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Sample records for abl sh3 domain

  1. Binding Mechanism of the N-Terminal SH3 Domain of CrkII and Proline-Rich Motifs in cAbl.

    PubMed

    Bhatt, Veer S; Zeng, Danyun; Krieger, Inna; Sacchettini, James C; Cho, Jae-Hyun

    2016-06-21

    The N-terminal Src homology 3 (nSH3) domain of a signaling adaptor protein, CT-10 regulator of kinase II (CrkII), recognizes proline-rich motifs (PRMs) of binding partners, such as cAbl kinase. The interaction between CrkII and cAbl kinase is involved in the regulation of cell spreading, microbial pathogenesis, and cancer metastasis. Here, we report the detailed biophysical characterizations of the interactions between the nSH3 domain of CrkII and PRMs in cAbl. We identified that the nSH3 domain of CrkII binds to three PRMs in cAbl with virtually identical affinities. Structural studies, by using x-ray crystallography and NMR spectroscopy, revealed that the binding modes of all three nSH3:PRM complexes are highly similar to each other. Van 't Hoff analysis revealed that nSH3:PRM interaction is associated with favorable enthalpy and unfavorable entropy change. The combination of experimentally determined thermodynamic parameters, structure-based calculations, and (15)N NMR relaxation analysis highlights the energetic contribution of conformational entropy change upon the complex formation, and water molecules structured in the binding interface of the nSH3:PRM complex. Understanding the molecular basis of nSH3:PRM interaction will provide, to our knowledge, new insights for the rational design of small molecules targeting the interaction between CrkII and cAbl. PMID:27332121

  2. Evolution of the SH3 Domain Specificity Landscape in Yeasts.

    PubMed

    Verschueren, Erik; Spiess, Matthias; Gkourtsa, Areti; Avula, Teja; Landgraf, Christiane; Mancilla, Victor Tapia; Huber, Aline; Volkmer, Rudolf; Winsor, Barbara; Serrano, Luis; Hochstenbach, Frans; Distel, Ben

    2015-01-01

    To explore the conservation of Src homology 3 (SH3) domain-mediated networks in evolution, we compared the specificity landscape of these domains among four yeast species, Saccharomyces cerevisiae, Ashbya gossypii, Candida albicans, and Schizosaccharomyces pombe, encompassing 400 million years of evolution. We first aligned and catalogued the families of SH3-containing proteins in these four species to determine the relationships between homologous domains. Then, we tagged and purified all soluble SH3 domains (82 in total) to perform a quantitative peptide assay (SPOT) for each SH3 domain. All SPOT readouts were hierarchically clustered and we observed that the organization of the SH3 specificity landscape in three distinct profile classes remains conserved across these four yeast species. We also produced a specificity profile for each SH3 domain from manually aligned top SPOT hits and compared the within-family binding motif consensus. This analysis revealed a striking example of binding motif divergence in a C. albicans Rvs167 paralog, which cannot be explained by overall SH3 sequence or interface residue divergence, and we validated this specificity change with a yeast two-hybrid (Y2H) assay. In addition, we show that position-weighted matrices (PWM) compiled from SPOT assays can be used for binding motif screening in potential binding partners and present cases where motifs are either conserved or lost among homologous SH3 interacting proteins. Finally, by comparing pairwise SH3 sequence identity to binding profile correlation we show that for ~75% of all analyzed families the SH3 specificity profile was remarkably conserved over a large evolutionary distance. Thus, a high sequence identity within an SH3 domain family predicts conserved binding specificity, whereas divergence in sequence identity often coincided with a change in binding specificity within this family. As such, our results are important for future studies aimed at unraveling complex specificity

  3. Identification of Src, Fyn, and Lyn SH3-binding proteins: implications for a function of SH3 domains.

    PubMed Central

    Weng, Z; Thomas, S M; Rickles, R J; Taylor, J A; Brauer, A W; Seidel-Dugan, C; Michael, W M; Dreyfuss, G; Brugge, J S

    1994-01-01

    Src homology 3 (SH3) domains mediate protein-protein interactions necessary for the coupling of cellular proteins involved in intracellular signal transduction. We previously established solution-binding conditions that allow affinity isolation of Src SH3-binding proteins from cellular extracts (Z. Weng, J. A. Taylor, C. E. Turner, J. S. Brugge, and C. Seidel-Dugan, J. Biol. Chem. 268:14956-14963, 1993). In this report, we identified three of these proteins: Shc, a signaling protein that couples membrane tyrosine kinases with Ras; p62, a protein which can bind to p21rasGAP; and heterogeneous nuclear ribonucleoprotein K, a pre-mRNA-binding protein. All of these proteins contain proline-rich peptide motifs that could serve as SH3 domain ligands, and the binding of these proteins to the Src SH3 domain was inhibited with a proline-rich Src SH3 peptide ligand. These three proteins, as well as most of the other Src SH3 ligands, also bound to the SH3 domains of the closely related protein tyrosine kinases Fyn and Lyn. However, Src- and Lyn-specific SH3-binding proteins were also detected, suggesting subtle differences in the binding specificity of the SH3 domains from these related proteins. Several Src SH3-binding proteins were phosphorylated in Src-transformed cells. The phosphorylation of these proteins was not detected in cells transformed by a mutant variant of Src lacking the SH3 domain, while there was little change in tyrosine phosphorylation of other Src-induced phosphoproteins. In addition, the coprecipitation of v-Src with two tyrosyl-phosphorylated proteins with M(r)s of 62,000 and 130,000 was inhibited by incubation with a Src SH3 peptide ligand, suggesting that the binding of these substrate proteins is dependent on interactions with the SH3 domain. These results strongly suggest a role for the Src SH3 domain in the recruitment of substrates to this protein tyrosine kinase, either through direct interaction with the SH3 domain or indirectly through

  4. SH3 Domains Differentially Stimulate Distinct Dynamin I Assembly Modes and G Domain Activity

    PubMed Central

    Krishnan, Sai; Collett, Michael; Robinson, Phillip J.

    2015-01-01

    Dynamin I is a highly regulated GTPase enzyme enriched in nerve terminals which mediates vesicle fission during synaptic vesicle endocytosis. One regulatory mechanism involves its interactions with proteins containing Src homology 3 (SH3) domains. At least 30 SH3 domain-containing proteins bind dynamin at its proline-rich domain (PRD). Those that stimulate dynamin activity act by promoting its oligomerisation. We undertook a systematic parallel screening of 13 glutathione-S-transferase (GST)-tagged endocytosis-related SH3 domains on dynamin binding, GTPase activity and oligomerisation. No correlation was found between dynamin binding and their potency to stimulate GTPase activity. There was limited correlation between the extent of their ability to stimulate dynamin activity and the level of oligomerisation, indicating an as yet uncharacterised allosteric coupling of the PRD and G domain. We examined the two variants, dynamin Iab and Ibb, which differ in the alternately splice middle domain α2 helix. They responded differently to the panel of SH3s, with the extent of stimulation between the splice variants varying greatly between the SH3s. This study reveals that SH3 binding can act as a heterotropic allosteric regulator of the G domain via the middle domain α2 helix, suggesting an involvement of this helix in communicating the PRD-mediated allostery. This indicates that SH3 binding both stabilises multiple conformations of the tetrameric building block of dynamin, and promotes assembly of dynamin-SH3 complexes with distinct rates of GTP hydrolysis. PMID:26659814

  5. Ubiquitin binds to and regulates a subset of SH3 domains

    PubMed Central

    Stamenova, Svetoslava D.; French, Michael E.; He, Yuan; Francis, Smitha A.; Kramer, Zachary B.; Hicke, Linda

    2009-01-01

    Summary SH3 domains are modules of 50-70 amino acids that promote interactions among proteins, often participating in the assembly of large dynamic complexes. These domains bind to peptide ligands, which usually contain a core Pro-X-X-Pro (PXXP) sequence. Here we identify a class of SH3 domains that binds to ubiquitin. The yeast endocytic protein Sla1, as well as the mammalian proteins CIN85 and amphiphysin, carry ubiquitin-binding SH3 domains. Ubiquitin and peptide ligands bind to the same hydrophobic groove on the SH3 domain surface, and ubiquitin and a PXXP-containing protein fragment compete for binding to SH3 domains. We conclude that a subset of SH3 domains constitutes a distinct type of ubiquitin-binding domain, and that ubiquitin-binding can negatively regulate interaction of SH3 domains with canonical proline-rich ligands. PMID:17244534

  6. 3D domain swapping in a chimeric c-Src SH3 domain takes place through two hinge loops.

    PubMed

    Cámara-Artigas, Ana; Martínez-Rodríguez, Sergio; Ortiz-Salmerón, Emilia; Martín-García, José M

    2014-04-01

    In the Src Homology 3 domain (SH3) the RT and n-Src loops form a pocket that accounts for the specificity and affinity in binding of proline rich motifs (PRMs), while the distal and diverging turns play a key role in the folding of the protein. We have solved the structure of a chimeric mutant c-Src-SH3 domain where specific residues at the RT- and n-Src-loops have been replaced by those present in the corresponding Abl-SH3 domain. Crystals of the chimeric protein show a single molecule in the asymmetric unit, which appears in an unfolded-like structure that upon generation of the symmetry related molecules reveals the presence of a domain swapped dimer where both, RT- and n-Src loops, act as hinge loops. In contrast, the fold of the diverging type II β-turn and the distal loop are well conserved. Our results are the first evidence for the presence of a structured diverging type II β-turn in an unfolded-like intermediate of the c-Src-SH3 domain, which can be stabilized by interactions from the β-strands of the same polypeptide chain or from a neighboring one. Futhermore, this crystallographic structure opens a unique opportunity to study the effect of the amino acid sequence of the hinge loops on the 3D domain swapping process of c-Src-SH3. PMID:24556574

  7. Bayesian Modeling of the Yeast SH3 Domain Interactome Predicts Spatiotemporal Dynamics of Endocytosis Proteins

    PubMed Central

    Gfeller, David; Landgraf, Christiane; Panni, Simona; Paoluzi, Serena; Castagnoli, Luisa; Currell, Bridget; Seshagiri, Somasekar; Yu, Haiyuan; Winsor, Barbara; Vidal, Marc; Gerstein, Mark B.; Bader, Gary D.; Volkmer, Rudolf; Cesareni, Gianni; Drubin, David G.; Kim, Philip M.; Sidhu, Sachdev S.; Boone, Charles

    2009-01-01

    SH3 domains are peptide recognition modules that mediate the assembly of diverse biological complexes. We scanned billions of phage-displayed peptides to map the binding specificities of the SH3 domain family in the budding yeast, Saccharomyces cerevisiae. Although most of the SH3 domains fall into the canonical classes I and II, each domain utilizes distinct features of its cognate ligands to achieve binding selectivity. Furthermore, we uncovered several SH3 domains with specificity profiles that clearly deviate from the two canonical classes. In conjunction with phage display, we used yeast two-hybrid and peptide array screening to independently identify SH3 domain binding partners. The results from the three complementary techniques were integrated using a Bayesian algorithm to generate a high-confidence yeast SH3 domain interaction map. The interaction map was enriched for proteins involved in endocytosis, revealing a set of SH3-mediated interactions that underlie formation of protein complexes essential to this biological pathway. We used the SH3 domain interaction network to predict the dynamic localization of several previously uncharacterized endocytic proteins, and our analysis suggests a novel role for the SH3 domains of Lsb3p and Lsb4p as hubs that recruit and assemble several endocytic complexes. PMID:19841731

  8. Predicting physiologically relevant SH3 domain mediated protein–protein interactions in yeast

    PubMed Central

    Jain, Shobhit; Bader, Gary D.

    2016-01-01

    Motivation: Many intracellular signaling processes are mediated by interactions involving peptide recognition modules such as SH3 domains. These domains bind to small, linear protein sequence motifs which can be identified using high-throughput experimental screens such as phage display. Binding motif patterns can then be used to computationally predict protein interactions mediated by these domains. While many protein–protein interaction prediction methods exist, most do not work with peptide recognition module mediated interactions or do not consider many of the known constraints governing physiologically relevant interactions between two proteins. Results: A novel method for predicting physiologically relevant SH3 domain-peptide mediated protein–protein interactions in S. cerevisae using phage display data is presented. Like some previous similar methods, this method uses position weight matrix models of protein linear motif preference for individual SH3 domains to scan the proteome for potential hits and then filters these hits using a range of evidence sources related to sequence-based and cellular constraints on protein interactions. The novelty of this approach is the large number of evidence sources used and the method of combination of sequence based and protein pair based evidence sources. By combining different peptide and protein features using multiple Bayesian models we are able to predict high confidence interactions with an overall accuracy of 0.97. Availability and implementation: Domain-Motif Mediated Interaction Prediction (DoMo-Pred) command line tool and all relevant datasets are available under GNU LGPL license for download from http://www.baderlab.org/Software/DoMo-Pred. The DoMo-Pred command line tool is implemented using Python 2.7 and C ++. Contact: gary.bader@utoronto.ca Supplementary information: Supplementary data are available at Bioinformatics online. PMID:26861823

  9. Dynamics of the Tec-family tyrosine kinase SH3 domains.

    PubMed

    Roberts, Justin M; Tarafdar, Sreya; Joseph, Raji E; Andreotti, Amy H; Smithgall, Thomas E; Engen, John R; Wales, Thomas E

    2016-04-01

    The Src Homology 3 (SH3) domain is an important regulatory domain found in many signaling proteins. X-ray crystallography and NMR structures of SH3 domains are generally conserved but other studies indicate that protein flexibility and dynamics are not. We previously reported that based on hydrogen exchange mass spectrometry (HX MS) studies, there is variable flexibility and dynamics among the SH3 domains of the Src-family tyrosine kinases and related proteins. Here we have extended our studies to the SH3 domains of the Tec family tyrosine kinases (Itk, Btk, Tec, Txk, Bmx). The SH3 domains of members of this family augment the variety in dynamics observed in previous SH3 domains. Txk and Bmx SH3 were found to be highly dynamic in solution by HX MS and Bmx was unstructured by NMR. Itk and Btk SH3 underwent a clear EX1 cooperative unfolding event, which was localized using pepsin digestion and mass spectrometry after hydrogen exchange labeling. The unfolding was localized to peptide regions that had been previously identified in the Src-family and related protein SH3 domains, yet the kinetics of unfolding were not. Sequence alignment does not provide an easy explanation for the observed dynamics behavior, yet the similarity of location of EX1 unfolding suggests that higher-order structural properties may play a role. While the exact reason for such dynamics is not clear, such motions can be exploited in intra- and intermolecular binding assays of proteins containing the domains. PMID:26808198

  10. Crystallographic studies on protein misfolding: Domain swapping and amyloid formation in the SH3 domain.

    PubMed

    Cámara-Artigas, Ana

    2016-07-15

    Oligomerization by 3D domain swapping is found in a variety of proteins of diverse size, fold and function. In the early 1960s this phenomenon was postulated for the oligomers of ribonuclease A, but it was not until the 1990s that X-ray diffraction provided the first experimental evidence of this special manner of oligomerization. Nowadays, structural information has allowed the identification of these swapped oligomers in over one hundred proteins. Although the functional relevance of this phenomenon is not clear, this alternative folding of protomers into intertwined oligomers has been related to amyloid formation. Studies on proteins that develop 3D domain swapping might provide some clues on the early stages of amyloid formation. The SH3 domain is a small modular domain that has been used as a model to study the basis of protein folding. Among SH3 domains, the c-Src-SH3 domain emerges as a helpful model to study 3D domain swapping and amyloid formation. PMID:26924596

  11. Parallel Chemical Protein Synthesis on a Surface Enables the Rapid Analysis of the Phosphoregulation of SH3 Domains.

    PubMed

    Zitterbart, Robert; Seitz, Oliver

    2016-06-13

    Analysis of postranslationally modified protein domains is complicated by an availability problem, as recombinant methods rarely allow site-specificity at will. Although total synthesis enables full control over posttranslational and other modifications, chemical approaches are limited to shorter peptides. To solve this problem, we herein describe a method that combines a) immobilization of N-terminally thiolated peptide hydrazides by hydrazone ligation, b) on-surface native chemical ligation with self-purified peptide thioesters, c) radical-induced desulfurization, and d) a surface-based fluorescence binding assay for functional characterization. We used the method to rapidly investigate 20 SH3 domains, with a focus on their phosphoregulation. The analysis suggests that tyrosine phosphorylation of SH3 domains found in Abl kinases act as a switch that can induce both the loss and, unexpectedly, gain of affinity for proline-rich ligands. PMID:27161995

  12. Structure of the SH3 domain of human osteoclast-stimulating factor at atomic resolution

    SciTech Connect

    Chen, Liqing Wang, Yujun; Wells, David; Toh, Diana; Harold, Hunt; Zhou, Jing; DiGiammarino, Enrico; Meehan, Edward J.

    2006-09-01

    The crystal structure of the SH3 domain of human osteoclast-stimulating factor has been determined and refined to the ultrahigh resolution of 1.07 Å. The structure at atomic resolution provides an accurate framework for structure-based design of its inhibitors. Osteoclast-stimulating factor (OSF) is an intracellular signaling protein, produced by osteoclasts themselves, that enhances osteoclast formation and bone resorption. It is thought to act via an Src-related signaling pathway and contains SH3 and ankyrin-repeat domains which are involved in protein–protein interactions. As part of a structure-based anti-bone-loss drug-design program, the atomic resolution X-ray structure of the recombinant human OSF SH3 domain (hOSF-SH3) has been determined. The domain, residues 12–72, yielded crystals that diffracted to the ultrahigh resolution of 1.07 Å. The overall structure shows a characteristic SH3 fold consisting of two perpendicular β-sheets that form a β-barrel. Structure-based sequence alignment reveals that the putative proline-rich peptide-binding site of hOSF-SH3 consists of (i) residues that are highly conserved in the SH3-domain family, including residues Tyr21, Phe23, Trp49, Pro62, Asn64 and Tyr65, and (ii) residues that are less conserved and/or even specific to hOSF, including Thr22, Arg26, Thr27, Glu30, Asp46, Thr47, Asn48 and Leu60, which might be key to designing specific inhibitors for hOSF to fight osteoporosis and related bone-loss diseases. There are a total of 13 well defined water molecules forming hydrogen bonds with the above residues in and around the peptide-binding pocket. Some of those water molecules might be important for drug-design approaches. The hOSF-SH3 structure at atomic resolution provides an accurate framework for structure-based design of its inhibitors.

  13. New approaches to high-throughput structure characterization of SH3 complexes: the example of Myosin-3 and Myosin-5 SH3 domains from S. cerevisiae.

    PubMed

    Musi, Valeria; Birdsall, Berry; Fernandez-Ballester, Gregorio; Guerrini, Remo; Salvatori, Severo; Serrano, Luis; Pastore, Annalisa

    2006-04-01

    SH3 domains are small protein modules that are involved in protein-protein interactions in several essential metabolic pathways. The availability of the complete genome and the limited number of clearly identifiable SH3 domains make the yeast Saccharomyces cerevisae an ideal proteomic-based model system to investigate the structural rules dictating the SH3-mediated protein interactions and to develop new tools to assist these studies. In the present work, we have determined the solution structure of the SH3 domain from Myo3 and modeled by homology that of the highly homologous Myo5, two myosins implicated in actin polymerization. We have then implemented an integrated approach that makes use of experimental and computational methods to characterize their binding properties. While accommodating their targets in the classical groove, the two domains have selectivity in both orientation and sequence specificity of the target peptides. From our study, we propose a consensus sequence that may provide a useful guideline to identify new natural partners and suggest a strategy of more general applicability that may be of use in other structural proteomic studies. PMID:16600966

  14. A summary of staphylococcal C-terminal SH3b_5 cell wall binding domains.

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Staphylococcal peptidoglycan hydrolases are a potential new source of antimicrobials. A large subset of these proteins contain a C-terminal SH3b_5 cell wall binding domain that has been shown for some to be essential for accurate cell wall recognition and subsequent staphylolytic activity, propert...

  15. Allosteric N-WASP activation by an inter-SH3 domain linker in Nck

    PubMed Central

    Okrut, Julia; Prakash, Sumit; Wu, Qiong; Kelly, Mark J. S.; Taunton, Jack

    2015-01-01

    Actin filament networks assemble on cellular membranes in response to signals that locally activate neural Wiskott–Aldrich-syndrome protein (N-WASP) and the Arp2/3 complex. An inactive conformation of N-WASP is stabilized by intramolecular contacts between the GTPase binding domain (GBD) and the C helix of the verprolin-homology, connector-helix, acidic motif (VCA) segment. Multiple SH3 domain-containing adapter proteins can bind and possibly activate N-WASP, but it remains unclear how such binding events relieve autoinhibition to unmask the VCA segment and activate the Arp2/3 complex. Here, we have used purified components to reconstitute a signaling cascade driven by membrane-localized Src homology 3 (SH3) adapters and N-WASP, resulting in the assembly of dynamic actin networks. Among six SH3 adapters tested, Nck was the most potent activator of N-WASP–driven actin assembly. We identify within Nck a previously unrecognized activation motif in a linker between the first two SH3 domains. This linker sequence, reminiscent of bacterial virulence factors, directly engages the N-WASP GBD and competes with VCA binding. Our results suggest that animals, like pathogenic bacteria, have evolved peptide motifs that allosterically activate N-WASP, leading to localized actin nucleation on cellular membranes. PMID:26554011

  16. Directed Evolution of a Highly Specific FN3 Monobody to the SH3 Domain of Human Lyn Tyrosine Kinase.

    PubMed

    Huang, Renhua; Fang, Pete; Hao, Zengping; Kay, Brian K

    2016-01-01

    Affinity reagents of high affinity and specificity are very useful for studying the subcellular locations and quantities of individual proteins. To generate high-quality affinity reagents for human Lyn tyrosine kinase, a phage display library of fibronectin type III (FN3) monobodies was affinity selected with a recombinant form of the Lyn SH3 domain. While a highly specific monobody, TA8, was initially isolated, we chose to improve its affinity through directed evolution. A secondary library of 1.2 × 109 variants was constructed and screened by affinity selection, yielding three variants, two of which have affinities of ~ 40 nM, a 130-fold increase over the original TA8 monobody. One of the variants, 2H7, displayed high specificity to the Lyn SH3 domain, as shown by ELISA and probing arrays of 150 SH3 domains. Furthermore, the 2H7 monobody was able to pull down endogenous Lyn from a lysate of Burkitt's lymphoma cells, thereby demonstrating its utility as an affinity reagent for detecting Lyn in a complex biological mixture. PMID:26731115

  17. Directed Evolution of a Highly Specific FN3 Monobody to the SH3 Domain of Human Lyn Tyrosine Kinase

    PubMed Central

    Huang, Renhua; Fang, Pete; Hao, Zengping; Kay, Brian K.

    2016-01-01

    Affinity reagents of high affinity and specificity are very useful for studying the subcellular locations and quantities of individual proteins. To generate high-quality affinity reagents for human Lyn tyrosine kinase, a phage display library of fibronectin type III (FN3) monobodies was affinity selected with a recombinant form of the Lyn SH3 domain. While a highly specific monobody, TA8, was initially isolated, we chose to improve its affinity through directed evolution. A secondary library of 1.2 × 109 variants was constructed and screened by affinity selection, yielding three variants, two of which have affinities of ~ 40 nM, a 130-fold increase over the original TA8 monobody. One of the variants, 2H7, displayed high specificity to the Lyn SH3 domain, as shown by ELISA and probing arrays of 150 SH3 domains. Furthermore, the 2H7 monobody was able to pull down endogenous Lyn from a lysate of Burkitt's lymphoma cells, thereby demonstrating its utility as an affinity reagent for detecting Lyn in a complex biological mixture. PMID:26731115

  18. Mixed-lineage kinase 2-SH3 domain binds dynamin and greatly enhances activation of GTPase by phospholipid.

    PubMed Central

    Rasmussen, R K; Rusak, J; Price, G; Robinson, P J; Simpson, R J; Dorow, D S

    1998-01-01

    Mixed-lineage kinase 2 (MLK2) is a cytoplasmic protein kinase expressed at high levels in mammalian brain. The MLK2 structure is composed of a Src homology 3 (SH3) domain, two leucine zippers, a basic motif, a Cdc42/Rac interactive binding motif and a large C-terminal domain rich in proline, serine and threonine residues. To begin to define the role of MLK2 in mammalian brain, we used an MLK2-SH3 domain-glutathione S-transferase fusion protein (GST-MLK2-SH3) to isolate MLK2-binding proteins from rat brain extract. This analysis revealed that the major MLK2-SH3-domain-binding protein in rat brain is the GTPase dynamin. By using two different forms of the dynamin proline-rich domain as affinity ligands, the binding site for MLK2-SH3 was mapped to the C-terminal region of dynamin between residues 832 and 864. In GTPase assays, the addition of MLK2-SH3 stimulated the activity of purified dynamin I by 3-fold over the basal level, whereas the addition of a known dynamin activator, phosphatidylserine (PtdSer), stimulated a 6-fold increase. When MLK2-SH3 was added to the assay together with PtdSer, however, dynamin GTPase activity accelerated by more than 23-fold over basal level. An MLK2 mutant (MLK2-W59A-SH3), with alanine replacing a conserved tryptophan residue in the SH3 domain consensus motif, had no effect on dynamin activity, either alone or in the presence of PtdSer. In the same assay the SH3 domain from the regulatory subunit of phosphatidylinositol 3'-kinase stimulated a similar synergistic acceleration of dynamin GTPase activity in the presence of PtdSer. These results suggest that synergy between phospholipid and SH3 domain binding might be a general mechanism for the regulation of GTP hydrolysis by dynamin. PMID:9742220

  19. The Binding of Syndapin SH3 Domain to Dynamin Proline-rich Domain Involves Short and Long Distance Elements.

    PubMed

    Luo, Lin; Xue, Jing; Kwan, Ann; Gamsjaeger, Roland; Wielens, Jerome; von Kleist, Lisa; Cubeddu, Liza; Guo, Zhong; Stow, Jennifer L; Parker, Michael W; Mackay, Joel P; Robinson, Phillip J

    2016-04-29

    Dynamin is a GTPase that mediates vesicle fission during synaptic vesicle endocytosis. Its long C-terminal proline-rich domain contains 13 PXXP motifs, which orchestrate its interactions with multiple proteins. The SH3 domains of syndapin and endophilin bind the PXXP motifs called Site 2 and 3 (Pro-786-Pro-793) at the N-terminal end of the proline-rich domain, whereas the amphiphysin SH3 binds Site 9 (Pro-833-Pro-836) toward the C-terminal end. In some proteins, SH3/peptide interactions also involve short distance elements, which are 5-15 amino acid extensions flanking the central PXXP motif for high affinity binding. Here we found two previously unrecognized elements in the central and the C-terminal end of the dynamin proline-rich domain that account for a significant increase in syndapin binding affinity compared with a previously reported Site 2 and Site 3 PXXP peptide alone. The first new element (Gly-807-Gly-811) is short distance element on the C-terminal side of Site 2 PXXP, which might contact a groove identified under the RT loop of the SH3 domain. The second element (Arg-838-Pro-844) is located about 50 amino acids downstream of Site 2. These two elements provide additional specificity to the syndapin SH3 domain outside of the well described polyproline-binding groove. Thus, the dynamin/syndapin interaction is mediated via a network of multiple contacts outside the core PXXP motif over a previously unrecognized extended region of the proline-rich domain. To our knowledge this is the first example among known SH3 interactions to involve spatially separated and extended long-range elements that combine to provide a higher affinity interaction. PMID:26893375

  20. SH3-domain binding protein 1 in the tumor microenvironment promotes hepatocellular carcinoma metastasis through WAVE2 pathway

    PubMed Central

    Tao, Yiming; Hu, Kuan; Tan, Fengbo; Zhang, Sai; Zhou, Ming; Luo, Jia; Wang, Zhiming

    2016-01-01

    SH3-domain binding protein-1 (SH3BP1) specifically inactivating Rac1 and its target WAVE2 is required for cell motility. The present study shows SH3BP1 expression patterns in human HCC tissues and cell lines were examined. The regulation of SH3BP1 on HCC cell migration and invasion related to Rac1-WAVE2 signaling was characterized using in vitro and in vivo models. SH3BP1 overexpressed in HCC tissues and highly metastatic HCC cells was significantly associated vascular invasion (VI). SH3BP1 promoted VEGF secretion via Rac1-WAVE2 signaling, so as to exert an augmentation on cell invasion and microvessel formation. In three study cohorts with a total of 516 HCC patients, high SH3BP1 expression combined with high microvessel density (MVD) was confirmed as a powerful independent predictor of HCC prognosis in both training cohorts and validation cohort. Being an important angiogenic factor of HCC through Rac1-WAVE2 signaling, SH3BP1 promotes tumor invasion and microvessel formation contributing to HCC metastasis and recurrence. SH3BP1 is a novel WAVE2 regulator, a prognostic marker and a potential therapeutic target of HCC. PMID:26933917

  1. Insights into Substrate Specificity of NlpC/P60 Cell Wall Hydrolases Containing Bacterial SH3 Domains

    PubMed Central

    Xu, Qingping; Liu, Xueqian W.; Patin, Delphine; Farr, Carol L.; Grant, Joanna C.; Chiu, Hsiu-Ju; Jaroszewski, Lukasz; Knuth, Mark W.; Godzik, Adam; Lesley, Scott A.; Elsliger, Marc-André; Deacon, Ashley M.

    2015-01-01

    ABSTRACT Bacterial SH3 (SH3b) domains are commonly fused with papain-like Nlp/P60 cell wall hydrolase domains. To understand how the modular architecture of SH3b and NlpC/P60 affects the activity of the catalytic domain, three putative NlpC/P60 cell wall hydrolases were biochemically and structurally characterized. These enzymes all have γ-d-Glu-A2pm (A2pm is diaminopimelic acid) cysteine amidase (or dl-endopeptidase) activities but with different substrate specificities. One enzyme is a cell wall lysin that cleaves peptidoglycan (PG), while the other two are cell wall recycling enzymes that only cleave stem peptides with an N-terminal l-Ala. Their crystal structures revealed a highly conserved structure consisting of two SH3b domains and a C-terminal NlpC/P60 catalytic domain, despite very low sequence identity. Interestingly, loops from the first SH3b domain dock into the ends of the active site groove of the catalytic domain, remodel the substrate binding site, and modulate substrate specificity. Two amino acid differences at the domain interface alter the substrate binding specificity in favor of stem peptides in recycling enzymes, whereas the SH3b domain may extend the peptidoglycan binding surface in the cell wall lysins. Remarkably, the cell wall lysin can be converted into a recycling enzyme with a single mutation. PMID:26374125

  2. SH3b Cell wall binding domains can enhance anti-staphylococcal activity of endolysin lytic domains.

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Bacteriophage endolysins are peptidoglycan hydrolases and a potential new source of antimicrobials. A large subset of these proteins contain a C-terminal SH3b_5 cell wall binding domain that has been shown [for some] to be essential for accurate cell wall recognition and subsequent staphylolytic ac...

