item-context and item-item binding. This associative-binding deficit has been attributed to senescent changes in frontal-hippocampal circuitry but has not been formally linked to impaired neuromodulation involving this circuitry. Previous neurocomputational work showed that impaired neuromodulation could result in less distinct neurocognitive representations. Here we extend this computational principle to simulate aging-related deficits in associative binding. As expected, networks with simulated deficiency in neuromodulation resulted in less distinct internal representations than did networks simulating the processing and performance of young adults, and were also more impaired under task conditions that required associative binding. The findings suggest that senescent changes in neuromodulatory mechanisms may play a basic role in aging-related impairment in associative binding by reducing the efficacy of distributed conjunctive coding. PMID:15943670

  4. DNA binding studies of Vinca alkaloids: experimental and computational evidence.

    Science.gov (United States)

    Pandya, Prateek; Gupta, Surendra P; Pandav, Kumud; Barthwal, Ritu; Jayaram, B; Kumar, Surat

    2012-03-01

    Fluorescence studies on the indole alkaloids vinblastine sulfate, vincristine sulfate, vincamine and catharanthine have demonstrated the DNA binding ability of these molecules. The binding mode of these molecules in the minor groove of DNA is non-specific. A new parameter of the purine-pyrimidine base sequence specificty was observed in order to define the non-specific DNA binding of ligands. Catharanthine had shown 'same' pattern of 'Pu-Py' specificity while evaluating its DNA binding profile. The proton resonances of a DNA decamer duplex were assigned. The models of the drug:DNA complexes were analyzed for DNA binding features. The effect of temperature on the DNA binding was also evaluated. PMID:22545401

  5. Negative Example Aided Transcription Factor Binding Site Search

    OpenAIRE

    Lee, Chih; Huang, Chun-Hsi

    2011-01-01

    Computational approaches to transcription factor binding site identification have been actively researched for the past decade. Negative examples have long been utilized in de novo motif discovery and have been shown useful in transcription factor binding site search as well. However, understanding of the roles of negative examples in binding site search is still very limited. We propose the 2-centroid and optimal discriminating vector methods, taking into account negative examples. Cross-val...

  6. LASAGNA: A novel algorithm for transcription factor binding site alignment

    OpenAIRE

    Lee, Chih; Huang, Chun-Hsi

    2013-01-01

    Background Scientists routinely scan DNA sequences for transcription factor (TF) binding sites (TFBSs). Most of the available tools rely on position-specific scoring matrices (PSSMs) constructed from aligned binding sites. Because of the resolutions of assays used to obtain TFBSs, databases such as TRANSFAC, ORegAnno and PAZAR store unaligned variable-length DNA segments containing binding sites of a TF. These DNA segments need to be aligned to build a PSSM. While the TRANSFAC database provid...

  7. Searching for transcription factor binding sites in vector spaces

    OpenAIRE

    Lee Chih; Huang Chun-Hsi

    2012-01-01

    Abstract Background Computational approaches to transcription factor binding site identification have been actively researched in the past decade. Learning from known binding sites, new binding sites of a transcription factor in unannotated sequences can be identified. A number of search methods have been introduced over the years. However, one can rarely find one single method that performs the best on all the transcription factors. Instead, to identify the best method for a particular trans...

  8. Biomimetic supramolecular metallohosts for binding and activation of dioxygen

    OpenAIRE

    Sprakel, Vera Stefanie Irene

    2004-01-01

    Host-guest chemistry involves the binding of a specific substrate in a receptor via molecular recognition based on supramolecular interactions. Metal-containing derivatives of receptors for the selective supramolecular binding of dihydroxybenzene substrates, which receptors model oxygen binding enzymes both in structure and in function are described in this thesis with the ultimate goal to realize biomimetic catalysis. A PY2-appended receptor 1 and a TPA-appended receptor 2 and the bis-copper...

  9. AFM studies of nonspecific binding of enzyme on DNA

    Institute of Scientific and Technical Information of China (English)

    张益; 谢恒月; 等

    1996-01-01

    Atomic force microscope(AFM) is used to study restriction endonuclease digestion of plasmid DNA,pWRr plasmid DNA is digested by Hind Ⅲ,and the specific and the nonspecific binding of the restriction endonuclease are imaged,and the biological function of the enzyme binding to nonspecific sites is discussed.In addition,it is found that nonspecific binding of Hind ǚ could not induce the DNA characteristic bending angle.

  10. Probabilistic Inference of Transcription Factor Binding from Multiple Data Sources

    OpenAIRE

    Lähdesmäki, Harri; Rust, Alistair G.; Shmulevich, Ilya

    2008-01-01

    An important problem in molecular biology is to build a complete understanding of transcriptional regulatory processes in the cell. We have developed a flexible, probabilistic framework to predict TF binding from multiple data sources that differs from the standard hypothesis testing (scanning) methods in several ways. Our probabilistic modeling framework estimates the probability of binding and, thus, naturally reflects our degree of belief in binding. Probabilistic modeling also allows for ...

  11. Cobalamin and folate binding proteins in human tumour tissue.

    OpenAIRE

    Sheppard, K; Bradbury, D A; Davies, J. M.; Ryrie, D. R.

    1984-01-01

    The serum of an 84 year old man with disseminated carcinoma was found to contain extremely high concentrations of cobalamin and of a cobalamin binding protein with trans-cobalamin I characteristics. Tumour tissue samples obtained at necropsy contained considerably higher concentrations of cobalamin binding protein (R-binder) than normal tissues. Tumour tissues also contained increased concentrations of specific folate binding protein. In all tissues studied a close correlation existed between...

  12. Effect of Nuclear Binding Energy to K Factor

    Institute of Scientific and Technical Information of China (English)

    HOU Zhao-Yu; GUO Ai-Qiang

    2007-01-01

    We modify the square of virtual photon four-momentum by using nuclear binding energy formula,and calculate the effect of nuclear binding energy to K factor and Compton subprocess and annihilate subprocess in A-A collision Drell-Yan process.The outcome indicates that the effect of nuclear binding energy to K factor is obvious in little x region and it would disappear gradually as x increases.

  13. Ligand Binding Analysis and Screening by Chemical Denaturation Shift

    OpenAIRE

    Sch n, Arne; Brown, Richard K; Hutchins, Burleigh M.; Freire, Ernesto

    2013-01-01

    The identification of small molecule ligands is an important first step in drug development, especially drugs that target proteins with no intrinsic activity. Towards this goal, it is important to have access to technologies that are able to measure binding affinities for a large number of potential ligands in a fast and accurate way. Since ligand binding stabilizes the protein structure in a manner dependent on concentration and binding affinity, the magnitude of the protein stabilization ef...

  14. Why the binding theory doesn’t apply at LF

    OpenAIRE

    Hicks, Glyn

    2008-01-01

    This article argues that the relegation of the binding theory to the C-I interface (LF) is theoretically undesirable and empirically unwarranted. Recent Minimalist research has sought to eliminate the binding theory from UG by reducing its conditions to narrow-syntactic operations (Hornstein 2000, 2006; Reuland 2001, 2006; Kayne 2002; Zwart 2002, 2006; Hicks 2006). This approach remains controversial since the canonical Minimalist binding theory (Chomsky 1993; Chomsky and Lasnik 1993) views t...

  15. Structural analysis of inhibitor binding to human carbonic anhydrase II.

    OpenAIRE

    Boriack-Sjodin, P. A.; Zeitlin, S; Chen, H H; Crenshaw, L.; Gross, S.; Dantanarayana, A.; P. Delgado; May, J. A.; Dean, T.; Christianson, D. W.

    1998-01-01

    X-ray crystal structures of carbonic anhydrase II (CAII) complexed with sulfonamide inhibitors illuminate the structural determinants of high affinity binding in the nanomolar regime. The primary binding interaction is the coordination of a primary sulfonamide group to the active site zinc ion. Secondary interactions fine-tune tight binding in regions of the active site cavity >5 A away from zinc, and this work highlights three such features: (1) advantageous conformational restraints of a bi...

  16. High-Fidelity DNA Sensing by Protein Binding Fluctuations

    CERN Document Server

    Tlusty, Tsvi; Libchaber, Albert; 10.1103/PhysRevLett.93.258103

    2010-01-01

    One of the major functions of RecA protein in the cell is to bind single-stranded DNA exposed upon damage, thereby triggering the SOS repair response.We present fluorescence anisotropy measurements at the binding onset, showing enhanced DNA length discrimination induced by adenosine triphosphate consumption. Our model explains the observed DNA length sensing as an outcome of out-of equilibrium binding fluctuations, reminiscent of microtubule dynamic instability. The cascade architecture of the binding fluctuations is a generalization of the kinetic proofreading mechanism. Enhancement of precision by an irreversible multistage pathway is a possible design principle in the noisy biological environment.

  17. Factor XII binding to endothelial cells depends on caveolae

    DEFF Research Database (Denmark)

    Schousboe, Inger; Thomsen, Peter; van Deurs, Bo

    2004-01-01

    It is now generally accepted that factor XII (FXII) binds to cellular surfaces in the vascular system. One of the suggested receptors of this binding is the glycosylphosphatidylinositol-anchored urokinase-like plasminogen activator (u-PAR) harbored in caveolae/lipid rafts. However, binding of FXII...... FXII binding to caveolae were performed by sucrose density-gradient centrifugations. This showed that the majority of FXII, chemically cross-linked to HUVEC, could be identified in the same fractions of the gradient as caveolin-1, a marker of caveolae, while the majority of u-PAR was identified in...

  18. Oligosaccharide binding to barley alpha-amylase 1

    DEFF Research Database (Denmark)

    Robert, X.; Haser, R.; Mori, H.;

    2005-01-01

    Enzymatic subsite mapping earlier predicted 10 binding subsites in the active site substrate binding cleft of barley alpha-amylase isozymes. The three-dimensional structures of the oligosaccharide complexes with barley alpha-amylase isozyme 1 (AMY1) described here give for the first time a thorough...... in barley alpha-amylase isozyme 2 (AMY2), and the sugar binding modes are compared between the two isozymes. The "sugar tongs" surface binding site discovered in the AMY1-thio-DP4 complex is confirmed in the present work. A site that putatively serves as an entrance for the substrate to the active...

  19. Penicillin-binding site on the Escherichia coli cell envelope

    International Nuclear Information System (INIS)

    The binding of 35S-labeled penicillin to distinct penicillin-binding proteins (PBPs) of the cell envelope obtained from the sonication of Escherichia coli was studied at different pHs ranging from 4 to 11. Experiments distinguishing the effect of pH on penicillin binding by PBP 5/6 from its effect on beta-lactamase activity indicated that although substantial binding occurred at the lowest pH, the amount of binding increased with pH, reaching a maximum at pH 10. Based on earlier studies, it is proposed that the binding at high pH involves the formation of a covalent bond between the C-7 of penicillin and free epsilon amino groups of the PBPs. At pHs ranging from 4 to 8, position 1 of penicillin, occupied by sulfur, is considered to be the site that establishes a covalent bond with the sulfhydryl groups of PBP 5. The use of specific blockers of free epsilon amino groups or sulfhydryl groups indicated that wherever the presence of each had little or no effect on the binding of penicillin by PBP 5, the presence of both completely prevented binding. The specific blocker of the hydroxyl group of serine did not affect the binding of penicillin

  20. Temperature and pressure adaptation of the binding site of acetylcholinesterase.

    Science.gov (United States)

    Hochachka, P W

    1974-12-01

    1. Studies with a carbon substrate analogue, 3,3-dimethylbutyl acetate, indicate that the hydrophobic contribution to binding at the anionic site of acetylcholinesterase is strongly disrupted at low temperatures and high pressures. 2. Animals living in different physical environments circumvent this problem by adjusting the enthalpic and entropic contributions to binding. 3. An extreme example of this adaptational strategy is supplied by brain acetylcholinesterase extracted from an abyssal fish living at 2 degrees C and up to several hundred atmospheres of pressure. This acetylcholinesterase appears to have a smaller hydrophobic binding region in the anionic site, playing a measurably decreased role in ligand binding. PMID:4462739

  1. Inhibition of tristetraprolin deadenylation by poly(A) binding protein

    OpenAIRE

    Rowlett, Robert M.; Chrestensen, Carol A.; Schroeder, Melanie J.; Harp, Mary G.; Pelo, Jared W.; Shabanowitz, Jeffery; DeRose, Robert; Hunt, Donald F.; Sturgill, Thomas W.; Worthington, Mark T.

    2008-01-01

    Tristetraprolin (TTP) is the prototype for a family of RNA binding proteins that bind the tumor necrosis factor (TNF) messenger RNA AU-rich element (ARE), causing deadenylation of the TNF poly(A) tail, RNA decay, and silencing of TNF protein production. Using mass spectrometry sequencing we identified poly(A) binding proteins-1 and -4 (PABP1 and PABP4) in high abundance and good protein coverage from TTP immunoprecipitates. PABP1 significantly enhanced TNF ARE binding by RNA EMSA and prevente...

  2. Drugs That Bind to α-Synuclein: Neuroprotective or Neurotoxic?

    Science.gov (United States)

    Kakish, Joe; Lee, Dongsoo; Lee, Jeremy S

    2015-12-16

    The misfolding of α-synuclein is a critical event in the death of dopaminergic neurons and the progression of Parkinson's disease. Drugs that bind to α-synuclein and form a loop structure between the N- and C-terminus tend to be neuroprotective, whereas others that cause a more compact structure tend to be neurotoxic. The binding of several natural products and other drugs that are involved in dopamine metabolism were investigated by nanopore analysis and isothermal titration calorimetry. The antinausea drugs, cinnarizine and metoclopramide, do not bind to α-synuclein, whereas amphetamine and the herbicides, paraquat and rotenone, bind tightly and cause α-synuclein to adopt a more compact conformation. The recreational drug, cocaine, binds to α-synuclein, whereas heroin and methadone do not. Metformin, which is prescribed for diabetes and is neuroprotective, binds well without causing α-synuclein to adopt a more compact conformation. Methylphenidate (ritalin) binds to sites in both the N- and C-terminus and causes α-synuclein to adopt a loop conformation. In contrast, amphetamine only binds to the N-terminus. Except for cinnarizine and metoclopramide, there is a good correlation between the mode of binding to α-synuclein and whether a drug is neuroprotective or neurotoxic. PMID:26378986

  3. Bladder endothelin-1 receptor binding of bosentan and ambrisentan.

    Science.gov (United States)

    Osano, Ayaka; Yokoyama, Yoshinari; Hayashi, Hideki; Itoh, Kunihiko; Okura, Takashi; Deguchi, Yoshiharu; Ito, Yoshihiko; Yamada, Shizuo

    2014-01-01

    The present study aimed to characterize bladder endothelin-1 (ET-1) receptor binding of clinically used ET-1 receptor antagonists by using [(125)I]ET-1. The inhibition of specific [(125)I]ET-1 binding was measured in the presence of ET-1 and its receptor antagonists. Specific binding of [(125)I]ET-1 in rat bladder was saturable and of high affinity, which characterized selective labeling of bladder ET-1 receptors. ET-1, bosentan, ambrisentan, and CI-1020 inhibited specific [(125)I]ET-1 binding in a concentration-dependent manner at nanomolar ranges of IC50. Nonlinear least squares regression analysis revealed the presence of high- and low-affinity ET-1 receptor sites for ambrisentan and CI-1020. Bosentan and ambrisentan significantly increased the dissociation constant for bladder [(125)I]ET-1 binding without affecting maximal number of binding sites (Bmax). Thus, bosentan and ambrisentan seem to bind to bladder ET-1 receptor in a competitive and reversible manner. Oral administration of bosentan caused a dose-dependent decrease in Bmax for bladder [(125)I]ET-1 binding, suggesting significant binding of bladder ET-1 receptors in vivo. A significant amount of pharmacologically relevant ET-1 receptors may exist in the bladder. These receptors may be implicated in the pathogenesis of lower urinary tract symptoms and may also be promising targets for the development of therapeutic agents. PMID:24389822

  4. Mutations in the RNA Binding Domain of Stem-Loop Binding Protein Define Separable Requirements for RNA Binding and for Histone Pre-mRNA Processing

    OpenAIRE

    Dominski, Zbigniew; Erkmann, Judith A.; Greenland, John A.; Marzluff, William F

    2001-01-01

    Expression of replication-dependent histone genes at the posttranscriptional level is controlled by stem-loop binding protein (SLBP). One function of SLBP is to bind the stem-loop structure in the 3′ untranslated region of histone pre-mRNAs and facilitate 3′ end processing. Interaction of SLBP with the stem-loop is mediated by the centrally located RNA binding domain (RBD). Here we identify several highly conserved amino acids in the RBD mutation of which results in complete or substantial lo...

  5. Experience with Legally Binding and Non-Binding International Nuclear Instruments

    International Nuclear Information System (INIS)

    Over the past several decades, a range of international legal instruments - both binding and non-binding - have been developed to address issues concerning the safety and security of nuclear and other radioactive materials and associated facilities. The following instruments and documents will be discussed as most directly relevant to the development and enhancement of a global nuclear safety and security regime: the Convention on the Physical Protection of Nuclear Material and its 2005 Amendment; the Convention on Nuclear Safety; the Convention on Early Notification of a Nuclear Accident; Convention on Assistance in the Event of a Nuclear Accident; the Joint Convention on the Safety of Spent Nuclear Fuel and Radioactive Waste Management; the International Convention for the Suppression of Nuclear Terrorism; United Nations Security Council Resolution 1540 (2004); Code of Conduct on the Safety of Research Reactors; Code of Conduct on the Safety and Security of Radioactive Sources and Guidance on Import and Export of Radioactive Sources; and the IAEA Nuclear Security Series. Implementation of these instruments is primarily the responsibility of the States that have either adhered to them or made a political commitment to apply them in implementing their national nuclear programmes. Given the widespread diversity among States in levels of nuclear development, as well as differing legal, economic, industrial, social, scientific and technical practices and conditions, assessing the overall experience with these instruments poses significant challenges. However, most of the relevant instruments contain provisions for periodic reviews by their States parties to assess implementation. For some instruments that do not explicitly include a review mechanism, review procedures have been established through agreement of the parties or interested States. These periodic reviews confirm that both binding and non-binding nuclear instruments and documents have provided a basis for

  6. Comprehensive human transcription factor binding site map for combinatory binding motifs discovery.

    Directory of Open Access Journals (Sweden)

    Arnoldo J Müller-Molina

    Full Text Available To know the map between transcription factors (TFs and their binding sites is essential to reverse engineer the regulation process. Only about 10%-20% of the transcription factor binding motifs (TFBMs have been reported. This lack of data hinders understanding gene regulation. To address this drawback, we propose a computational method that exploits never used TF properties to discover the missing TFBMs and their sites in all human gene promoters. The method starts by predicting a dictionary of regulatory "DNA words." From this dictionary, it distills 4098 novel predictions. To disclose the crosstalk between motifs, an additional algorithm extracts TF combinatorial binding patterns creating a collection of TF regulatory syntactic rules. Using these rules, we narrowed down a list of 504 novel motifs that appear frequently in syntax patterns. We tested the predictions against 509 known motifs confirming that our system can reliably predict ab initio motifs with an accuracy of 81%-far higher than previous approaches. We found that on average, 90% of the discovered combinatorial binding patterns target at least 10 genes, suggesting that to control in an independent manner smaller gene sets, supplementary regulatory mechanisms are required. Additionally, we discovered that the new TFBMs and their combinatorial patterns convey biological meaning, targeting TFs and genes related to developmental functions. Thus, among all the possible available targets in the genome, the TFs tend to regulate other TFs and genes involved in developmental functions. We provide a comprehensive resource for regulation analysis that includes a dictionary of "DNA words," newly predicted motifs and their corresponding combinatorial patterns. Combinatorial patterns are a useful filter to discover TFBMs that play a major role in orchestrating other factors and thus, are likely to lock/unlock cellular functional clusters.

  7. Subtilases and metal binding - the weak binding site of sutilisins revisited

    Czech Academy of Sciences Publication Activity Database

    Dohnálek, Jan; MacAuley, K.; Brzozowski, A. M.; Borchert, T. V.; Wilson, K. S.

    2007-01-01

    Roč. 14, č. 1 (2007), s. 28. ISSN 1211-5894. [Discussions in Structural Molecular Biology and Bioinformatics /6./. 29.03.2007-31.03.2007, Nové Hrady] R&D Projects: GA MŠk 1K05008 Institutional research plan: CEZ:AV0Z40500505 Keywords : subtilisin-like proteases * metal binding Subject RIV: CF - Physical ; Theoretical Chemistry

  8. Mapping convulsants’ binding to the GABA-A receptor chloride ionophore: a proposed model for channel binding sites

    OpenAIRE

    Kalueff, A.V.

    2006-01-01

    Gamma aminobutyric acid (GABA) type A receptors play a key role in brain inhibitory neurotransmission, and are ligand-activated chloride channels blocked by numerous convulsant ligands. Here we summarize data on binding of picrotoxin, tetrazoles, β-lactams, bicyclophosphates, butyrolactones and neurotoxic pesticides to GABA-A ionophore, and discuss functional and structural overlapping of their binding sites. The paper reviews data on convulsants’ binding sensitivity to different point mutati...

  9. Fatty Acid- and Retinoid-binding Proteins Have Distinct Binding Pockets for the Two Types of Cargo*

    OpenAIRE

    Jordanova, Rositsa; Groves, Matthew R.; Kostova, Elena; Woltersdorf, Christian; Liebau, Eva; Tucker, Paul A.

    2009-01-01

    Parasitic nematodes cause serious diseases in humans, animals, and plants. They have limited lipid metabolism and are reliant on lipid-binding proteins to acquire these metabolites from their hosts. Several structurally novel families of lipid-binding proteins in nematodes have been described, including the fatty acid- and retinoid-binding protein family (FAR). In Caenorhabditis elegans, used as a model for studying parasitic nematodes, eight C. elegans FAR proteins have been described. The c...

  10. A Genetic Screen Identifies Putative Targets and Binding Partners of CREB-Binding Protein in the Developing Drosophila Eye

    OpenAIRE

    Anderson, Jason; Bhandari, Rohan; Kumar, Justin P.