  3. Regulation of dynamin-2 assembly-disassembly and function through the SH3A domain of intersectin-1s.

    PubMed

    Knezevic, Ivana; Predescu, Dan; Bardita, Cristina; Wang, Minhua; Sharma, Tiffany; Keith, Barbara; Neamu, Radu; Malik, Asrar B; Predescu, Sanda

    2011-11-01

    Intersectin-1s (ITSN-1s), a five Src homology 3 (SH3) domain-containing protein, is critically required for caveolae and clathrin-mediated endocytosis (CME), due to its interactions with dynamin (dyn). Of the five SH3A-E domains, SH3A is unique because of its high affinity for dyn and potent inhibition of CME. However, the molecular mechanism by which SH3A integrates in the overall function of ITSN-1s to regulate the endocytic process is not understood. Using biochemical and functional approaches as well as high-resolution electron microscopy, we show that SH3A exogenously expressed in human lung endothelial cells caused abnormal endocytic structures, distorted caveolae clusters, frequent staining-dense rings around the caveolar necks and 60% inhibition of caveolae internalization. In vitro studies further revealed that SH3A, similar to full-length ITSN-1s stimulates dyn2 oligomerization and guanosine triphosphatase (GTP)ase activity, effects not detected when other SH3 domains of ITSN-1s were used as controls. Strikingly, in the presence of SH3A, dyn2-dyn2 interactions are stabilized and despite continuous GTP hydrolysis, dyn2 oligomers cannot disassemble. SH3A may hold up caveolae release from the plasma membrane and formation of free-transport vesicles, by prolonging the lifetime of assembled dyn2. Altogether, our results indicate that ITSN-1s, via its SH3A has the unique ability to regulate dyn2 assembly-disassembly and function during endocytosis. PMID:21129155

  4. Regulation of dynamin-2 assembly–disassembly and function through the SH3A domain of intersectin-1s

    PubMed Central

    Knezevic, Ivana; Predescu, Dan; Bardita, Cristina; Wang, Minhua; Sharma, Tiffany; Keith, Barbara; Neamu, Radu; Malik, Asrar B; Predescu, Sanda

    2011-01-01

    Abstract Intersectin-1s (ITSN-1s), a five Src homology 3 (SH3) domain-containing protein, is critically required for caveolae and clathrin-mediated endocytosis (CME), due to its interactions with dynamin (dyn). Of the five SH3A-E domains, SH3A is unique because of its high affinity for dyn and potent inhibition of CME. However, the molecular mechanism by which SH3A integrates in the overall function of ITSN-1s to regulate the endocytic process is not understood. Using biochemical and functional approaches as well as high-resolution electron microscopy, we show that SH3A exogenously expressed in human lung endothelial cells caused abnormal endocytic structures, distorted caveolae clusters, frequent staining-dense rings around the caveolar necks and 60% inhibition of caveolae internalization. In vitro studies further revealed that SH3A, similar to full-length ITSN-1s stimulates dyn2 oligomerization and guanosine triphosphatase (GTP)ase activity, effects not detected when other SH3 domains of ITSN-1s were used as controls. Strikingly, in the presence of SH3A, dyn2–dyn2 interactions are stabilized and despite continuous GTP hydrolysis, dyn2 oligomers cannot disassemble. SH3A may hold up caveolae release from the plasma membrane and formation of free-transport vesicles, by prolonging the lifetime of assembled dyn2. Altogether, our results indicate that ITSN-1s, via its SH3A has the unique ability to regulate dyn2 assembly–disassembly and function during endocytosis. PMID:21129155

  5. The SH3 domain of UNC-89 (obscurin) interacts with paramyosin, a coiled-coil protein, in Caenorhabditis elegans muscle

    PubMed Central

    Qadota, Hiroshi; Mayans, Olga; Matsunaga, Yohei; McMurry, Jonathan L.; Wilson, Kristy J.; Kwon, Grace E.; Stanford, Rachel; Deehan, Kevin; Tinley, Tina L.; Ngwa, Verra M.; Benian, Guy M.

    2016-01-01

    UNC-89 is a giant polypeptide located at the sarcomeric M-line of Caenorhabditis elegans muscle. The human homologue is obscurin. To understand how UNC-89 is localized and functions, we have been identifying its binding partners. Screening a yeast two-hybrid library revealed that UNC-89 interacts with paramyosin. Paramyosin is an invertebrate-specific coiled-coil dimer protein that is homologous to the rod portion of myosin heavy chains and resides in thick filament cores. Minimally, this interaction requires UNC-89’s SH3 domain and residues 294–376 of paramyosin and has a KD of ∼1.1 μM. In unc-89 loss-of-function mutants that lack the SH3 domain, paramyosin is found in accumulations. When the SH3 domain is overexpressed, paramyosin is mislocalized. SH3 domains usually interact with a proline-rich consensus sequence, but the region of paramyosin that interacts with UNC-89’s SH3 is α-helical and lacks prolines. Homology modeling of UNC-89’s SH3 suggests structural features that might be responsible for this interaction. The SH3-binding region of paramyosin contains a “skip residue,” which is likely to locally unwind the coiled-coil and perhaps contributes to the binding specificity. PMID:27009202

  6. Binding of a proline-independent hydrophobic motif by the Candida albicans Rvs167-3 SH3 domain.

    PubMed

    Gkourtsa, Areti; van den Burg, Janny; Avula, Teja; Hochstenbach, Frans; Distel, Ben

    2016-09-01

    Src-homology 3 (SH3) domains are small protein-protein interaction modules. While most SH3 domains bind to proline-x-x-proline (PxxP) containing motifs in their binding partners, some SH3 domains recognize motifs other than proline-based sequences. Recently, we showed that the SH3 domain of Candida albicans Rvs167-3 binds peptides enriched in hydrophobic residues and containing a single proline residue (RΦxΦxΦP, where x is any amino acid and Φ is a hydrophobic residue). Here, we demonstrate that the proline in this motif is not required for Rvs167-3 SH3 recognition. Through mutagenesis studies we show that binding of the peptide ligand involves the conserved tryptophan in the canonical PxxP binding pocket as well as residues in the extended n-Src loop of Rvs167-3 SH3. Our studies establish a novel, proline-independent, binding sequence for Rvs167-3 SH3 (RΦxΦxΦ) that is comprised of a positively charged residue (arginine) and three hydrophobic residues. PMID:27393996

  7. Structure-Based Identification, Characterization, and Disruption of Human Securin-Binding SH3 Domains in Lung Cancer.

    PubMed

    Wang, Keping; Qiu, Tiefeng; Li, Xianwen

    2016-05-27

    The human securin is an oncogenic transcription factor that has been found to promote migration and invasion of lung cancer and many other tumors. The protein contains a PxxP motif that can be recognized and bound by diverse cellular partners via Src homology (SH3) domain to regulate biological and pathological events. The motif is covered by a decapeptide segment (161)LGPPSPVKMP(170) (SecPeptide) as the potential binding site of SH3 domains. Here, we attempted to systemically identify the SH3 binding partners of human securin in lung cancer and to characterize the intermolecular interaction between SecPeptide and the identified SH3 domains. A bioinformatics protocol that integrated literature curation, complex structural modeling, and binding affinity analysis was described to perform systematic search against an array of SH3-containing proteins involved in lung cancer signaling pathway and, consequently, three putative domains, namely GRB2, CRK, and RasGAP, were identified that have high potential to recognize and bind SecPeptide. The molecular mechanism and biological implication underlying the intermolecular interaction between these domains and SecPetide were investigated at structural and energetic level. Surface plasmon resonance assay revealed a high or moderate affinity of SecPeptide and its two mutants binding to CRK-SH3 domain with dissociation constants Kd = 79.8, 24.2, and 64.6 µM, respectively. PMID:27210447

  8. Structural recognition mechanisms between human Src homology domain 3 (SH3) and ALG-2-interacting protein X (Alix).

    PubMed

    Shi, Xiaoli; Betzi, Stephane; Lugari, Adrien; Opi, Sandrine; Restouin, Audrey; Parrot, Isabelle; Martinez, Jean; Zimmermann, Pascale; Lecine, Patrick; Huang, Mingdong; Arold, Stefan T; Collette, Yves; Morelli, Xavier

    2012-06-21

    The functions of Src family kinases are tightly regulated through Src homology (SH) domain-mediated protein-protein interactions. We previously reported the biophysical characteristics of the apoptosis-linked gene 2-interacting protein X (Alix) in complex with the haemopoietic cell kinase (Hck) SH3 domain. In the current study, we have combined ITC, NMR, SAXS and molecular modeling to determine a 3D model of the complex. We demonstrate that Hck SH3 recognizes an extended linear proline-rich region of Alix. This particular binding mode enables Hck SH3 to sense a specific non-canonical residue situated in the SH3 RT-loop of the kinase. The resulting model helps clarify the mechanistic insights of Alix-Hck interaction. PMID:22641034

  9. Paramagnetic relaxation enhancements in unfolded proteins: Theory and application to drkN SH3 domain

    PubMed Central

    Xue, Yi; Podkorytov, Ivan S; Rao, D Krishna; Benjamin, Nathan; Sun, Honglei; Skrynnikov, Nikolai R

    2009-01-01

    Site-directed spin labeling in combination with paramagnetic relaxation enhancement (PRE) measurements is one of the most promising techniques for studying unfolded proteins. Since the pioneering work of Gillespie and Shortle (J Mol Biol 1997;268:158), PRE data from unfolded proteins have been interpreted using the theory that was originally developed for rotational spin relaxation. At the same time, it can be readily recognized that the relative motion of the paramagnetic tag attached to the peptide chain and the reporter spin such as 1HN is best described as a translation. With this notion in mind, we developed a number of models for the PRE effect in unfolded proteins: (i) mutual diffusion of the two tethered spheres, (ii) mutual diffusion of the two tethered spheres subject to a harmonic potential, (iii) mutual diffusion of the two tethered spheres subject to a simulated mean-force potential (Smoluchowski equation); (iv) explicit-atom molecular dynamics simulation. The new models were used to predict the dependences of the PRE rates on the 1HN residue number and static magnetic field strength; the results are appreciably different from the Gillespie–Shortle model. At the same time, the Gillespie–Shortle approach is expected to be generally adequate if the goal is to reconstruct the distance distributions between 1HN spins and the paramagnetic center (provided that the characteristic correlation time is known with a reasonable accuracy). The theory has been tested by measuring the PRE rates in three spin-labeled mutants of the drkN SH3 domain in 2M guanidinium chloride. Two modifications introduced into the measurement scheme—using a reference compound to calibrate the signals from the two samples (oxidized and reduced) and using peak volumes instead of intensities to determine the PRE rates—lead to a substantial improvement in the quality of data. The PRE data from the denatured drkN SH3 are mostly consistent with the model of moderately expanded random

  10. Lipid binding by the Unique and SH3 domains of c-Src suggests a new regulatory mechanism

    PubMed Central

    Pérez, Yolanda; Maffei, Mariano; Igea, Ana; Amata, Irene; Gairí, Margarida; Nebreda, Angel R.; Bernadó, Pau; Pons, Miquel

    2013-01-01

    c-Src is a non-receptor tyrosine kinase involved in numerous signal transduction pathways. The kinase, SH3 and SH2 domains of c-Src are attached to the membrane-anchoring SH4 domain through the flexible Unique domain. Here we show intra- and intermolecular interactions involving the Unique and SH3 domains suggesting the presence of a previously unrecognized additional regulation layer in c-Src. We have characterized lipid binding by the Unique and SH3 domains, their intramolecular interaction and its allosteric modulation by a SH3-binding peptide or by Calcium-loaded calmodulin binding to the Unique domain. We also show reduced lipid binding following phosphorylation at conserved sites of the Unique domain. Finally, we show that injection of full-length c-Src with mutations that abolish lipid binding by the Unique domain causes a strong in vivo phenotype distinct from that of wild-type c-Src in a Xenopus oocyte model system, confirming the functional role of the Unique domain in c-Src regulation. PMID:23416516

  11. Crystal structure of Src-like adaptor protein 2 reveals close association of SH3 and SH2 domains through β-sheet formation.

    PubMed

    Wybenga-Groot, Leanne E; McGlade, C Jane

    2013-12-01

    The Src-like adaptor proteins (SLAP/SLAP2) are key components of Cbl-dependent downregulation of antigen receptor, cytokine receptor, and receptor tyrosine kinase signaling in hematopoietic cells. SLAP and SLAP2 consist of adjacent SH3 and SH2 domains that are most similar in sequence to Src family kinases (SFKs). Notably, the SH3-SH2 connector sequence is significantly shorter in SLAP/SLAP2 than in SFKs. To understand the structural implication of a short SH3-SH2 connector sequence, we solved the crystal structure of a protein encompassing the SH3 domain, SH3-SH2 connector, and SH2 domain of SLAP2 (SLAP2-32). While both domains adopt typical folds, the short SH3-SH2 connector places them in close association. Strand βe of the SH3 domain interacts with strand βA of the SH2 domain, resulting in the formation of a continuous β sheet that spans the length of the protein. Disruption of the SH3/SH2 interface through mutagenesis decreases SLAP-32 stability in vitro, consistent with inter-domain binding being an important component of SLAP2 structure and function. The canonical peptide binding pockets of the SH3 and SH2 domains are fully accessible, in contrast to other protein structures that display direct interaction between SH3 and SH2 domains, in which either peptide binding surface is obstructed by the interaction. Our results reveal potential sites of novel interaction for SH3 and SH2 domains, and illustrate the adaptability of SH2 and SH3 domains in mediating interactions. As well, our results suggest that the SH3 and SH2 domains of SLAP2 function interdependently, with implications on their mode of substrate binding. PMID:24018043

  12. Selection of recombinant anti-SH3 domain antibodies by high-throughput phage display.

    PubMed

    Huang, Haiming; Economopoulos, Nicolas O; Liu, Bernard A; Uetrecht, Andrea; Gu, Jun; Jarvik, Nick; Nadeem, Vincent; Pawson, Tony; Moffat, Jason; Miersch, Shane; Sidhu, Sachdev S

    2015-11-01

    Antibodies are indispensable tools in biochemical research and play an expanding role as therapeutics. While hybridoma technology is the dominant method for antibody production, phage display is an emerging technology. Here, we developed and employed a high-throughput pipeline that enables selection of antibodies against hundreds of antigens in parallel. Binding selections using a phage-displayed synthetic antigen-binding fragment (Fab) library against 110 human SH3 domains yielded hundreds of Fabs targeting 58 antigens. Affinity assays demonstrated that representative Fabs bind tightly and specifically to their targets. Furthermore, we developed an efficient affinity maturation strategy adaptable to high-throughput, which increased affinity dramatically but did not compromise specificity. Finally, we tested Fabs in common cell biology applications and confirmed recognition of the full-length antigen in immunoprecipitation, immunoblotting and immunofluorescence assays. In summary, we have established a rapid and robust high-throughput methodology that can be applied to generate highly functional and renewable antibodies targeting protein domains on a proteome-wide scale. PMID:26332758

  13. Structural insights into the specific binding of huntingtin proline-rich region with the SH3 and WW domains.

    PubMed

    Gao, Yong-Guang; Yan, Xian-Zhong; Song, Ai-Xin; Chang, Yong-Gang; Gao, Xue-Chao; Jiang, Nan; Zhang, Qi; Hu, Hong-Yu

    2006-12-01

    The interactions of huntingtin (Htt) with the SH3 domain- or WW domain-containing proteins have been implicated in the pathogenesis of Huntington's disease (HD). We report the specific interactions of Htt proline-rich region (PRR) with the SH3GL3-SH3 domain and HYPA-WW1-2 domain pair by NMR. The results show that Htt PRR binds with the SH3 domain through nearly its entire chain, and that the binding region on the domain includes the canonical PxxP-binding site and the specificity pocket. The C terminus of PRR orients to the specificity pocket, whereas the N terminus orients to the PxxP-binding site. Htt PRR can also specifically bind to WW1-2; the N-terminal portion preferentially binds to WW1, while the C-terminal portion binds to WW2. This study provides structural insights into the specific interactions between Htt PRR and its binding partners as well as the alteration of these interactions that involve PRR, which may have implications for the understanding of HD. PMID:17161366

  14. Crystal Structure of the SH3 Domain of beta PIX in Complex with a High Affinity Peptide from PAK2

    SciTech Connect

    Hoelz,A.; Janz, J.; Lawrie, S.; Corwin, B.; Lee, A.; Sakmar, T.

    2006-01-01

    The p21-activated kinases (PAKs) are important effector proteins of the small GTPases Cdc42 and Rac and control cytoskeletal rearrangements and cell proliferation. The direct interaction of PAKs with guanine nucleotide exchange factors from the PIX/Cool family, which is responsible for the localization of PAK kinases to focal complexes in the cell, is mediated by a 24-residue peptide segment in PAKs and an N-terminal src homology 3 (SH3) domain in PIX/Cool. The SH3-binding segment of PAK contains the atypical consensus-binding motif PxxxPR, which is required for unusually high affinity binding. In order to understand the structural basis for the high affinity and specificity of the PIX-PAK interaction, we solved crystal structures for the N-terminal SH3 domain of {beta}PIX and for the complex of the atypical binding segment of PAK2 with the N-terminal SH3 domain of {beta}PIX at 0.92 Angstroms and 1.3 Angstroms resolution, respectively. The asymmetric unit of the crystal contains two SH3 domains and two peptide ligands. The bound peptide adopts a conformation that allows for intimate contacts with three grooves on the surface of the SH3 domain that lie between the n-Src and RT-loops. Most notably, the arginine residue of the PxxxPR motif forms a salt-bridge and is tightly coordinated by a number of residues in the SH3 domain. This arginine-specific interaction appears to be the key determinant for the high affinity binding of PAK peptides. Furthermore, C-terminal residues of the peptide engage in additional interactions with the surface of the RT-loop, which significantly increases binding specificity. Compared to a recent NMR structure of a similar complex, our crystal structure reveals an alternate binding mode. Finally, we compare our crystal structure with the recently published {beta}PIX/Cbl-b complex structure, and suggest the existence of a molecular switch.

  15. Insights into Substrate Specificity of NlpC/P60 Cell Wall Hydrolases Containing Bacterial SH3 Domains

    SciTech Connect

    Xu, Qingping; Mengin-Lecreulx, Dominique; Liu, Xueqian W.; Patin, Delphine; Farr, Carol L.; Grant, Joanna C.; Chiu, Hsiu-Ju; Jaroszewski, Lukasz; Knuth, Mark W.; Godzik, Adam; Lesley, Scott A.; Elsliger, Marc-André; Deacon, Ashley M.; Wilson, Ian A.

    2015-09-15

    ABSTRACT

    Bacterial SH3 (SH3b) domains are commonly fused with papain-like Nlp/P60 cell wall hydrolase domains. To understand how the modular architecture of SH3b and NlpC/P60 affects the activity of the catalytic domain, three putative NlpC/P60 cell wall hydrolases were biochemically and structurally characterized. These enzymes all have γ-d-Glu-A2pm (A2pm is diaminopimelic acid) cysteine amidase (ordl-endopeptidase) activities but with different substrate specificities. One enzyme is a cell wall lysin that cleaves peptidoglycan (PG), while the other two are cell wall recycling enzymes that only cleave stem peptides with an N-terminall-Ala. Their crystal structures revealed a highly conserved structure consisting of two SH3b domains and a C-terminal NlpC/P60 catalytic domain, despite very low sequence identity. Interestingly, loops from the first SH3b domain dock into the ends of the active site groove of the catalytic domain, remodel the substrate binding site, and modulate substrate specificity. Two amino acid differences at the domain interface alter the substrate binding specificity in favor of stem peptides in recycling enzymes, whereas the SH3b domain may extend the peptidoglycan binding surface in the cell wall lysins. Remarkably, the cell wall lysin can be converted into a recycling enzyme with a single mutation.

    IMPORTANCEPeptidoglycan is a meshlike polymer that envelops the bacterial plasma membrane and bestows structural integrity. Cell wall lysins and recycling enzymes are part of a set of lytic enzymes that target covalent bonds connecting the amino acid and amino sugar building blocks of the PG network. These hydrolases are involved in processes such as cell growth and division, autolysis, invasion, and PG turnover and recycling. To avoid cleavage of unintended substrates, these enzymes have very selective substrate specificities. Our biochemical and structural

  16. Insights into substrate specificity of NlpC/P60 cell wall hydrolases containing bacterial SH3 domains

    DOE PAGESBeta

    Xu, Qingping; Mengin-Lecreulx, Dominique; Liu, Xueqian W.; Patin, Delphine; Farr, Carol L.; Grant, Joanna C.; Chiu, Hsiu -Ju; Jaroszewski, Lukasz; Knuth, Mark W.; Godzik, Adam; et al

    2015-09-15

    Bacterial SH3 (SH3b) domains are commonly fused with papain-like Nlp/P60 cell wall hydrolase domains. To understand how the modular architecture of SH3b and NlpC/P60 affects the activity of the catalytic domain, three putative NlpC/P60 cell wall hydrolases were biochemically and structurally characterized. In addition, these enzymes all have γ-d-Glu-A2pm (A2pm is diaminopimelic acid) cysteine amidase (ordl-endopeptidase) activities but with different substrate specificities. One enzyme is a cell wall lysin that cleaves peptidoglycan (PG), while the other two are cell wall recycling enzymes that only cleave stem peptides with an N-terminall-Ala. Their crystal structures revealed a highly conserved structure consisting ofmore » two SH3b domains and a C-terminal NlpC/P60 catalytic domain, despite very low sequence identity. Interestingly, loops from the first SH3b domain dock into the ends of the active site groove of the catalytic domain, remodel the substrate binding site, and modulate substrate specificity. Two amino acid differences at the domain interface alter the substrate binding specificity in favor of stem peptides in recycling enzymes, whereas the SH3b domain may extend the peptidoglycan binding surface in the cell wall lysins. Remarkably, the cell wall lysin can be converted into a recycling enzyme with a single mutation.Peptidoglycan is a meshlike polymer that envelops the bacterial plasma membrane and bestows structural integrity. Cell wall lysins and recycling enzymes are part of a set of lytic enzymes that target covalent bonds connecting the amino acid and amino sugar building blocks of the PG network. These hydrolases are involved in processes such as cell growth and division, autolysis, invasion, and PG turnover and recycling. To avoid cleavage of unintended substrates, these enzymes have very selective substrate specificities. Our biochemical and structural analysis of three modular NlpC/P60 hydrolases, one lysin, and two recycling enzymes, show

  17. Insights into substrate specificity of NlpC/P60 cell wall hydrolases containing bacterial SH3 domains

    SciTech Connect

    Xu, Qingping; Mengin-Lecreulx, Dominique; Liu, Xueqian W.; Patin, Delphine; Farr, Carol L.; Grant, Joanna C.; Chiu, Hsiu -Ju; Jaroszewski, Lukasz; Knuth, Mark W.; Godzik, Adam; Lesley, Scott A.; Elsliger, Marc -André; Deacon, Ashley M.; Wilson, Ian A.

    2015-09-15

    Bacterial SH3 (SH3b) domains are commonly fused with papain-like Nlp/P60 cell wall hydrolase domains. To understand how the modular architecture of SH3b and NlpC/P60 affects the activity of the catalytic domain, three putative NlpC/P60 cell wall hydrolases were biochemically and structurally characterized. In addition, these enzymes all have γ-d-Glu-A2pm (A2pm is diaminopimelic acid) cysteine amidase (ordl-endopeptidase) activities but with different substrate specificities. One enzyme is a cell wall lysin that cleaves peptidoglycan (PG), while the other two are cell wall recycling enzymes that only cleave stem peptides with an N-terminall-Ala. Their crystal structures revealed a highly conserved structure consisting of two SH3b domains and a C-terminal NlpC/P60 catalytic domain, despite very low sequence identity. Interestingly, loops from the first SH3b domain dock into the ends of the active site groove of the catalytic domain, remodel the substrate binding site, and modulate substrate specificity. Two amino acid differences at the domain interface alter the substrate binding specificity in favor of stem peptides in recycling enzymes, whereas the SH3b domain may extend the peptidoglycan binding surface in the cell wall lysins. Remarkably, the cell wall lysin can be converted into a recycling enzyme with a single mutation.

    Peptidoglycan is a meshlike polymer that envelops the bacterial plasma membrane and bestows structural integrity. Cell wall lysins and recycling enzymes are part of a set of lytic enzymes that target covalent bonds connecting the amino acid and amino sugar building blocks of the PG network. These hydrolases are involved in processes such as cell growth and division, autolysis, invasion, and PG turnover and recycling. To avoid cleavage of unintended substrates, these enzymes have very selective substrate specificities. Our biochemical and structural analysis of three modular NlpC/P60

  18. Associations among PH and SH3 domain-containing proteins and Rho-type GTPases in Yeast.

    PubMed

    Bender, L; Lo, H S; Lee, H; Kokojan, V; Peterson, V; Bender, A

    1996-05-01

    The src homology region 3 (SH3) domain-bearing protein Bem1p and the Rho-type GTPase Cdc42p are important for bud emergence in Saccharomyces cervisiae. Here, we present evidence that through its second SH3 domain, Bem1p binds to the structurally and functionally similar proteins Boi1p and Boi2p, each of which contain an SH3 and pleckstrin homology (PH) domain. Deletion of BOI1 and BO12 together leads to impaired morphogenesis and poor ability. A PH domain-bearing segment of Boi1p that lacks the Bem1p-binding site is necessary and sufficient for function. This segment of Boi1p displays a two-hybrid interaction with Cdc42p, suggesting that Boi1p either binds directly to or is part of a larger complex that contains Cdc42p. Consistent with these possibilities, overexpression of Boi1p inhibits bud emergence, but this inhibition is counteracted by cooverexpression of Cdc42p. Increased expression of the Rho-type GTPase Rho3p, which is implicated in bud growth defects of boil boi2 mutants, suggesting that Boi1p and Boi2p may also play roles in the activation or function of Rho3p. These findings provide an example of a tight coupling in function between PH domain-bearing proteins and both Rho-type GTPases and SH3 domain-containing proteins, and they raise the possibility that Boi1p and Boi2 play a role in linking the actions of Cdc42p and Rho3p. PMID:8666672

  19. Expression, purification and preliminary crystallographic studies of the C-terminal SH3 domain of human Tks4.

    PubMed

    Huang, Yuxin; Qian, Huolian; Wang, Xiaoying; Cheng, Zhong; Ren, Jixia; Zhao, Weichen; Xie, Yong

    2014-03-01

    The Src homology 3 (SH3) domain is a small, noncatalytic domain with a conserved sequence of about 60 amino-acid residues that interacts with proline-rich peptides to form a protein complex. In this study, the C-terminal SH3 domain of human Tks4 (residues 853-911) was expressed, purified and crystallized. X-ray diffraction data were collected to 2.3 Å resolution. The crystal belonged to the trigonal space group P3121 (or P3221), with unit-cell parameters a = b = 83.87, c = 108.44 Å, α = β = 90, γ = 120°. Calculating the self-rotation and the native Patterson function did not lead to the detection of any noncrystallographic translational symmetry. Six, seven or eight protein molecules are likely to be present in the asymmetric unit, resulting in a Matthews coefficient and approximate solvent content of 2.71 Å(3) Da(-1) and 55%, 2.32 Å(3) Da(-1) and 47%, and 2.03 Å(3) Da(-1) and 39%, respectively. To solve the crystal structure of the C-terminal SH3 domain of human Tks4, the isomorphous replacement method is presently being utilized. PMID:24598923

  20. Proteins recruited by SH3 domains of Ruk/CIN85 adaptor identified by LC-MS/MS

    PubMed Central

    Havrylov, Serhiy; Rzhepetskyy, Yuriy; Malinowska, Agata; Drobot, Lyudmyla; Redowicz, Maria Jolanta

    2009-01-01

    Background Ruk/CIN85 is a mammalian adaptor molecule with three SH3 domains. Using its SH3 domains Ruk/CIN85 can cluster multiple proteins and protein complexes, and, consequently, facilitates organisation of elaborate protein interaction networks with diverse regulatory roles. Previous research linked Ruk/CIN85 with the regulation of vesicle-mediated transport and cancer cell invasiveness. Despite the recent findings, precise molecular functions of Ruk/CIN85 in these processes remain largely elusive and further research is hampered by a lack of complete lists of its partner proteins. Results In the present study we employed a LC-MS/MS-based experimental pipeline to identify a considerable number (over 100) of proteins recruited by the SH3 domains of Ruk/CIN85 in vitro. Most of these identifications are novel Ruk/CIN85 interaction candidates. The identified proteins have diverse molecular architectures and can interact with other proteins, as well as with lipids and nucleic acids. Some of the identified proteins possess enzymatic activities. Functional profiling analyses and literature mining demonstrate that many of the proteins recruited by the SH3 domains of Ruk/CIN85 identified in this work were involved in the regulation of membranes and cytoskeletal structures necessary for vesicle-mediated transport and cancer cell invasiveness. Several groups of the proteins were also associated with few other cellular processes not previously related to Ruk/CIN85, most prominently with cell division. Conclusion Obtained data support the notion that Ruk/CIN85 regulates vesicle-mediated transport and cancer cell invasiveness through the assembly of multimeric protein complexes governing coordinated remodelling of membranes and underlying cytoskeletal structures, and imply its important roles in formation of coated vesicles and biogenesis of invadopodia. In addition, this study points to potential involvement of Ruk/CIN85 in other cellular processes, chiefly in cell division

  1. HIV-1 Tat binds to SH3 domains: cellular and viral outcome of Tat/Grb2 interaction.

    PubMed

    Rom, Slava; Pacifici, Marco; Passiatore, Giovanni; Aprea, Susanna; Waligorska, Agnieszka; Del Valle, Luis; Peruzzi, Francesca

    2011-10-01

    The Src-homology 3 (SH3) domain is one of the most frequent protein recognition modules (PRMs), being represented in signal transduction pathways and in several pathologies such as cancer and AIDS. Grb2 (growth factor receptor-bound protein 2) is an adaptor protein that contains two SH3 domains and is involved in receptor tyrosine kinase (RTK) signal transduction pathways. The HIV-1 transactivator factor Tat is required for viral replication and it has been shown to bind directly or indirectly to several host proteins, deregulating their functions. In this study, we show interaction between the cellular factor Grb2 and the HIV-1 trans-activating protein Tat. The binding is mediated by the proline-rich sequence of Tat and the SH3 domain of Grb2. As the adaptor protein Grb2 participates in a wide variety of signaling pathways, we characterized at least one of the possible downstream effects of the Tat/Grb2 interaction on the well-known IGF-1R/Raf/MAPK cascade. We show that the binding of Tat to Grb2 impairs activation of the Raf/MAPK pathway, while potentiating the PKA/Raf inhibitory pathway. The Tat/Grb2 interaction affects also viral function by inhibiting the Tat-mediated transactivation of HIV-1 LTR and viral replication in infected primary microglia. PMID:21745501

  2. The nebulin SH3 domain is dispensable for normal skeletal muscle structure but is required for effective active load bearing in mouse

    PubMed Central

    Yamamoto, Daniel L.; Vitiello, Carmen; Zhang, Jianlin; Gokhin, David S.; Castaldi, Alessandra; Coulis, Gerald; Piaser, Fabio; Filomena, Maria Carmela; Eggenhuizen, Peter J.; Kunderfranco, Paolo; Camerini, Serena; Takano, Kazunori; Endo, Takeshi; Crescenzi, Marco; Luther, Pradeep K. L.; Lieber, Richard L.; Chen, Ju; Bang, Marie-Louise

    2013-01-01

    Summary Nemaline myopathy (NM) is a congenital myopathy with an estimated incidence of 1∶50,000 live births. It is caused by mutations in thin filament components, including nebulin, which accounts for about 50% of the cases. The identification of NM cases with nonsense mutations resulting in loss of the extreme C-terminal SH3 domain of nebulin suggests an important role of the nebulin SH3 domain, which is further supported by the recent demonstration of its role in IGF-1-induced sarcomeric actin filament formation through targeting of N-WASP to the Z-line. To provide further insights into the functional significance of the nebulin SH3 domain in the Z-disk and to understand the mechanisms by which truncations of nebulin lead to NM, we took two approaches: (1) an affinity-based proteomic screening to identify novel interaction partners of the nebulin SH3 domain; and (2) generation and characterization of a novel knockin mouse model with a premature stop codon in the nebulin gene, eliminating its C-terminal SH3 domain (NebΔSH3 mouse). Surprisingly, detailed analyses of NebΔSH3 mice revealed no structural or histological skeletal muscle abnormalities and no changes in gene expression or localization of interaction partners of the nebulin SH3 domain, including myopalladin, palladin, zyxin and N-WASP. Also, no significant effect on peak isometric stress production, passive tensile stress or Young's modulus was found. However, NebΔSH3 muscle displayed a slightly altered force–frequency relationship and was significantly more susceptible to eccentric contraction-induced injury, suggesting that the nebulin SH3 domain protects against eccentric contraction-induced injury and possibly plays a role in fine-tuning the excitation–contraction coupling mechanism. PMID:24046450

  3. Conformational change of Sos-derived proline-rich peptide upon binding Grb2 N-terminal SH3 domain probed by NMR

    NASA Astrophysics Data System (ADS)

    Ogura, Kenji; Okamura, Hideyasu

    2013-10-01

    Growth factor receptor-bound protein 2 (Grb2) is a small adapter protein composed of a single SH2 domain flanked by two SH3 domains. The N-terminal SH3 (nSH3) domain of Grb2 binds a proline-rich region present in the guanine nucleotide releasing factor, son of sevenless (Sos). Using NMR relaxation dispersion and chemical shift analysis methods, we investigated the conformational change of the Sos-derived proline-rich peptide during the transition between the free and Grb2 nSH3-bound states. The chemical shift analysis revealed that the peptide does not present a fully random conformation but has a relatively rigid structure. The relaxation dispersion analysis detected conformational exchange of several residues of the peptide upon binding to Grb2 nSH3.