    2005-01-01

    Drosophila CREB-binding protein (dCBP) is a very large multidomain protein, which belongs to the CBP/p300 family of proteins that were first identified by their ability to bind the CREB transcription factor and the adenoviral protein E1. Since then CBP has been shown to bind to >100 additional proteins and functions in a multitude of different developmental contexts. Among other activities, CBP is known to influence development by remodeling chromatin, by serving as a transcriptional coactiva...

  11. The Cobalamin-binding Protein in Zebrafish is an Intermediate Between the Three Cobalamin-binding Proteins in Human

    OpenAIRE

    Greibe, Eva Holm; Fedosov, Sergey; Nexø, Ebba

    2012-01-01

    In humans, three soluble extracellular cobalamin-binding proteins; transcobalamin (TC), intrinsic factor (IF), and haptocorrin (HC), are involved in the uptake and transport of cobalamin. In this study, we investigate a cobalamin-binding protein from zebrafish (Danio rerio) and summarize current knowledge concerning the phylogenetic evolution of kindred proteins. We identified a cobalamin binding capacity in zebrafish protein extracts (8.2 pmol/fish) and ambient water (13.5 pmol/fish) associa...

  12. Alpha-Amylase Starch Binding Domains: Cooperative Effects of Binding to Starch Granules of Multiple Tandemly Arranged Domains▿

    OpenAIRE

    Guillén, D.; Santiago, M.; Linares, L; Pérez, R; Morlon, J.; Ruiz, B; Sánchez, S.; Rodríguez-Sanoja, R.

    2007-01-01

    The Lactobacillus amylovorus alpha-amylase starch binding domain (SBD) is a functional domain responsible for binding to insoluble starch. Structurally, this domain is dissimilar from other reported SBDs because it is composed of five identical tandem modules of 91 amino acids each. To understand adsorption phenomena specific to this SBD, the importance of their modular arrangement in relationship to binding ability was investigated. Peptides corresponding to one, two, three, four, or five mo...

  13. Paracetamol and cytarabine binding competition in high affinity binding sites of transporting protein

    Science.gov (United States)

    Sułkowska, A.; Bojko, B.; Równicka, J.; Sułkowski, W. W.

    2006-07-01

    Paracetamol (acetaminophen, AA) the most popular analgesic drug is commonly used in the treatment of pain in patients suffering from cancer. In our studies, we evaluated the competition in binding with serum albumin between paracetamol (AA) and cytarabine, antyleukemic drug (araC). The presence of one drug can alter the binding affinity of albumin towards the second one. Such interaction can result in changing of the free fraction of the one of these drugs in blood. Two spectroscopic methods were used to determine high affinity binding sites and the competition of the drugs. Basing on the change of the serum albumin fluorescence in the presence of either of the drugs the quenching ( KQ) constants for the araC-BSA and AA-BSA systems were calculated. Analysis of UV difference spectra allowed us to describe the changes in drug-protein complexes (araC-albumin and AA-albumin) induced by the presence of the second drug (AA and araC, respectively). The mechanism of competition between araC and AA has been proposed.

  14. Study on Synthesis and Binding Ability of a New Anion Receptor Containing NH Binding Sites

    Institute of Scientific and Technical Information of China (English)

    QIAO,Yan-Hong; LIN,Hai; LIN,Hua-Kuan

    2007-01-01

    A new colorimetric recognition receptor 1 based on the dual capability containing NH binding sites of selectively sensing anionic guest species has been synthesized. Compared with other halide anions, its UV/Vis absorption spectrum in dimethyl sulfoxide showed the response toward the presence of fluoride anion with high selectivity,and also displayed dramatic color changes from colorless to yellow in the presence of TBAF (5 × 10-5 mol/L). The similar UV/Vis absorption spectrum change also occurred when 1 was treated with AcO- while a little change with H2PO-4 and OH-. Receptor 1 has almost not affinity abilities to Cl-, Br- and I-. The binding ability of receptor 1to fluoride with high selectivity over other halides contributes to the anion size and the ability of forming hydrogen bonding. While the different ability of binding with geometrically triangular (AcO-), tetrahedral (H2PO-4 ) and linear (OH-) anions maybe result from their geometry configuration.

  15. Functional zinc-binding motifs in enzymes and DNA-binding proteins.

    Science.gov (United States)

    Vallee, B L; Auld, D S

    1992-01-01

    Zinc is now known to be an integral component of a large number and variety of enzymes and proteins involved in virtually all aspects of metabolism, thus accounting for the fact that this element is essential for growth and development. The chemistry of zinc, superficially bland, in reality has turned out to be ideally appropriate and versatile for the unexpected development of multiple and unique chemical structures which biology has used for specific life processes. The present discussion will centre on those distinctive zinc-binding motifs that are critical both to enzyme function and the expression of the genetic message. X-Ray diffraction structure determination of 15 zinc enzymes belonging to IUB classes I-IV provide absolute standards of reference for the identity and nature of zinc ligands in their families. Three types of zinc enzyme binding motifs emerge through analysis of these: catalytic, coactive or cocatalytic, and structural. In contrast to zinc enzymes virtually all DNA-binding proteins contain multiple zinc atoms. With the availability of NMR and X-ray structure analyses three distinct motifs now emerge for those: zinc fingers, twists and clusters. PMID:1290939

  16. Unusual Heme Binding in the Bacterial Iron Response Regulator Protein (Irr): Spectral Characterization of Heme Binding to Heme Regulatory Motif

    OpenAIRE

    Ishikawa, Haruto; Nakagaki, Megumi; Bamba, Ai; Uchida, Takeshi; Hori, Hiroshi; O'Brian, Mark R.; Iwai, Kazuhiro; Ishimori, Koichiro

    2011-01-01

    We characterized heme binding in the bacterial iron response regulator (Irr) protein, which is a simple heme-regulated protein having a single “heme-regulatory motif”, HRM, and plays a key role in the iron homeostasis of a nitrogen fixing bacterium. The heme titration to wild-type and mutant Irr clearly showed that Irr has two heme binding sites: one of the heme binding sites is in the HRM, where 29Cys is the axial ligand, and the other one, the secondary heme binding site, is located outside...

  17. The human enhancer-binding protein Gata3 binds to several T-cell receptor regulatory elements.

    OpenAIRE

    Marine, J; Winoto, A

    1991-01-01

    The tissue-specific developmental regulation of the alpha, beta, gamma and delta T-cell antigen receptor (TCR) genes is controlled by the corresponding distinct enhancers and their enhancer-binding proteins. To find a common TCR regulatory element, we have studied the ability of the newly described enhancer-binding protein Gata3 to bind to the sequence motif (A/T)GATA(G/A) shared between enhancer elements of all four TCR genes. Gata3 was shown in the chicken to be an enhancer-binding protein ...

  18. Whole-genome cartography of estrogen receptor alpha binding sites.

    Directory of Open Access Journals (Sweden)

    Chin-Yo Lin

    2007-06-01

    Full Text Available Using a chromatin immunoprecipitation-paired end diTag cloning and sequencing strategy, we mapped estrogen receptor alpha (ERalpha binding sites in MCF-7 breast cancer cells. We identified 1,234 high confidence binding clusters of which 94% are projected to be bona fide ERalpha binding regions. Only 5% of the mapped estrogen receptor binding sites are located within 5 kb upstream of the transcriptional start sites of adjacent genes, regions containing the proximal promoters, whereas vast majority of the sites are mapped to intronic or distal locations (>5 kb from 5' and 3' ends of adjacent transcript, suggesting transcriptional regulatory mechanisms over significant physical distances. Of all the identified sites, 71% harbored putative full estrogen response elements (EREs, 25% bore ERE half sites, and only 4% had no recognizable ERE sequences. Genes in the vicinity of ERalpha binding sites were enriched for regulation by estradiol in MCF-7 cells, and their expression profiles in patient samples segregate ERalpha-positive from ERalpha-negative breast tumors. The expression dynamics of the genes adjacent to ERalpha binding sites suggest a direct induction of gene expression through binding to ERE-like sequences, whereas transcriptional repression by ERalpha appears to be through indirect mechanisms. Our analysis also indicates a number of candidate transcription factor binding sites adjacent to occupied EREs at frequencies much greater than by chance, including the previously reported FOXA1 sites, and demonstrate the potential involvement of one such putative adjacent factor, Sp1, in the global regulation of ERalpha target genes. Unexpectedly, we found that only 22%-24% of the bona fide human ERalpha binding sites were overlapping conserved regions in whole genome vertebrate alignments, which suggest limited conservation of functional binding sites. Taken together, this genome-scale analysis suggests complex but definable rules governing ERalpha

  19. Evolution of Metal(Loid) Binding Sites in Transcriptional Regulators

    Energy Technology Data Exchange (ETDEWEB)

    Ordonez, E.; Thiyagarajan, S.; Cook, J.D.; Stemmler, T.L.; Gil, J.A.; Mateos, L.M.; Rosen, B.P.

    2009-05-22

    Expression of the genes for resistance to heavy metals and metalloids is transcriptionally regulated by the toxic ions themselves. Members of the ArsR/SmtB family of small metalloregulatory proteins respond to transition metals, heavy metals, and metalloids, including As(III), Sb(III), Cd(II), Pb(II), Zn(II), Co(II), and Ni(II). These homodimeric repressors bind to DNA in the absence of inducing metal(loid) ion and dissociate from the DNA when inducer is bound. The regulatory sites are often three- or four-coordinate metal binding sites composed of cysteine thiolates. Surprisingly, in two different As(III)-responsive regulators, the metalloid binding sites were in different locations in the repressor, and the Cd(II) binding sites were in two different locations in two Cd(II)-responsive regulators. We hypothesize that ArsR/SmtB repressors have a common backbone structure, that of a winged helix DNA-binding protein, but have considerable plasticity in the location of inducer binding sites. Here we show that an As(III)-responsive member of the family, CgArsR1 from Corynebacterium glutamicum, binds As(III) to a cysteine triad composed of Cys{sup 15}, Cys{sup 16}, and Cys{sup 55}. This binding site is clearly unrelated to the binding sites of other characterized ArsR/SmtB family members. This is consistent with our hypothesis that metal(loid) binding sites in DNA binding proteins evolve convergently in response to persistent environmental pressures.

  20. Heterogeneity of binding subunits of the human 150K insulin-like growth factor binding protein.

    Science.gov (United States)

    Gelato, M C; Gaynes, L A; Greenstein, L A; Nissley, S P

    1990-04-01

    Models for the structure of the GH-dependent 150K insulin-like growth factor-binding protein (IGF-BP) complex include 1) a binding subunit of 40-60K mol wt associated with a larger nonbinding component, and 2) an oligomeric structure simply made up of six 25-28K monomeric IGF-BP complexes. To evaluate these alternative models we examined the IGF-binding characteristics and behavior on an SP-Sephadex ion exchange column of BP species identified by chemically cross-linking [125I]IGF-I and [125I]IGF-II. In addition, human serum was gel filtered on Sephadex G-200 in 0.05 M NH4HCO3, pH 8.0, and the 150K BP identified by binding of [125I]IGF-II to column fractions. When [125I]IGF-I or [125I]IGF-II was cross-linked to the 150K BP with disuccinimidyl suberate and analyzed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (10-15%) and autoradiography, four specifically labeled complexes of 20K, 24K, 33K, and 47K mol wt were identified. We examined the IGF-binding characteristics of these species by cross-linking [125I]IGF-I and [125I]IGF-II after incubation in the presence of increasing concentrations of unlabeled IGF-I or IGF-II. Formation of the 24K complex was inhibited more potently by IGF-II than IGF-I, whereas the relative potency of IGF-I vs. IGF-II for inhibition of the formation of the other complexes depended upon whether [125I]IGF-II or [125I]IGF-I was used. When the 150K BP complex generated from gel filtration on Sephadex G-200 was acid stripped, the only species seen with chemical cross-linking of either [125I]IGF-I or [125I]IGF-II was the 47K complex. By both conventional competitive binding studies and cross-linking [125I]IGF-I and [125I]IGF-II after incubation with increasing concentrations of unlabeled IGF-I or IGF-II, the formation of the 47K complex was usually more potently inhibited by IGF-I than IGF-II. When Cohn fraction IV extract was chromatographed on a SP-Sephadex column (pH 3) and cross-linking performed on the flow-through, the 47K

  1. Complete Genome Sequence of a J Subgroup Avian Leukosis Virus Isolated from Local Commercial Broilers

    OpenAIRE

    Li, Hongxin; Xue, Chunyi; Ji, Jun; CHANG, SHUANG; Shang, Huiqin; Zhang, Lingjun; Ma, Jingyun; Bi, Yingzuo; Xie, Qingmei

    2012-01-01

    Subgroup J avian leukosis virus (ALV-J) isolate GDKP1202 was isolated from a 50-day-old local yellow commercial broiler in the Guangdong province of China in 2012. Here we report the complete genomic sequence of the GDKP1202 isolate, which caused high mortality, serious growth suppression, thymic atrophy, and liver enlargement in commercial broilers. A novel potential binding site (5′-GGCACCTCC-3′) for c-myb was identified in the GDKP1202 genome. These findings will provide additional insight...

  2. STRUCTURAL FEATURES OF PLANT CHITINASES AND CHITIN-BINDING PROTEINS

    NARCIS (Netherlands)

    BEINTEMA, JJ

    1994-01-01

    Structural features of plant chitinases and chitin-binding proteins are discussed. Many of these proteins consist of multiple domains,of which the chitin-binding hevein domain is a predominant one. X-ray and NMR structures of representatives of the major classes of these proteins are available now,

  3. Binding of reactive organophosphate by oximes via hydrogen bond

    Indian Academy of Sciences (India)

    Andrea Pappalardo; Maria E Amato; Francesco P Ballistreri; Valentina La Paglia Fragola; Gaetano A Tomaselli; Rosa Maria Toscano; Giuseppe Trusso Sfrazzetto

    2013-07-01

    In this contribution, the ability of simple oximes to bind a well-known nerve agent simulant (dimethylmethylphosphonate, DMMP) via hydrogen bond is reported. UV/Vis measurements indicate the formation of 1:1 complexes. 1H-, 31P-NMR titrations and T-ROESY experiments confirm that oximes bind the organophosphate via hydrogen bond.

  4. Kinetic and Stoichiometric Characterisation of Streptavidin-Binding Aptamers

    NARCIS (Netherlands)

    Ruigrok, V.J.B.; Duijn, van E.; Barendregt, A.; Dyer, K.; Tainer, J.A.; Stoltenburg, R.; Strehlitz, B.; Levisson, M.; Smidt, H.; Oost, van der J.

    2012-01-01

    Aptamers are oligonucleotide ligands that are selected for high-affinity binding to molecular targets. Only limited knowledge relating to relations between structural and kinetic properties that define aptamer-target interactions is available. To this end, streptavidin-binding aptamers were isolated

  5. Mannan-binding lectin in astma and allergy

    DEFF Research Database (Denmark)

    Kaur, S.; Thiel, Steffen; Sarma, P.U.;

    2006-01-01

    Mannan-binding lectin (MBL) is a vital and versatile component of innate immunity. It is present in serum and may bind to a plethora of microbial pathogens and mediate opsonization of these by complement-dependent and/or independent mechanisms. Low-MBL levels in serum, attributed to certain genetic...

  6. Mannose-binding lectin genetics: from A to Z

    DEFF Research Database (Denmark)

    Garred, Peter

    2008-01-01

    MBL (mannose-binding lectin) is primarily a liver-derived collagen-like serum protein. It binds sugar structures on micro-organisms and on dying host cells and is one of the four known mediators that initiate activation of the complement system via the lectin pathway. Common variant alleles...

  7. Binding energy calculations using the molecular orbital wave function

    International Nuclear Information System (INIS)

    The molecular orbital wave function is used in describing the 4 N-nuclei internal wave function. Using the variational technique the binding energies of the nuclei 12C, 16O, 20Ne and 24Mg are calculated using different Skyrm interaction parameters. Both v.m.s. radii and binding energies obtained in this work are comparable with the corresponding experimental values. (author)

  8. Human DC-SIGN Binds Specific Human Milk Glycans

    Science.gov (United States)

    Noll, Alexander J.; Yu, Ying; Lasanajak, Yi; Duska-McEwen, Geralyn; Buck, Rachael H.; Smith, David F.; Cummings, Richard D.

    2016-01-01

    Human milk glycans (HMGs) are prebiotics, pathogen receptor decoys, and regulators of host physiology and immune responses. Mechanistically, human lectins (glycan-binding proteins, hGBPs) expressed by dendritic cells (DC) are of major interest, as these cells directly contact HMGs. To explore such interactions, we screened many C-type lectins and Siglecs expressed by DC for glycan binding on microarrays presenting over 200 HMGs. Unexpectedly, DC-SIGN showed robust binding to many HMGs, whereas other C-type lectins failed to bind, and Siglecs-5 and -9 showed weak binding to a few glycans. By contrast, most hGBPs bound to multiple glycans on other microarrays lacking HMGs. An α-linked fucose residue was characteristic of HMGs bound by DC-SIGN. Binding of DC-SIGN to the simple HMGs 2′-fucosyllactose (2′-FL) and 3-fucosyllactose (3-FL) was confirmed by flow cytometry to beads conjugated with 2′-FL or 3-FL, as well as the ability of the free glycans to inhibit DC-SIGN binding. 2′-FL had an IC50 of ~1 mM for DC-SIGN, which is within the physiological concentration of 2′-FL in human milk. These results demonstrate that DC-SIGN among the many hGBPs expressed by DC binds to α-fucosylated HMGs, and suggest that such interactions may be important in influencing immune responses in the developing infant. PMID:26976925

  9. Cue integration and the perception of action in intentional binding

    DEFF Research Database (Denmark)

    Wolpe, Noham; Haggard, Patrick; Siebner, Hartwig R;

    2013-01-01

    'Intentional binding' describes the perceived temporal attraction between a voluntary action and its sensory consequence. Binding has been used in health and disease as an indirect measure of awareness of action or agency, that is, the sense that one controls one's own actions. It has been proposed...

  10. Copper binding to soil fulvic and humic acids

    NARCIS (Netherlands)

    Xu, Jinling; Tan, Wenfeng; Xiong, Juan; Wang, Mingxia; Fang, Linchuan; Koopal, Luuk K.

    2016-01-01

    Binding of Cu(II) to soil fulvic acid (JGFA), soil humic acids (JGHA, JLHA), and lignite-based humic acid (PAHA) was investigated through NICA-Donnan modeling and conditional affinity spectrum (CAS). It is to extend the knowledge of copper binding by soil humic substances (HS) both in respect of

  11. Improved assay for measuring heparin binding to bull sperm

    International Nuclear Information System (INIS)

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

  12. Does antibody binding to diverse antigens predict future infection?

    Science.gov (United States)

    Owen, J P; Waite, J L; Holden, K Z; Clayton, D H

    2014-11-01

    We studied diverse antigen binding in hosts and the outcome of parasitism. We used captive-bred F1 descendants of feral rock pigeons (Columba livia) challenged with blood-feeding flies (Hippoboscidae) and a protozoan parasite (Haemoproteus). Enzyme-linked immunosorbent assays (ELISAs) and immunoblots were used to test (i) whether pre-infection IgY antigen binding predicts parasite fitness and (ii) whether antigen binding changes after infection. Assays used extracts from three pigeon parasites (northern fowl mite, Salmonella bacteria and avian pox virus), as well as nonparasitic molecules from cattle, chicken and keyhole limpet. Binding to hippoboscid and S. enterica extracts were predictive of hippoboscid fly fitness. Binding to extracts from hippoboscids, pox virus and nonparasitic organisms was predictive of Haemoproteus infection levels. Antigen binding to all extracts increased after parasite challenge, despite the fact that birds were only exposed to flies and Haemoproteus. Immunoblots suggested innate Ig binding to parasite-associated molecular markers and revealed that new antigens were bound in extracts after infection. These data suggest that host antibody binding to diverse antigens predicts parasite fitness even when the antigens are not related to the infecting parasite. We discuss the implications of these data for the study of host-parasite immunological interaction. PMID:25313676

  13. 3H-spiroperidol binding sites in blood platelets

    International Nuclear Information System (INIS)

    3H-spiroperidol, an antagonist of dopamine receptors in brain (striatum), was found to bind to human and rat platelet membrane preparations. The binding was rapid, reversible, saturable and specific. Unlabelled haloperidol displaced the specifically bound 3H-spiroperidol. Binding equilibrium was attained in 15 min at pH 7.4 and 37 degrees C. Scatchard analysis of 3H-spiroperidol binding revealed a single population of binding site with Kd of 7.6 nM in rat platelet membrane and Kd of 15 nM in human platelet membrane. Unlabelled 5-hydroxytryptamine produced no significant effect on 3H-spiroperidol binding to rat or human blood platelet membranes in the presence or absence of haloperidol. Some dopaminergic agents, known to inhibit spiroperidol binding in corpus striatum, also inhibited the same in rat and human blood platelet membranes under in vitro conditions. This study suggests the presence of specific 3H-spiroperidol binding sites in blood platelets

  14. Binding-Induced Fluorescence of Serotonin Transporter Ligands

    DEFF Research Database (Denmark)

    Wilson, James; Ladefoged, Lucy Kate; Babinchak, Michael;

    2014-01-01

    The binding-induced fluorescence of 4-(4-(dimethylamino)-phenyl)-1-methylpyridinium (APP(+)) and two new serotonin transporter (SERT)-binding fluorescent analogues, 1-butyl-4-[4-(1-dimethylamino)phenyl]-pyridinium bromide (BPP(+)) and 1-methyl-4-[4-(1-piperidinyl)phenyl]-pyridinium (PPP(+)), has...

  15. Extrapolations of nuclear binding energies from new linear mass relations

    DEFF Research Database (Denmark)

    Hove, D.; Jensen, A. S.; Riisager, K.

    2013-01-01

    We present a method to extrapolate nuclear binding energies from known values for neighboring nuclei. We select four specific mass relations constructed to eliminate smooth variation of the binding energy as function nucleon numbers. The fast odd-even variations are avoided by comparing nuclei...