  4. Conformational change of Sos-derived proline-rich peptide upon binding Grb2 N-terminal SH3 domain probed by NMR.

    PubMed

    Ogura, Kenji; Okamura, Hideyasu

    2013-01-01

    Growth factor receptor-bound protein 2 (Grb2) is a small adapter protein composed of a single SH2 domain flanked by two SH3 domains. The N-terminal SH3 (nSH3) domain of Grb2 binds a proline-rich region present in the guanine nucleotide releasing factor, son of sevenless (Sos). Using NMR relaxation dispersion and chemical shift analysis methods, we investigated the conformational change of the Sos-derived proline-rich peptide during the transition between the free and Grb2 nSH3-bound states. The chemical shift analysis revealed that the peptide does not present a fully random conformation but has a relatively rigid structure. The relaxation dispersion analysis detected conformational exchange of several residues of the peptide upon binding to Grb2 nSH3. PMID:24105423

  5. Crystal structure of the N-terminal SH3 domain of mouse {beta}PIX, p21-activated kinase-interacting exchange factor

    SciTech Connect

    Li Xiaofeng; Liu Xueqi; Sun Fei; Gao Jia; Zhou Hongwei; Gao, George F.; Bartlam, Mark; Rao Zihe . E-mail: raozh@xtal.tsinghua.edu.cn

    2006-01-06

    The mouse {beta}PIX-SH3 domain, residues 8-63 of P21-activated kinase interacting exchange factor, has been characterized by X-ray diffraction. Crystals belonging to space group P3{sub 2}21 diffracted to 2.0 A and the structure was phased by the single-wavelength anomalous diffraction method. The domain is a compact {beta}-barrel with an overall conformation similar to the general SH3 structure. The X-ray structure shows mouse {beta}PIX-SH3 domain binding the way in which the {beta}PIX characteristic amino acids do so for an unconventional ligand binding surface. This arrangement provides a rationale for the unusual ligand recognition motif exhibited by mouse {beta}PIX-SH3 domain. Comparison with another SH3/peptide complex shows that the recognition mode of the mouse {beta}PIX-SH3 domain should be very similar to the RXXK ligand binding mode. The unique large and planar hydrophobic pocket may contribute to the promiscuity of {beta}PIX-SH3 domain resulting in its multiple biological functions.

  6. Proline Substitutions and Threonine Pseudophosphorylation of the SH3 Ligand of 18.5-kDa Myelin Basic Protein Decrease Its Affinity for the Fyn-SH3 Domain and Alter Process Development and Protein Localization in Oligodendrocytes

    PubMed Central

    Smith, Graham S.T.; De Avila, Miguel; Paez, Pablo M.; Spreuer, Vilma; Wills, Melanie K.B.; Jones, Nina; Boggs, Joan M.; Harauz, George

    2012-01-01

    The developmentally regulated myelin basic proteins (MBPs), which arise from the golli (gene of oligodendrocyte lineage) complex, are highly positively charged, intrinsically disordered, multifunctional proteins having several alternatively spliced isoforms and posttranslational modifications, and they play key roles in myelin compaction. The classic 18.5-kDa MBP isoform has a proline-rich region comprising amino acids 92–99 (murine sequence –T92PRTPPPS99–) that contains a minimal SH3 ligand domain. We have previously shown that 18.5-kDa MBP binds to several SH3 domains, including that of Fyn, a member of the Src family of tyrosine kinases involved in a number of signaling pathways during CNS development. To determine the physiological role of this binding as well as the role of phosphorylation of Thr92 and Thr95, in the current study we have produced several MBP variants specifically targeting phosphorylation sites and key structural regions of MBP’s SH3 ligand domain. Using isothermal titration calorimetry, we have demonstrated that, compared with the wild-type protein, these variants have lower affinity for the SH3 domain of Fyn. Moreover, overexpression of N-terminal-tagged GFP versions in immortalized oligodendroglial N19 and N20.1 cell cultures results in aberrant elongation of membrane processes and increased branching complexity and inhibits the ability of MBP to decrease Ca2+ influx. Phosphorylation of Thr92 can also cause MBP to traffic to the nucleus, where it may participate in additional protein–protein interactions. Coexpression of MBP with a constitutively active form of Fyn kinase resulted in membrane process elaboration, a phenomenon that was abolished by point amino acid substitutions in MBP’s SH3 ligand domain. These results suggest that MBP’s SH3 ligand domain plays a key role in intracellular protein interactions in vivo and may be required for proper membrane elaboration of developing oligodendrocytes and, further, that

  7. A BAR-Domain Protein SH3P2, Which Binds to Phosphatidylinositol 3-Phosphate and ATG8, Regulates Autophagosome Formation in Arabidopsis[C][W

    PubMed Central

    Zhuang, Xiaohong; Wang, Hao; Lam, Sheung Kwan; Gao, Caiji; Wang, Xiangfeng; Cai, Yi; Jiang, Liwen

    2013-01-01

    Autophagy is a well-defined catabolic mechanism whereby cytoplasmic materials are engulfed into a structure termed the autophagosome. In plants, little is known about the underlying mechanism of autophagosome formation. In this study, we report that SH3 DOMAIN-CONTAINING PROTEIN2 (SH3P2), a Bin-Amphiphysin-Rvs domain–containing protein, translocates to the phagophore assembly site/preautophagosome structure (PAS) upon autophagy induction and actively participates in the membrane deformation process. Using the SH3P2–green fluorescent protein fusion as a reporter, we found that the PAS develops from a cup-shaped isolation membranes or endoplasmic reticulum–derived omegasome-like structures. Using an inducible RNA interference (RNAi) approach, we show that RNAi knockdown of SH3P2 is developmentally lethal and significantly suppresses autophagosome formation. An in vitro membrane/lipid binding assay demonstrates that SH3P2 is a membrane-associated protein that binds to phosphatidylinositol 3-phosphate. SH3P2 may facilitate membrane expansion or maturation in coordination with the phosphatidylinositol 3-kinase (PI3K) complex during autophagy, as SH3P2 promotes PI3K foci formation, while PI3K inhibitor treatment inhibits SH3P2 from translocating to autophagosomes. Further interaction analysis shows that SH3P2 associates with the PI3K complex and interacts with ATG8s in Arabidopsis thaliana, whereby SH3P2 may mediate autophagy. Thus, our study has identified SH3P2 as a novel regulator of autophagy and provided a conserved model for autophagosome biogenesis in Arabidopsis. PMID:24249832

  8. The SH3 regulatory domain of the hematopoietic cell kinase Hck binds ELMO via its polyproline motif.

    PubMed

    Awad, Rida; Sévajol, Marion; Ayala, Isabel; Chouquet, Anne; Frachet, Philippe; Gans, Pierre; Reiser, Jean-Baptiste; Kleman, Jean-Philippe

    2015-01-01

    Eukaryotic EnguLfment and cell MOtility (ELMO) proteins form an evolutionary conserved family of regulators involved in small GTPase dependent actin remodeling processes that regulates the guanine exchange factor activity of some of the Downstream Of CrK (DOCK) family members. Gathered data strongly suggest that DOCK activation by ELMO and the subsequent signaling result from a subtle balance in the binding of partners to ELMO. Among its putative upward modulators, the Hematopoietic cell kinase (Hck), a member of the Src kinase superfamily, has been identified as a binding partner and a specific tyrosine kinase for ELMO1. Indeed, Hck is implicated in distinct molecular signaling pathways governing phagocytosis, cell adhesion, and migration of hematopoietic cells. Although ELMO1 has been shown to interact with the regulatory Src Homology 3 (SH3) domain of Hck, no direct evidence indicating the mode of interaction between Hck and ELMO1 have been provided in the literature. In the present study, we report convergent pieces of evidence that demonstrate the specific interaction between the SH3 domain of Hck and the polyproline motif of ELMO1. Our results also suggest that the tyrosine-phosphorylation state of ELMO1 tail might act as a putative modulator of Hck kinase activity towards ELMO1 that in turn participates in DOCK180 activation and further triggers subsequent signaling towards actin remodeling. PMID:25737835

  9. The SH3 regulatory domain of the hematopoietic cell kinase Hck binds ELMO via its polyproline motif

    PubMed Central

    Awad, Rida; Marion, Sévajol; Isabel, Ayala; Anne, Chouquet; Philippe, Frachet; Pierre, Gans; Jean-Baptiste, Reiser; Jean-Philippe, Kleman

    2015-01-01

    Eukaryotic EnguLfment and cell MOtility (ELMO) proteins form an evolutionary conserved family of regulators involved in small GTPase dependent actin remodeling processes that regulates the guanine exchange factor activity of some of the Downstream Of CrK (DOCK) family members. Gathered data strongly suggest that DOCK activation by ELMO and the subsequent signaling result from a subtle balance in the binding of partners to ELMO. Among its putative upward modulators, the Hematopoietic cell kinase (Hck), a member of the Src kinase superfamily, has been identified as a binding partner and a specific tyrosine kinase for ELMO1. Indeed, Hck is implicated in distinct molecular signaling pathways governing phagocytosis, cell adhesion, and migration of hematopoietic cells. Although ELMO1 has been shown to interact with the regulatory Src Homology 3 (SH3) domain of Hck, no direct evidence indicating the mode of interaction between Hck and ELMO1 have been provided in the literature. In the present study, we report convergent pieces of evidence that demonstrate the specific interaction between the SH3 domain of Hck and the polyproline motif of ELMO1. Our results also suggest that the tyrosine-phosphorylation state of ELMO1 tail might act as a putative modulator of Hck kinase activity towards ELMO1 that in turn participates in DOCK180 activation and further triggers subsequent signaling towards actin remodeling. PMID:25737835

  10. Metallofullerenol Gd@C82(OH)22 distracts the proline-rich-motif from putative binding on the SH3 domain

    NASA Astrophysics Data System (ADS)

    Kang, Seung-Gu; Huynh, Tien; Zhou, Ruhong

    2013-03-01

    Biocompatibility is often regarded as one important aspect of de novo designed nanomaterials for biosafety. However, the toxicological effect, appearing along with its latency, is much more difficult to address by linearly mapping physicochemical properties of related nanomaterials with biological effects such as immune or cellular regulatory responses due to the complicated protein-protein interactions. Here, we investigate a potential interference of a metallofullerenol, Gd@C82(OH)22, on the function of SH3 domain, a highly promiscuous protein-protein interaction mediator involved in signaling and regulatory pathways through its binding with the proline-rich motif (PRM) peptides, using the atomistic molecular dynamics simulation. Our study shows that when only Gd@C82(OH)22 and the SH3 domain are present (without the PRM ligand), Gd@C82(OH)22 can interact with the SH3 domain by either directly blocking the hydrophobic active site or binding with a hydrophilic off-site with almost equal probability, which can be understood from its intrinsic amphiphilic nature. In a binding competition with the PRM onto the SH3 domain, however, the on-site binding mode is depleted while Gd@C82(OH)22 effectively intercepts the PRM from the putative binding site of the SH3 domain, implying that Gd@C82(OH)22 can disturb protein-protein interactions mediated by the SH3 domain. Despite a successful surface modification in an aqueous biological medium and a more recent demonstration as potential de novo cancer therapeutics, our study indicates that greater attention is needed in assessing the potential cytotoxicity of these nanomaterials.Biocompatibility is often regarded as one important aspect of de novo designed nanomaterials for biosafety. However, the toxicological effect, appearing along with its latency, is much more difficult to address by linearly mapping physicochemical properties of related nanomaterials with biological effects such as immune or cellular regulatory responses

  11. The role of water molecules in the binding of class I and II peptides to the SH3 domain of the Fyn tyrosine kinase.

    PubMed

    Camara-Artigas, Ana; Ortiz-Salmeron, Emilia; Andujar-Sánchez, Montserrrat; Bacarizo, Julio; Martin-Garcia, Jose Manuel

    2016-09-01

    Interactions of proline-rich motifs with SH3 domains are present in signal transduction and other important cell processes. Analysis of structural and thermodynamic data suggest a relevant role of water molecules in these protein-protein interactions. To determine whether or not the SH3 domain of the Fyn tyrosine kinase shows the same behaviour, the crystal structures of its complexes with two high-affinity synthetic peptides, VSL12 and APP12, which are class I and II peptides, respectively, have been solved. In the class I complexes two water molecules were found at the binding interface that were not present in the class II complexes. The structures suggest a role of these water molecules in facilitating conformational changes in the SH3 domain to allow the binding of the class I or II peptides. In the third binding pocket these changes modify the cation-π and salt-bridge interactions that determine the affinity of the binding. Comparison of the water molecules involved in the binding of the peptides with previous reported hydration spots suggests a different pattern for the SH3 domains of the Src tyrosine kinase family. PMID:27599862

  12. Nck Binds to the T Cell Antigen Receptor Using Its SH3.1 and SH2 Domains in a Cooperative Manner, Promoting TCR Functioning.

    PubMed

    Paensuwan, Pussadee; Hartl, Frederike A; Yousefi, O Sascha; Ngoenkam, Jatuporn; Wipa, Piyamaporn; Beck-Garcia, Esmeralda; Dopfer, Elaine P; Khamsri, Boonruang; Sanguansermsri, Donruedee; Minguet, Susana; Schamel, Wolfgang W; Pongcharoen, Sutatip

    2016-01-01

    Ligand binding to the TCR causes a conformational change at the CD3 subunits to expose the CD3ε cytoplasmic proline-rich sequence (PRS). It was suggested that the PRS is important for TCR signaling and T cell activation. It has been shown that the purified, recombinant SH3.1 domain of the adaptor molecule noncatalytic region of tyrosine kinase (Nck) can bind to the exposed PRS of CD3ε, but the molecular mechanism of how full-length Nck binds to the TCR in cells has not been investigated so far. Using the in situ proximity ligation assay and copurifications, we show that the binding of Nck to the TCR requires partial phosphorylation of CD3ε, as it is based on two cooperating interactions. First, the SH3.1(Nck) domain has to bind to the nonphosphorylated and exposed PRS, that is, the first ITAM tyrosine has to be in the unphosphorylated state. Second, the SH2(Nck) domain has to bind to the second ITAM tyrosine in the phosphorylated state. Likewise, mutations of the SH3.1 and SH2 domains in Nck1 resulted in the loss of Nck1 binding to the TCR. Furthermore, expression of an SH3.1-mutated Nck impaired TCR signaling and T cell activation. Our data suggest that the exact pattern of CD3ε phosphorylation is critical for TCR functioning. PMID:26590318

  13. Breast Cancer Anti-estrogen Resistance 3 (BCAR3) Protein Augments Binding of the c-Src SH3 Domain to Crk-associated Substrate (p130cas)*

    PubMed Central

    Makkinje, Anthony; Vanden Borre, Pierre; Near, Richard I.; Patel, Prayag S.; Lerner, Adam

    2012-01-01

    The focal adhesion adapter protein p130cas regulates adhesion and growth factor-related signaling, in part through Src-mediated tyrosine phosphorylation of p130cas. AND-34/BCAR3, one of three NSP family members, binds the p130cas carboxyl terminus, adjacent to a bipartite p130cas Src-binding domain (SBD) and induces anti-estrogen resistance in breast cancer cell lines as well as phosphorylation of p130cas. Only a subset of the signaling properties of BCAR3, specifically augmented motility, are dependent upon formation of the BCAR3-p130cas complex. Using GST pull-down and immunoprecipitation studies, we show that among NSP family members, only BCAR3 augments the ability of p130cas to bind the Src SH3 domain through an RPLPSPP motif in the p130cas SBD. Although our prior work identified phosphorylation of the serine within the p130cas RPLPSPP motif, mutation of this residue to alanine or glutamic acid did not alter BCAR3-induced Src SH3 domain binding to p130cas. The ability of BCAR3 to augment Src SH3 binding requires formation of a BCAR3-p130cas complex because mutations that reduce association between these two proteins block augmentation of Src SH3 domain binding. Similarly, in MCF-7 cells, BCAR3-induced tyrosine phosphorylation of the p130cas substrate domain, previously shown to be Src-dependent, was reduced by an R743A mutation that blocks BCAR3 association with p130cas. Immunofluorescence studies demonstrate that BCAR3 expression alters the intracellular location of both p130cas and Src and that all three proteins co-localize. Our work suggests that BCAR3 expression may regulate Src signaling in a BCAR3-p130cas complex-dependent fashion by altering the ability of the Src SH3 domain to bind the p130cas SBD. PMID:22711540

  14. A graph kernel approach for alignment-free domain–peptide interaction prediction with an application to human SH3 domains

    PubMed Central

    Kundu, Kousik; Costa, Fabrizio; Backofen, Rolf

    2013-01-01

    Motivation: State-of-the-art experimental data for determining binding specificities of peptide recognition modules (PRMs) is obtained by high-throughput approaches like peptide arrays. Most prediction tools applicable to this kind of data are based on an initial multiple alignment of the peptide ligands. Building an initial alignment can be error-prone, especially in the case of the proline-rich peptides bound by the SH3 domains. Results: Here, we present a machine-learning approach based on an efficient graph-kernel technique to predict the specificity of a large set of 70 human SH3 domains, which are an important class of PRMs. The graph-kernel strategy allows us to (i) integrate several types of physico-chemical information for each amino acid, (ii) consider high-order correlations between these features and (iii) eliminate the need for an initial peptide alignment. We build specialized models for each human SH3 domain and achieve competitive predictive performance of 0.73 area under precision-recall curve, compared with 0.27 area under precision-recall curve for state-of-the-art methods based on position weight matrices. We show that better models can be obtained when we use information on the noninteracting peptides (negative examples), which is currently not used by the state-of-the art approaches based on position weight matrices. To this end, we analyze two strategies to identify subsets of high confidence negative data. The techniques introduced here are more general and hence can also be used for any other protein domains, which interact with short peptides (i.e. other PRMs). Availability: The program with the predictive models can be found at http://www.bioinf.uni-freiburg.de/Software/SH3PepInt/SH3PepInt.tar.gz. We also provide a genome-wide prediction for all 70 human SH3 domains, which can be found under http://www.bioinf.uni-freiburg.de/Software/SH3PepInt/Genome-Wide-Predictions.tar.gz. Contact: backofen@informatik.uni-freiburg.de Supplementary

  15. Oxygen as a paramagnetic probe of clustering and solvent exposure in folded and unfolded states of an SH3 domain.

    PubMed

    Bezsonova, Irina; Evanics, Ferenc; Marsh, Joseph A; Forman-Kay, Julie D; Prosser, R Scott

    2007-02-14

    The N-terminal SH3 domain of the Drosophila modular protein Drk undergoes slow exchange between a folded (Fexch) and highly populated unfolded (Uexch) state under nondenaturing buffer conditions, enabling both Fexch and Uexch states to be simultaneously monitored. The addition of dissolved oxygen, equilibrated to a partial pressure of either 30 atm or 60 atm, provides the means to study solvent exposure with atomic resolution via 13C NMR paramagnetic shifts in 1H,13C HSQC (heteronuclear single quantum coherence) spectra. Absolute differences in these paramagnetic shifts between the Fexch and Uexch states allow the discrimination of regions of the protein which undergo change in solvent exposure upon unfolding. Contact with dissolved oxygen for both the Fexch and Uexch states could also be assessed through 13C paramagnetic shifts which were normalized based on the corresponding paramagnetic shifts seen in the free amino acids. In the Fexch state, the 13C nuclei belonging to the hydrophobic core of the protein exhibited very weak normalized paramagnetic shifts while those with greater solvent accessible surface area exhibited significantly larger normalized shifts. The Uexch state displayed less varied 13C paramagnetic shifts although distinct regions of protection from solvent exposure could be identified by a lack of such shifts. These regions, which included Phe9, Thr12, Ala13, Lys21, Thr22, Ile24, Ile27, and Arg38, overlapped with those found to have residual nativelike and non-native structures in previous studies and in some cases provided novel information. Thus, the paramagnetic shifts from dissolved oxygen are highly useful in the study of a transient structure or clustering in disordered systems, where conventional NMR measurements (couplings, chemical shift deviations from random coil values, and NOEs) may give little information. PMID:17253684

  16. Identification of a Src kinase SH3 binding site in the C-terminal domain of the human ErbB2 receptor tyrosine kinase.

    PubMed

    Bornet, Olivier; Nouailler, Matthieu; Feracci, Michaël; Sebban-Kreuzer, Corinne; Byrne, Deborah; Halimi, Hubert; Morelli, Xavier; Badache, Ali; Guerlesquin, Françoise

    2014-06-01

    Overexpression of the ErbB2 receptor tyrosine kinase is associated with most aggressive tumors in breast cancer patients and is thus one of the main investigated therapeutic targets. Human ErbB2 C-terminal domain is an unstructured anchor that recruits specific adaptors for signaling cascades resulting in cell growth, differentiation and migration. Herein, we report the presence of a SH3 binding motif in the proline rich unfolded ErbB2 C-terminal region. NMR analysis of this motif supports a PPII helix conformation and the binding to Fyn-SH3 domain. The interaction of a kinase of the Src family with ErbB2 C-terminal domain could contribute to synergistic intracellular signaling and enhanced oncogenesis. PMID:24815698

  17. Post-synaptic density perturbs insulin-induced Kv1.3 channel modulation via a clustering mechanism involving the SH3 domain

    PubMed Central

    Marks, D. R.; Fadool, D. A.

    2009-01-01

    The olfactory bulb (OB) contains the highest concentration of the insulin receptor (IR) kinase in the central nervous system; however, its functional role and modulation in this region remains poorly understood. IR kinase contains a number of proline-rich motifs, making it an excellent candidate for modulation by SH3 domain-containing adaptor proteins. Kv1.3, a voltage-gated Shaker potassium channel and tyrosine phosphorylation substrate of IR kinase, contains several prolinerich sequences and a canonical post-synaptic density 95 (PSD-95)/discs large/zO-1 domain (PDZ) recognition motif common to most Shaker family members. We sought to determine if a functional relationship existed between Kv1.3, IR kinase, and the SH3/PDZ adaptor protein PSD-95. Through patch-clamp electrophysiology, immunochemistry, and co-immunoprecipitation, we found that while Kv1.3 and PSD-95 alone interact via the canonical C-terminal PDZ recognition motif of the channel, this molecular site of interaction acts to cluster the channels but the PSD-95 SH3-guanylate kinase domain functionally modulates Kv1.3 activity via two prolinerich domains in its N- and C-terminal. Therefore, these data suggest that adaptor domains responsible for ion-channel clustering and functional modulation are not necessarily coupled. Moreover, IR kinase and Kv1.3 can only be co-immunoprecipitated in the presence of PSD-95 as the adapting linker. Functionally, insulin-dependent Kv1.3 phosphorylation that causes channel current suppression is blocked via interaction with the PSD-95 SH3-guanylate kinase domain. Because all the three proteins co-localize in multiple lamina of the OB that are known to be rich in synaptic connections, membrane excitability and synaptic transmission at critical locations in the OB have the capacity to be finely regulated. PMID:17854350

  18. GTPase Activating Protein (Sh3 Domain) Binding Protein 1 Regulates the Processing of MicroRNA-1 during Cardiac Hypertrophy

    PubMed Central

    He, Minzhen; Yang, Zhi; Abdellatif, Maha; Sayed, Danish

    2015-01-01

    Background MicroRNAs (miR) are small, posttranscriptional regulators, expressed as part of a longer primary transcript, following which they undergo nuclear and cytoplasmic processing by Drosha and Dicer, respectively, to form the functional mature ~20mer that gets incorporated into the silencing complex. Others and we have shown that mature miR-1 levels decrease with pressure-induced cardiac hypertrophy, however, there is little or no change in the primary transcript encompassing miR-1 stem-loop, suggesting critical regulatory step in microRNA processing. The objective of this study was to investigate the underlying mechanisms regulating miR-1 expression in cardiomyocytes. Results Here we report that GTPase–activating protein (SH3 domain) binding protein 1 (G3bp1), an endoribonuclease regulates miR-1 processing in cardiomyocytes. G3bp1 is upregulated during cardiac hypertrophy and restricts miR-1 processing by binding to its consensus sequence in the pre-miR-1-2 stem-loop. In accordance, exogenous G3bp1 is sufficient to reduce miR-1 levels, along with derepression of miR-1 targets; General transcription factor IIB (Gtf2b), cyclin dependent factor 9 (Cdk9) and eukaryotic initiation factor 4E (Eif4e). While Cdk9 and Gtf2b are essential for transcription, Eif4e is required for translation. Thus, downregulation of miR-1 is necessary for increase in these molecules. Similar to miR-1 knockdown, G3bp1 overexpression is not sufficient for development of cardiac hypertrophy. Conversely, knockdown of G3bp1 in hypertrophying cardiomyocytes inhibited downregulation of miR-1 and upregulation of its targets along with restricted hypertrophy, suggesting that G3bp1 is necessary for development of cardiac hypertrophy. These results indicate that G3bp1-mediated inhibition of miR-1 processing with growth stimulation results in decrease in mature miR-1 and, thereby, an increase of its targets, which play fundamental roles in the development of hypertrophy. Conclusion G3bp1

  19. The SLE variant Ala71Thr of BLK severely decreases protein abundance and binding to BANK1 through impairment of the SH3 domain function.

    PubMed

    Díaz-Barreiro, A; Bernal-Quirós, M; Georg, I; Marañón, C; Alarcón-Riquelme, M E; Castillejo-López, C

    2016-03-01

    The B-lymphocyte kinase (BLK) gene is associated genetically with several human autoimmune diseases including systemic lupus erythematosus. We recently described that the genetic risk is given by two haplotypes: one covering several strongly linked single-nucleotide polymorphisms within the promoter of the gene that correlated with low transcript levels, and a second haplotype that includes a rare nonsynonymous variant (Ala71Thr). Here we show that this variant, located within the BLK SH3 domain, is a major determinant of protein levels. In vitro analyses show that the 71Thr isoform is hyperphosphorylated and promotes kinase activation. As a consequence, BLK is ubiquitinated, its proteasomal degradation enhanced and the average life of the protein is reduced by half. Altogether, these findings suggest that an intrinsic autoregulatory mechanism previously unappreciated in BLK is disrupted by the 71Thr substitution. Because the SH3 domain is also involved in protein interactions, we sought for differences between the two isoforms in trafficking and binding to protein partners. We found that binding of the 71Thr variant to the adaptor protein BANK1 is severely reduced. Our study provides new insights on the intrinsic regulation of BLK activation and highlights the dominant role of its SH3 domain in BANK1 binding. PMID:26821283

  20. Electrostatic Effects in the Folding of the SH3 Domain of the c-Src Tyrosine Kinase: pH-Dependence in 3D-Domain Swapping and Amyloid Formation

    PubMed Central

    Bacarizo, Julio; Martinez-Rodriguez, Sergio; Martin-Garcia, Jose Manuel; Andujar-Sanchez, Montserrat; Ortiz-Salmeron, Emilia; Neira, Jose Luis; Camara-Artigas, Ana

    2014-01-01

    The SH3 domain of the c-Src tyrosine kinase (c-Src-SH3) aggregates to form intertwined dimers and amyloid fibrils at mild acid pHs. In this work, we show that a single mutation of residue Gln128 of this SH3 domain has a significant effect on: (i) its thermal stability; and (ii) its propensity to form amyloid fibrils. The Gln128Glu mutant forms amyloid fibrils at neutral pH but not at mild acid pH, while Gln128Lys and Gln128Arg mutants do not form these aggregates under any of the conditions assayed. We have also solved the crystallographic structures of the wild-type (WT) and Gln128Glu, Gln128Lys and Gln128Arg mutants from crystals obtained at different pHs. At pH 5.0, crystals belong to the hexagonal space group P6522 and the asymmetric unit is formed by one chain of the protomer of the c-Src-SH3 domain in an open conformation. At pH 7.0, crystals belong to the orthorhombic space group P212121, with two molecules at the asymmetric unit showing the characteristic fold of the SH3 domain. Analysis of these crystallographic structures shows that the residue at position 128 is connected to Glu106 at the diverging β-turn through a cluster of water molecules. Changes in this hydrogen-bond network lead to the displacement of the c-Src-SH3 distal loop, resulting also in conformational changes of Leu100 that might be related to the binding of proline rich motifs. Our findings show that electrostatic interactions and solvation of residues close to the folding nucleation site of the c-Src-SH3 domain might play an important role during the folding reaction and the amyloid fibril formation. PMID:25490095

  1. Sample Optimization and Identification of Signal Patterns of Amino Acid Side Chains in 2D RFDR Spectra of the α-Spectrin SH3 Domain

    NASA Astrophysics Data System (ADS)

    Pauli, Jutta; van Rossum, Barth; Förster, Hans; de Groot, Huub J. M.; Oschkinat, Hartmut

    2000-04-01

    Future structural investigations of proteins by solid-state CPMAS NMR will rely on uniformly labeled protein samples showing spectra with an excellent resolution. NMR samples of the solid α-spectrin SH3 domain were generated in four different ways, and their 13C CPMAS spectra were compared. The spectrum of a [u-13C, 15N]-labeled sample generated by precipitation shows very narrow 13C signals and resolved scalar carbon-carbon couplings. Linewidths of 16-19 Hz were found for the three alanine Cβ signals of a selectively labeled [70% 3-13C]alanine-enriched SH3 sample. The signal pattern of the isoleucine, of all prolines, valines, alanines, and serines, and of three of the four threonines were identified in 2D 13C-13C RFDR spectra of the [u-13C,15N]-labeled SH3 sample. A comparison of the 13C chemical shifts of the found signal patterns with the 13C assignment obtained in solution shows an intriguing match.

  2. Four mutations in SH2 and SH3 domains of Bruton`s tyrosine kinase (BTK) resulting in classic X-linked agammaglobulinemia (XLA)

    SciTech Connect

    Chen, S.H.; Zhang, M.; Zhu, Q.; Scott, C.R.; Och, H.D.

    1994-09-01

    XLA is an X-linked immunodeficient disease in man resulted from mutations in the BTK gene. BTK contains a unique amino-region of unknown function, SH2 and SH3 (src homology) domains, and a carboxyl-terminal kinase (SH1) domain. We have studied the normal genomic organizations of the SH2 and SH3 domains and found the regions containing 6 exons are about 3000 bp in length. We also carried out sequence analyses of cDNA and genomic DNA of XLA patients to identify mutations. Four of fourteen families with XLA were found to have mutations within the regions. (1) A point mutation G to T in codon 240 resulted in a stop codon. (2) A transition mutation (g to a) at first nucleotide of intron 8 resulted in exon 8 skipping, missing 21 codons and shorter polypeptide but with normal kinase activity and ATP binding ability. (3) An a to t transversion at one of the invariant dinucleotides (ag) of the 3{prime} end of intron 11 resulted in alternative splicing at a position 13 nucleotides downstream from the normal one. The mutation produced mRNA with 13 nucleotide deletion and presumably resulted in a frameshift at codon 372 leading to a stop codon at 398. (4) A 16 nucleotide duplication (1248 to 1263 of the cDNA sequence) consistently present in mRNA of three brothers with XLA. However, genomic sequence of patient DNA of the regions did not reveal the anormaly. The observation that mutations within SH2 and SH3 causing severe B-cell defects typical for XLA suggests that these two domains are crucial for the function of BTK, possibly by regulating the interaction of cytoplasmic proteins involved in signal transduction.