  16. Studies of the silencing of Baculovirus DNA binding protein

    NARCIS (Netherlands)

    Quadt, I.; Lent, van J.W.M.; Knebel-Morsdorf, D.

    2007-01-01

    Baculovirus DNA binding protein (DBP) binds preferentially single-stranded DNA in vitro and colocalizes with viral DNA replication sites. Here, its putative role as viral replication factor has been addressed by RNA interference. Silencing of DBP in Autographa californica multiple nucleopolyhedrovir

  17. Binding of Divalent Magnesium by Escherichia coli Phosphoribosyl Diphosphate Synthetase

    DEFF Research Database (Denmark)

    Willemoës, Martin; Hove-Jensen, Bjarne

    1997-01-01

    The mechanism of binding of the substrates MgATP and ribose 5-phosphate as well as Mg2+ to the enzyme 5-phospho-d-ribosyl a-1-diphosphate synthetase from Escherichia coli has been analyzed. By use of the competive inhibitors of ATP and ribose 5-phosphate binding, a,ß-methylene ATP and (+)-1-a,2-a...

  18. Binding of divalent magnesium by Escherichia coli phosphoribosyl diphosphate synthetase

    DEFF Research Database (Denmark)

    Willemoës, Martin; Hove-Jensen, Bjarne

    1997-01-01

    The mechanism of binding of the substrates Mg x ATP and ribose 5-phosphate as well as Mg2+ to the enzyme 5-phospho-D-ribosyl (alpha-1-diphosphate synthetase from Escherichia coli has been analyzed. By use of the competive inhibitors of ATP and ribose 5-phosphate binding, alpha,beta-methylene ATP ...

  19. The Audiovisual Temporal Binding Window Narrows in Early Childhood

    Science.gov (United States)

    Lewkowicz, David J.; Flom, Ross

    2014-01-01

    Binding is key in multisensory perception. This study investigated the audio-visual (A-V) temporal binding window in 4-, 5-, and 6-year-old children (total N = 120). Children watched a person uttering a syllable whose auditory and visual components were either temporally synchronized or desynchronized by 366, 500, or 666 ms. They were asked…

  20. Experimental Binding Energies in Supramolecular Complexes.

    Science.gov (United States)

    Biedermann, Frank; Schneider, Hans-Jörg

    2016-05-11

    On the basis of many literature measurements, a critical overview is given on essential noncovalent interactions in synthetic supramolecular complexes, accompanied by analyses with selected proteins. The methods, which can be applied to derive binding increments for single noncovalent interactions, start with the evaluation of consistency and additivity with a sufficiently large number of different host-guest complexes by applying linear free energy relations. Other strategies involve the use of double mutant cycles, of molecular balances, of dynamic combinatorial libraries, and of crystal structures. Promises and limitations of these strategies are discussed. Most of the analyses stem from solution studies, but a few also from gas phase. The empirically derived interactions are then presented on the basis of selected complexes with respect to ion pairing, hydrogen bonding, electrostatic contributions, halogen bonding, π-π-stacking, dispersive forces, cation-π and anion-π interactions, and contributions from the hydrophobic effect. Cooperativity in host-guest complexes as well as in self-assembly, and entropy factors are briefly highlighted. Tables with typical values for single noncovalent free energies and polarity parameters are in the Supporting Information. PMID:27136957

  1. Assessment Criteria of Bentonite Binding Properties

    Directory of Open Access Journals (Sweden)

    S. Żymankowska-Kumon

    2012-09-01

    Full Text Available The criteria, with which one should be guided at the assessment of the binding properties of bentonites used for moulding sands, areproposed in the paper. Apart from the standard parameter which is the active bentonite content, the unrestrained growth indicator should be taken into account since it seems to be more adequate in the estimation of the sand compression strength. The investigations performed for three kinds of bentonites, applied in the Polish foundry plants, subjected to a high temperature influences indicate, that the pathway of changes of the unrestrained growth indicator is very similar to the pathway of changes of the sand compression strength. Instead, the character of changes of the montmorillonite content in the sand in dependence of the temperature is quite different. The sand exhibits the significant active bentonite content, and the sand compression strength decreases rapidly. The montmorillonite content in bentonite samples was determined by the modern copper complex method of triethylenetetraamine (Cu(II-TET. Tests were performed for bentonites and for sands with those bentonites subjected to high temperatures influences in a range: 100-700ºC.

  2. Neptunium Binding Kinetics with Arsenazo(III)

    Energy Technology Data Exchange (ETDEWEB)

    Leigh R. Martin; Aaron T. Johnson; Stephen P. Mezyk

    2014-08-01

    This document has been prepared to meet FCR&D level 2 milestone M2FT-14IN0304021, “Report on the results of actinide binding kinetics with aqueous phase complexants” This work was carried out under the auspices of the Thermodynamics and Kinetics of Advanced Separations Systems FCR&D work package. The report details kinetics experiments that were performed to measure rates of aqueous phase complexation for pentavalent neptunium with the chromotropic dye Arsenazo III (AAIII). The studies performed were designed to determine how pH, ionic strength and AAIII concentration may affect the rate of the reaction. A brief comparison with hexavalent neptunium is also made. It was identified that as pH was increased the rate of reaction also increased, however increasing the ionic strength and concentration of AAIII had the opposite effect. Interestingly, the rate of reaction of Np(VI) with AAIII was found to be slower than that of the Np(V) reaction.

  3. Nuclear binding near a quantum phase transition

    CERN Document Server

    Elhatisari, Serdar; Rokash, Alexander; Alarcón, Jose Manuel; Du, Dechuan; Klein, Nico; Lu, Bing-nan; Meißner, Ulf-G; Epelbaum, Evgeny; Krebs, Hermann; Lähde, Timo A; Lee, Dean; Rupak, Gautam

    2016-01-01

    How do protons and neutrons bind to form nuclei? This is the central question of ab initio nuclear structure theory. While the answer may seem as simple as the fact that nuclear forces are attractive, the full story is more complex and interesting. In this work we present numerical evidence from ab initio lattice simulations showing that nature is near a quantum phase transition, a zero-temperature transition driven by quantum fluctuations. Using lattice effective field theory, we perform Monte Carlo simulations for systems with up to twenty nucleons. For even and equal numbers of protons and neutrons, we discover a first-order transition at zero temperature from a Bose-condensed gas of alpha particles (4He nuclei) to a nuclear liquid. Whether one has an alpha-particle gas or nuclear liquid is determined by the strength of the alpha-alpha interactions, and we show that the alpha-alpha interactions depend on the strength and locality of the nucleon-nucleon interactions. The existence of the nearby first-order ...

  4. 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; Hinds, Terry D

    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

  5. 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.

  6. Babel Fortran 2003 Binding for Structured Data Types

    Energy Technology Data Exchange (ETDEWEB)

    Muszala, S; Epperly, T; Wang, N

    2008-05-02

    Babel is a tool aimed at the high-performance computing community that addresses the need for mixing programming languages (Java, Python, C, C++, Fortran 90, FORTRAN 77) in order to leverage the specific benefits of those languages. Scientific codes often rely on structured data types (structs, derived data types) to encapsulate data, and Babel has been lacking in this type of support until recently. We present a new language binding that focuses on their interoperability of C/C++ with Fortran 2003. The new binding builds on the existing Fortran 90 infrastructure by using the iso-c-binding module defined in the Fortran 2003 standard as the basis for C/C++ interoperability. We present the technical approach for the new binding and discuss our initial experiences in applying the binding in FACETS (Framework Application for Core-Edge Transport Simulations) to integrate C++ with legacy Fortran codes.

  7. Flavonoid-DNA binding studies and thermodynamic parameters

    Science.gov (United States)

    Janjua, Naveed Kausar; Shaheen, Amber; Yaqub, Azra; Perveen, Fouzia; Sabahat, Sana; Mumtaz, Misbah; Jacob, Claus; Ba, Lalla Aicha; Mohammed, Hamdoon A.

    2011-09-01

    Interactional studies of new flavonoid derivatives (Fl) with chicken blood ds.DNA were investigated spectrophotometrically in DMSO-H 2O (9:1 v/v) at various temperatures. Spectral parameters suggest considerable binding between the flavonoid derivatives studied and ds.DNA. The binding constant values lie in the enhanced-binding range. Thermodynamic parameters obtained from UV studies also point to strong spontaneous binding of Fl with ds.DNA. Viscometric studies complimented the UV results where a small linear increase in relative viscosity of the DNA solution was observed with added optimal flavonoid concentration. An overall mixed mode of interaction (intercalative plus groove binding) is proposed between DNA and flavonoids. Conclusively, investigated flavonoid derivatives are found to be strong DNA binders and seem to be promising drug candidates like their natural analogues.

  8. Identification and characterization of anion binding sites in RNA

    Energy Technology Data Exchange (ETDEWEB)

    Kieft, Jeffrey S.; Chase, Elaine; Costantino, David A.; Golden, Barbara L. (Purdue); (Colorado)

    2010-05-24

    Although RNA molecules are highly negatively charged, anions have been observed bound to RNA in crystal structures. It has been proposed that anion binding sites found within isolated RNAs represent regions of the molecule that could be involved in intermolecular interactions, indicating potential contact points for negatively charged amino acids from proteins or phosphate groups from an RNA. Several types of anion binding sites have been cataloged based on available structures. However, currently there is no method for unambiguously assigning anions to crystallographic electron density, and this has precluded more detailed analysis of RNA-anion interaction motifs and their significance. We therefore soaked selenate into two different types of RNA crystals and used the anomalous signal from these anions to identify binding sites in these RNA molecules unambiguously. Examination of these sites and comparison with other suspected anion binding sites reveals features of anion binding motifs, and shows that selenate may be a useful tool for studying RNA-anion interactions.

  9. Clinical relevance of drug binding to plasma proteins

    Science.gov (United States)

    Ascenzi, Paolo; Fanali, Gabriella; Fasano, Mauro; Pallottini, Valentina; Trezza, Viviana

    2014-12-01

    Binding to plasma proteins highly influences drug efficacy, distribution, and disposition. Serum albumin, the most abundant protein in plasma, is a monomeric multi-domain macromolecule that displays an extraordinary ligand binding capacity, providing a depot and carrier for many endogenous and exogenous compounds, such as fatty acids and most acidic drugs. α-1-Acid glycoprotein, the second main plasma protein, is a glycoprotein physiologically involved in the acute phase reaction and is the main carrier for basic and neutral drugs. High- and low-density lipoproteins play a limited role in drug binding and are natural drug delivery system only for few lipophilic drugs or lipid-based formulations. Several factors influence drug binding to plasma proteins, such as pathological conditions, concurrent administration of drugs, sex, and age. Any of these factors, in turn, influences drug efficacy and toxicity. Here, biochemical, biomedical, and biotechnological aspects of drug binding to plasma proteins are reviewed.

  10. Oligosaccharide binding to barley alpha-amylase 1

    DEFF Research Database (Denmark)

    Robert, X.; Haser, R.; Mori, H.; Svensson, Birte; Aghajari, N.

    2005-01-01

    insight into the substrate binding by describing residues defining 9 subsites, namely -7 through +2. These structures support that the pseudotetrasaccharide inhibitor acarbose is hydrolyzed by the active enzymes. Moreover, sugar binding was observed to the starch granule-binding site previously determined...... in barley alpha-amylase isozyme 2 (AMY2), and the sugar binding modes are compared between the two isozymes. The "sugar tongs" surface binding site discovered in the AMY1-thio-DP4 complex is confirmed in the present work. A site that putatively serves as an entrance for the substrate to the active...... active site for polysaccharide chains. Moreover, the sugar tongs surface site could also perform the unraveling of amylose chains, with the aid of Tyr-380 acting as "molecular tweezers"....

  11. Conformational flexibility of avidin: the influence of biotin binding

    International Nuclear Information System (INIS)

    Ligand binding to proteins is a key process in cell biochemistry. The interaction usually induces modifications in the unfolding thermodynamic parameters of the macromolecule due to the coupling of unfolding and binding equilibria. In addition, these modifications can be attended by changes in protein structure and/or conformational flexibility induced by ligand binding. In this work, we have explored the effect of biotin binding on conformation and dynamic properties of avidin by using infrared spectroscopy including kinetics of hydrogen/deuterium exchange. Our results, along with previously thermodynamic published data, indicate a clear correlation between thermostability and protein compactness. In addition, our results also help to interpret the thermodynamic binding parameters of the exceptionally stable biotin:AVD complex

  12. A sequential binding mechanism in a PDZ domain

    DEFF Research Database (Denmark)

    Chi, Celestine N; Bach, Anders; Engström, Åke; Wang, Huiqun; Strømgaard, Kristian; Gianni, Stefano; Jemth, Per

    2009-01-01

    Conformational selection and induced fit are two well-known mechanisms of allosteric protein-ligand interaction. Some proteins, like ubiquitin, have recently been found to exist in multiple conformations at equilibrium, suggesting that the conformational selection may be a general mechanism of...... interaction, in particular for single-domain proteins. Here, we found that the PDZ2 domain of SAP97 binds its ligand via a sequential (induced fit) mechanism. We performed binding experiments using SAP97 PDZ2 and peptide ligands and observed biphasic kinetics with the stopped-flow technique, indicating that...... ligand binding involves at least a two-step process. By using an ultrarapid continuous-flow mixer, we then detected a hyperbolic dependence of binding rate constants on peptide concentration, corroborating the two-step binding mechanism. Furthermore, we found a similar dependence of the rate constants on...

  13. Stereoselective binding of chiral drugs to plasma proteins

    Institute of Scientific and Technical Information of China (English)

    Qi SHEN; Lu WANG; Hui ZHOU; Hui-di JIANG; Lu-shan YU; Su ZENG

    2013-01-01

    Chiral drugs show distinct biochemical and pharmacological behaviors in the human body.The binding of chiral drugs to plasma proteins usually exhibits stereoselectivity,which has a far-reaching influence on their pharmacological activities and pharmacokinetic profiles.In this review,the stereoselective binding of chiral drugs to human serum albumin (HSA),α1-acid glycoprotein (AGP)and lipoprotein,three most important proteins in human plasma,are detailed.Furthermore,the application of AGP variants and recombinant fragments of HSA for studying enantiomer binding properties is also discussed.Apart from the stereoselectivity of enantiomer-protein binding,enantiomer-enantiomer interactions that may induce allosteric effects are also described.Additionally,the techniques and methods used to determine drug-protein binding parameters are briefly reviewed.

  14. 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....

  15. Sex Differences in Serotonin 1 Receptor Binding in Rat Brain

    Science.gov (United States)

    Fischette, Christine T.; Biegon, Anat; McEwen, Bruce S.

    1983-10-01

    Male and female rats exhibit sex differences in binding by serotonin 1 receptors in discrete areas of the brain, some of which have been implicated in the control of ovulation and of gonadotropin release. The sex-specific changes in binding, which occur in response to the same hormonal (estrogenic) stimulus, are due to changes in the number of binding sites. Castration alone also affects the number of binding sites in certain areas. The results lead to the conclusion that peripheral hormones modulate binding by serotonin 1 receptors. The status of the serotonin receptor system may affect the reproductive capacity of an organism and may be related to sex-linked emotional disturbances in humans.

  16. Electrochemistry of heparin binding to tau protein on Au surfaces

    International Nuclear Information System (INIS)

    Highlights: • Anionic heparin binds tau protein film on Au • N-terminal of tau protein is critical for heparin binding • Negatively charged heparin binds positively charged tau domains • Heparin binding to tau increases charge transfer resistance - ABSTRACT: The tau protein is a neurodegenerative disease biomarker. The in vitro aggregation of tau is triggered by electrostatic charge imbalance induced by an anionic inducing agent, such as heparin. The binding of the tau-heparin complex is based on electrostatic interactions, but the exact binding mode of heparin to the tau protein has not been fully identified. In this work, the effects of the tau protein orientation on gold (Au) electrode to heparin were explored by the cyclic voltammetry and electrochemical impedance spectroscopy. To modulate the accessibility of N-terminal of the tau to heparin, the tau films on Au surfaces were fabricated in two ways: immobilization of tau via the N-terminal of tau protein (N-tau-Au) or by the Cys291/Cys322 residues, located in the R-repeat domains of the tau protein (Cys-tau-Au). The sulfur-Au bonding was characterized by X-ray photoelectron spectroscopy. The charge transfer resistance was measured for N-tau-Au and Cys-tau-Au as a function of heparin concentration. The heparin concentration range was varied from 0.2 pM to 216 μM with the optimal binding concentration at 21 nM (the highest charge transfer resistance value). The heparin binding to tau films was investigated in the presence of [Fe(CN)6]3−/4− or benzoquinone redox probes. The tau-heparin binding was greater for the Cys-tau-Au surface over N-tau-Au, indicating specific tau domains may be required for optimal heparin binding

  17. Quantitative modeling of transcription factor binding specificities using DNA shape.

    Science.gov (United States)

    Zhou, Tianyin; Shen, Ning; Yang, Lin; Abe, Namiko; Horton, John; Mann, Richard S; Bussemaker, Harmen J; Gordân, Raluca; Rohs, Remo

    2015-04-14

    DNA binding specificities of transcription factors (TFs) are a key component of gene regulatory processes. Underlying mechanisms that explain the highly specific binding of TFs to their genomic target sites are poorly understood. A better understanding of TF-DNA binding requires the ability to quantitatively model TF binding to accessible DNA as its basic step, before additional in vivo components can be considered. Traditionally, these models were built based on nucleotide sequence. Here, we integrated 3D DNA shape information derived with a high-throughput approach into the modeling of TF binding specificities. Using support vector regression, we trained quantitative models of TF binding specificity based on protein binding microarray (PBM) data for 68 mammalian TFs. The evaluation of our models included cross-validation on specific PBM array designs, testing across different PBM array designs, and using PBM-trained models to predict relative binding affinities derived from in vitro selection combined with deep sequencing (SELEX-seq). Our results showed that shape-augmented models compared favorably to sequence-based models. Although both k-mer and DNA shape features can encode interdependencies between nucleotide positions of the binding site, using DNA shape features reduced the dimensionality of the feature space. In addition, analyzing the feature weights of DNA shape-augmented models uncovered TF family-specific structural readout mechanisms that were not revealed by the DNA sequence. As such, this work combines knowledge from structural biology and genomics, and suggests a new path toward understanding TF binding and genome function. PMID:25775564

  18. Microbes bind complement inhibitor factor H via a common site.

    Science.gov (United States)

    Meri, T; Amdahl, H; Lehtinen, M J; Hyvärinen, S; McDowell, J V; Bhattacharjee, A; Meri, S; Marconi, R; Goldman, A; Jokiranta, T S

    2013-01-01

    To cause infections microbes need to evade host defense systems, one of these being the evolutionarily old and important arm of innate immunity, the alternative pathway of complement. It can attack all kinds of targets and is tightly controlled in plasma and on host cells by plasma complement regulator factor H (FH). FH binds simultaneously to host cell surface structures such as heparin or glycosaminoglycans via domain 20 and to the main complement opsonin C3b via domain 19. Many pathogenic microbes protect themselves from complement by recruiting host FH. We analyzed how and why different microbes bind FH via domains 19-20 (FH19-20). We used a selection of FH19-20 point mutants to reveal the binding sites of several microbial proteins and whole microbes (Haemophilus influenzae, Bordetella pertussis, Pseudomonas aeruginosa, Streptococcus pneumonia, Candida albicans, Borrelia burgdorferi, and Borrelia hermsii). We show that all studied microbes use the same binding region located on one side of domain 20. Binding of FH to the microbial proteins was inhibited with heparin showing that the common microbial binding site overlaps with the heparin site needed for efficient binding of FH to host cells. Surprisingly, the microbial proteins enhanced binding of FH19-20 to C3b and down-regulation of complement activation. We show that this is caused by formation of a tripartite complex between the microbial protein, FH, and C3b. In this study we reveal that seven microbes representing different phyla utilize a common binding site on the domain 20 of FH for complement evasion. Binding via this site not only mimics the glycosaminoglycans of the host cells, but also enhances function of FH on the microbial surfaces via the novel mechanism of tripartite complex formation. This is a unique example of convergent evolution resulting in enhanced immune evasion of important pathogens via utilization of a "superevasion site." PMID:23637600

  19. Genetic influences on mannan-binding lectin (MBL) and mannan-binding lectin associated serine protease-2 (MASP-2) activity

    DEFF Research Database (Denmark)

    Sørensen, Grith Lykke; Petersen, Inge; Thiel, Steffen; Fenger, Mogens; Christensen, Kaare; Kyvik, Kirsten O; Sørensen, Thorkild I A; Holmskov, Uffe; Jensenius, Jens Christian

    2007-01-01

    The lectin pathway of the complement system is activated when Mannan-binding lectin (MBL) in complex with MASP-2 binds microorganisms. Polymorphisms in both genes are responsible for low serum levels, which associate with increased risk of infection and autoimmune disease. The present study...

  20. Isothermal titration calorimetry and surface plasmon resonance allow quantifying substrate binding to different binding sites of Bacillus subtilis xylanase

    DEFF Research Database (Denmark)

    Cuyvers, Sven; Dornez, Emmie; Abou Hachem, Maher;

    2012-01-01

    Isothermal titration calorimetry and surface plasmon resonance were tested for their ability to study substrate binding to the active site (AS) and to the secondary binding site (SBS) of Bacillus subtilis xylanase A separately. To this end, three enzyme variants were compared. The first was a...

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

    DEFF Research Database (Denmark)

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

    2001-01-01

    of the cell-binding domain of FN with integrin is sufficient to rescue rat embryo fibroblasts (REFs) from detachment-induced apoptosis. REFs attached and spread normally after plating on substrates coated with either intact FN or a FN fragment, FN120, that contains the cell-binding domain but lacks the C-terminal...