  3. Changing the topology of protein backbone: the effect of backbone cyclization on the structure and dynamics of a SH3 domain

    PubMed Central

    Schumann, Frank H.; Varadan, Ranjani; Tayakuniyil, Praveen P.; Grossman, Jennifer H.; Camarero, Julio A.; Fushman, David

    2015-01-01

    Understanding of the effects of the backbone cyclization on the structure and dynamics of a protein is essential for using protein topology engineering to alter protein stability and function. Here we have determined, for the first time, the structure and dynamics of the linear and various circular constructs of the N-SH3 domain from protein c-Crk. These constructs differ in the length and amino acid composition of the cyclization region. The backbone cyclization was carried out using intein-mediated intramolecular chemical ligation between the juxtaposed N- and the C-termini. The structure and backbone dynamics studies were performed using solution NMR. Our data suggest that the backbone cyclization has little effect on the overall three-dimensional structure of the SH3 domain: besides the termini, only minor structural changes were found in the proximity of the cyclization region. In contrast to the structure, backbone dynamics are significantly affected by the cyclization. On the subnanosecond time scale, the backbone of all circular constructs on average appears more rigid than that of the linear SH3 domain; this effect is observed over the entire backbone and is not limited to the cyclization site. The backbone mobility of the circular constructs becomes less restricted with increasing length of the circularization loop. In addition, significant conformational exchange motions (on the sub-millisecond time scale) were found in the N-Src loop and in the adjacent β-strands in all circular constructs studied in this work. These effects of backbone cyclization on protein dynamics have potential implications for the stability of the protein fold and for ligand binding. PMID:25905098

  4. Roles for SH2 and SH3 Domains in Lyn Kinase Association with Activated FcεRI in RBL Mast Cells Revealed by Patterned Surface Analysis

    PubMed Central

    Hammond, Stephanie; Wagenknecht-Wiesner, Alice; Veatch, Sarah L.; Holowka, David; Baird, Barbara

    2009-01-01

    In mast cells, antigen-mediated cross-linking of IgE bound to its high affinity surface receptor, FcεRI, initiates a signaling cascade that culminates in degranulation and release of allergic mediators. Antigen-patterned surfaces, in which the antigen is deposited in micron-sized features on a silicon substrate, were used to examine the spatial relationship between clustered IgE-FcεRI complexes and Lyn, the signal-initiating tyrosine kinase. RBL mast cells expressing wild-type Lyn-EGFP showed co-redistribution of this protein with clustered IgE receptors on antigen-patterned surfaces, whereas Lyn-EGFP containing an inhibitory point mutation in its SH2 domain did not significantly accumulate with the patterned antigen, and Lyn-EGFP with an inhibitory point mutation in its SH3 domain exhibited reduced interactions. Our results using antigen-patterned surfaces and quantitative cross-correlation image analysis reveal that both the SH2 and SH3 domains contribute to interactions between Lyn kinase and cross-linked IgE receptors in stimulated mast cells. PMID:19427382

  5. A single amino acid in the SH3 domain of Hck determines its high affinity and specificity in binding to HIV-1 Nef protein.

    PubMed Central

    Lee, C H; Leung, B; Lemmon, M A; Zheng, J; Cowburn, D; Kuriyan, J; Saksela, K

    1995-01-01

    We have examined the differential binding of Hck and Fyn to HIV-1 Nef to elucidate the structural basis of SH3 binding affinity and specificity. Full-length Nef bound to Hck SH3 with the highest affinity reported for an SH3-mediated interaction (KD 250 nM). In contrast to Hck, affinity of the highly homologous Fyn SH3 for Nef was too weak (KD > 20 microM) to be accurately determined. We show that this distinct specificity lies in a variable loop, the 'RT loop', positioned close to conserved SH3 residues implicated in the binding of proline-rich (PxxP) motifs. A mutant Fyn SH3 with a single amino acid substitution (R96I) in its RT loop had an affinity (KD 380 nM) for Nef comparable with that of Hck SH3. Based on additional mutagenesis studies we propose that the selective recognition of Nef by Hck SH3 is determined by hydrophobic interactions involving an isoleucine residue in its RT loop. Although Nef contains a PxxP motif which is necessary for the interaction with Hck SH3, high affinity binding was only observed for intact Nef protein. The binding of a peptide containing the Nef PxxP motif showed > 300-fold weaker affinity for Hck SH3 than full-length Nef. Images PMID:7588629

  6. Metalloprotease-disintegrin ADAM 12 binds to the SH3 domain of Src and activates Src tyrosine kinase in C2C12 cells.

    PubMed Central

    Kang, Q; Cao, Y; Zolkiewska, A

    2000-01-01

    ADAM 12, a member of the ADAM (protein containing a disintegrin and metalloprotease) family of metalloprotease-disintegrins, has been implicated in the differentiation and fusion of skeletal myoblasts, and its expression is dramatically up-regulated in many cancer cells. While the extracellular portion of ADAM 12 contains an active metalloprotease and a cell-adhesion domain, the function of the cytoplasmic portion is much less clear. In this paper, we show that the cytoplasmic tail of ADAM 12 mediates interactions with the non-receptor protein tyrosine kinase Src. The interaction is direct, specific, and involves the N-terminal proline-rich region in the cytoplasmic tail of ADAM 12 and the Src homology 3 (SH3) domain of Src. ADAM 12 and Src co-immunoprecipitate from transfected C2C12 cells, suggesting that the two proteins form a complex in vivo. Co-expression of Src and ADAM 12, but not ADAM 9, in C2C12 cells results in activation of the recombinant Src. Moreover, endogenous ADAM 12 associates with and activates endogenous Src in differentiating C2C12 cells. These results indicate that ADAM 12 may mediate adhesion-induced signalling during myoblast differentiation. PMID:11104699

  7. Resveratrol induces apoptosis by directly targeting Ras-GTPase activating protein SH3 domain binding protein 1 (G3BP1)

    PubMed Central

    Oi, Naomi; Yuan, Jian; Malakhova, Margarita; Luo, Kuntian; Li, Yunhui; Ryu, Joohyun; Zhang, Lei; Bode, Ann M.; Xu, Zengguang; Li, Yan; Lou, Zhenkun; Dong, Zigang

    2014-01-01

    Resveratrol possesses a strong anticancer activity exhibited as the induction of apoptosis through p53 activation. However, the molecular mechanism and direct target(s) of resveratrol-induced p53 activation remain elusive. Here, the Ras-GTPase activating protein SH3 domain binding protein 1 (G3BP1) was identified as a potential target of resveratrol, and in vitro binding assay results using resveratrol (RSVL)-conjugated Sepharose 4B beads confirmed their direct binding. Depletion of G3BP1 significantly diminishes resveratrol-induced p53 expression and apoptosis. We also found that G3BP1 negatively regulates p53 expression by interacting with ubiquitin-specific protease 10 (USP10), a deubiquitinating enzyme of p53. Disruption of the interaction of p53 with USP10 by G3BP1 interference leads to suppression of p53 deubiquitination. Resveratrol, on the other hand, directly binds to G3BP1 and prevents the G3BP1/USP10 interaction, resulting in enhanced USP10-mediated deubiquitination of p53 and consequently increased p53 expression. These findings disclose a novel mechanism of resveratrol-induced p53 activation and resveratrol-induced apoptosis by direct targeting of G3BP1. PMID:24998844

  8. Binding of the proline-rich region of the epithelial Na+ channel to SH3 domains and its association with specific cellular proteins.

    PubMed Central

    McDonald, F J; Welsh, M J

    1995-01-01

    The amiloride-sensitive epithelial Na+ channel (ENaC) is an important component of the Na(+)-reabsorption pathway in many epithelia. The identification of three subunits of ENaC (alpha, beta and gamma), as well as results from a number of functional and biochemical studies, suggests that functional Na+ channels are composed of a complex of proteins. To learn about possible interactions of the channel with other proteins, we studied the alpha-subunit of rat and human ENaC. We found that the proline-rich C-terminal domains of both rat and human alpha-ENaC, expressed as glutathione S-transferase fusion proteins, bound to SH3 domains in vitro. A 116 kDa protein from a human lung adenocarcinoma cell line (H441) was specifically bound by the human alpha-ENaC C-terminal fusion protein and by a shorter 18-amino acid proline-rich peptide derived from the larger fusion protein. The 116 kDa protein was not glycosylated and was not phosphorylated on tyrosine or by cyclic AMP-dependent protein kinase (PKA). A 134 kDa protein which was also bound by the human alpha-ENaC C-terminal fusion protein was a substrate for phosphorylation by PKA. These data suggest that the proline-rich C-terminal tail of alpha-ENaC may interact with other proteins that control its function, regulation or localization. Images Figure 2 Figure 3 Figure 4 Figure 5 Figure 6 PMID:8526861

  9. Expression patterns of SH3BGR family members in zebrafish development.

    PubMed

    Tong, Fang; Zhang, Mingming; Guo, Xiaoling; Shi, Hongshun; Li, Li; Guan, Wen; Wang, Haihe; Yang, Shulan

    2016-07-01

    SH3 domain-binding glutamic acid-rich (SH3BGR) gene family is composed of SH3BGR, SH3BGRL, SH3BGRL2, and SH3BGRL3 which encodes a cluster of small thioredoxin-like proteins and shares a Src homology 3 (SH3) domain. However, biological functions of SH3BGR family members are largely elusive. Given that zebrafish (Danio rerio) sh3bgrl, sh3bgrl2, sh3bgrl3, and sh3bgr are evolutionally identical to their corresponding human orthologues, we analyzed the spatiotemporal expression of SH3BGR family members in zebrafish embryonic development stages by in situ hybridization. Our results revealed that except sh3bgrl, other members are all maternally expressed, especially for sh3bgrl3 that is strongly expressed from one-cell stage to juvenile fishes. In situ expression patterns of SH3BGR members are similar in the very early developmental stages, including with commonly strong expression in intestines, olfactory bulbs, and neuromasts for neural system building up. Organ-specific expressions are also demonstrated, of which sh3bgr is uniquely expressed in sarcomere, and sh3bgrl3 in liver. sh3bgrl and sh3bgrl2 are similarly expressed in intestines, notochords, and neuromasts after 12-h post-fertilization of embryos. Eventually, messenger RNAs (mRNAs) of all sh3bgr members are mainly constrained into intestines of juvenile fishes. Collectively, our study clarified the expression patterns of sh3bgr family members in diverse organogenesis in embryonic development and indicates that SH3BGR members may play predominant roles in neural system development and in maintenance of normal function of digestive organs, especially for intestine homeostasis. However, their expression patterns are varied with the development stages and organ types, suggesting that the aberrant expression of these members would result in multiple diseases. PMID:27233781

  10. Predicted structure of the extracellular region of ligand-gated ion-channel receptors shows SH2-like and SH3-like domains forming the ligand-binding site.

    PubMed Central

    Gready, J. E.; Ranganathan, S.; Schofield, P. R.; Matsuo, Y.; Nishikawa, K.

    1997-01-01

    Fast synaptic neurotransmission is mediated by ligand-gated ion-channel (LGIC) receptors, which include receptors for acetylcholine, serotonin, GABA, glycine, and glutamate. LGICs are pentamers with extracellular ligand-binding domains and form integral membrane ion channels that are selective for cations (acetylcholine and serotonin 5HT3 receptors) or anions (GABAA and glycine receptors and the invertebrate glutamate-binding chloride channel). They form a protein superfamily with no sequence similarity to any protein of known structure. Using a 1D-3D structure mapping approach, we have modeled the extracellular ligand-binding domain based on a significant match with the SH2 and SH3 domains of the biotin repressor structure. Refinement of the model based on knowledge of the large family of SH2 and SH3 structures, sequence alignments, and use of structure templates for loop building, allows the prediction of both monomer and pentamer models. These are consistent with medium-resolution electron microscopy structures and with experimental structure/function data from ligand-binding, antibody-binding, mutagenesis, protein-labeling and subunit-linking studies, and glycosylation sites. Also, the predicted polarity of the channel pore calculated from electrostatic potential maps of pentamer models of superfamily members is consistent with known ion selectivities. Using the glycine receptor alpha 1 subunit, which forms homopentamers, the monomeric and pentameric models define the agonist and antagonist (strychnine) binding sites to a deep crevice formed by an extended loop, which includes the invariant disulfide bridge, between the SH2 and SH3 domains. A detailed binding site for strychnine is reported that is in strong agreement with known structure/function data. A site for interaction of the extracellular ligand-binding domain with the activation of the M2 transmembrane helix is also suggested. PMID:9144769

  11. The Vps27/Hrs/STAM (VHS) Domain of the Signal-transducing Adaptor Molecule (STAM) Directs Associated Molecule with the SH3 Domain of STAM (AMSH) Specificity to Longer Ubiquitin Chains and Dictates the Position of Cleavage.

    PubMed

    Baiady, Nardeen; Padala, Prasanth; Mashahreh, Bayan; Cohen-Kfir, Einav; Todd, Emily A; Du Pont, Kelly E; Berndsen, Christopher E; Wiener, Reuven

    2016-01-22

    The deubiquitinating enzyme associated molecule with the SH3 domain of STAM (AMSH) is crucial for the removal of ubiquitin molecules during receptor-mediated endocytosis and lysosomal receptor sorting. AMSH interacts with signal transducing adapter molecule (STAM) 1 or 2, which enhances the activity of AMSH through an unknown mechanism. This stimulation is dependent on the ubiquitin-interacting motif of STAM. Here we investigate the specific mechanism of AMSH stimulation by STAM proteins and the role of the STAM Vps27/Hrs/STAM domain. We show that, in the presence of STAM, the length of the ubiquitin chains affects the apparent cleavage rate. Through measurement of the chain cleavage kinetics, we found that, although the kcat of Lys(63)-linked ubiquitin chain cleavage was comparable for di- and tri-ubiquitin, the Km value was lower for tri-ubiquitin. This increased affinity for longer chains was dependent on the Vps27/Hrs/STAM domain of STAM and required that the substrate ubiquitin chain contain homogenous Lys(63)-linkages. In addition, STAM directed AMSH cleavage toward the distal isopeptide bond in tri-ubiquitin chains. Finally, we generated a structural model of AMSH-STAM to show how the complex binds Lys(63)-linked ubiquitin chains and cleaves at the distal end. These data show how a deubiquitinating enzyme-interacting protein dictates the efficiency and specificity of substrate cleavage. PMID:26601948

  12. Dual-faced SH3BGRL: oncogenic in mice, tumor suppressive in humans

    PubMed Central

    Wang, H; Liu, B; Al-Aidaroos, A Q O; Shi, H; Li, L; Guo, K; Li, J; Tan, B C P; Loo, J M; Tang, J P; Thura, M; Zeng, Q

    2016-01-01

    Despite abundant data supporting c-Src as a metastasis-promoting oncogene, activating mutations of c-Src are rare. This suggests that trans-interacting proteins may have a critical role in regulating c-Src activation. Here, we first report the discovery of Src homology 3 (SH3) domain-binding glutamic acid-rich-like protein (SH3BGRL), a novel c-Src activator in mice. Ectopic expression of murine SH3BGRL (mSH3BGRL) strongly promoted both tumor cell invasion and lung metastasis. Molecularly, mSH3BGRL specifically bound the inactive form of c-Src phosphorylated at Tyr527, promoting Tyr416 phosphorylation of c-Src and subsequent FAK-mediated activation of ERK and AKT signaling pathways. Targeting endogenous c-Src alone was sufficient to abolish mSH3BGRL-induced cancer metastasis in vivo. Unexpectedly, human SH3BGRL (hSH3BGRL) in turn suppressed tumorigenesis and metastasis in nature. We attempted site-specific reversion of hSH3BGRL amino-acid sequence to mSH3BGRL and found V108A substitution sufficient to restore SH3BGRL function as a c-Src activator and metastasis promoter. Notably, the somatic mutation R76C of hSH3BGRL can similarly act as hSH3BGRL-V108A and mSH3BGRL in tumorigenesis and metastasis. Our results uncover an evolutionarily controversial role of SH3BGRL in driving tumor metastasis through c-Src activation, and suggests that hSH3BGRL mutation status could be relevant to cancer diagnosis and therapy. PMID:26455318

  13. Dual-faced SH3BGRL: oncogenic in mice, tumor suppressive in humans.

    PubMed

    Wang, H; Liu, B; Al-Aidaroos, A Q O; Shi, H; Li, L; Guo, K; Li, J; Tan, B C P; Loo, J M; Tang, J P; Thura, M; Zeng, Q

    2016-06-23

    Despite abundant data supporting c-Src as a metastasis-promoting oncogene, activating mutations of c-Src are rare. This suggests that trans-interacting proteins may have a critical role in regulating c-Src activation. Here, we first report the discovery of Src homology 3 (SH3) domain-binding glutamic acid-rich-like protein (SH3BGRL), a novel c-Src activator in mice. Ectopic expression of murine SH3BGRL (mSH3BGRL) strongly promoted both tumor cell invasion and lung metastasis. Molecularly, mSH3BGRL specifically bound the inactive form of c-Src phosphorylated at Tyr527, promoting Tyr416 phosphorylation of c-Src and subsequent FAK-mediated activation of ERK and AKT signaling pathways. Targeting endogenous c-Src alone was sufficient to abolish mSH3BGRL-induced cancer metastasis in vivo. Unexpectedly, human SH3BGRL (hSH3BGRL) in turn suppressed tumorigenesis and metastasis in nature. We attempted site-specific reversion of hSH3BGRL amino-acid sequence to mSH3BGRL and found V108A substitution sufficient to restore SH3BGRL function as a c-Src activator and metastasis promoter. Notably, the somatic mutation R76C of hSH3BGRL can similarly act as hSH3BGRL-V108A and mSH3BGRL in tumorigenesis and metastasis. Our results uncover an evolutionarily controversial role of SH3BGRL in driving tumor metastasis through c-Src activation, and suggests that hSH3BGRL mutation status could be relevant to cancer diagnosis and therapy. PMID:26455318

  14. Crystal structures of the BsPif1 helicase reveal that a major movement of the 2B SH3 domain is required for DNA unwinding.

    PubMed

    Chen, Wei-Fei; Dai, Yang-Xue; Duan, Xiao-Lei; Liu, Na-Nv; Shi, Wei; Li, Na; Li, Ming; Dou, Shou-Xing; Dong, Yu-Hui; Rety, Stephane; Xi, Xu-Guang

    2016-04-01

    Pif1 helicases are ubiquitous members of the SF1B family and are essential for maintaining genome stability. It was speculated that Pif1-specific motifs may fold in specific structures, conferring distinct activities upon it. Here, we report the crystal structures of the Pif1 helicase from Bacteroides spp with and without adenosine triphosphate (ATP) analog/ssDNA. BsPif1 shares structural similarities with RecD2 and Dda helicases but has specific features in the 1B and 2B domains. The highly conserved Pif1 family specific sequence motif interacts with and constraints a putative pin-loop in domain 1B in a precise conformation. More importantly, we found that the 2B domain which contains a specific extended hairpin undergoes a significant rotation and/or movement upon ATP and DNA binding, which is absolutely required for DNA unwinding. We therefore propose a mechanism for DNA unwinding in which the 2B domain plays a predominant role. The fact that the conformational change regulates Pif1 activity may provide insight into the puzzling observation that Pif1 becomes highly processive during break-induced replication in association with Polδ, while the isolated Pif1 has low processivity. PMID:26809678

  15. Crystal structures of the BsPif1 helicase reveal that a major movement of the 2B SH3 domain is required for DNA unwinding

    PubMed Central

    Chen, Wei-Fei; Dai, Yang-Xue; Duan, Xiao-Lei; Liu, Na-Nv; Shi, Wei; Li, Na; Li, Ming; Dou, Shou-Xing; Dong, Yu-Hui; Rety, Stephane; Xi, Xu-Guang

    2016-01-01

    Pif1 helicases are ubiquitous members of the SF1B family and are essential for maintaining genome stability. It was speculated that Pif1-specific motifs may fold in specific structures, conferring distinct activities upon it. Here, we report the crystal structures of the Pif1 helicase from Bacteroides spp with and without adenosine triphosphate (ATP) analog/ssDNA. BsPif1 shares structural similarities with RecD2 and Dda helicases but has specific features in the 1B and 2B domains. The highly conserved Pif1 family specific sequence motif interacts with and constraints a putative pin-loop in domain 1B in a precise conformation. More importantly, we found that the 2B domain which contains a specific extended hairpin undergoes a significant rotation and/or movement upon ATP and DNA binding, which is absolutely required for DNA unwinding. We therefore propose a mechanism for DNA unwinding in which the 2B domain plays a predominant role. The fact that the conformational change regulates Pif1 activity may provide insight into the puzzling observation that Pif1 becomes highly processive during break-induced replication in association with Polδ, while the isolated Pif1 has low processivity. PMID:26809678

  16. Role of Electrostatic Interactions in Binding of Peptides and Intrinsically Disordered Proteins to Their Folded Targets: 2. The Model of Encounter Complex Involving the Double Mutant of the c-Crk N-SH3 Domain and Peptide Sos.

    PubMed

    Yuwen, Tairan; Xue, Yi; Skrynnikov, Nikolai R

    2016-03-29

    In the first part of this work (paper 1, Xue, Y. et al. Biochemistry 2014 , 53 , 6473 ), we have studied the complex between the 10-residue peptide Sos and N-terminal SH3 domain from adaptor protein c-Crk. In the second part (this paper), we designed the double mutant of the c-Crk N-SH3 domain, W169F/Y186L, with the intention to eliminate the interactions responsible for tight peptide-protein binding, while retaining the interactions that create the initial electrostatic encounter complex. The resulting system was characterized experimentally by measuring the backbone and side-chain (15)N relaxation rates, as well as binding shifts and (1)H(N) temperature coefficients. In addition, it was also modeled via a series of ∼5 μs molecular dynamics (MD) simulations recorded in a large water box under an Amber ff99SB*-ILDN force field. Similar to paper 1, we have found that the strength of arginine-aspartate and arginine-glutamate salt bridges is overestimated in the original force field. To address this problem we have applied the empirical force-field correction described in paper 1. Specifically, the Lennard-Jones equilibrium distance for the nitrogen-oxygen pair across Arg-to-Asp/Glu salt bridges has been increased by 3%. This modification led to MD models in good agreement with the experimental data. The emerging picture is that of a fuzzy complex, where the peptide "dances" over the surface of the protein, making transient contacts via salt-bridge interactions. Every once in a while the peptide assumes a certain more stable binding pose, assisted by a number of adventitious polar and nonpolar contacts. On the other hand, occasionally Sos flies off the protein surface; it is then guided by electrostatic steering to quickly reconnect with the protein. The dynamic interaction between Sos and the double mutant of c-Crk N-SH3 gives rise to only small binding shifts. The peptide retains a high degree of conformational mobility, although it is appreciably slowed down due

  17. The proline-rich region of 18.5 kDa myelin basic protein binds to the SH3-domain of Fyn tyrosine kinase with the aid of an upstream segment to form a dynamic complex in vitro

    PubMed Central

    De Avila, Miguel; Vassall, Kenrick A.; Smith, Graham S. T.; Bamm, Vladimir V.; Harauz, George

    2014-01-01

    The intrinsically disordered 18.5 kDa classic isoform of MBP (myelin basic protein) interacts with Fyn kinase during oligodendrocyte development and myelination. It does so primarily via a central proline-rich SH3 (Src homology 3) ligand (T92–R104, murine 18.5 kDa MBP sequence numbering) that is part of a molecular switch due to its high degree of conservation and modification by MAP (mitogen-activated protein) and other kinases, especially at residues T92 and T95. Here, we show using co-transfection experiments of an early developmental oligodendroglial cell line (N19) that an MBP segment upstream of the primary ligand is involved in MBP–Fyn–SH3 association in cellula. Using solution NMR spectroscopy in vitro, we define this segment to comprise MBP residues (T62–L68), and demonstrate further that residues (V83–P93) are the predominant SH3-target, assessed by the degree of chemical shift change upon titration. We show by chemical shift index analysis that there is no formation of local poly-proline type II structure in the proline-rich segment upon binding, and by NOE (nuclear Overhauser effect) and relaxation measurements that MBP remains dynamic even while complexed with Fyn–SH3. The association is a new example first of a non-canonical SH3-domain interaction and second of a fuzzy MBP complex. PMID:25343306

  18. Iterative tyrosine phosphorylation controls non-canonical domain utilization in Crk.

    PubMed

    Sriram, G; Jankowski, W; Kasikara, C; Reichman, C; Saleh, T; Nguyen, K-Q; Li, J; Hornbeck, P; Machida, K; Liu, T; Li, H; Kalodimos, C G; Birge, R B

    2015-08-01

    Crk, the prototypical member of a class of Src homology-2 (SH2) and Src homology-3 (SH3) domain containing proteins that controls the coordinated assembly of signaling complexes, is regulated by phosphorylation of Y221 in the linker region, which forms an intramolecular SH2-pY221 auto-clamp to interrupt SH2-N-terminal SH3 domain (SH3N) signaling. Here, we show using LC-MS/MS and by generating phospho-specific antibodies that, iteratively with Y221, the Crk C-terminal SH3 domain (SH3C) is routinely phosphorylated on Y239 and/or Y251 by several extracellular stimuli known to engage Crk. Although phosphorylation at Y221 auto-inhibits the Crk SH2, phosphorylation of the SH3C generates an unconventional phosphoSH3C-SH3N unit in which the SH3N is fully functional to bind polyproline type II ligands and the phosphoSH3C binds de novo to other SH2 domains. Using high-throughput SH2 domain profiling, artificial neural network and position-specific scoring matrix-based bioinformatics approaches, and unbiased mass spectometry, we found that the phosphoSH3C binds several SH2 domain containing proteins, including specific non-receptor tyrosine kinases-Abl via pY251 and C-terminal Src kinase via pY239. Functionally, we show that the phosphoSH3C modulates the Abl-mediated phenotypes of cell spreading and motility. Together, these studies describe a versatile mechanism wherein phosphorylation of Crk at Y221 is not an off switch but redirects signaling from the SH2-SH3N axis to a phosphoSH3C-SH3N axis, with the SH3N as a common denominator. PMID:25381819

  19. HBV X protein interacts with cytoskeletal signaling proteins through SH3 binding.

    PubMed

    Feng, Huixing; Tan, Tuan Lin; Niu, Dandan; Chen, Wei Ning

    2010-01-01

    The aim of this study was to investigate interactions between cellular SH3-containing proteins and the proline-rich domain in Hepatitis B Virus (HBV) X protein (HBx) The proline-rich domain of HBx (amino acids 19-58) as well as the relevant site-directed mutagenesis (proline to alanine residues) were cloned into pGEX-5X-1 and expressed as GST-PXXP and GST-AXXA probes. Panomics SH3 domain arrays were probed using both GST-PXXP and GST-AXXA to identify potential interacting SH3 domain containing proteins. The specific interactions were confirmed by the immunoprecipitation of the full-length SH3 domain-containing protein. We report here the binding assay which demonstrated interaction between PXXP domain in HBx and the SH3-domain containing proteins, in particular various signaling proteins involved in cytoskeletal reorganization. Our findings were consistent with similar virus-host interactions via SH3 binding for other viruses such as hepatitis C virus (HCV) and human immunodeficiency virus (HIV) Further characterization of the proline-rich binding to SH3 domains could yield important information for the design of novel therapeutic measures against downstream disease causative effects of HBx in the liver cells. PMID:20036864

  20. Fluorescence Polarization Screening Assays for Small Molecule Allosteric Modulators of ABL Kinase Function

    PubMed Central

    Grover, Prerna; Shi, Haibin; Baumgartner, Matthew; Camacho, Carlos J.; Smithgall, Thomas E.