  2. The liver fatty acid binding protein--comparison of cavity properties of intracellular lipid-binding proteins.

    Science.gov (United States)

    Thompson, J; Ory, J; Reese-Wagoner, A; Banaszak, L

    1999-02-01

    The crystal and solution structures of all of the intracellular lipid binding proteins (iLBPs) reveal a common beta-barrel framework with only small local perturbations. All existing evidence points to the binding cavity and a poorly delimited 'portal' region as defining the function of each family member. The importance of local structure within the cavity appears to be its influence on binding affinity and specificity for the lipid. The portal region appears to be involved in the regulation of ligand exchange. Within the iLBP family, liver fatty acid binding protein or LFABP, has the unique property of binding two fatty acids within its internalized binding cavity rather than the commonly observed stoichiometry of one. Furthermore, LFABP will bind hydrophobic molecules larger than the ligands which will associate with other iLBPs. The crystal structure of LFABP contains two bound oleate molecules and provides the explanation for its unusual stoichiometry. One of the bound fatty acids is completely internalized and has its carboxylate interacting with an arginine and two serines. The second oleate represents an entirely new binding mode with the carboxylate on the surface of LFABP. The two oleates also interact with each other. Because of this interaction and its inner location, it appears the first oleate must be present before the second more external molecule is bound. PMID:10331654

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

    Science.gov (United States)

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

    2015-01-01

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

  4. In vitro binding of selenium by rat liver mitochondrial selenium-binding protein

    International Nuclear Information System (INIS)

    Last year the authors reported that upon freezing and thawing mitochondria from rats injected with [75Se]Na2SeO3 (75Se-selenite), a 75Se-binding protein (SeBP) was released. They have studied further in vitro labelling of SeBP. This matrix protein was labelled in vitro when lysed mitochondria (containing non-matrix material) were incubated with 75Se-selenite but not when matrix material alone was incubated with 75Se-selenite. Thus, there are one or more promoters of in vitro SeBP labelling in the non-matrix fraction. SeBP was also labelled in vitro when 75Se-selenite was added to matrix alone and dialyzed. Dialysis tubing, and not the dialysis process, promoted labelling by affecting SeBP and not by affecting 75Se-selenite. Labelling did not occur when matrix alone and 75Se-selenite were incubated (not dialyzed) in a glass test tube but did occur in a polystyrene test tube. They hypothesize that non-covalent interactions occur between SeBP and dialysis tubing or polystyrene that expose Se binding sites on the protein. A similar mechanism involving mitochondrial non-matrix material may function in vivo. Non-denaturing disc gel electrophoresis of partially purified SeBP labelled in vivo or in vitro suggested that the same protein was labelled in both conditions. Using in vitro binding techniques, SeBP was also found in sheep liver mitochondrial matrix. This supports the theory that SeBP is important in Se metabolism

  5. L-(TH)glutamate binds to kainate-, NMDA- and AMPA-sensitive binding sites: an autoradiographic analysis

    Energy Technology Data Exchange (ETDEWEB)

    Monaghan, D.T.; Yao, D.; Cotman, C.W.

    1985-08-12

    The anatomical distribution of L-(TH)glutamate binding sites was determined in the presence of various glutamate analogues using quantitative autoradiography. The binding of L-(TH)glutamate is accounted for by the presence of 3 distinct binding sites when measured in the absence of CaS , Cl and Na ions. The anatomical distribution and pharmacological specificity of these binding sites correspond to that reported for the 3 excitatory amino acid binding sites selectively labelled by D-(TH)2-amino-5-phosphonopentanoate (D-(TH)AP5), (TH)kainate ((TH)KA) and (TH) -amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid ((TH)AMPA) which are thought to be selective ligands for the N-methyl-D-aspartate (NMDA), KA and quisqualate (QA) receptors, respectively. (Auth.). 29 refs.; 1 figure; 1 table.

  6. Alpha-amylase starch binding domains: cooperative effects of binding to starch granules of multiple tandemly arranged domains.

    Science.gov (United States)

    Guillén, D; Santiago, M; Linares, L; Pérez, R; Morlon, J; Ruiz, B; Sánchez, S; Rodríguez-Sanoja, R

    2007-06-01

    The Lactobacillus amylovorus alpha-amylase starch binding domain (SBD) is a functional domain responsible for binding to insoluble starch. Structurally, this domain is dissimilar from other reported SBDs because it is composed of five identical tandem modules of 91 amino acids each. To understand adsorption phenomena specific to this SBD, the importance of their modular arrangement in relationship to binding ability was investigated. Peptides corresponding to one, two, three, four, or five modules were expressed as His-tagged proteins. Protein binding assays showed an increased capacity of adsorption as a function of the number of modules, suggesting that each unit of the SBD may act in an additive or synergic way to optimize binding to raw starch. PMID:17468268

  7. The actin binding protein adseverin regulates osteoclastogenesis.

    Science.gov (United States)

    Hassanpour, Siavash; Jiang, Hongwei; Wang, Yongqiang; Kuiper, Johannes W P; Glogauer, Michael

    2014-01-01

    Adseverin (Ads), a member of the Gelsolin superfamily of actin binding proteins, regulates the actin cytoskeleton architecture by severing and capping existing filamentous actin (F-actin) strands and nucleating the assembly of new F-actin filaments. Ads has been implicated in cellular secretion, exocytosis and has also been shown to regulate chondrogenesis and megakaryoblastic leukemia cell differentiation. Here we report for the first time that Ads is involved in regulating osteoclastogenesis (OCG). Ads is induced during OCG downstream of RANK-ligand (RANKL) stimulation and is highly expressed in mature osteoclasts. The D5 isoform of Ads is not involved in regulating OCG, as its expression is not induced in response to RANKL. Three clonal Ads knockdown RAW264.7 (RAW) macrophage cell lines with varying degrees of Ads expression and OCG deficiency were generated. The most drastic OCG defect was noted in the clonal cell line with the greatest degree of Ads knockdown as indicated by a lack of TRAcP staining and multinucleation. RNAi mediated knockdown of Ads in osteoclast precursors resulted in distinct morphological changes characterized by altered F-actin distribution and increased filopodia formation. Ads knockdown precursor cells experienced enhanced migration while fusion of knockdown precursors cells was limited. Transient reintroduction of de novo Ads back into the knockdown system was capable of rescuing TRAcP expression but not osteoclast multinucleation most likely due to the transient nature of Ads expression. This preliminary study allows us to conclude that Ads is a RANKL induced early regulator of OCG with a potential role in pre-osteoclast differentiation and fusion. PMID:25275604

  8. The actin binding protein adseverin regulates osteoclastogenesis.

    Directory of Open Access Journals (Sweden)

    Siavash Hassanpour

    Full Text Available Adseverin (Ads, a member of the Gelsolin superfamily of actin binding proteins, regulates the actin cytoskeleton architecture by severing and capping existing filamentous actin (F-actin strands and nucleating the assembly of new F-actin filaments. Ads has been implicated in cellular secretion, exocytosis and has also been shown to regulate chondrogenesis and megakaryoblastic leukemia cell differentiation. Here we report for the first time that Ads is involved in regulating osteoclastogenesis (OCG. Ads is induced during OCG downstream of RANK-ligand (RANKL stimulation and is highly expressed in mature osteoclasts. The D5 isoform of Ads is not involved in regulating OCG, as its expression is not induced in response to RANKL. Three clonal Ads knockdown RAW264.7 (RAW macrophage cell lines with varying degrees of Ads expression and OCG deficiency were generated. The most drastic OCG defect was noted in the clonal cell line with the greatest degree of Ads knockdown as indicated by a lack of TRAcP staining and multinucleation. RNAi mediated knockdown of Ads in osteoclast precursors resulted in distinct morphological changes characterized by altered F-actin distribution and increased filopodia formation. Ads knockdown precursor cells experienced enhanced migration while fusion of knockdown precursors cells was limited. Transient reintroduction of de novo Ads back into the knockdown system was capable of rescuing TRAcP expression but not osteoclast multinucleation most likely due to the transient nature of Ads expression. This preliminary study allows us to conclude that Ads is a RANKL induced early regulator of OCG with a potential role in pre-osteoclast differentiation and fusion.

  9. TIM-4 structures identify a Metal Ion-dependent Ligand Binding Site where phosphatidylserine binds

    OpenAIRE

    Santiago, Cesar; Ballesteros, Angela; Martinez-Muñoz, Laura; Mellado, Mario; Kaplan, Gerardo G.; Freeman, Gordon J.; Casasnovas, José M.

    2007-01-01

    The T-cell immunoglobulin and mucin domain (TIM) proteins are important regulators of T cell responses. They have been linked to autoimmunity and cancer. Structures of the murine TIM-4 identified a Metal Ion-dependent Ligand Binding Site (MILIBS) in the immunoglobulin (Ig) domain of the TIM family. The characteristic CC’ loop of the TIM domain and the hydrophobic FG loop shaped a narrow cavity where acidic compounds penetrate and coordinate to a metal ion bound to conserved residues in the TI...

  10. Characterization of 6-mercaptopurine binding to bovine serum albumin and its displacement from the binding sites by quercetin and rutin

    Energy Technology Data Exchange (ETDEWEB)

    Ehteshami, Mehdi [Nutrition Research Center, School of Health and Nutrition, Tabriz University of Medical Sciences, Tabriz 51644-14766 (Iran, Islamic Republic of); Rasoulzadeh, Farzaneh [Drug Applied Research Center, Tabriz University of Medical Sciences, Tabriz 51644-14766 (Iran, Islamic Republic of); Mahboob, Soltanali [Nutrition Research Center, School of Health and Nutrition, Tabriz University of Medical Sciences, Tabriz 51644-14766 (Iran, Islamic Republic of); Rashidi, Mohammad-Reza, E-mail: rashidi@tbzmed.ac.ir [Research Center for Pharmaceutical Nanotechnology, Tabriz University of Medical Sciences, Tabriz 51644-14766 (Iran, Islamic Republic of)

    2013-03-15

    Binding of a drug to the serum albumins as major serum transport proteins can be influenced by other ligands leading to alteration of its pharmacological properties. In the present study, binding characteristics of 6-mercaptopurine (6-MP) with bovine serum albumin (BSA) together with its displacement from its binding site by quercetin and rutin have been investigated by the spectroscopic method. According to the binding parameters, a static quenching component in overall dynamic quenching process is operative in the interaction between 6-MP and BSA. The binding of 6-MP to BSA occurred spontaneously due to entropy-driven hydrophobic interactions. The synchronous fluorescence spectroscopy study revealed that the secondary structure of BSA is changed in the presence of 6-MP and both Tyr and Trp residues participate in the interaction between 6-MP and BSA with the later one being more dominant. The binding constant value of 6-MP-BSA in the presence of quercetin and rutin increased. 6-MP was displaced by ibuprofen indicating that the binding site of 6-MP on albumin is site II. Therefore, the change of the pharmacokinetic and pharmacodynamic properties of 6-MP by quercetin and rutin through alteration of binding capacity of 6-MP to the serum albumin cannot be ruled out. In addition, the displacement study showed that 6-MP is located in site II of BSA. - Highlights: Black-Right-Pointing-Pointer Participation of both Tyr and particularly Trp residues in the interaction between 6-MP and BSA. Black-Right-Pointing-Pointer Involvement of a static quenching component in an overall dynamic quenching process. Black-Right-Pointing-Pointer Ability of quercetin and rutin to change the binding constants of 6-MP-BSA complex. Black-Right-Pointing-Pointer Binding of 6-MP to BSA through entropy-driven hydrophobic interactions.