    2015-01-01

    The ABL protein-tyrosine kinase regulates intracellular signaling pathways controlling diverse cellular processes and contributes to several forms of cancer. The kinase activity of ABL is repressed by intramolecular interactions involving its regulatory Ncap, SH3 and SH2 domains. Small molecules that allosterically regulate ABL kinase activity through its non-catalytic domains may represent selective probes of ABL function. Here we report a screening assay for chemical modulators of ABL kinase activity that target the regulatory interaction of the SH3 domain with the SH2-kinase linker. This fluorescence polarization (FP) assay is based on a purified recombinant ABL protein consisting of the N-cap, SH3 and SH2 domains plus the SH2-kinase linker (N32L protein) and a short fluorescein-labeled probe peptide that binds to the SH3 domain. In assay development experiments, we found that the probe peptide binds to the recombinant ABL N32L protein in vitro, producing a robust FP signal that can be competed with an excess of unlabeled peptide. The FP signal is not observed with control N32L proteins bearing either an inactivating mutation in the SH3 domain or enhanced SH3:linker interaction. A pilot screen of 1200 FDA-approved drugs identified four compounds that specifically reduced the FP signal by at least three standard deviations from the untreated controls. Secondary assays showed that one of these hit compounds, the antithrombotic drug dipyridamole, enhances ABL kinase activity in vitro to a greater extent than the previously described ABL agonist, DPH. Docking studies predicted that this compound binds to a pocket formed at the interface of the SH3 domain and the linker, suggesting that it activates ABL by disrupting this regulatory interaction. These results show that screening assays based on the non-catalytic domains of ABL can identify allosteric small molecule regulators of kinase function, providing a new approach to selective drug discovery for this important

  1. Proline-rich sequences that bind to Src homology 3 domains with individual specificities.

    PubMed Central

    Alexandropoulos, K; Cheng, G; Baltimore, D

    1995-01-01

    To study the binding specificity of Src homology 3 (SH3) domains, we have screened a mouse embryonic expression library for peptide fragments that interact with them. Several clones were identified that express fragments of proteins which, through proline-rich binding sites, exhibit differential binding specificity to various SH3 domains. Src-SH3-specific binding uses a sequence of 7 aa of the consensus RPLPXXP, in which the N-terminal arginine is very important. The SH3 domains of the Src-related kinases Fyn, Lyn, and Hck bind to this sequence with the same affinity as that of the Src SH3. In contrast, a quite different proline-rich sequence from the Btk protein kinase binds to the Fyn, Lyn, and Hck SH3 domains, but not to the Src SH3. Specific binding of the Abl SH3 requires a longer, more proline-rich sequence but no arginine. One clone that binds to both Src and Abl SH3 domains through a common site exhibits reversed binding orientation, in that an arginine indispensable for binding to all tested SH3 domains occurs at the C terminus. Another clone contains overlapping yet distinct Src and Abl SH3 binding sites. Binding to the SH3 domains is mediated by a common PXXP amino acid sequence motif present on all ligands, and specificity comes about from other interactions, often ones involving arginine. The rules governing in vivo usage of particular sites by particular SH3 domains are not clear, but one binding orientation may be more specific than another. Images Fig. 1 Fig. 2 Fig. 3 PMID:7536925

  2. From Binding-Induced Dynamic Effects in SH3 Structures to Evolutionary Conserved Sectors.

    PubMed

    Zafra Ruano, Ana; Cilia, Elisa; Couceiro, José R; Ruiz Sanz, Javier; Schymkowitz, Joost; Rousseau, Frederic; Luque, Irene; Lenaerts, Tom

    2016-05-01

    Src Homology 3 domains are ubiquitous small interaction modules known to act as docking sites and regulatory elements in a wide range of proteins. Prior experimental NMR work on the SH3 domain of Src showed that ligand binding induces long-range dynamic changes consistent with an induced fit mechanism. The identification of the residues that participate in this mechanism produces a chart that allows for the exploration of the regulatory role of such domains in the activity of the encompassing protein. Here we show that a computational approach focusing on the changes in side chain dynamics through ligand binding identifies equivalent long-range effects in the Src SH3 domain. Mutation of a subset of the predicted residues elicits long-range effects on the binding energetics, emphasizing the relevance of these positions in the definition of intramolecular cooperative networks of signal transduction in this domain. We find further support for this mechanism through the analysis of seven other publically available SH3 domain structures of which the sequences represent diverse SH3 classes. By comparing the eight predictions, we find that, in addition to a dynamic pathway that is relatively conserved throughout all SH3 domains, there are dynamic aspects specific to each domain and homologous subgroups. Our work shows for the first time from a structural perspective, which transduction mechanisms are common between a subset of closely related and distal SH3 domains, while at the same time highlighting the differences in signal transduction that make each family member unique. These results resolve the missing link between structural predictions of dynamic changes and the domain sectors recently identified for SH3 domains through sequence analysis. PMID:27213566

  3. From Binding-Induced Dynamic Effects in SH3 Structures to Evolutionary Conserved Sectors

    PubMed Central

    Ruiz Sanz, Javier; Schymkowitz, Joost; Rousseau, Frederic

    2016-01-01

    Src Homology 3 domains are ubiquitous small interaction modules known to act as docking sites and regulatory elements in a wide range of proteins. Prior experimental NMR work on the SH3 domain of Src showed that ligand binding induces long-range dynamic changes consistent with an induced fit mechanism. The identification of the residues that participate in this mechanism produces a chart that allows for the exploration of the regulatory role of such domains in the activity of the encompassing protein. Here we show that a computational approach focusing on the changes in side chain dynamics through ligand binding identifies equivalent long-range effects in the Src SH3 domain. Mutation of a subset of the predicted residues elicits long-range effects on the binding energetics, emphasizing the relevance of these positions in the definition of intramolecular cooperative networks of signal transduction in this domain. We find further support for this mechanism through the analysis of seven other publically available SH3 domain structures of which the sequences represent diverse SH3 classes. By comparing the eight predictions, we find that, in addition to a dynamic pathway that is relatively conserved throughout all SH3 domains, there are dynamic aspects specific to each domain and homologous subgroups. Our work shows for the first time from a structural perspective, which transduction mechanisms are common between a subset of closely related and distal SH3 domains, while at the same time highlighting the differences in signal transduction that make each family member unique. These results resolve the missing link between structural predictions of dynamic changes and the domain sectors recently identified for SH3 domains through sequence analysis. PMID:27213566

  4. Bone Marrow Transplantation Improves Autoinflammation and Inflammatory Bone Loss in SH3BP2 Knock-In Cherubism Mice

    PubMed Central

    Yoshitaka, Teruhito; Kittaka, Mizuho; Ishida, Shu; Mizuno, Noriyoshi; Mukai, Tomoyuki; Ueki, Yasuyoshi

    2014-01-01

    Cherubism (OMIM#118400) is a genetic disorder in children characterized by excessive jawbone destruction with proliferation of fibro-osseous lesions containing a large number of osteoclasts. Mutations in the SH3-domain binding protein 2 (SH3BP2) are responsible for cherubism. Analysis of the knock-in (KI) mouse model of cherubism showed that homozygous cherubism mice (Sh3bp2KI/KI) spontaneously develop systemic autoinflammation and inflammatory bone loss and that cherubism is a TNF-α-dependent hematopoietic disorder. In this study, we investigated whether bone marrow transplantation (BMT) is effective for the treatment of inflammation and bone loss in Sh3bp2KI/KI mice. Bone marrow (BM) cells from wild-type (Sh3bp2+/+) mice were transplanted to 6-week-old Sh3bp2KI/KI mice with developing inflammation and to 10-week-old Sh3bp2KI/KI mice with established inflammation. Six-week-old Sh3bp2KI/KI mice transplanted with Sh3bp2+/+ BM cells exhibited improved body weight loss, facial swelling, and survival rate. Inflammatory lesions in the liver and lung as well as bone loss in calvaria and mandibula were ameliorated at 10 weeks after BMT compared to Sh3bp2KI/KI mice transplanted with Sh3bp2KI/KI BM cells. Elevation of serum TNF-α levels was not detected after BMT. BMT was effective for up to 20 weeks in 6-week-old Sh3bp2KI/KI mice transplanted with Sh3bp2+/+ BM cells. BMT also ameliorated the inflammation and bone loss in 10-week-old Sh3bp2KI/KI mice. Thus our study demonstrates that BMT improves the inflammation and bone loss in cherubism mice. BMT may be effective for the treatment of cherubism patients. PMID:25445458

  5. A potent and highly specific FN3 monobody inhibitor of the Abl SH2 domain

    SciTech Connect

    Wojcik, John; Hantschel, Oliver; Grebien, Florian; Kaupe, Ines; Bennett, Keiryn L.; Barkinge, John; Jones, Richard B.; Koide, Akiko; Superti-Furga, Giulio; Koide, Shohei

    2010-09-02

    Interactions between Src homology 2 (SH2) domains and phosphotyrosine sites regulate tyrosine kinase signaling networks. Selective perturbation of these interactions is challenging due to the high homology among the 120 human SH2 domains. Using an improved phage-display selection system, we generated a small antibody mimic (or 'monobody'), termed HA4, that bound to the Abelson (Abl) kinase SH2 domain with low nanomolar affinity. SH2 protein microarray analysis and MS of intracellular HA4 interactors showed HA4's specificity, and a crystal structure revealed how this specificity is achieved. HA4 disrupted intramolecular interactions of Abl involving the SH2 domain and potently activated the kinase in vitro. Within cells, HA4 inhibited processive phosphorylation activity of Abl and also inhibited STAT5 activation. This work provides a design guideline for highly specific and potent inhibitors of a protein interaction domain and shows their utility in mechanistic and cellular investigations.

  6. Nonenzymatic domains of Kalirin7 contribute to spine morphogenesis through interactions with phosphoinositides and Abl

    PubMed Central

    Ma, Xin-Ming; Miller, Megan B.; Vishwanatha, K. S.; Gross, Maegan J.; Wang, Yanping; Abbott, Thomas; Lam, TuKiet T.; Mains, Richard E.; Eipper, Betty A.

    2014-01-01

    Like several Rho GDP/GTP exchange factors (GEFs), Kalirin7 (Kal7) contains an N-terminal Sec14 domain and multiple spectrin repeats. A natural splice variant of Kalrn lacking the Sec14 domain and four spectrin repeats is unable to increase spine formation; our goal was to understand the function of the Sec14 and spectrin repeat domains. Kal7 lacking its Sec14 domain still increased spine formation, but the spines were short. Strikingly, Kal7 truncation mutants containing only the Sec14 domain and several spectrin repeats increased spine formation. The Sec14 domain bound phosphoinositides, a minor but crucial component of cellular membranes, and binding was increased by a phosphomimetic mutation. Expression of KalSec14-GFP in nonneuronal cells impaired receptor-mediated endocytosis, linking Kal7 to membrane trafficking. Consistent with genetic studies placing Abl, a non–receptor tyrosine kinase, and the Drosophila orthologue of Kalrn into the same signaling pathway, Abl1 phosphorylated two sites in the fourth spectrin repeat of Kalirin, increasing its sensitivity to calpain-mediated degradation. Treating cortical neurons of the wild-type mouse, but not the Kal7KO mouse, with an Abl inhibitor caused an increase in linear spine density. Phosphorylation of multiple sites in the N-terminal Sec14/spectrin region of Kal7 may allow coordination of the many signaling pathways contributing to spine morphogenesis. PMID:24600045

  7. The SH2 domain of Abl kinases regulates kinase autophosphorylation by controlling activation loop accessibility

    NASA Astrophysics Data System (ADS)

    Lamontanara, Allan Joaquim; Georgeon, Sandrine; Tria, Giancarlo; Svergun, Dmitri I.; Hantschel, Oliver

    2014-11-01

    The activity of protein kinases is regulated by multiple molecular mechanisms, and their disruption is a common driver of oncogenesis. A central and almost universal control element of protein kinase activity is the activation loop that utilizes both conformation and phosphorylation status to determine substrate access. In this study, we use recombinant Abl tyrosine kinases and conformation-specific kinase inhibitors to quantitatively analyse structural changes that occur after Abl activation. Allosteric SH2-kinase domain interactions were previously shown to be essential for the leukemogenesis caused by the Bcr-Abl oncoprotein. We find that these allosteric interactions switch the Abl activation loop from a closed to a fully open conformation. This enables the trans-autophosphorylation of the activation loop and requires prior phosphorylation of the SH2-kinase linker. Disruption of the SH2-kinase interaction abolishes activation loop phosphorylation. Our analysis provides a molecular mechanism for the SH2 domain-dependent activation of Abl that may also regulate other tyrosine kinases.

  8. Heterologous SH3-p85β inhibits influenza A virus replication

    PubMed Central

    2010-01-01

    Phosphatidylinositol 3-kinase (PI3K)/Akt signalling pathway can support the replication of influenza A virus through binding of viral NS1 protein to the Src homology 3 (SH3) domain of p85β regulatory subunit of PI3K. Here we investigated the effect of heterologously overexpressed SH3 on the replication of different influenza A virus subtypes/strains, and on the phosphorylation of Akt in the virus-infected cells. We found that heterologous SH3 reduced replication of influenza A viruses at varying degrees in a subtype/strain-dependent manner and SH3 overexpression reduced the induction of the phosphorylation of Akt in the cells infected with PR8(H1N1) and ST364(H3N2), but not with ST1233(H1N1), Ph2246(H9N2), and Qa199(H9N2). Our results suggest that interference with the NS1-p85β interaction by heterologous SH3 can be served as a useful antiviral strategy against influenza A virus infection. PMID:20653952

  9. BCR first exon sequences specifically activate the BCR/ABL tyrosine kinase oncogene of Philadelphia chromosome-positive human leukemias

    SciTech Connect

    Muller, A.J.; Witte, O.N. ); Young, J.C.; Pendergast, A.; Pondel, M. ); Landau, N.R.; Littman, D.R. )

    1991-04-01

    The c-abl proto-oncogene encodes a cytoplasmic tyrosine kinase which is homologous to the src gene product in its kinase domain and in the upstream kinase regulatory domains SH2 (src homology region 2) and SH3 (src homology region 3). The murine v-abl oncogene product has lost the SH3 domain as a consequence of N-terminal fusion of gag sequences. Deletion of the SH3 domain is sufficient to render the murine c-abl proto-oncogene product transforming when myristylated N-terminal membrane localization sequences are also present. In contrast, the human BCR/ABL oncogene of the Philadelphia chromosome translocation has an intact SH3 domain and its product is not myristylated at the N terminus. To analyze the contribution of BCR-encoded sequences to BCR/ABL-mediated transformation, the effects of a series of deletions and substitutions were assessed in fibroblast and hematopoietic-cell transformation assays. BCR first-exon sequences specifically potentiate transformation and tyrosine kinase activation when they are fused to the second exon of otherwise intact c-ABL. This suggests that BCR-encoded sequences specifically interfere with negative regulation of the ABL-encoded tyrosine kinase, which would represent a novel mechanism for the activation of nonreceptor tyrosine kinase-encoding proto-oncogenes.

  10. Mutations in the BCR-ABL1 Kinase Domain and Elsewhere in Chronic Myeloid Leukemia.

    PubMed

    Soverini, Simona; de Benedittis, Caterina; Mancini, Manuela; Martinelli, Giovanni

    2015-06-01

    Chronic myeloid leukemia (CML) has been the first human malignancy to be associated, more than 50 years ago, with a consistent chromosomal abnormality--the t(9;22)(q34;q11) chromosomal translocation. The resulting BCR-ABL1 fusion gene, encoding a tyrosine kinase with deregulated activity, has a central role in the pathogenesis of CML. Ancestral or additional genetic events necessary for CML to develop have long been hypothesized but never really demonstrated. CML can successfully be treated with tyrosine kinase inhibitors (TKIs). Mutations in the BCR-ABL1 kinase domain might arise, however, that confer resistance to 1 or more of the currently available TKIs. Hence, the critical role of BCR-ABL1 mutation screening for optimal therapeutic management, with the current gold standard technique, conventional sequencing, likely to be replaced soon by ultra-deep sequencing. Mutations in genes other than BCR-ABL1 include ASXL1, TET2, RUNX1, DNMT3A, EZH2, and TP53 in chronic phase patients and RUNX1, ASXL1, IKZF1, WT1, TET2, NPM1, IDH1, IDH2, NRAS, KRAS, CBL, TP53, CDKN2A, RB1, and GATA-2 mutations in advanced phase patients. The latter also display additional cytogenetic abnormalities, including submicroscopic regions of gain or loss that only single nucleotide polymorphism arrays or array comparative genomic hybridization can detect. Whether whole genome/exome sequencing studies will uncover novel mutations relevant for pathogenesis, progression, and risk-adapted therapy is still unclear. PMID:26297264

  11. The SH3/PH domain protein AgBoi1/2 collaborates with the Rho-type GTPase AgRho3 to prevent nonpolar growth at hyphal tips of Ashbya gossypii.

    PubMed

    Knechtle, Philipp; Wendland, Jürgen; Philippsen, Peter

    2006-10-01

    Unlike most other cells, hyphae of filamentous fungi permanently elongate and lack nonpolar growth phases. We identified AgBoi1/2p in the filamentous ascomycete Ashbya gossypii as a component required to prevent nonpolar growth at hyphal tips. Strains lacking AgBoi1/2p frequently show spherical enlargement at hyphal tips with concomitant depolarization of actin patches and loss of tip-located actin cables. These enlarged tips can repolarize and resume hyphal tip extension in the previous polarity axis. AgBoi1/2p permanently localizes to hyphal tips and transiently to sites of septation. Only the tip localization is important for sustained elongation of hyphae. In a yeast two-hybrid experiment, we identified the Rho-type GTPase AgRho3p as an interactor of AgBoi1/2p. AgRho3p is also required to prevent nonpolar growth at hyphal tips, and strains deleted for both AgBOI1/2 and AgRHO3 phenocopied the respective single-deletion strains, demonstrating that AgBoi1/2p and AgRho3p function in a common pathway. Monitoring the polarisome of growing hyphae using AgSpa2p fused to the green fluorescent protein as a marker, we found that polarisome disassembly precedes the onset of nonpolar growth in strains lacking AgBoi1/2p or AgRho3p. AgRho3p locked in its GTP-bound form interacts with the Rho-binding domain of the polarisome-associated formin AgBni1p, implying that AgRho3p has the capacity to directly activate formin-driven actin cable nucleation. We conclude that AgBoi1/2p and AgRho3p support polarisome-mediated actin cable formation at hyphal tips, thereby ensuring permanent polar tip growth. PMID:16950929

  12. Dimerization is required for SH3PX1 tyrosine phosphorylation in response to epidermal growth factor signalling and interaction with ACK2

    PubMed Central

    Childress, Chandra; Lin, Qiong; Yang, Wannian

    2005-01-01

    SH3PX1 [SNX9 (sorting nexin 9)] is a member of SNX super-family that is recognized by sharing a PX (phox homology) domain. We have previously shown that SH3PX1, phosphorylated by ACK2 (activated Cdc42-associated tyrosine kinase 2), regulates the degradation of EGF (epidermal growth factor) receptor. In mapping the tyrosine phosphorylation region, we found that the C-terminus of SH3PX1 is required for its tyrosine phosphorylation. Further analysis indicates that this region, known as the coiled-coil domain or the BAR (Bin–amphiphysin–Rvs homology) domain, is the dimerization domain of SH3PX1. Truncation of as little as 13 amino acid residues at the very C-terminus in the coiled-coil/BAR domain of SH3PX1 resulted in no dimerization, no ACK2-catalysed and EGF-stimulated tyrosine phosphorylation and no interaction with ACK2. The intracellular localization of SH3PX1 became dysfunctional upon truncation in the BAR domain. Taken together, our results indicate that the dimerization, which is mediated by the BAR domain, is essential for the intracellular function of SH3PX1. PMID:16316319

  13. Dimerization is required for SH3PX1 tyrosine phosphorylation in response to epidermal growth factor signalling and interaction with ACK2.

    PubMed

    Childress, Chandra; Lin, Qiong; Yang, Wannian

    2006-03-15

    SH3PX1 [SNX9 (sorting nexin 9)] is a member of SNX super-family that is recognized by sharing a PX (phox homology) domain. We have previously shown that SH3PX1, phosphorylated by ACK2 (activated Cdc42-associated tyrosine kinase 2), regulates the degradation of EGF (epidermal growth factor) receptor. In mapping the tyrosine phosphorylation region, we found that the C-terminus of SH3PX1 is required for its tyrosine phosphorylation. Further analysis indicates that this region, known as the coiled-coil domain or the BAR (Bin-amphiphysin-Rvs homology) domain, is the dimerization domain of SH3PX1. Truncation of as little as 13 amino acid residues at the very C-terminus in the coiled-coil/BAR domain of SH3PX1 resulted in no dimerization, no ACK2-catalysed and EGF-stimulated tyrosine phosphorylation and no interaction with ACK2. The intracellular localization of SH3PX1 became dysfunctional upon truncation in the BAR domain. Taken together, our results indicate that the dimerization, which is mediated by the BAR domain, is essential for the intracellular function of SH3PX1. PMID:16316319

  14. Characterization of a novel weak interaction between MUC1 and Src-SH3 using nuclear magnetic resonance spectroscopy

    SciTech Connect

    Gunasekara, Nirosha; Sykes, Brian; Hugh, Judith

    2012-05-18

    Highlights: Black-Right-Pointing-Pointer MUC1 binds the Src-SH3 domain potentially triggering Src dependent cell migration. Black-Right-Pointing-Pointer NMR Spectroscopy was used to monitor MUC1-CD and Src SH3 domain titrations. Black-Right-Pointing-Pointer MUC1-CD peptides bind with a low affinity (K{sub d} of 2-3 mM) to a non-canonical site. Black-Right-Pointing-Pointer Weak interactions may mediate dynamic processes like migration. Black-Right-Pointing-Pointer The MUC1-CD and Src-SH3 interaction may be a prime target to inhibit cell migration. -- Abstract: Breast cancer causes death through cancer cell migration and subsequent metastasis to distant organs. In vitro, the MUC1 mucin can mediate breast cancer cell migration by binding to intercellular adhesion molecule-1 (ICAM-1). This migration is dependent on MUC1 cytoplasmic domain (MUC1-CD) activation of the non-receptor tyrosine kinase, Src, possibly through competitive displacement of an inhibitory Src intramolecular SH3 binding. Therefore, we characterized the binding site and affinity of the MUC1-CD for Src-SH3 using multidimensional nuclear magnetic resonance (NMR) spectroscopy to monitor the titration of the {sup 15}N labeled Src-SH3 domain with synthetic native and mutant peptides of MUC1-CD. The results revealed that the dissociation constant (K{sub d}) for the interaction of the native MUC1-CD peptides and Src-SH3 domain was weak with a K{sub d} of 2-3 mM. Notably, the SH3 residues most perturbed upon peptide binding were located outside the usual hydrophobic binding cleft in a previously described alternate binding site on the Src-SH3, suggesting that MUC1-CD binds to a non-canonical site. The binding characteristics outlined here suggest that the interaction between Src-SH3 and MUC1-CD represents a novel weak electrostatic interaction of the type which is increasingly recognized as important in transient and dynamic protein complexes required for cell migration and signal transduction. As such, this

  15. Structural Basis for Autoinhibition of c-Abl Tyrosine Kinase

    SciTech Connect

    Nagar, Bhushan; Hantschel, Oliver; Young, Matthew A.; Scheffzek,Klaus; Veach, Darren; Bornmann, William; Clarkson, Bayard; Superti-Furga,Giulio; Kuriyan, John

    2003-03-21

    c-Abl is normally regulated by an autoinhibitory mechanism, the disruption of which leads to chronic myelogenous leukemia. The details of this mechanism have been elusive because c-Abl lacks aphosphotyrosine residue that triggers the assembly of the autoinhibited form of the closely related Src kinases by internally engaging the SH2 domain. Crystal structures of c-Abl show that the N-terminal myristoyl modification of c-Abl 1b binds to the kinase domain and induces conformational changes that allow the SH2 and SH3 domains to dock onto it. Autoinhibited c-Abl forms an assembly that is strikingly similar to that of inactive Src kinases but with specific differences that explain the differential ability of the drug STI-571/Gleevec/imatinib (STI-571)to inhibit the catalytic activity of Abl, but not that of c-Src.

  16. c-Abl Tyrosine Kinase Adopts Multiple Active Conformational States in Solution

    PubMed Central

    2016-01-01

    Protein tyrosine kinases of the Abl family have diverse roles in normal cellular regulation and drive several forms of leukemia as oncogenic fusion proteins. In the crystal structure of the inactive c-Abl kinase core, the SH2 and SH3 domains dock onto the back of the kinase domain, resulting in a compact, assembled state. This inactive conformation is stabilized by the interaction of the myristoylated N-cap with a pocket in the C-lobe of the kinase domain. Mutations that perturb these intramolecular interactions result in kinase activation. Here, we present X-ray scattering solution structures of multidomain c-Abl kinase core proteins modeling diverse active states. Surprisingly, the relative positions of the regulatory N-cap, SH3, and SH2 domains in an active myristic acid binding pocket mutant (A356N) were virtually identical to those of the assembled wild-type kinase core, indicating that Abl kinase activation does not require dramatic reorganization of the downregulated core structure. In contrast, the positions of the SH2 and SH3 domains in a clinically relevant imatinib-resistant gatekeeper mutant (T315I) appear to be reconfigured relative to their positions in the wild-type protein. Our results demonstrate that c-Abl kinase activation can occur either with (T315I) or without (A356N) global allosteric changes in the core, revealing the potential for previously unrecognized signaling diversity. PMID:27166638

  17. Mutant forms of growth factor-binding protein-2 reverse BCR-ABL-induced transformation.

    PubMed Central

    Gishizky, M L; Cortez, D; Pendergast, A M

    1995-01-01

    Growth factor-binding protein 2 (Grb2) is an adaptor protein that links tyrosine kinases to Ras. BCR-ABL is a tyrosine kinase oncoprotein that is implicated in the pathogenesis of Philadelphia chromosome (Ph1)-positive leukemias. Grb2 forms a complex with BCR-ABL and the nucleotide exchange factor Sos that leads to the activation of the Ras protooncogene. In this report we demonstrate that Grb2 mutant proteins lacking amino- or carboxyl-terminal src homology SH3 domains suppress BCR-ABL-induced Ras activation and reverse the oncogenic phenotype. The Grb2 SH3-deletion mutant proteins bind to BCR-ABL and do not impair tyrosine kinase activity. Expression of the Grb2 SH3-deletion mutant proteins in BCR-ABL-transformed Rat-1 fibroblasts and in the human Ph1-positive leukemic cell line K562 inhibits their ability to grow as foci in soft agar and form tumors in nude mice. Furthermore, expression of the Grb2 SH3-deletion mutants in K562 cells induced their differentiation. Because Ras plays an important role in signaling by receptor and nonreceptor tyrosine kinases, the use of interfering mutant Grb2 proteins may be applied to block the proliferation of other cancers that depend in part on activated tyrosine kinases for growth. Images Fig. 1 Fig. 2 Fig. 3 PMID:7479904

  18. The SH3BGR/STAT3 Pathway Regulates Cell Migration and Angiogenesis Induced by a Gammaherpesvirus MicroRNA

    PubMed Central

    Ding, Xiangya; Shen, Chenyou; Hu, Minmin; Zhu, Ying; Qin, Di; Lu, Hongmei; Krueger, Brian J.; Renne, Rolf; Gao, Shou-Jiang; Lu, Chun

    2016-01-01

    Kaposi’s sarcoma (KS)-associated herpesvirus (KSHV) is a gammaherpesvirus etiologically associated with KS, a highly disseminated angiogenic tumor of hyperproliferative spindle endothelial cells. KSHV encodes 25 mature microRNAs but their roles in KSHV-induced tumor dissemination and angiogenesis remain unknown. Here, we investigated KSHV-encoded miR-K12-6-3p (miR-K6-3p) promotion of endothelial cell migration and angiogenesis, which are the underlying mechanisms of tumor dissemination and angiogenesis. We found that ectopic expression of miR-K6-3p promoted endothelial cell migration and angiogenesis. Mass spectrometry, bioinformatics and luciferase reporter analyses revealed that miR-K6-3p directly targeted sequence in the 3’ untranslated region (UTR) of SH3 domain binding glutamate-rich protein (SH3BGR). Overexpression of SH3BGR reversed miR-K6-3p induction of cell migration and angiogenesis. Mechanistically, miR-K6-3p downregulated SH3BGR, hence relieved STAT3 from SH3BGR direct binding and inhibition, which was required for miR-K6-3p maximum activation of STAT3 and induction of cell migration and angiogenesis. Finally, deletion of miR-K6 from the KSHV genome abrogated its effect on the SH3BGR/STAT3 pathway, and KSHV-induced migration and angiogenesis. Our results illustrated that, by inhibiting SH3BGR, miR-K6-3p enhances cell migration and angiogenesis by activating the STAT3 pathway, and thus contributes to the dissemination and angiogenesis of KSHV-induced malignancies. PMID:27128969

  19. Mathematical model of the SH-3G helicopter

    NASA Technical Reports Server (NTRS)

    Phillips, J. D.

    1982-01-01

    A mathematical model of the Sikorsky SH-3G helicopter based on classical nonlinear, quasi-steady rotor theory was developed. The model was validated statically and dynamically by comparison with Navy flight-test data. The model incorporates ad hoc revisions which address the ideal assumptions of classical rotor theory and improve the static trim characteristics to provide a more realistic simulation, while retaining the simplicity of the classical model.

  20. The Structure of Dasatinib (BNS-354825) Bound to Activated ABL Kinase Domain Elucidates its Inhibitory Activity Against Imatinib-Resistant ABL Mutants

    SciTech Connect

    Tokarski,J.; Newitt, J.; Chang, C.; Cheng, J.; Wittekind, M.; Kiefer, S.; Kish, K.; Lee, F.; Borzilerri, R.; et al.

    2006-01-01

    Chronic myeloid leukemia (CML) is caused by the constitutively activated tyrosine kinase breakpoint cluster (BCR)-ABL. Current frontline therapy for CML is imatinib, an inhibitor of BCR-ABL. Although imatinib has a high rate of clinical success in early phase CML, treatment resistance is problematic, particularly in later stages of the disease, and is frequently mediated by mutations in BCR-ABL. Dasatinib (BMS-354825) is a multitargeted tyrosine kinase inhibitor that targets oncogenic pathways and is a more potent inhibitor than imatinib against wild-type BCR-ABL. It has also shown preclinical activity against all but one of the imatinib-resistant BCR-ABL mutants tested to date. Analysis of the crystal structure of dasatinib-bound ABL kinase suggests that the increased binding affinity of dasatinib over imatinib is at least partially due to its ability to recognize multiple states of BCR-ABL. The structure also provides an explanation for the activity of dasatinib against imatinib-resistant BCR-ABL mutants.

  1. Characterization of a pH and detergent-tolerant, cold-adapted type I pullulanase from Exiguobacterium sp. SH3.

    PubMed

    Rajaei, Sarah; Noghabi, Kambiz Akbari; Sadeghizadeh, Majid; Zahiri, Hossein Shahbani

    2015-11-01

    A pullulanase-encoding gene from psychrotrophic Exiguobacterium sp. SH3 was cloned and expressed in both E. coli and Bacillus subtilis. The functional recombinant enzyme (Pul-SH3) was purified as a His-tagged protein. Pul-SH3 was characterized to be a cold-adapted type I pullulanase with maximum activity at 45 °C. Using fluorescence spectroscopy, the melting temperature of Pul-SH3 was determined to be about 52 °C. The enzyme was able to hydrolyze pullulan, soluble starch, potato starch, and rice flour. It was exceptionally tolerant in the pH range of 4-11, exhibiting maximum activity at pH 8.5 and more than 60% of the activity in the pH range of 5-11. Being a detergent-tolerant pullulanase, Pul-SH3 retained 99, 89, and 54% of its activity at 10% concentration of Triton-X100, Tween 20, and SDS, respectively. The enzyme also exhibited an activity of 80.4 and 93.7% in the presence of two commercial detergents, Rika (7.5% v/v) and Fadisheh (2.5% w/v), respectively. The enzyme was even able to remain active by 54.5 and 85% after 10-day holding with the commercial detergents. Thermal stability of the enzyme could w on silica. Pul-SH3 with several industrially important characteristics seems desirable for cold hydrolysis of starch. PMID:26349928

  2. Kinase Domain Point Mutations in Ph+ Acute Lymphoblastic Leukemia (ALL) Emerge Following Therapy with BCR-ABL Kinase Inhibitors

    PubMed Central

    Jones, Dan; Thomas, Deborah; Yin, C. Cameron; O'Brien, Susan; Cortes, Jorge E.; Jabbour, Elias; Breeden, Megan; Giles, Francis J.; Zhao, Weiqiang; Kantarjian, Hagop M.

    2008-01-01

    Background BCR-ABL kinase domain (KD) mutations are detected in approximately 45% of imatinib-resistant CML. Patterns of KD mutations in Philadelphia chromosome (Ph)+ acute lymphoblastic leukemia (ALL) are less well-studied. Methods We assessed KD mutations in relapsed Ph+ ALL following treatments that included one or more kinase inhibitors (n = 24) or no prior KI therapy (n = 12). Results ABL KD mutations were detected by direct sequencing in 15 of 17 (88%) relapsed Ph+ ALL with prior imatinib (n = 16) or dasatinib (n = 1) treatment, and in 6 of 7 (86%) resistant/relapsed tumors treated with 2 or more KIs, compared with 0 of 12 relapsed Ph+ ALL never treated with KI. A restricted set of mutations was seen, mostly Y253H and T315I, detected on average 13 months following KI initiation, and mutations were not detected in the initial tumor samples prior to KI therapy in 12 patients assessed. Using a more sensitive pyrosequencing method, we did not detect mutations at codons 315 and 253 in the diagnostic samples from these 12 patients or in 30 Ph+ ALL patients who never relapsed. Conclusions ABL KD mutations, especially at codons 315 and 253, emerge upon relapse in the vast majority of patients with Ph+ ALL receiving maintenance KI therapy. Ongoing KI exposure may thus alter the patterns of relapse and favor outgrowth of clones with KI-resistant mutations. PMID:18615627

  3. The cyanobacterial cytochrome b6f subunit PetP adopts an SH3 fold in solution.

    PubMed

    Veit, Sebastian; Nagadoi, Aritaka; Rögner, Matthias; Rexroth, Sascha; Stoll, Raphael; Ikegami, Takahisa

    2016-06-01

    PetP is a peripheral subunit of the cytochrome b(6)f complex (b(6)f) present in both, cyanobacteria and red algae. It is bound to the cytoplasmic surface of this membrane protein complex where it greatly affects the efficiency of the linear photosynthetic electron flow although it is not directly involved in the electron transfer reactions. Despite the crystal structures of the b(6)f core complex, structural information for the transient regulatory b(6)f subunits is still missing. Here we present the first structure of PetP at atomic resolution as determined by solution NMR. The protein adopts an SH3 fold, which is a common protein motif in eukaryotes but comparatively rare in prokaryotes. The structure of PetP enabled the identification of the potential interaction site for b(6)f binding by conservation mapping. The interaction surface is mainly formed by two large loop regions and one short 310 helix which also exhibit an increased flexibility as indicated by heteronuclear steady-state {(1)H}-(15)N NOE and random coil index parameters. The properties of this potential b(6)f binding site greatly differ from the canonical peptide binding site which is highly conserved in eukaryotic SH3 domains. Interestingly, three other proteins of the photosynthetic electron transport chain share this SH3 fold with PetP: NdhS of the photosynthetic NADH dehydrogenase-like complex (NDH-1), PsaE of the photosystem 1 and subunit α of the ferredoxin-thioredoxin reductase have, similar to PetP, a great impact on the photosynthetic electron transport. Finally, a model is presented to illustrate how SH3 domains modulate the photosynthetic electron transport processes in cyanobacteria. PMID:27033306

  4. Etanercept Administration to Neonatal SH3BP2 Knock-In Cherubism Mice Prevents TNF-α-induced Inflammation and Bone Loss

    PubMed Central

    Yoshitaka, Teruhito; Ishida, Shu; Mukai, Tomoyuki; Kittaka, Mizuho; Reichenberger, Ernst J.; Ueki, Yasuyoshi

    2014-01-01

    Cherubism is a genetic disorder of the craniofacial skeleton caused by gain-of-function mutations in the signaling adaptor protein, SH3-domain binding protein 2 (SH3BP2). In a knock-in mouse model for cherubism, we previously demonstrated that homozygous mutant mice develop T/B cell-independent systemic macrophage inflammation leading to bone erosion and joint destruction. Homozygous mice develop multiostotic bone lesions while cherubism lesions in humans are limited to jawbones. We identified a critical role of TNF-α in the development of autoinflammation by creating homozygous TNF-α-deficient cherubism mutants, where systemic inflammation and bone destruction were rescued. In the current study, we examined whether postnatal administration of an anti-TNF-α antagonist can prevent or ameliorate the disease progression in cherubism mice. Neonatal homozygous mutants, where active inflammation has not yet developed, were treated with a high dose of etanercept (25 mg/kg, twice/week) for 7 weeks. Etanercept-treated neonatal mice showed strong rescue of facial swelling and bone loss in jaws and calvariae. Destruction of joints was fully rescued in the high dose group. Moreover, the high dose treatment group showed a significant decrease in lung and liver inflammatory lesions. However, inflammation and bone loss, which were successfully treated by etanercept administration recurred after etanercept discontinuation. No significant effect was observed in low dose- (0.5 mg/kg, twice/week) and vehicle-treated groups. In contrast, when 10-week-old cherubism mice with fully active inflammation were treated with etanercept for 7 weeks, even the high dose administration did not decrease bone loss, lung or liver inflammation. Taken together, the results suggest that anti-TNF-α therapy may be effective in young cherubism patients, if treated before the inflammatory phase or bone resorption occurs. Therefore, early genetic diagnosis and early treatment with anti

  5. Localization of SH3PXD2B in human eyes and detection of rare variants in patients with anterior segment diseases and glaucoma

    PubMed Central

    Mao, Mao; Solivan-Timpe, Frances; Roos, Ben R.; Mullins, Robert F.; Oetting, Thomas A.; Kwon, Young H.; Brzeskiewicz, Peter M.; Stone, Edwin M.; Alward, Wallace L.M.; Anderson, Michael G.

    2012-01-01

    Purpose Analysis of mutant mouse strains and linkage analysis with human families have both demonstrated that mutations influencing the podosomal adaptor protein SH3 and PX domains 2B (SH3PXD2B) can result in a congenital form of glaucoma. Here, we use immunohistochemistry to describe localization of the SH3PXD2B protein throughout the adult human eye and test whether sequence variants in SH3PXD2B occur in multiple other forms of glaucoma. Methods In immunohistochemical experiments, cryosections of human donor eyes were evaluated for SH3PXD2B immunoreactivity with a polyclonal antibody. In genetic experiments, exon sequences of SH3PXD2B from patients with primary congenital glaucoma (n=21), Axenfeld-Rieger syndrome (n=30), and primary open angle glaucoma (n=127) were compared to control subjects (n=89). The frequency of non-synonymous SH3PXD2B coding sequence variants were compared between patient cohorts and controls using Fisher’s exact test. Results Varying intensities of SH3PXD2B immunoreactivity were detected in almost all ocular tissues. Among tissues important to glaucoma, immunoreactivity was detected in the drainage structures of the iridocorneal angle, ciliary body, and retinal ganglion cells. Intense immunoreactivity was present in photoreceptor inner segments. From DNA analysis, a total of 11 non-synonymous variants were detected. By Fisher’s Exact test, there was not a significant skew in the overall frequency of these changes in any patient cohort versus controls (p-value >0.05). Each cohort contained unique variants not detected in other cohorts or patients. Conclusions SH3PXD2B is widely distributed in the adult human eye, including several tissues important to glaucoma pathogenesis. Analysis of DNA variants in three forms of glaucoma detected multiple variants unique to each patient cohort. While statistical analysis failed to support a pathogenic role for these variants, some of them may be rare disease-causing variants whose biologic

  6. Biochemical properties of Glu-SH3 as a family 13 glycoside hydrolase with remarkable substrate specificity for trehalose: Implications to sequence-based classification of CAZymes.

    PubMed

    Ghadikolaei, Kamran Khalili; Shojaei, Maral; Ghaderi, Armin; Hojjati, Farzaneh; Noghabi, Kambiz Akbari; Zahiri, Hossein Shahbani

    2016-08-01

    A novel glycoside hydrolase from Exiguobacterium sp. SH3 was characterized. The enzyme, designated as Glu-SH3, was predicted by in silico analysis to have structural similarity with members of oligo-1,6-glucosidase and trehalose-6-phosphate hydrolase subfamilies in the GH-13 family of glycoside hydrolases. The gene was expressed in Escherichia coli and the recombinant enzyme was purified as a His-tagged protein of about 60 kDa. The enzyme was shown to have remarkable substrate specificity for trehalose. The characteristic ability of Glu-SH3 to hydrolyze trehalose was ascertained by zymography, thin layer chromatography, and NMR spectroscopy. The maximum activity of Glu-SH3 was obtained at 35 °C and pH 7, but it was able to exhibit more than 90% of the activity within the pH range of 5-8. The Vmax and Km values were estimated to be 170 U and 4.5 mg ml(-1), respectively. By comparison with trehalases, Glu-SH3 with Kcat and Kcat/Km values of 1552 s(-1) and 119.4 mM(-1) s(-1) can be recognized as a very efficient trehalose-hydrolyzing glycosidase. Given the phylogeny and the substrate specificity of Glu-SH3, it may be assumed that the enzyme shares a common ancestor with oligo-1,6-glucosidases but have evolved distinctly to serve a physiological function in trehalose metabolism. PMID:27177969

  7. Computer-assisted re-design of spectrin SH3 residue clusters.

    PubMed

    Angrand, I; Serrano, L; Lacroix, E

    2001-10-15

    We have developed a protein design computer program, called Perla, which performs searches in sequence space to uncover optimal amino acid sequences for desired protein three-dimensional structures. Optimal sequences are localised at the minima of a sequence-structure energy landscape defined using a complex scoring function (an all-atom molecular mechanics force field plus statistical terms including entropy and solvation) measured with respect to a reference state simulating a denatured protein. Sequence choices eventually optimise side chain packing, secondary structure propensities, and hydrogen bonding and electrostatics interactions. Perla was used to re-design clusters of residues of the SH3 domain of alpha-spectrin. Several mutant proteins were produced and characterised. Some of our designed proteins have significantly higher stabilities (stability enhancements about 0.25, 0.70 and 1.0 kcal mol(-1)) than the wild-type protein. These successful protein re-designs, and similar examples found in the literature, establish the quality of the structure-based computational approach to protein design. PMID:11566604

  8. Sh3pxd2b Mice Are a Model for Craniofacial Dysmorphology and Otitis Media

    PubMed Central

    Yang, Bin; Tian, Cong; Zhang, Zhi-guang; Han, Feng-chan; Azem, Rami; Yu, Heping; Zheng, Ye; Jin, Ge; Arnold, James E.; Zheng, Qing Y.

    2011-01-01

    Craniofacial defects that occur through gene mutation during development increase vulnerability to eustachian tube dysfunction. These defects can lead to an increased incidence of otitis media. We examined the effects of a mutation in the Sh3pxd2b gene (Sh3pxd2bnee) on the progression of otitis media and hearing impairment at various developmental stages. We found that all mice that had the Sh3pxd2bnee mutation went on to develop craniofacial dysmorphologies and subsequently otitis media, by as early as 11 days of age. We found noteworthy changes in cilia and goblet cells of the middle ear mucosa in Sh3pxd2bnee mutant mice using scanning electronic microscopy. By measuring craniofacial dimensions, we determined for the first time in an animal model that this mouse has altered eustachian tube morphology consistent with a more horizontal position of the eustachian tube. All mutants were found to have hearing impairment. Expression of TNF-α and TLR2, which correlates with inflammation in otitis media, was up-regulated in the ears of mutant mice when examined by immunohistochemistry and semi-quantitative RT-PCR. The mouse model with a mutation in the Sh3pxd2b gene (Sh3pxd2bnee) mirrors craniofacial dysmorphology and otitis media in humans. PMID:21818352

  9. Sh3pxd2b mice are a model for craniofacial dysmorphology and otitis media.

    PubMed

    Yang, Bin; Tian, Cong; Zhang, Zhi-guang; Han, Feng-chan; Azem, Rami; Yu, Heping; Zheng, Ye; Jin, Ge; Arnold, James E; Zheng, Qing Y

    2011-01-01

    Craniofacial defects that occur through gene mutation during development increase vulnerability to eustachian tube dysfunction. These defects can lead to an increased incidence of otitis media. We examined the effects of a mutation in the Sh3pxd2b gene (Sh3pxd2b(nee)) on the progression of otitis media and hearing impairment at various developmental stages. We found that all mice that had the Sh3pxd2b(nee) mutation went on to develop craniofacial dysmorphologies and subsequently otitis media, by as early as 11 days of age. We found noteworthy changes in cilia and goblet cells of the middle ear mucosa in Sh3pxd2b(nee) mutant mice using scanning electronic microscopy. By measuring craniofacial dimensions, we determined for the first time in an animal model that this mouse has altered eustachian tube morphology consistent with a more horizontal position of the eustachian tube. All mutants were found to have hearing impairment. Expression of TNF-α and TLR2, which correlates with inflammation in otitis media, was up-regulated in the ears of mutant mice when examined by immunohistochemistry and semi-quantitative RT-PCR. The mouse model with a mutation in the Sh3pxd2b gene (Sh3pxd2b(nee)) mirrors craniofacial dysmorphology and otitis media in humans. PMID:21818352

  10. Functional phosphoproteomic analysis reveals cold-shock domain protein A to be a Bcr-Abl effector-regulating proliferation and transformation in chronic myeloid leukemia

    PubMed Central

    Sears, D; Luong, P; Yuan, M; Nteliopoulos, G; Man, Y K S; Melo, J V; Basu, S

    2010-01-01

    One proposed strategy to suppress the proliferation of imatinib-resistant cells in chronic myeloid leukemia (CML) is to inhibit key proteins downstream of Bcr-Abl. The PI3K/Akt pathway is activated by Bcr-Abl and is specifically required for the growth of CML cells. To identify targets of this pathway, we undertook a proteomic screen and identified several proteins that differentially bind 14-3-3, dependent on Bcr-Abl kinase activity. An siRNA screen of candidates selected by bioinformatics analysis reveals cold-shock domain protein A (CSDA), shown previously to regulate cell cycle progression in epithelial cells, to be a positive regulator of proliferation in a CML cell line. We show that Akt can phosphorylate the serine 134 residue of CSDA but, downstream of Bcr-Abl activity, this modification is mediated through the activation of MEK/p90 ribosomal S6 kinase (RSK) signaling. Inhibition of RSK, similarly to treatment with imatinib, blocked proliferation specifically in Bcr-Abl-positive leukemia cell lines, as well as cells from CML patients. Furthermore, these primary CML cells showed an increase in CSDA phosphorylation. Expression of a CSDA phospho-deficient mutant resulted in the decrease of Bcr-Abl-dependent transformation in Rat1 cells. Our results support a model whereby phosphorylation of CSDA downstream of Bcr-Abl enhances proliferation in CML cells to drive leukemogenesis. PMID:21368869

  11. BCR-ABL kinase domain mutations, including 2 novel mutations in imatinib resistant Malaysian chronic myeloid leukemia patients-Frequency and clinical outcome.

    PubMed

    Elias, Marjanu Hikmah; Baba, Abdul Aziz; Azlan, Husin; Rosline, Hassan; Sim, Goh Ai; Padmini, Menon; Fadilah, S Abdul Wahid; Ankathil, Ravindran

    2014-04-01

    Discovery of imatinib mesylate (IM) as the targeted BCR-ABL protein tyrosine kinase inhibitor (TKI) has resulted in its use as the frontline therapy for chronic myeloid leukemia (CML) across the world. Although high response rates are observed in CML patients who receive IM treatment, a significant number of patients develop resistance to IM. Resistance to IM in patients has been associated with a heterogeneous array of mechanisms of which point mutations within the ABL tyrosine kinase domain (TKD) are the frequently documented. The types and frequencies of mutations reported in different population studies have shown wide variability. We screened 125 Malaysian CML patients on IM therapy who showed either TKI refractory or resistance to IM to investigate the frequency and pattern of BCR-ABL kinase domain mutations among Malaysian CML patients undergoing IM therapy and to determine the clinical significance. Mutational screening using denaturing high performance liquid chromatography (dHPLC) followed by DNA sequencing was performed on 125 IM resistant Malaysian CML patients. Mutations were detected in 28 patients (22.4%). Fifteen different types of mutations (T315I, E255K, G250E, M351T, F359C, G251E, Y253H, V289F, E355G, N368S, L387M, H369R, A397P, E355A, D276G), including 2 novel mutations were identified, with T315I as the predominant type of mutation. The data generated from clinical and molecular parameters studied were correlated with the survival of CML patients. Patients with Y253H, M351T and E355G TKD mutations showed poorer prognosis compared to those without mutation. Interestingly, when the prognostic impact of the observed mutations was compared inter-individually, E355G and Y253H mutations were associated with more adverse prognosis and shorter survival (P=0.025 and 0.005 respectively) than T315I mutation. Results suggest that apart from those mutations occurring in the three crucial regions (catalytic domain, P-loop and activation-loop), other rare

  12. SH3BP2 is an activator of NFAT activity and osteoclastogenesis

    SciTech Connect

    Lietman, Steven A. Yin Lihong; Levine, Michael A.

    2008-07-11

    Heterozygous activating mutations in exon 9 of SH3BP2 have been found in most patients with cherubism, an unusual genetic syndrome characterized by excessive remodeling of the mandible and maxilla due to spontaneous and excessive osteoclastic bone resorption. Osteoclasts differentiate after binding of sRANKL to RANK induces a number of downstream signaling effects, including activation of the calcineurin/NFAT (nuclear factor of activated T cells) pathway. Here, we have investigated the functional significance of SH3BP2 protein on osteoclastogenesis in the presence of sRANKL. Our results indicate that SH3BP2 both increases nuclear NFATc1 in sRANKL treated RAW 264.7 preosteoclast cells and enhances expression of tartrate resistant acid phosphatase (TRAP), a specific marker of osteoclast differentiation. Moreover, overexpression of SH3BP2 in RAW 264.7 cells potentiates sRANKL-stimulated phosphorylation of PLC{gamma}1 and 2, thus providing a mechanistic pathway for the rapid translocation of NFATc1 into the nucleus and increased osteoclastogenesis in cherubism.

  13. Results of the flight noise measurement program using a standard and modified SH-3A helicopter

    NASA Technical Reports Server (NTRS)

    Pegg, R. J.; Henderson, H. R.; Hilton, D. A.

    1973-01-01

    A field noise measurement program has been conducted using both a standard SH-3A helicopter and an SH-3A helicopter modified to reduce external noise levels. Modifications included reducing rotor speed, increasing the number of rotor blades, modifying the blade-tip shapes, and acoustically treating the engine air intakes and exhaust. The purpose of this study was to document the noise characteristics recorded on the ground of each helicopter during flyby, hover, landing, and take-off operations. Based on an analysis of the measured results, the average of the overhead, overall, ontrack noise levels was approximately 4 db lower for the modified helicopter than for the standard helicopter. The improved in-flight noise characteristics, and associated small footprint areas and time durations, were judged to be mainly due to tail-rotor noise reductions. The noise reductions were obtained at the expense of required power increases at airspeeds greater than 70 knots for the modified helicopter.

  14. Angiotensin II down-regulates nephrin–Akt signaling and induces podocyte injury: roleof c-Abl

    PubMed Central

    Yang, Qian; Ma, Yiqiong; Liu, Yipeng; Liang, Wei; Chen, Xinghua; Ren, Zhilong; Wang, Huiming; Singhal, Pravin C.; Ding, Guohua

    2016-01-01

    Recent studies have shown that nephrin plays a vital role in angiotensin II (Ang II)–induced podocyte injury and thus contributes to the onset of proteinuria and the progression of renal diseases, but its specific mechanism remains unclear. c-Abl is an SH2/SH3 domain–containing nonreceptor tyrosine kinase that is involved in cell survival and regulation of the cytoskeleton. Phosphorylated nephrin is able to interact with molecules containing SH2/SH3 domains, suggesting that c-Abl may be a downstream molecule of nephrin signaling. Here we report that Ang II–infused rats developed proteinuria and podocyte damage accompanied by nephrin dephosphorylation and minimal interaction between nephrin and c-Abl. In vitro, Ang II induced podocyte injury and nephrin and Akt dephosphorylation, which occurred in tandem with minimal interaction between nephrin and c-Abl. Moreover, Ang II promoted c-Abl phosphorylation and interaction between c-Abl and SH2 domain–containing 5′-inositol phosphatase 2 (SHIP2). c-Abl small interfering RNA (siRNA) and STI571 (c-Abl inhibitor) provided protection against Ang II–induced podocyte injury, suppressed the Ang II-induced c-Abl–SHIP2 interaction and SHIP2 phosphorylation, and maintained a stable level of nephrin phosphorylation. These results indicate that c-Abl is a molecular chaperone of nephrin signaling and the SHIP2-Akt pathway and that the released c-Abl contributes to Ang II–induced podocyte injury. PMID:26510503

  15. BAR-SH3 sorting nexins are conserved interacting proteins of Nervous wreck that organize synapses and promote neurotransmission

    PubMed Central

    Ukken, Fiona P.; Bruckner, Joseph J.; Weir, Kurt L.; Hope, Sarah J.; Sison, Samantha L.; Birschbach, Ryan M.; Hicks, Lawrence; Taylor, Kendra L.; Dent, Erik W.; Gonsalvez, Graydon B.; O'Connor-Giles, Kate M.

    2016-01-01

    ABSTRACT Nervous wreck (Nwk) is a conserved F-BAR protein that attenuates synaptic growth and promotes synaptic function in Drosophila. In an effort to understand how Nwk carries out its dual roles, we isolated interacting proteins using mass spectrometry. We report a conserved interaction between Nwk proteins and BAR-SH3 sorting nexins, a family of membrane-binding proteins implicated in diverse intracellular trafficking processes. In mammalian cells, BAR-SH3 sorting nexins induce plasma membrane tubules that localize NWK2, consistent with a possible functional interaction during the early stages of endocytic trafficking. To study the role of BAR-SH3 sorting nexins in vivo, we took advantage of the lack of genetic redundancy in Drosophila and employed CRISPR-based genome engineering to generate null and endogenously tagged alleles of SH3PX1. SH3PX1 localizes to neuromuscular junctions where it regulates synaptic ultrastructure, but not synapse number. Consistently, neurotransmitter release was significantly diminished in SH3PX1 mutants. Double-mutant and tissue-specific-rescue experiments indicate that SH3PX1 promotes neurotransmitter release presynaptically, at least in part through functional interactions with Nwk, and might act to distinguish the roles of Nwk in regulating synaptic growth and function. PMID:26567222

  16. Fitness Conferred by BCR-ABL Kinase Domain Mutations Determines the Risk of Pre-Existing Resistance in Chronic Myeloid Leukemia

    PubMed Central

    Skaggs, Brian; Gorre, Mercedes; Sawyers, Charles L.; Michor, Franziska

    2011-01-01

    Chronic myeloid leukemia (CML) is the first human malignancy to be successfully treated with a small molecule inhibitor, imatinib, targeting a mutant oncoprotein (BCR-ABL). Despite its successes, acquired resistance to imatinib leads to reduced drug efficacy and frequent progression of disease. Understanding the characteristics of pre-existing resistant cells is important for evaluating the benefits of first-line combination therapy with second generation inhibitors. However, due to limitations of assay sensitivity, determining the existence and characteristics of resistant cell clones at the start of therapy is difficult. Here we combined a mathematical modeling approach using branching processes with experimental data on the fitness changes (i.e., changes in net reproductive rate) conferred by BCR-ABL kinase domain mutations to investigate the likelihood, composition, and diversity of pre-existing resistance. Furthermore, we studied the impact of these factors on the response to tyrosine kinase inhibitors. Our approach predicts that in most patients, there is at most one resistant clone present at the time of diagnosis of their disease. Interestingly, patients are no more likely to harbor the most aggressive, pan-resistant T315I mutation than any other resistance mutation; however, T315I cells on average establish larger-sized clones at the time of diagnosis. We established that for patients diagnosed late, the relative benefit of combination therapy over monotherapy with imatinib is significant, while this benefit is modest for patients with a typically early diagnosis time. These findings, after pre-clinical validation, will have implications for the clinical management of CML: we recommend that patients with advanced-phase disease be treated with combination therapy with at least two tyrosine kinase inhibitors. PMID:22140458

  17. Skb5, an SH3 adaptor protein, regulates Pmk1 MAPK signaling by controlling the intracellular localization of the MAPKKK Mkh1.

    PubMed

    Kanda, Yuki; Satoh, Ryosuke; Matsumoto, Saki; Ikeda, Chisato; Inutsuka, Natsumi; Hagihara, Kanako; Matzno, Sumio; Tsujimoto, Sho; Kita, Ayako; Sugiura, Reiko

    2016-08-15

    The mitogen-activated protein kinase (MAPK) cascade is a highly conserved signaling module composed of MAPK kinase kinases (MAPKKKs), MAPK kinases (MAPKK) and MAPKs. The MAPKKK Mkh1 is an initiating kinase in Pmk1 MAPK signaling, which regulates cell integrity in fission yeast (Schizosaccharomyces pombe). Our genetic screen for regulators of Pmk1 signaling identified Shk1 kinase binding protein 5 (Skb5), an SH3-domain-containing adaptor protein. Here, we show that Skb5 serves as an inhibitor of Pmk1 MAPK signaling activation by downregulating Mkh1 localization to cell tips through its interaction with the SH3 domain. Consistent with this, the Mkh1(3PA) mutant protein, with impaired Skb5 binding, remained in the cell tips, even when Skb5 was overproduced. Intriguingly, Skb5 needs Mkh1 to localize to the growing ends as Mkh1 deletion and disruption of Mkh1 binding impairs Skb5 localization. Deletion of Pck2, an upstream activator of Mkh1, impaired the cell tip localization of Mkh1 and Skb5 as well as the Mkh1-Skb5 interaction. Interestingly, both Pck2 and Mkh1 localized to the cell tips at the G1/S phase, which coincided with Pmk1 MAPK activation. Taken together, Mkh1 localization to cell tips is important for transmitting upstream signaling to Pmk1, and Skb5 spatially regulates this process. PMID:27451356

  18. A Discovery Strategy for Selective Inhibitors of c-Src in Complex with the Focal Adhesion Kinase SH3/SH2-binding Region

    PubMed Central

    Moroco, Jamie A.; Baumgartner, Matthew P.; Rust, Heather L.; Choi, Hwan Geun; Hur, Wooyoung; Gray, Nathanael S.; Camacho, Carlos J.; Smithgall, Thomas E.

    2015-01-01

    The c-Src tyrosine kinase co-operates with the focal adhesion kinase to regulate cell adhesion and motility. Focal adhesion kinase engages the regulatory SH3 and SH2 domains of c-Src, resulting in localized kinase activation that contributes to tumor cell metastasis. Using assay conditions where c-Src kinase activity required binding to a tyrosine phosphopeptide based on the focal adhesion kinase SH3-SH2 docking sequence, we screened a kinase-biased library for selective inhibitors of the Src/focal adhesion kinase peptide complex versus c-Src alone. This approach identified an aminopyrimidinyl carbamate compound, WH-4-124-2, with nanomolar inhibitory potency and fivefold selectivity for c-Src when bound to the phospho-focal adhesion kinase peptide. Molecular docking studies indicate that WH-4-124-2 may preferentially inhibit the ‘DFG-out’ conformation of the kinase active site. These findings suggest that interaction of c-Src with focal adhesion kinase induces a unique kinase domain conformation amenable to selective inhibition. PMID:25376742

  19. c-Abl kinase regulates the protein binding activity of c-Crk.

    PubMed Central

    Feller, S M; Knudsen, B; Hanafusa, H

    1994-01-01

    c-Crk is a proto-oncogene product composed largely of Src homology (SH) 2 and 3 domains. We have identified a kinase activity, which binds to the first Crk SH3 domain and phosphorylates c-Crk on tyrosine 221 (Y221), as c-Abl. c-Abl has a strong preference for c-Crk, when compared with common tyrosine kinase substrates. The phosphorylation of c-Crk Y221 creates a binding site for the Crk SH2 domain. Bacterially expressed c-Crk protein lacks phosphorylation on Y221 and can bind specifically to several proteins, while mammalian c-Crk, which is phosphorylated on tyrosine, remains uncomplexed. The protein binding activity of c-Crk is therefore likely regulated by a mechanism similar to that of the Src family kinases. v-Crk is truncated before c-Crk Y221 and forms constitutive complexes with c-Abl and other proteins. Our results suggest that c-Abl regulates c-Crk function and that it could be involved in v-Crk transformation. Images PMID:8194526

  20. Characterizing of Four Common BCR-ABL Kinase Domain Mutations (T315I, Y253H, M351T and E255K) in Iranian Chronic Myelogenous Leukemia Patients With Imatinib Resistance

    PubMed Central

    Rejali, Leili; Poopak, Behzad; Hasanzad, Mandana; Sheikhsofla, Fatemeh; Varnoosfaderani, Ameneh Saadat; Safari, Nazila; Rabieipoor, Saghar

    2015-01-01

    Background: Chronic myelogenous leukemia (CML) is a kind of hematopoietic stem-cell cancer. A significant number of CML patients who do not achieve an acceptable response to therapy, show acquired resistance against Imatinib. One of the most considerable causes of resistance against Imatinib as the first line of therapy, are BCR-ABL kinase domain mutations. Objectives: One of the most considerable causes of resistance against Imatinib as the first line of therapy, are BCR-ABL kinase domain mutations. Patients and Methods: The study was performed on 39 CML patients with Imatinib resistance. Basic hematologic parameters in blood samples were checked to identify hematologic response. To identify molecular response, BCR-ABL/ABL ratio was assessed by Real-time PCR. The ABL kinase domain amplification was performed by PCR. Restriction fragment length polymorphism (RFLP) was performed to detect four common mutations (T315I, Y253H, E255K and M351T). Finally the results were approved by direct sequencing. Results: In this study, the Y253H mutation, detected by RFLP method and confirmed by direct sequencing, was the prevalent ABL kinase domain mutation in these 39 CML patients. The G250E, V379I and L384M mutations were found in three different cases with failure molecular response. CML patients with these four ABL kinase domain mutations cannot achieve major molecular response (MMR). In addition, complete hematologic response (CHR) was observed only in the V379I mutated case and not in other mutated patients. Conclusions: Identification of ABL kinase domain mutations may be used as a proper and useful method for improving therapeutic strategies, avoiding delay in treatment and excessive expenditure in CML patients with Imatinib resistance. PMID:26413254

  1. An SH3 binding motif within the nucleocapsid protein of porcine reproductive and respiratory syndrome virus interacts with the host cellular signaling proteins STAMI, TXK, Fyn, Hck, and cortactin.

    PubMed

    Kenney, Scott P; Meng, Xiang-Jin

    2015-06-01

    Porcine reproductive and respiratory syndrome virus (PRRSV) causes an economically important global swine disease, and has a complicated virus-host immunomodulation that often leads to a weak Th2 immune response and viral persistence. In this study, we identified a Src homology 3 (SH3) binding motif, PxxPxxP, that is conserved within the N protein of PRRSV strains. Subsequently, we identified five host cellular proteins [signal transducing adaptor molecule (STAM)I, TXK tyrosine kinase (TXK), protein tyrosine kinase fyn (Fyn), hematopoietic cell kinase (Hck), and cortactin] that interact with this SH3 motif. We demonstrated that binding of SH3 proteins with PRRSV N protein depends on at least one intact PxxP motif as disruption of P53 within the motif significantly reduced interaction of each of the 5 proteins. The first PxxP motif appears to be more important for STAMI-N protein interactions whereas the second PxxP motif was more important for Hck interaction. Both STAMI and Hck interactions with PRRSV N protein required an unhindered C-terminal domain as the interaction was only observed with STAMI and Hck proteins with N-terminal but not C-terminal fluorescent tags. We showed that the P56 residue within the SH3 motif is critical for virus lifecycle as mutation resulted in a loss of virus infectivity, however the P50 and P53 mutations did not abolish virus infectivity suggesting that these highly conserved proline residues within the SH3 motif may provide a selective growth advantage through interactions with the host rather than a vital functional element. These results have important implications in understanding PRRSV-host interactions. PMID:25882913

  2. Phylogeny of Tec family kinases identification of a premetazoan origin of Btk, Bmx, Itk, Tec, Txk, and the Btk regulator SH3BP5.