  11. 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.

  12. Characterization of 6-mercaptopurine binding to bovine serum albumin and its displacement from the binding sites by quercetin and rutin

    International Nuclear Information System (INIS)

    Binding of a drug to the serum albumins as major serum transport proteins can be influenced by other ligands leading to alteration of its pharmacological properties. In the present study, binding characteristics of 6-mercaptopurine (6-MP) with bovine serum albumin (BSA) together with its displacement from its binding site by quercetin and rutin have been investigated by the spectroscopic method. According to the binding parameters, a static quenching component in overall dynamic quenching process is operative in the interaction between 6-MP and BSA. The binding of 6-MP to BSA occurred spontaneously due to entropy-driven hydrophobic interactions. The synchronous fluorescence spectroscopy study revealed that the secondary structure of BSA is changed in the presence of 6-MP and both Tyr and Trp residues participate in the interaction between 6-MP and BSA with the later one being more dominant. The binding constant value of 6-MP–BSA in the presence of quercetin and rutin increased. 6-MP was displaced by ibuprofen indicating that the binding site of 6-MP on albumin is site II. Therefore, the change of the pharmacokinetic and pharmacodynamic properties of 6-MP by quercetin and rutin through alteration of binding capacity of 6-MP to the serum albumin cannot be ruled out. In addition, the displacement study showed that 6-MP is located in site II of BSA. - Highlights: ► Participation of both Tyr and particularly Trp residues in the interaction between 6-MP and BSA. ► Involvement of a static quenching component in an overall dynamic quenching process. ► Ability of quercetin and rutin to change the binding constants of 6-MP–BSA complex. ► Binding of 6-MP to BSA through entropy-driven hydrophobic interactions

  13. Importance of a Hydrophobic Pocket for Peptide Binding in Lactococcal OppA▿

    OpenAIRE

    Berntsson, Ronnie P-A; Thunnissen, Andy-Mark W H; Poolman, Bert; Slotboom, Dirk-Jan

    2011-01-01

    Lactococcal oligopeptide-binding protein A (OppA) binds peptides with widely varied lengths and sequences. We previously hypothesized that a hydrophobic pocket in OppA preferentially binds a hydrophobic peptide side chain and thus determines its binding register. Two crystal structures of OppA with different nonapeptides now indeed show binding in different registers.

  14. Chronic exercise increases insulin binding in muscles but not liver

    International Nuclear Information System (INIS)

    It has been postulated that the improved glucose tolerance provoked by chronic exercise is primarily attributable to increased insulin binding in skeletal muscle. Therefore, the authors investigated the effects of progressively increased training (6 wk) on insulin binding by five hindlimb skeletal muscles and in liver. In the trained animals serum insulin levels at rest were lower either in a fed or fasted state and after an oral glucose tolerance test. Twenty-four hours after the last exercise bout sections of the liver, soleus (S), plantaris (P), extensor digitorum longus (EDL), and red (RG) and white gastrocnemius (WG) muscles were pooled from four to six rats. Insulin binding to plasma membranes increased in S, P, and EDL but not in WG or in liver. There were insulin binding differences among muscles. Comparison of rank orders of insulin binding data with published glucose transport data for the same muscles revealed that these parameters do not correspond well. In conclusion, insulin binding to muscle is shown to be heterogeneous and training can increase insulin binding to selected muscles but not liver

  15. Thermodynamic characterization of proflavine–DNA binding through microcalorimetric studies

    International Nuclear Information System (INIS)

    Highlights: • Energetics of the interaction of proflavine with DNA has been studied. • The binding reaction was favored by both negative enthalpy and positive entropy. • Enthalpy–entropy compensation phenomenon was observed. • Non-polyelectrolytic forces played a dominant role in the binding process. • Proflavine enhanced the thermal stability of DNA remarkably. - Abstract: The interaction of an important acridine dye, proflavine hydrochloride, with double stranded DNA was investigated using isothermal titration calorimetry and differential scanning calorimetry. The equilibrium constant for the binding reaction was calculated to be (1.60 ± 0.04) · 105 · M−1 at T = 298.15 K. The binding of proflavine hydrochloride to DNA was favored by both negative enthalpy and positive entropy contributions to the Gibbs energy. The equilibrium constant for the binding reaction decreased with increasing temperature. The standard molar enthalpy change became increasingly negative while the standard molar entropy change became less positive with rise in temperature. However, the standard molar Gibbs free energy change varied marginally suggesting the occurrence of enthalpy–entropy compensation phenomenon. The binding reaction was dominated by non-polyelectrolytic forces which remained virtually unchanged at all the salt concentrations studied. The binding also significantly increased the thermal stability of DNA against thermal denaturation

  16. In vitro gibberellin A1 binding in Zea mays L

    International Nuclear Information System (INIS)

    The first and second leaf sheaths of Zea mays L. cv Golden Jubilee were extracted and the extract centrifuged at 100,000g to yield a supernatant or cytosol fraction. Binding of [3H]gibberellin A1 (GA1) to a soluble macromolecular component present in the cytosol was demonstrated at 40C by Sephadex G-200 chromatography. The binding component was of high molecular weight (HMW) and greater than 500 kilodaltons. The HMW component was shown to be a protein and the 3H-activity bound to this protein was largely [3H]GA1 and not a metabolite. Binding was pH sensitive but only a small percentage (20%) appeared to be exchangeable on addition of unlabeled GA1. Both biologically active and inactive GAs and non-GAs were able to inhibit GA1 binding. [3H]GA1 binding to an intermediate molecular weight (IMW) fraction (40-100 kilodaltons) was also detected, provided cytosol was first desalted using Sephadex G-200 chromatography. Gel filtration studies suggest that the HMW binding component is an aggregate derived from the IMW fraction. The HMW binding fraction can be separated into two components using anion exchange chromatography

  17. Photochemical Microscale Electrophoresis Allows Fast Quantification of Biomolecule Binding.

    Science.gov (United States)

    Möller, Friederike M; Kieß, Michael; Braun, Dieter

    2016-04-27

    Intricate spatiotemporal patterns emerge when chemical reactions couple to physical transport. We induce electrophoretic transport by a confined photochemical reaction and use it to infer the binding strength of a second, biomolecular binding reaction under physiological conditions. To this end, we use the photoactive compound 2-nitrobenzaldehyde, which releases a proton upon 375 nm irradiation. The charged photoproducts locally perturb electroneutrality due to differential diffusion, giving rise to an electric potential Φ in the 100 μV range on the micrometer scale. Electrophoresis of biomolecules in this field is counterbalanced by back-diffusion within seconds. The biomolecule concentration is measured by fluorescence and settles proportionally to exp(-μ/D Φ). Typically, binding alters either the diffusion coefficient D or the electrophoretic mobility μ. Hence, the local biomolecule fluorescence directly reflects the binding state. A fit to the law of mass action reveals the dissociation constant of the binding reaction. We apply this approach to quantify the binding of the aptamer TBA15 to its protein target human-α-thrombin and to probe the hybridization of DNA. Dissociation constants in the nanomolar regime were determined and match both results in literature and in control experiments using microscale thermophoresis. As our approach is all-optical, isothermal and requires only nanoliter volumes at nanomolar concentrations, it will allow for the fast screening of biomolecule binding in low volume multiwell formats. PMID:27042755

  18. Lipid A binding proteins in macrophages detected by ligand blotting

    International Nuclear Information System (INIS)

    Endotoxin (LPS) stimulates a variety of eukaryotic cells. These actions are involved in the pathogenesis of Gram-negative septicemia. The site of action of the LPS toxic moiety, lipid A (LA), is unclear. Their laboratory has previously identified a bioactive LA precursor lipid IV/sub A/, which can be enzymatically labeled with 32P/sub i/ (109 dpm/nmole) and purified (99%). They now show that this ligand binds to specific proteins immobilized on nitrocellulose (NC) from LPS-sensitive RAW 264.7 cultured macrophages. NC blots were incubated with [32P]-IV/sub A/ in a buffer containing BSA, NaCl, polyethylene glycol, and azide. Binding was assessed using autoradiography or scintillation counting. Dot blot binding of the radioligand was inhibited by excess cold IV/sub A/, LA, or ReLPS but not by phosphatidylcholine, cardiolipin, phosphatidylinositol, or phosphatidic acid. Binding was trypsin-sensitive and dependent on protein concentration. Particulate macrophage proteins were subjected to SDS-PAGE and then electroblotted onto NC. Several discrete binding proteins were observed. Identical treatment of fetal bovine serum or molecular weight standards revealed no detectable binding. By avoiding high nonspecific binding of intact membranes, this ligand blotting assay may be useful in elucidating the molecular actions of LPS

  19. Binding cells of 125I-iodoamphetamine in rat liver

    International Nuclear Information System (INIS)

    We recently reported that transrectal or intestinal portal scintigraphy with 123I-iodoamphetamine (IMP) could be a useful method for the non-invasive and quantitative evaluation of the portosystemic shunt in portal hypertension, but what cells in the liver trap IMP has not been clarified. This study was aimed at elucidating whether IMP was extracted by parenchymal cells, sinusoidal endothelial cells, Kupffer cells or fat storing cells. Each type of liver cell was isolated from rats and cultured. The cells were incubated with 125I-IMP and the radioactivity of the lysate was determined. Nonspecific binding was assessed in the presence of an excess of unlabeled IMP, and specific binding was determined by subtracting the nonspecific from total binding. Specific binding observed in parenchymal cells, endothelial cells and Kupffer cells was 70.2±0.4, 4.2±1.4 and 2.3±0.8 pmol/well, respectively, but no specific binding was observed in fat storing cells. The binding in parenchymal cells was much higher than that in endothelial cells or Kupffer cells (p<0.005). In addition, the binding to parenchymal cells reached equilibrium within 20 min and was not saturable over the concentration range tested (0.5-10 μM). These findings indicate that IMP is mostly extracted by parenchymal cells in the liver. (author)

  20. Relative Binding Affinities of Monolignols to Horseradish Peroxidase.

    Science.gov (United States)

    Sangha, Amandeep K; Petridis, Loukas; Cheng, Xiaolin; Smith, Jeremy C

    2016-08-11

    Monolignol binding to the peroxidase active site is the first step in lignin polymerization in plant cell walls. Using molecular dynamics, docking, and free energy perturbation calculations, we investigate the binding of monolignols to horseradish peroxidase C. Our results suggest that p-coumaryl alcohol has the strongest binding affinity followed by sinapyl and coniferyl alcohol. Stacking interactions between the monolignol aromatic rings and nearby phenylalanine residues play an important role in determining the calculated relative binding affinities. p-Coumaryl and coniferyl alcohols bind in a pose productive for reaction in which a direct H-bond is formed between the phenolic -OH group and a water molecule (W2) that may facilitate proton transfer during oxidation. In contrast, in the case of sinapyl alcohol there is no such direct interaction, the phenolic -OH group instead interacting with Pro139. Since proton and electron transfer is the rate-limiting step in monolignol oxidation by peroxidase, the binding pose (and thus the formation of near attack conformation) appears to play a more important role than the overall binding affinity in determining the oxidation rate. PMID:27447548