    PubMed

    Ortutay, Csaba; Nore, Beston F; Vihinen, Mauno; Smith, C I Edvard

    2008-01-01

    It is generally considered mammals and birds have five Tec family kinases (TFKs): Btk, Bmx (also known as Etk), Itk, Tec, and Txk (also known as Rlk). Here, we discuss the domains and their functions and regulation in TFKs. Over the last few years, a large number of genomes from various phyla have been sequenced making it possible to study evolutionary relationships at the molecular and sequence level. Using bioinformatics tools, we for the first time demonstrate that a TFK ancestor exists in the unicellular choanoflagellate Monosiga brevicollis, which is the closest known relative to metazoans with a sequenced genome. The analysis of the genomes for sponges, insects, hagfish, and frogs suggests that these species encode a single TFK. The insect form has a divergent and unique N-terminal region. Duplications generating the five members took place prior to the emergence of vertebrates. Fishes have two or three forms and the platypus, Ornithorhynchus anatinus, has four (lacks Txk). Thus, not all mammals have all five TFKs. The single identified TFK in frogs is an ortholog of Tec. Bmx seems to be unique to mammals and birds. SH3BP5 is a negative regulator of Btk. It is conserved in choanoflagellates and interestingly exists also in nematodes, which do not express TFKs, suggesting a broader function in addition to Btk regulation. The related SH3BP5-like protein is not found in Nematodes. PMID:19161832

  3. SH3BP2 Gain-Of-Function Mutation Exacerbates Inflammation and Bone Loss in a Murine Collagen-Induced Arthritis Model

    PubMed Central

    Mukai, Tomoyuki; Gallant, Richard; Ishida, Shu; Yoshitaka, Teruhito; Kittaka, Mizuho; Nishida, Keiichiro; Fox, David A.; Morita, Yoshitaka; Ueki, Yasuyoshi

    2014-01-01

    Objective SH3BP2 is a signaling adapter protein which regulates immune and skeletal systems. Gain-of-function mutations in SH3BP2 cause cherubism, characterized by jawbone destruction. This study was aimed to examine the role of SH3BP2 in inflammatory bone loss using a collagen-induced arthritis (CIA) model. Methods CIA was induced in wild-type (Sh3bp2+/+) and heterozygous P416R SH3BP2 cherubism mutant knock-in (Sh3bp2KI/+) mice, an SH3BP2 gain-of-function model. Severity of the arthritis was determined by assessing the paw swelling and histological analyses of the joints. Micro-CT analysis was used to determine the levels of bone loss. Inflammation and osteoclastogenesis in the joints were evaluated by quantitating the gene expression of inflammatory cytokines and osteoclast markers. Furthermore, involvement of the T- and B-cell responses was determined by draining lymph node cell culture and measurement of the serum anti-mouse type II collagen antibody levels, respectively. Finally, roles of the SH3BP2 mutation in macrophage activation and osteoclastogenesis were determined by evaluating the TNF-α production levels and osteoclast formation in bone marrow-derived M-CSF-dependent macrophage (BMM) cultures. Results Sh3bp2KI/+ mice exhibited more severe inflammation and bone loss, accompanying an increased number of osteoclasts. The mRNA levels for TNF-α and osteoclast marker genes were higher in the joints of Sh3bp2KI/+ mice. Lymph node cell culture showed that lymphocyte proliferation and IFN-γ and IL-17 production were comparable between Sh3bp2+/+ and Sh3bp2KI/+ cells. Serum anti-type II collagen antibody levels were comparable between Sh3bp2+/+ and Sh3bp2KI/+ mice. In vitro experiments showed that TNF-α production in Sh3bp2KI/+ BMMs is elevated compared with Sh3bp2+/+ BMMs and that RANKL-induced osteoclastogenesis is enhanced in Sh3bp2KI/+ BMMs associated with increased NFATc1 nuclear localization. Conclusion Gain-of-function of SH3BP2 augments inflammation

  4. Epithelial junction formation requires confinement of Cdc42 activity by a novel SH3BP1 complex

    PubMed Central

    Elbediwy, Ahmed; Zihni, Ceniz; Terry, Stephen J.; Clark, Peter

    2012-01-01

    Epithelial cell–cell adhesion and morphogenesis require dynamic control of actin-driven membrane remodeling. The Rho guanosine triphosphatase (GTPase) Cdc42 regulates sequential molecular processes during cell–cell junction formation; hence, mechanisms must exist that inactivate Cdc42 in a temporally and spatially controlled manner. In this paper, we identify SH3BP1, a GTPase-activating protein for Cdc42 and Rac, as a regulator of junction assembly and epithelial morphogenesis using a functional small interfering ribonucleic acid screen. Depletion of SH3BP1 resulted in loss of spatial control of Cdc42 activity, stalled membrane remodeling, and enhanced growth of filopodia. SH3BP1 formed a complex with JACOP/paracingulin, a junctional adaptor, and CD2AP, a scaffolding protein; both were required for normal Cdc42 signaling and junction formation. The filamentous actin–capping protein CapZ also associated with the SH3BP1 complex and was required for control of actin remodeling. Epithelial junction formation and morphogenesis thus require a dual activity complex, containing SH3BP1 and CapZ, that is recruited to sites of active membrane remodeling to guide Cdc42 signaling and cytoskeletal dynamics. PMID:22891260

  5. BCR-ABL1 compound mutations combining key kinase domain positions confer clinical resistance to ponatinib in Ph chromosome-positive leukemia.

    PubMed

    Zabriskie, Matthew S; Eide, Christopher A; Tantravahi, Srinivas K; Vellore, Nadeem A; Estrada, Johanna; Nicolini, Franck E; Khoury, Hanna J; Larson, Richard A; Konopleva, Marina; Cortes, Jorge E; Kantarjian, Hagop; Jabbour, Elias J; Kornblau, Steven M; Lipton, Jeffrey H; Rea, Delphine; Stenke, Leif; Barbany, Gisela; Lange, Thoralf; Hernández-Boluda, Juan-Carlos; Ossenkoppele, Gert J; Press, Richard D; Chuah, Charles; Goldberg, Stuart L; Wetzler, Meir; Mahon, Francois-Xavier; Etienne, Gabriel; Baccarani, Michele; Soverini, Simona; Rosti, Gianantonio; Rousselot, Philippe; Friedman, Ran; Deininger, Marie; Reynolds, Kimberly R; Heaton, William L; Eiring, Anna M; Pomicter, Anthony D; Khorashad, Jamshid S; Kelley, Todd W; Baron, Riccardo; Druker, Brian J; Deininger, Michael W; O'Hare, Thomas

    2014-09-01

    Ponatinib is the only currently approved tyrosine kinase inhibitor (TKI) that suppresses all BCR-ABL1 single mutants in Philadelphia chromosome-positive (Ph(+)) leukemia, including the recalcitrant BCR-ABL1(T315I) mutant. However, emergence of compound mutations in a BCR-ABL1 allele may confer ponatinib resistance. We found that clinically reported BCR-ABL1 compound mutants center on 12 key positions and confer varying resistance to imatinib, nilotinib, dasatinib, ponatinib, rebastinib, and bosutinib. T315I-inclusive compound mutants confer high-level resistance to TKIs, including ponatinib. In vitro resistance profiling was predictive of treatment outcomes in Ph(+) leukemia patients. Structural explanations for compound mutation-based resistance were obtained through molecular dynamics simulations. Our findings demonstrate that BCR-ABL1 compound mutants confer different levels of TKI resistance, necessitating rational treatment selection to optimize clinical outcome. PMID:25132497

  6. Loss of SH3BP2 function suppresses bone destruction in TNF-driven and collagen-induced arthritis mouse models

    PubMed Central

    Mukai, Tomoyuki; Gallant, Richard; Ishida, Shu; Kittaka, Mizuho; Yoshitaka, Teruhito; Fox, David A.; Morita, Yoshitaka; Nishida, Keiichiro; Rottapel, Robert; Ueki, Yasuyoshi

    2014-01-01

    Objective SH3BP2 is a signaling adapter protein which regulates immune and skeletal systems. The purpose of this study was to investigate the role of SH3BP2 in arthritis in human TNF-α transgenic (hTNFtg) and collagen-induced arthritis (CIA) models. Methods First, SH3BP2-deficient (Sh3bp2–/–) and wild-type (Sh3bp2+/+) mice were crossed with hTNFtg mice. Inflammation and bone loss were examined by clinical inspection and histological and micro-CT analyses. Osteoclastogenesis was evaluated with primary bone marrow-derived M-CSF-dependent macrophages (BMMs). Second, CIA was induced in Sh3bp2–/– and Sh3bp2+/+ mice, and the incidence and severity of arthritis were evaluated. Anti-mouse type II collagen (CII) antibody levels were measured by ELISA. Lymph node cell responses to CII were also determined. Results SH3BP2-deficiency did not alter the severity of joint swelling but suppressed bone erosion in the hTNFtg model. Bone loss of talus and tibia was prevented in Sh3bp2–/–/hTNFtg mice compared to Sh3bp2+/+/hTNFtg mice. RANKL- and TNF-α-induced osteoclastogenesis was suppressed in Sh3bp2–/– BMM cultures. NFATc1 nuclear localization in response to TNF-α was decreased in Sh3bp2–/– BMMs compared to Sh3bp2+/+ BMMs. In the CIA model, SH3BP2-deficiency suppressed the incidence of arthritis, which was associated with decreased anti-CII antibody production, while the antigen-specific T-cell responses in lymph nodes were not significantly different between Sh3bp2+/+ and Sh3bp2–/– mice. Conclusion SH3BP2-deficiency prevents bone loss via impaired osteoclastogenesis in the hTNFtg model and suppresses the induction of arthritis via decreased autoantibody production in the CIA model. Therefore, SH3BP2 could be a therapeutic target for rheumatoid arthritis. PMID:25470448

  7. Is Spectral-Domain Optical Coherence Tomography Always Able to Detect the Anti-Vascular Endothelial Growth Factor Action on Neovascular Membrane?

    PubMed Central

    Borgia, Luigi; Del Noce, Chiara; Iester, Michele

    2016-01-01

    Purpose To show the presence of an active neovascular membrane in age-related macular degeneration even if optical coherence tomography (OCT) does not detect intra- or subretinal edema. Methods This is a retrospective case report. During the follow-up after the intravitreal injection, 3 patients showed no intraretinal or subretinal edema by OCT; however, there was a progressive reduction in their visual acuity; thus, a fluorangiography (FA) examination was performed. Results In these 3 cases, FA showed an active neovascular network. Conclusion OCT could show a real reduction in the edema, but it is not always able to detect neovessel presence. Intravitreal injection could improve the vessel permeability without care and delete the neovascular network. PMID:27462260

  8. SH2 domains prevent tyrosine dephosphorylation of the EGF receptor: identification of Tyr992 as the high-affinity binding site for SH2 domains of phospholipase C gamma.

    PubMed Central

    Rotin, D; Margolis, B; Mohammadi, M; Daly, R J; Daum, G; Li, N; Fischer, E H; Burgess, W H; Ullrich, A; Schlessinger, J

    1992-01-01

    Several cytoplasmic tyrosine kinases contain a conserved, non-catalytic stretch of approximately 100 amino acids called the src homology 2 (SH2) domain, and a region of approximately 50 amino acids called the SH3 domain. SH2/SH3 domains are also found in several other proteins, including phospholipase C-gamma (PLC gamma). Recent studies indicate that SH2 domains promote association between autophosphorylated growth factor receptors such as the epidermal growth factor (EGF) receptor and signal transducing molecules such as PLC gamma. Because SH2 domains bind specifically to protein sequences containing phosphotyrosine, we examined their capacity to prevent tyrosine dephosphorylation of the EGF and other receptors with tyrosine kinase activity. For this purpose, various SH2/SH3 constructs of PLC gamma were expressed in Escherichia coli as glutathione-S-transferase fusion proteins. Our results show that purified SH2 domains of PLC gamma are able to prevent tyrosine dephosphorylation of the EGF receptor and other receptors with tyrosine activity. The inhibition of tyrosine dephosphorylation paralleled the capacity of various SH2-containing constructs to bind to the EGF receptor, suggesting that the tyrosine phosphatase and the SH2 domain compete for the same tyrosine phosphorylation sites in the carboxy-terminal tail of the EGF receptor. Analysis of the phosphorylation sites protected from dephosphorylation by PLC gamma-SH2 revealed substantial inhibition of dephosphorylation of Tyr992 at 1 microM SH2. This indicates that Tyr992 and its flanking sequence is the high-affinity binding site for SH2 domains of PLC gamma.(ABSTRACT TRUNCATED AT 250 WORDS) Images PMID:1537335

  9. Anterior Segment Dysgenesis and Early-Onset Glaucoma in nee Mice with Mutation of Sh3pxd2b

    PubMed Central

    Mao, Mao; Hedberg-Buenz, Adam; Koehn, Demelza; John, Simon W. M.

    2011-01-01

    Purpose. Mutations in SH3PXD2B cause Frank-Ter Haar syndrome, a rare condition characterized by congenital glaucoma, as well as craniofacial, skeletal, and cardiac anomalies. The nee strain of mice carries a spontaneously arising mutation in Sh3pxd2b. The purpose of this study was to test whether nee mice develop glaucoma. Methods. Eyes of nee mutants and strain-matched controls were comparatively analyzed at multiple ages by slit lamp examination, intraocular pressure recording, and histologic analysis. Cross sections of the optic nerve were analyzed to confirm glaucomatous progression. Results. Slit lamp examination showed that, from an early age, nee mice uniformly exhibited severe iridocorneal adhesions around the entire circumference of the eye. Presumably as a consequence of aqueous humor outflow blockage, they rapidly developed multiple indices of glaucoma. By 3 to 4 months of age, they exhibited high intraocular pressure (30.8 ± 12.5 mm Hg; mean ± SD), corneal opacity, and enlarged anterior chambers. Although histologic analyses at P17 did not reveal any indices of damage, similar analysis at 3 to 4 months of age revealed a course of progressive retinal ganglion cell loss, optic nerve head excavation, and axon loss. Conclusions. Eyes of nee mice exhibit anterior segment dysgenesis and early-onset glaucoma. Because SH3PXD2B is predicted to be a podosome adaptor protein, these findings implicate podosomes in normal development of the iridocorneal angle and the genes influencing podosomes as candidates in glaucoma. Because of the early-onset, high-penetrance glaucoma, nee mice offer many potential advantages as a new mouse model of the disease. PMID:21282566

  10. SH3BP2 cherubism mutation potentiates TNF-α-induced osteoclastogenesis via NFATc1 and TNF-α-mediated inflammatory bone loss

    PubMed Central

    Mukai, Tomoyuki; Ishida, Shu; Ishikawa, Remi; Yoshitaka, Teruhito; Kittaka, Mizuho; Gallant, Richard; Lin, Yi-Ling; Rottapel, Robert; Brotto, Marco; Reichenberger, Ernst J.; Ueki, Yasuyoshi

    2014-01-01

    Cherubism (OMIM#118400) is a genetic disorder with excessive jawbone resorption caused by mutations in the signaling adaptor protein SH3BP2. Studies on the mouse model for cherubism carrying a P416R knock-in mutation have revealed that mutant SH3BP2 enhances TNF-α production and RANKL-induced osteoclast differentiation in myeloid cells. TNF-α is expressed in human cherubism lesions, which contain a large number of TRAP-positive multinucleated cells, and TNF-α plays a critical role in inflammatory bone destruction in homozygous cherubism mice (Sh3bp2KI/KI). The data suggest a pathophysiological relationship between mutant SH3BP2 and TNF-α-mediated bone loss by osteoclasts. Therefore, we investigated whether P416R mutant SH3BP2 is involved in TNF-α-mediated osteoclast formation and bone loss. Here, we show that bone marrow-derived M-CSF-dependent macrophages (BMMs) from the heterozygous cherubism mutant (Sh3bp2KI/+) mice are highly responsive to TNF-α and can differentiate into osteoclasts independently of RANKL in vitro by a mechanism that involves SYK and PLCγ2 phosphorylation, leading to increased nuclear translocation of NFATc1. The heterozygous cherubism mutation exacerbates bone loss with increased osteoclast formation in a mouse calvarial TNF-α injection model as well as in a human TNF-α transgenic mouse model (hTNFtg). SH3BP2 knockdown in RAW264.7 cells results in decreased TRAP-positive multinucleated cell formation. These findings suggest that the SH3BP2 cherubism mutation can cause jawbone destruction by promoting osteoclast formation in response to TNF-α expressed in cherubism lesions and that SH3BP2 is a key regulator for TNF-α-induced osteoclastogenesis. Inhibition of SH3BP2 expression in osteoclast progenitors could be a potential strategy for the treatment of bone loss in cherubism as well as in other inflammatory bone disorders. PMID:24916406

  11. Monkey Able After Recovery

    NASA Technical Reports Server (NTRS)

    1959-01-01

    On May 28, 1959, a Jupiter Intermediate Range Ballistic Missile provided by a U.S. Army team in Redstone Arsenal, Alabama, launched a nose cone carrying Baker, A South American squirrel monkey and Able, An American-born rhesus monkey. This photograph shows Able after recovery of the nose cone of the Jupiter rocket by U.S.S. Kiowa.

  12. Expressions of SH3BP5, LMO3, and SNAP25 in diffuse large B-cell lymphoma cells and their association with clinical features.

    PubMed

    Kobayashi, Kyoko; Yamaguchi, Motoko; Miyazaki, Kana; Imai, Hiroshi; Yokoe, Kaori; Ono, Ryoichi; Nosaka, Tetsuya; Katayama, Naoyuki

    2016-08-01

    Diffuse large B-cell lymphoma (DLBCL) is clinicopathologically and genetically heterogeneous with variable clinical outcomes. We previously identified signature genes overexpressed in CD5-positive (CD5(+) ) DLBCL, which is a poor prognostic subgroup of DLBCL. To elucidate the clinical significance of the protein expression of the signature genes overexpressed in CD5(+) DLBCL with regard to all DLBCL, not otherwise specified (NOS), 10 genes (SH3BP5, LMO3, SNAP25, SYT5, SV2C, CABP1, FGF1, FGFR2, NEUROD1, and SYN2) were selected and examined immunohistochemically with samples from 28 patients with DLBCL, NOS. Only three protein expressions, SH3BP5, LMO3, and SNAP25, were detected in DLBCL cells and then analyzed further with samples from 187 patients with DLBCL, NOS. The SH3BP5, LMO3, and SNAP25 proteins were expressed in 60% (103/173), 34% (59/175), and 46% (77/168) of DLBCL patients, respectively. These protein expressions were associated with CD5 expression, and only SH3BP5 was frequently expressed in activated B-cell-like DLBCL (P = 0.046). Compared to the SH3BP5-negative group, the SH3BP5(+) group was correlated with elderly onset (>60 years, P = 0.0096) and advanced-stage disease (stage III/IV, P = 0.037). The LMO3(+) group showed a worse performance status (>1, P = 0.0004). The SH3BP5(+) group and the LMO3(+) group had significantly worse overall survival than the negative groups (P = 0.030, 0.034; respectively) for the entire group. In a subgroup analysis of patients treated with rituximab-containing chemotherapy, there was no significant difference between groups. To the best of our knowledge, this is the first report showing the protein expressions of SH3BP5, LMO3, and SNAP25 in DLBCL cells and their clinical significance in patients with DLBCL. The SH3BP5 and LMO3 protein expressions are associated with the baseline clinical characteristics of DLBCL. PMID:27184832

  13. Reciprocal regulation of Abl kinase by Crk Y251 and Abi1 controls invasive phenotypes in glioblastoma

    PubMed Central

    Kumar, Sushil; Lu, Bin; Dixit, Updesh; Hossain, Sajjad; Liu, Yongzhang; Li, Jing; Hornbeck, Peter; Zheng, Weiming; Sowalsky, Adam G.; Kotula, Leszek; Birge, Raymond B.

    2015-01-01

    Crk is the prototypical member of a class of Src homology 2 (SH2) and Src homology 3 (SH3) domain-containing adaptor proteins that positively regulate cell motility via the activation of Rac1 and, in certain tumor types such as GBM, can promote cell invasion and metastasis by mechanisms that are not well understood. Here we demonstrate that Crk, via its phosphorylation at Tyr251, promotes invasive behavior of tumor cells, is a prominent feature in GBM, and correlating with aggressive glioma grade IV staging and overall poor survival outcomes. At the molecular level, Tyr251 phosphorylation of Crk is negatively regulated by Abi1, which competes for Crk binding to Abl and attenuates Abl transactivation. Together, these results show that Crk and Abi1 have reciprocal biological effects and act as a molecular rheostat to control Abl activation and cell invasion. Finally, these data suggest that Crk Tyr251 phosphorylation regulate invasive cell phenotypes and may serve as a biomarker for aggressive GBM. PMID:26473374

  14. SNP rs1049430 in the 3'-UTR of SH3GL2 regulates its expression: Clinical and prognostic implications in head and neck squamous cell carcinoma.

    PubMed

    Maiti, Guru Prasad; Ghosh, Amlan; Mondal, Pinaki; Baral, Aradhita; Datta, Sayantan; Samadder, Sudip; Nayak, Sandeep P; Chakrabarti, Jayanta; Biswas, Jaydeep; Sikdar, Nilabja; Chowdhury, Shantanu; Roy, Bidyut; Roychowdhury, Susanta; Panda, Chinmay Kumar

    2015-05-01

    Single nucleotide polymorphisms (SNPs) in the 3'-UTR region are emerging cis-regulatory factors associated with the occurrences of several human diseases. SH3GL2, which is located at chromosome 9p21-22, is associated with hyperplastic/mildly dysplastic lesions of the head and neck and has a long 3'-UTR with multiple SNPs. The aim of the present study was to determine the susceptible allele(s) in the 3'-UTR SNPs of SH3GL2 in head and neck squamous cell carcinoma (HNSCC). First, we screened the genotypes of all SNPs located in the 3'-UTR of SH3GL2 in 110 controls and 147 cases in Indian populations by sequencing. A SNP (rs1049430:>G/T) that showed only heterozygosity was further confirmed by genotyping with an Illumina GoldenGate platform in 530 controls and 764 cases. Genotype-specific survival analysis of the HNSCC patients was performed. In addition, genotype-specific mRNA stability, isoform expression and protein expression were analyzed. SNP rs1049430 was not associated with disease occurrence, but it was associated with poor patient outcome. The G allele was associated with decreased SH3GL2 mRNA stability, differential splicing and low protein expression. Thus, our data demonstrate that the presence of the susceptible G allele in SNP rs1049430 is associated with the inactivation of SH3GL2 and could be used as a prognostic marker of HNSCC. PMID:25728707

  15. Cyclophilin A promotes cell migration via the Abl-Crk signaling pathway

    PubMed Central

    Saleh, Tamjeed; Jankowski, Wojciech; Sriram, Ganapathy; Rossi, Paolo; Shah, Shreyas; Lee, Ki-Bum; Cruz, Lissette Alicia; Rodriguez, Alexis J.; Birge, Raymond B.

    2015-01-01

    Summary Cyclophilin A (CypA) is over-expressed in a number of human cancer types, but the mechanisms by which CypA promotes oncogenic properties of cells are not understood. Here we demonstrate that CypA binds to and prevents the CrkII adaptor protein from switching to the inhibited state. CrkII is involved in cell motility and invasion by mediating signaling through its SH2 and SH3 domains. CrkII Tyr221 phosphorylation by the Abl or EGFR kinases induces an inhibited state of CrkII, by means of an intramolecular SH2-pTyr221 interaction, causing signaling interruption. We show that the CrkII phosphorylation site constitutes a binging site for CypA. Recruitment of CypA sterically restricts the accessibility of Tyr221 to kinases, thereby suppressing CrkII phosphorylation and promoting the active state. Structural, biophysical, and in vivo data show that CypA augments CrkII-mediated signaling. A strong stimulation of cell migration is observed in cancer cells wherein both CypA and CrkII are greatly up-regulated. PMID:26656091

  16. Detailed conformation dynamics and activation process of wild type c-Abl and T315I mutant

    NASA Astrophysics Data System (ADS)

    Yang, Li-Jun; Zhao, Wen-Hua; Liu, Qian

    2014-10-01

    Bcr-Abl is an important target for therapy against chronic myelogenous leukemia (CML) and acute lymphocytic leukemia (ALL). The synergistic effect between myristyl pocket and the ATP pocket has been found. But its detailed information based on molecular level still has not been achieved. In this study, conventional molecular dynamics (CMD) and target molecular dynamics (TMD) simulations were performed to explore the effect of T315I mutation on dynamics and activation process of Abl containing the N-terminal cap (Ncap). The CMD simulation results reveal the increasing flexibility of ATP pocket in kinase domain (KD) after T315I mutation which confirms the disability of ATP-pocket inhibitors to the Abl-T315I mutant. On the contrary, the T315I mutation decreased the flexibility of remote helix αI which suggests the synergistic effect between them. The mobility of farther regions containing Ncap, SH3, SH2 and SH2-KD linker were not affected by T315I mutation. The TMD simulation results show that the activation process of wild type Abl and Abl-T315I mutant experienced global conformation change. Their differences were elucidated by the activation motion of subsegments including A-loop, P-loop and Ncap. Besides, the T315I mutation caused decreasing energy barrier and increasing intermediate number in activation process, which results easier activation process. The TMD and CMD results indicate that a drug targeting only the ATP pocket is not enough to inhibit the Abl-T315I mutant. An effective way to inhibit the abnormal activity of Abl-T315I mutant is to combine the ATP-pocket inhibitors with inhibitors binding at non-ATP pockets mainly related to Ncap, SH2-KD linker and myristyl pocket.

  17. Germyl cations with Ge-S bond: an experimental and theoretical study on the gaseous F(n)Ge(SH)(3-n)+ (n=0-2).

    PubMed

    Antoniotti, Paola; Benzi, Paola; Operti, Lorenzo; Rabezzana, Roberto; Borocco, Stefano; Giordani, Maria; Grandinetti, Felice

    2012-01-01

    The germyl cations F(2)Ge(SH)(+), FGe(SH)(2)(+) and Ge(SH)(3)(+) were obtained from ionized mixtures of GeF(4) and H(2)S. Ion trap mass spectrometry revealed the occurrence of three consecutive addition-HF elimination reactions between GeF(3)(+), F(2)Ge(SH)(+) and FGe(SH)(2)(+) and H(2)S. The structure and the mechanism of formation of the observed F(n)Ge(SH)(3-n)(+) (n = 0-2) were investigated by ab initio calculations performed at the MP2 and coupled cluster level of theory. It was also possible to note regular trends in the geometries and Lewis acidities of the F(n)Ge(SH)(3-n)(+) (n= 0-3). PMID:23221118

  18. Lytic activity of the staphylolytic Twort phage endolysin CHAP domain is enhanced by the SH3b cell wall binding domain

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Increases in the prevalence of antibiotic resistant strains of Staphylococcus (S.) aureus have elicited efforts to develop novel antimicrobials to treat these drug-resistant pathogens. One potential treatment repurposes the lytic enzymes produced by bacteriophages as antimicrobials. The phage Twor...

  19. Exclusive expression of the Rab11 effector SH3TC2 in Schwann cells links integrin-α6 and myelin maintenance to Charcot-Marie-Tooth disease type 4C.

    PubMed

    Vijay, Sauparnika; Chiu, Meagan; Dacks, Joel B; Roberts, Rhys C

    2016-07-01

    Charcot-Marie-Tooth disease type 4C (CMT4C) is one of the commonest autosomal recessive inherited peripheral neuropathies and is associated with mutations in the Rab11 effector, SH3TC2. Disruption of the SH3TC2-Rab11 interaction is the molecular abnormality underlying this disease. However, why SH3TC2 mutations cause an isolated demyelinating neuropathy remains unanswered. Here we show that SH3TC2 is an exclusive Schwann cell protein expressed late in myelination and is downregulated following denervation suggesting a functional role in myelin sheath maintenance. We support our data with an evolutionary cell biological analysis showing that the SH3TC2 gene, and its paralogue SH3TC1, are derived from an ancestral homologue, the duplication of which occurred in the common ancestor of jawed vertebrates, coincident with the appearance of Schwann cells and peripheral axon myelination. Furthermore, we report that SH3TC2 associates with integrin-α6, suggesting that aberrant Rab11-dependent endocytic trafficking of this critical laminin receptor in myelinated Schwann cells is connected to the demyelination seen in affected nerves. Our study therefore highlights the inherent evolutionary link between SH3TC2 and peripheral nerve myelination, pointing also towards a molecular mechanism underlying the specific demyelinating neuropathy that characterizes CMT4C. PMID:27068304

  20. Clinical spectrum of CMT4C disease in patients homozygous for the p.Arg1109X mutation in SH3TC2.

    PubMed

    Colomer, Jaume; Gooding, Rebecca; Angelicheva, Dora; King, Rosalind H M; Guillén-Navarro, Encarna; Parman, Yesim; Nascimento, Andres; Conill, Joan; Kalaydjieva, Luba

    2006-07-01

    We investigated the manifestations of CMT4C disease in a genetically homogeneous group of patients homozygous for the recently identified Gypsy founder mutation p.Arg1109X in SH3TC2. We observed a surprising degree of variation in age at onset, rate of progression, extent and severity of motor and sensory involvement, scoliosis, and cranial nerve involvement, suggesting that the phenotypic spectrum of CMT4C disease is much broader than the classical diagnostic criteria. Phenotype similarity in first degree relatives and increasing heterogeneity in more distantly related subjects point to the involvement of genetic modifiers, possibly variants in the genes encoding protein partners interacting with SH3TC2. PMID:16806930

  1. Clinical impact of ABL1 kinase domain mutations and IKZF1 deletion in adults under age 60 with Philadelphia chromosome-positive (Ph+) acute lymphoblastic leukemia (ALL): molecular analysis of CALGB (Alliance) 10001 and 9665.

    PubMed

    DeBoer, Rebecca; Koval, Gregory; Mulkey, Flora; Wetzler, Meir; Devine, Steven; Marcucci, Guido; Stone, Richard M; Larson, Richard A; Bloomfield, Clara D; Geyer, Susan; Mullighan, Charles G; Stock, Wendy

    2016-10-01

    Recent studies have identified oncogenic lesions in Philadelphia chromosome-positive (Ph+)  acute lymphoblastic leukemia (ALL) and ABL1 kinase mutations that confer resistance to tyrosine kinase inhibitors. We sought to determine the prevalence and clinical impact of these lesions in patients on CALGB 10001, a previously reported Phase II study of imatinib, chemotherapy, and hematopoietic cell transplant in adult Ph + ALL. Of the 58 enrolled, 22 relapsed. By direct sequencing, an ABL1 kinase mutation known to induce imatinib resistance was present at relapse in 13 of 20. Using quantitative PCR assays, the mutations were detectable at diagnosis or early during treatment in most (62%) relapsed patients. Aberrations in IKZF1, CDKN2A/B, and PAX5 were assessed in 28 samples using SNP arrays and genomic DNA sequencing. Of these, 22 (79%) had IKZF1 deletion. The combination of IKZF1 deletion and p210 BCR-ABL1 (p < 0.0001), high white blood cell count (p = 0.021), and minimal residual disease (p = 0.013) were associated with worse disease-free survival. PMID:26892479

  2. A non-chromatographic protein purification strategy using Src 3 homology domains as generalized capture domains.

    PubMed

    Kim, Heejae; Chen, Wilfred

    2016-09-20

    Protein purification using inverse phase transition of elastin-like polypeptide (ELP) domains is a useful alternative to chromatography. Genetic fusions of ELP domains to various proteins have the ability to reversibly transition between soluble monomers and micron-sized aggregates and this has been used to selectively purify many ELP fusions. Affinity domains can enhance this technology by using specific protein binding domains to enable ELP mediated affinity capture (EMAC) of proteins of interest (POI) that have been fused to corresponding affinity ligands. In this paper, we highlight the use of Src homology 3 (SH3) domains and corresponding peptide ligands in EMAC that have differential binding affinities towards SH3 for efficient capture and elution of proteins. Furthermore, differences between capture and elution of a monomeric and a multimeric protein were also studied. PMID:27457699

  3. Amphiphysin II (SH3P9; BIN1), a Member of the Amphiphysin/Rvs Family, Is Concentrated in the Cortical Cytomatrix of Axon Initial Segments and Nodes of Ranvier in Brain and around T Tubules in Skeletal Muscle

    PubMed Central

    Butler, Margaret Husta; David, Carol; Ochoa, Gian-Carlo; Freyberg, Zachary; Daniell, Laurie; Grabs, Detlev; Cremona, Ottavio; Camilli, Pietro De

    1997-01-01

    Amphiphysin (amphiphysin I), a dominant autoantigen in paraneoplastic Stiff-man syndrome, is a neuronal protein highly concentrated in nerve terminals, where it has a putative role in endocytosis. The yeast homologue of amphiphysin, Rvs167, has pleiotropic functions, including a role in endocytosis and in actin dynamics, suggesting that amphiphysin may also be implicated in the function of the presynaptic actin cytoskeleton. We report here the characterization of a second mammalian amphiphysin gene, amphiphysin II (SH3P9; BIN1), which encodes products primarily expressed in skeletal muscle and brain, as differentially spliced isoforms. In skeletal muscle, amphiphysin II is concentrated around T tubules, while in brain it is concentrated in the cytomatrix beneath the plasmamembrane of axon initial segments and nodes of Ranvier. In both these locations, amphiphysin II is colocalized with splice variants of ankyrin3 (ankyrinG), a component of the actin cytomatrix. In the same regions, the presence of clathrin has been reported. These findings support the hypothesis that, even in mammalian cells, amphiphysin/Rvs family members have a role both in endocytosis and in actin function and suggest that distinct amphiphysin isoforms contribute to define distinct domains of the cortical cytoplasm. Since amphiphysin II (BIN1) was reported to interact with Myc, it may also be implicated in a signaling pathway linking the cortical cytoplasm to nuclear function. PMID:9182667

  4. Enhanced TLR-MYD88 Signaling Stimulates Autoinflammation in SH3BP2 Cherubism Mice and Defines the Etiology of Cherubism

    PubMed Central

    Yoshitaka, Teruhito; Mukai, Tomoyuki; Kittaka, Mizuho; Alford, Lisa M.; Masrani, Salome; Ishida, Shu; Yamaguchi, Ken; Yamada, Motohiko; Mizuno, Noriyoshi; Olsen, Bjorn R.; Reichenberger, Ernst J.; Ueki, Yasuyoshi

    2014-01-01

    Summary Cherubism is caused by mutations in SH3BP2. Studies of cherubism mice showed that TNF-α-dependent autoinflammation is a major cause for the disorder, but failed to explain why human cherubism lesions are restricted to jaws and regress after puberty. We demonstrate that the inflammation in cherubism mice is MYD88-dependent and is rescued in the absence of TLR2 and TLR4. However, germ-free cherubism mice also develop inflammation. Mutant macrophages are hyper-responsive to PAMPs (pathogen-associated molecular patterns) and DAMPs (damage-associated molecular patterns) that activate TLRs, resulting in TNF-α overproduction. Phosphorylation of SH3BP2 at Y183 is critical for the TNF-α production. Finally, SYK depletion in macrophages prevents the inflammation. These data suggest that the presence of a large amount of TLR ligands, presumably oral bacteria and DAMPs during jawbone remodeling, may cause the jaw-specific development of human cherubism lesions. Reduced levels of DAMPs after stabilization of jaw remodeling may contribute to the age-dependent regression. PMID:25220465

  5. Regulation of ASPP2 Interaction with p53 Core Domain by an Intramolecular Autoinhibitory Mechanism

    PubMed Central

    Rotem-Bamberger, Shahar; Katz, Chen; Friedler, Assaf

    2013-01-01

    ASPP2 is a key protein in regulating apoptosis both in p53-dependent and-independent pathways. The C-terminal part of ASPP2 contains four ankyrin repeats and an SH3 domain (Ank-SH3) that mediate the interactions of ASPP2 with apoptosis related proteins such as p53, Bcl-2 and the p65 subunit of NFκB. p53 core domain (p53CD) binds the n-src loop and the RT loop of ASPP2 SH3. ASPP2 contains a disordered proline rich domain (ASPP2 Pro) that forms an intramolecular autoinhibitory interaction with the Ank-SH3 domains. Here we show how this intramolecular interaction affects the intermolecular interactions of ASPP2 with p53, Bcl-2 and NFkB. We used biophysical methods to obtain better understanding of the relationship between ASPP2 and its partners for getting a comprehensive view on ASPP2 pathways. Fluorescence anisotropy competition experiments revealed that both ASPP2 Pro and p53CD competed for binding the n-src loop of the ASPP2 SH3, indicating regulation of p53CD binding to this loop by ASPP2 Pro. Peptides derived from the ASPP2-binding interface of Bcl-2 did not compete with p53CD or NFkB peptides for binding the ASPP2 n-src loop. However, p53CD displaced the NFκB peptide (residues 303–332) from its complex with ASPP2 Ank-SH3, indicating that NFκB 303–332 and p53CD bind a partly overlapping site in ASPP2 SH3, mostly in the RT loop. These results are in agreement with previous docking studies, which showed that ASPP2 Ank-SH3 binds Bcl-2 and NFκB mostly via distinct sites from p53. However they show some overlap between the binding sites of p53CD and NFkB in ASPP2 Ank-SH3. Our results provide experimental evidence that the intramolecular interaction in ASPP2 regulates its binding to p53CD and that ASPP2 Ank-SH3 binds Bcl-2 and NFκB via distinct sites. PMID:23472201

  6. Disruption of Bcr-Abl Coiled Coil Oligomerization by Design*

    PubMed Central

    Dixon, Andrew S.; Pendley, Scott S.; Bruno, Benjamin J.; Woessner, David W.; Shimpi, Adrian A.; Cheatham, Thomas E.; Lim, Carol S.

    2011-01-01

    Oligomerization is an important regulatory mechanism for many proteins, including oncoproteins and other pathogenic proteins. The oncoprotein Bcr-Abl relies on oligomerization via its coiled coil domain for its kinase activity, suggesting that a designed coiled coil domain with enhanced binding to Bcr-Abl and reduced self-oligomerization would be therapeutically useful. Key mutations in the coiled coil domain of Bcr-Abl were identified that reduce homo-oligomerization through intermolecular charge-charge repulsion yet increase interaction with the Bcr-Abl coiled coil through additional salt bridges, resulting in an enhanced ability to disrupt the oligomeric state of Bcr-Abl. The mutations were modeled computationally to optimize the design. Assays performed in vitro confirmed the validity and functionality of the optimal mutations, which were found to exhibit reduced homo-oligomerization and increased binding to the Bcr-Abl coiled coil domain. Introduction of the mutant coiled coil into K562 cells resulted in decreased phosphorylation of Bcr-Abl, reduced cell proliferation, and increased caspase-3/7 activity and DNA segmentation. Importantly, the mutant coiled coil domain was more efficacious than the wild type in all experiments performed. The improved inhibition of Bcr-Abl through oligomeric disruption resulting from this modified coiled coil domain represents a viable alternative to small molecule inhibitors for therapeutic intervention. PMID:21659527

  7. The SH3-SAM adaptor HACS1 is up-regulated in B cell activation signaling cascades.

    PubMed

    Zhu, Yuan Xiao; Benn, Sally; Li, Zhi Hua; Wei, Ellen; Masih-Khan, Esther; Trieu, Young; Bali, Meenakshi; McGlade, C Jane; Claudio, Jaime O; Stewart, A Keith

    2004-09-20

    HACS1 is a Src homology 3 and sterile alpha motif domain-containing adaptor that is preferentially expressed in normal hematopoietic tissues and malignancies including myeloid leukemia, lymphoma, and myeloma. Microarray data showed HACS1 expression is up-regulated in activated human B cells treated with interleukin (IL)-4, CD40L, and anti-immunoglobulin (Ig)M and clustered with genes involved in signaling, including TNF receptor-associated protein 1, signaling lymphocytic activation molecule, IL-6, and DEC205. Immunoblot analysis demonstrated that HACS1 is up-regulated by IL-4, IL-13, anti-IgM, and anti-CD40 in human peripheral blood B cells. In murine spleen B cells, Hacs1 can also be up-regulated by lipopolysaccharide but not IL-13. Induction of Hacs1 by IL-4 is dependent on Stat6 signaling and can also be impaired by inhibitors of phosphatidylinositol 3-kinase, protein kinase C, and nuclear factor kappaB. HACS1 associates with tyrosine-phosphorylated proteins after B cell activation and binds in vitro to the inhibitory molecule paired Ig-like receptor B. Overexpression of HACS1 in murine spleen B cells resulted in a down-regulation of the activation marker CD23 and enhancement of CD138 expression, IgM secretion, and Xbp-1 expression. Knock down of HACS1 in a human B lymphoma cell line by small interfering ribonucleic acid did not significantly change IL-4-stimulated B cell proliferation. Our study demonstrates that HACS1 is up-regulated by B cell activation signals and is a participant in B cell activation and differentiation. PMID:15381729

  8. BCR-ABL1: Test

    MedlinePlus

    ... called p190), which is most frequently associated with Ph chromosome-positive ALL. The quantitative BCR-ABL1 molecular test measures either of the breakpoints in the fusion gene. It is used to establish a baseline value and then to monitor the person's response to ...

  9. Structure-based characterization of the binding of peptide to the human endophilin-1 Src homology 3 domain using position-dependent noncovalent potential analysis.

    PubMed

    Fu, Chunjiang; Wu, Gang; Lv, Fenglin; Tian, Feifei

    2012-05-01

    Many protein-protein interactions are mediated by a peptide-recognizing domain, such as WW, PDZ, or SH3. In the present study, we describe a new method called position-dependent noncovalent potential analysis (PDNPA), which can accurately characterize the nonbonding profile between the human endophilin-1 Src homology 3 (hEndo1 SH3) domain and its peptide ligands and quantitatively predict the binding affinity of peptide to hEndo1 SH3. In this procedure, structure models of diverse peptides in complex with the hEndo1 SH3 domain are constructed by molecular dynamics simulation and a virtual mutagenesis protocol. Subsequently, three noncovalent interactions associated with each position of the peptide ligand in the complexed state are analyzed using empirical potential functions, and the resulting potential descriptors are then correlated with the experimentally measured affinity on the basis of 1997 hEndo1 SH3-binding peptides with known activities, using linear partial least squares regression (PLS) and the nonlinear support vector machine (SVM). The results suggest that: (i) the electrostatics appears to be more important than steric properties and hydrophobicity in the formation of the hEndo1 SH3-peptide complex; (ii) P(-4) of the core decapeptide ligand with the sequence pattern P(-6)P(-5)P(-4)P(-3)P(-2)P(-1)P(0)P(1)P(2)P(3) is the most important position in terms of determining both the stability and specificity of the architecture of the complex, and; (iii) nonlinear SVM appears to be more effective than linear PLS for accurately predicting the binding affinity of a peptide ligand to hEndo1 SH3, whereas PLS models are straightforward and easy to interpret as compared to those built by SVM. PMID:21947444

  10. Molecular Detection of BCR-ABL in Chronic Myeloid Leukemia.

    PubMed

    Qin, Ya-Zhen; Huang, Xiao-Jun

    2016-01-01

    All chronic myeloid leukemia (CML) patients have the BCR-ABL fusion gene. The constitutively activated BCR-ABL tyrosine kinase is a critical pathogenetic event in CML. Tyrosine kinase inhibitors (TKIs), such as imatinib, are synthesized small molecules that primarily target BCR-ABL tyrosine kinases and have become a first-line treatment for CML. Detection of BCR-ABL transcript level by real-time quantitative polymerase chain reaction (RQ-PCR) is a clinical routine for evaluating TKI treatment efficacy and predicting long-term response. Furthermore, because they are a main TKI resistance mechanism, the BCR-ABL tyrosine kinase domain (TKD) point mutations that are detected by Sanger sequencing can help clinicians make decisions on subsequent treatment selections. Here, we present protocols for the two abovementioned molecular methods for CML analysis. PMID:27581134

  11. Specificity of Collybistin-Phosphoinositide Interactions: IMPACT OF THE INDIVIDUAL PROTEIN DOMAINS.

    PubMed

    Ludolphs, Michaela; Schneeberger, Daniela; Soykan, Tolga; Schäfer, Jonas; Papadopoulos, Theofilos; Brose, Nils; Schindelin, Hermann; Steinem, Claudia

    2016-01-01

    The regulatory protein collybistin (CB) recruits the receptor-scaffolding protein gephyrin to mammalian inhibitory glycinergic and GABAergic postsynaptic membranes in nerve cells. CB is tethered to the membrane via phosphoinositides. We developed an in vitro assay based on solid-supported 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine membranes doped with different phosphoinositides on silicon/silicon dioxide substrates to quantify the binding of various CB2 constructs using reflectometric interference spectroscopy. Based on adsorption isotherms, we obtained dissociation constants and binding capacities of the membranes. Our results show that full-length CB2 harboring the N-terminal Src homology 3 (SH3) domain (CB2SH3+) adopts a closed and autoinhibited conformation that largely prevents membrane binding. This autoinhibition is relieved upon introduction of the W24A/E262A mutation, which conformationally "opens" CB2SH3+ and allows the pleckstrin homology domain to properly bind lipids depending on the phosphoinositide species with a preference for phosphatidylinositol 3-monophosphate and phosphatidylinositol 4-monophosphate. This type of membrane tethering under the control of the release of the SH3 domain of CB is essential for regulating gephyrin clustering. PMID:26546675

  12. P130Cas Src-Binding and Substrate Domains Have Distinct Roles in Sustaining Focal Adhesion Disassembly and Promoting Cell Migration

    PubMed Central

    Meenderink, Leslie M.; Ryzhova, Larisa M.; Donato, Dominique M.; Gochberg, Daniel F.; Kaverina, Irina; Hanks, Steven K.

    2010-01-01

    The docking protein p130Cas is a prominent Src substrate found in focal adhesions (FAs) and is implicated in regulating critical aspects of cell motility including FA disassembly and protrusion of the leading edge plasma membrane. To better understand how p130Cas acts to promote these events we examined requirements for established p130Cas signaling motifs including the SH3-binding site of the Src binding domain (SBD) and the tyrosine phosphorylation sites within the substrate domain (SD). Expression of wild type p130Cas in Cas −/− mouse embryo fibroblasts resulted in enhanced cell migration associated with increased leading-edge actin flux, increased rates of FA assembly/disassembly, and uninterrupted FA turnover. Variants lacking either the SD phosphorylation sites or the SBD SH3-binding motif were able to partially restore the migration response, while only a variant lacking both signaling functions was fully defective. Notably, the migration defects associated with p130Cas signaling-deficient variants correlated with longer FA lifetimes resulting from aborted FA disassembly attempts. However the SD mutational variant was fully defective in increasing actin assembly at the protruding leading edge and FA assembly/disassembly rates, indicating that SD phosphorylation is the sole p130Cas signaling function in regulating these processes. Our results provide the first quantitative evidence supporting roles for p130Cas SD tyrosine phosphorylation in promoting both leading edge actin flux and FA turnover during cell migration, while further revealing that the p130Cas SBD has a function in cell migration and sustained FA disassembly that is distinct from its known role of promoting SD tyrosine phosphorylation. PMID:20976150

  13. Structure and Dynamic Regulation of Abl Kinases*

    PubMed Central

    Panjarian, Shoghag; Iacob, Roxana E.; Chen, Shugui; Engen, John R.; Smithgall, Thomas E.

    2013-01-01

    The c-abl proto-oncogene encodes a unique protein-tyrosine kinase (Abl) distinct from c-Src, c-Fes, and other cytoplasmic tyrosine kinases. In normal cells, Abl plays prominent roles in cellular responses to genotoxic stress as well as in the regulation of the actin cytoskeleton. Abl is also well known in the context of Bcr-Abl, the oncogenic fusion protein characteristic of chronic myelogenous leukemia. Selective inhibitors of Bcr-Abl, of which imatinib is the prototype, have had a tremendous impact on clinical outcomes in chronic myelogenous leukemia and revolutionized the field of targeted cancer therapy. In this minireview, we focus on the structural organization and dynamics of Abl kinases and how these features influence inhibitor sensitivity. PMID:23316053

  14. A c.1244G>A (p.Arg415Gln) mutation in SH3BP2 gene causes cherubism in a Turkish family: Report of a family with review of the literature

    PubMed Central

    Balta, Burhan; Hu, Ying; Reichenberger, Ernst J.; Etoz, Osman A.; Nazlim, Sinan; Bayrakdar, Ibrahim S.

    2014-01-01

    Objectives: The present study was aimed at advancing the understanding of the pathogenesis of cherubism by presenting a case study based on history, physical examination, typical radiological features, molecular and histopathological laboratory tests and a review of the literature. Study Design: This study began with a 7-year-old boy who was referred due to mandibular overgrowth. A panoramic radiograph revealed multilocular radiolucent lesions of the upper/lower jaws suggestive of cherubism. Overall, a total of four family members were tested for SH3BP2 mutations, namely two siblings and their parents. Both siblings had been clinically diagnosed with cherubism; however, the parents were clinically normal. Peripheral blood was collected from all participants and genomic DNA sequencing was carried out. Results: A missense mutation was found in the two affected siblings and their asymptomatic mother. The mutation was a 1244 G>A transversion which resulted in an amino acid substitution from arginine to glutamine (p.Arg415Gln) in exon 9. Conclusions: The present study emphasized the importance of further clinical and molecular investigation even when only a single case of cherubism is identified within a family. Genotype-phenotype association studies in individuals with cherubism are necessary to provide important insights into the molecular mechanisms associated with this disease. Key words:Cherubism, mandible, maxilla, SH3BP2, gene analysis, CBCT. PMID:24608212

  15. ABLE: The Future of Mechanical Aids.

    ERIC Educational Resources Information Center

    Lowell, Harvey; Finkelstein, Stan

    1989-01-01

    Advanced Bio-Mechanical Linkage Enablement (ABLE) technology constitutes a leap forward in physical aids for people with disabilities, as they integrate high technologies in rehabilitation to create individualized self-contained care systems. Described are the need for universal standards, barriers to acceptance of ABLE technologies, and ways to…

  16. Domain cooperativity in multidomain proteins: what can we learn from molecular alignment in anisotropic media?

    PubMed

    Yuwen, Tairan; Post, Carol Beth; Skrynnikov, Nikolai R

    2011-09-01

    Many proteins have modular design with multiple globular domains connected via flexible linkers. As a simple model of such system, we study a tandem construct consisting of two identical SH3 domains and a variable-length Gly/Ser linker. When the linker is short, this construct represents a dumbbell-shaped molecule with limited amount of domain-domain mobility. Due to its elongated shape, this molecule efficiently aligns in steric alignment media. As the length of the linker increases, the two domains become effectively uncoupled and begin to behave as independent entities. Consequently, their degree of alignment drops, approaching that found in the (near-spherical) isolated SH3 domains. To model the dependence of alignment parameters on the length of the interdomain linker, we have generated in silico a series of conformational ensembles representing SH3 tandems with different linker length. These ensembles were subsequently used as input for alignment prediction software PALES. The predicted alignment tensors were compared with the results of experimental measurements using a series of tandem-SH3 samples in PEG/hexanol alignment media. This comparison broadly confirmed the expected trends. At the same time, it has been found that the isolated SH3 domain aligns much stronger than expected. This finding can be attributed to complex morphology of the PEG/hexanol media and/or to weak site-specific interactions between the protein and the media. In the latter case, there are strong indications that electrostatic interactions may play a role. The fact that PEG/hexanol does not behave as a simple steric media should serve as a caution for studies that use PALES as a quantitative prediction tool (especially for disordered proteins). Further progress in this area depends on our ability to accurately model the anisotropic media and its site-specific interactions with protein molecules. Once this ability is improved, it should be possible to use the alignment parameters as a

  17. Monkey Able Being Ready for preflight Test

    NASA Technical Reports Server (NTRS)

    1959-01-01

    A squirrel monkey, Able, is being ready for placement into a capsule for a preflight test of Jupiter, AM-18 mission. AM-18 was launched on May 28, 1959 and also carried a rhesus monkey, Baker, into suborbit.

  18. Supporting the Digitally Able Beginning Teacher

    ERIC Educational Resources Information Center

    Starkey, Louise

    2010-01-01

    This article reports on research which explored the experiences six digitally able beginning teachers during their first year in secondary schools. Using a complexity theoretical framework, the barriers and enablers that influenced the integration of digital technologies into teaching practice were examined. The findings indicate that context…

  19. Planned HST observations of LSQ15abl

    NASA Astrophysics Data System (ADS)

    Quimby, R. M.; Cooke, J.; Pritchard, T.

    2015-04-01

    Following the discovery and classification of LSQ15abl as a SLSN-II (ATel #7412), we have triggered the Hubble Space Telescope program, "The First UV Spectra of a Hydrogen-Rich Superluminous Supernova," (GO-13784; PI=Quimby), as well as the Swift program, "Photometry of a Hydrogen-Rich Superluminous Supernova" (Swift-1114192; PI=Quimby).

  20. ABL and BAM Friction Analysis Comparison

    DOE PAGESBeta

    Warner, Kirstin F.; Sandstrom, Mary M.; Brown, Geoffrey W.; Remmers, Daniel L.; Phillips, Jason J.; Shelley, Timothy J.; Reyes, Jose A.; Hsu, Peter C.; Reynolds, John G.

    2014-12-29

    Here, the Integrated Data Collection Analysis (IDCA) program has conducted a proficiency study for Small-Scale Safety and Thermal (SSST) testing of homemade explosives (HMEs). Described here is a comparison of the Alleghany Ballistic Laboratory (ABL) friction data and Bundesanstalt fur Materialforschung und -prufung (BAM) friction data for 19 HEM and military standard explosives.

  1. Aleukemic bcr-abl positive granulocytic sarcoma.

    PubMed

    Kuan, Jew-Win; Pathmanathan, Rajadurai; Chang, Kian-Meng; Tan, Sen-Moi

    2009-11-01

    Granulocytic sarcoma (GS) can occur de novo or in association with intramedullary myeloid disorders. With the advent of sophisticated molecular detection techniques to detect diagnostic genes such as bcr-abl, PML-RARA and CBFB/MYH11 in bone marrow or peripheral blood, many cases of the so called 'primary' GS are questionable. We report a case of primary GS where the tumor mass bcr-abl translocation was demonstrated by fluorescent in situ hybridization in which there was no evidence of chronic myeloid leukemia (CML). This is an important finding as it highlights the possibility that CML may present as a sole extramedullary form, and illustrates potential treatment by tyrosine kinase inhibitor. PMID:19215983

  2. PRL-1 Protein Promotes ERK1/2 and RhoA Protein Activation through a Non-canonical Interaction with the Src Homology 3 Domain of p115 Rho GTPase-activating Protein

    SciTech Connect

    Bai, Yunpeng; Luo, Yong; Liu, Sijiu; Zhang, Lujuan; Shen, Kui; Dong, Yuanshu; Walls, Chad D.; Quilliam, Lawrence A.; Wells, Clark D.; Cao, Youjia; Zhang, Zhong-Yin

    2012-03-15

    Phosphatases of the regenerating liver (PRL) play oncogenic roles in cancer development and metastasis. Although previous studies indicate that PRL-1 promotes cell growth and migration by activating both the ERK1/2 and RhoA pathways, the mechanism by which it activates these signaling events remains unclear. We have identified a PRL-1-binding peptide (Peptide 1) that shares high sequence identity with a conserved motif in the Src homology 3 (SH3) domain of p115 Rho GTPase-activating protein (GAP). p115 RhoGAP directly binds PRL-1 in vitro and in cells via its SH3 domain. Structural analyses of the PRL-1 {center_dot} Peptide 1 complex revealed a novel protein-protein interaction whereby a sequence motif within the PxxP ligand-binding site of the p115 RhoGAP SH3 domain occupies a folded groove within PRL-1. This prevents the canonical interaction between the SH3 domain of p115 RhoGAP and MEKK1 and results in activation of ERK1/2. Furthermore, PRL-1 binding activates RhoA signaling by inhibiting the catalytic activity of p115 RhoGAP. The results demonstrate that PRL-1 binding to p115 RhoGAP provides a coordinated mechanism underlying ERK1/2 and RhoA activation.

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

    SciTech Connect

    Nomme, Julian; Fanning, Alan S.; Caffrey, Michael; Lye, Ming F.; Anderson, James M.; Lavie, Arnon

    2012-01-20

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

  4. Analysis of the binding of the Src homology 2 domain of Csk to tyrosine-phosphorylated proteins in the suppression and mitotic activation of c-Src.

    PubMed Central

    Sabe, H; Hata, A; Okada, M; Nakagawa, H; Hanafusa, H

    1994-01-01

    Csk (C-terminal Src kinase), a protein-tyrosine kinase, bearing the Src homology 2 and 3 (SH2 and SH3) domains, has been implicated in phosphorylation of c-Src Tyr-527, resulting in suppression of c-Src kinase activity. We found that mutations in the SH2 or SH3 domain of Csk, though they did not affect its kinase activity, resulted in a loss of suppression of c-Src activity in fibroblasts. In normal fibroblasts, tyrosine-phosphorylated paxillin and focal adhesion kinase pp125FAK, which colocalize at focal adhesion plaques, were the major proteins to which the Csk SH2 domain bound. Loss of binding to these proteins by the Csk SH2 mutants correlated with loss of the activity to suppress c-Src. Consistent with this observation, the levels of tyrosine phosphorylation of paxillin and pp125FAK were greatly reduced during mitosis, whereas the kinase activity of c-Src was elevated. We suggest that the SH2 domain is required for Csk to suppress c-Src, perhaps in combination with the SH3 domain, by anchoring Csk to a particular subcellular location where c-Src may exist. Our data also indicate that a certain fraction of the Csk and Src family kinases function at the focal adhesion plaques. The activity of the c-Src kinase localized at the focal adhesion plaques appears to be regulated by cell adhesion to the extracellular matrix. Images PMID:7513429

  5. Zebra finches are able to learn affixation-like patterns.

    PubMed

    Chen, Jiani; Jansen, Naomi; ten Cate, Carel

    2016-01-01

    Adding an affix to transform a word is common across the world languages, with the edges of words more likely to carry out such a function. However, detecting affixation patterns is also observed in learning tasks outside the domain of language, suggesting that the underlying mechanism from which affixation patterns have arisen may not be language or even human specific. We addressed whether a songbird, the zebra finch, is able to discriminate between, and generalize, affixation-like patterns. Zebra finches were trained and tested in a Go/Nogo paradigm to discriminate artificial song element sequences resembling prefixed and suffixed 'words.' The 'stems' of the 'words,' consisted of different combinations of a triplet of song elements, to which a fourth element was added as either a 'prefix' or a 'suffix.' After training, the birds were tested with novel stems, consisting of either rearranged familiar element types or novel element types. The birds were able to generalize the affixation patterns to novel stems with both familiar and novel element types. Hence, the discrimination resulting from the training was not based on memorization of individual stimuli, but on a shared property among Go or Nogo stimuli, i.e., affixation patterns. Remarkably, birds trained with suffixation as Go pattern showed clear evidence of using both prefix and suffix, while those trained with the prefix as the Go stimulus used primarily the prefix. This finding illustrates that an asymmetry in attending to different affixations is not restricted to human languages. PMID:26297477

  6. Evolution of domain-peptide interactions to coadapt specificity and affinity to functional diversity.

    PubMed

    Kelil, Abdellali; Levy, Emmanuel D; Michnick, Stephen W

    2016-07-01

    Evolution of complexity in eukaryotic proteomes has arisen, in part, through emergence of modular independently folded domains mediating protein interactions via binding to short linear peptides in proteins. Over 30 years, structural properties and sequence preferences of these peptides have been extensively characterized. Less successful, however, were efforts to establish relationships between physicochemical properties and functions of domain-peptide interactions. To our knowledge, we have devised the first strategy to exhaustively explore the binding specificity of protein domain-peptide interactions. We applied the strategy to SH3 domains to determine the properties of their binding peptides starting from various experimental data. The strategy identified the majority (∼70%) of experimentally determined SH3 binding sites. We discovered mutual relationships among binding specificity, binding affinity, and structural properties and evolution of linear peptides. Remarkably, we found that these properties are also related to functional diversity, defined by depth of proteins within hierarchies of gene ontologies. Our results revealed that linear peptides evolved to coadapt specificity and affinity to functional diversity of domain-peptide interactions. Thus, domain-peptide interactions follow human-constructed gene ontologies, which suggest that our understanding of biological process hierarchies reflect the way chemical and thermodynamic properties of linear peptides and their interaction networks, in general, have evolved. PMID:27317745

  7. Normal ABL1 is a tumor suppressor and therapeutic target in human and mouse leukemias expressing oncogenic ABL1 kinases.

    PubMed

    Dasgupta, Yashodhara; Koptyra, Mateusz; Hoser, Grazyna; Kantekure, Kanchan; Roy, Darshan; Gornicka, Barbara; Nieborowska-Skorska, Margaret; Bolton-Gillespie, Elisabeth; Cerny-Reiterer, Sabine; Müschen, Markus; Valent, Peter; Wasik, Mariusz A; Richardson, Christine; Hantschel, Oliver; van der Kuip, Heiko; Stoklosa, Tomasz; Skorski, Tomasz

    2016-04-28

    Leukemias expressing constitutively activated mutants of ABL1 tyrosine kinase (BCR-ABL1, TEL-ABL1, NUP214-ABL1) usually contain at least 1 normal ABL1 allele. Because oncogenic and normal ABL1 kinases may exert opposite effects on cell behavior, we examined the role of normal ABL1 in leukemias induced by oncogenic ABL1 kinases. BCR-ABL1-Abl1(-/-) cells generated highly aggressive chronic myeloid leukemia (CML)-blast phase-like disease in mice compared with less malignant CML-chronic phase-like disease from BCR-ABL1-Abl1(+/+) cells. Additionally, loss of ABL1 stimulated proliferation and expansion of BCR-ABL1 murine leukemia stem cells, arrested myeloid differentiation, inhibited genotoxic stress-induced apoptosis, and facilitated accumulation of chromosomal aberrations. Conversely, allosteric stimulation of ABL1 kinase activity enhanced the antileukemia effect of ABL1 tyrosine kinase inhibitors (imatinib and ponatinib) in human and murine leukemias expressing BCR-ABL1, TEL-ABL1, and NUP214-ABL1. Therefore, we postulate that normal ABL1 kinase behaves like a tumor suppressor and therapeutic target in leukemias expressing oncogenic forms of the kinase. PMID:26864341

  8. Structure of Sla1p homology domain 1 and interaction with the NPFxD endocytic internalization motif

    PubMed Central

    Mahadev, Ravi K; Di Pietro, Santiago M; Olson, John M; Piao, Hai Lan; Payne, Gregory S; Overduin, Michael

    2007-01-01

    Adaptor proteins play important endocytic roles including recognition of internalization signals in transmembrane cargo. Sla1p serves as the adaptor for uptake of transmembrane proteins containing the NPFxD internalization signal, and is essential for normal functioning of the actin cytoskeleton during endocytosis. The Sla1p homology domain 1 (SHD1) within Sla1p is responsible for recognition of the NPFxD signal. This study presents the NMR structure of the NPFxD-bound state of SHD1 and a model for the protein–ligand complex. The α+β structure of the protein reveals an SH3-like topology with a solvent-exposed hydrophobic ligand binding site. NMR chemical shift perturbations and effects of structure-based mutations on ligand binding in vitro define residues that are key for NPFxD binding. Mutations that abolish ligand recognition in vitro also abolish NPFxD-mediated receptor internalization in vivo. Thus, SHD1 is a novel functional domain based on SH3-like topology, which employs a unique binding site to recognize the NPFxD endocytic internalization signal. Its distant relationship with the SH3 fold endows this superfamily with a new role in endocytosis. PMID:17363896

  9. Caspase-dependent cleavage of c-Abl contributes to apoptosis.

    PubMed

    Barilà, Daniela; Rufini, Alessandra; Condò, Ivano; Ventura, Natascia; Dorey, Karel; Superti-Furga, Giulio; Testi, Roberto

    2003-04-01

    The nonreceptor tyrosine kinase c-Abl may contribute to the regulation of apoptosis. c-Abl activity is induced in the nucleus upon DNA damage, and its activation is required for execution of the apoptotic program. Recently, activation of nuclear c-Abl during death receptor-induced apoptosis has been reported; however, the mechanism remains largely obscure. Here we show that c-Abl is cleaved by caspases during tumor necrosis factor- and Fas receptor-induced apoptosis. Cleavage at the very C-terminal region of c-Abl occurs mainly in the cytoplasmic compartment and generates a 120-kDa fragment that lacks the nuclear export signal and the actin-binding region but retains the intact kinase domain, the three nuclear localization signals, and the DNA-binding domain. Upon caspase cleavage, the 120-kDa fragment accumulates in the nucleus. Transient-transfection experiments show that cleavage of c-Abl may affect the efficiency of Fas-induced cell death. These data reveal a novel mechanism by which caspases can recruit c-Abl to the nuclear compartment and to the mammalian apoptotic program. PMID:12665579

  10. Dogs are able to solve a means-end task

    PubMed Central

    Range, Friederike; Hentrup, Marleen; Viranyi, Zsofia

    2014-01-01

    Dogs, although very skilled in social communicative tasks, have shown limited abilities in the domain of physical cognition. Consequently, several researchers hypothesized that domestication enhanced dogs’ cognitive abilities in the social realm, but relaxed selection on the physical one. For instance, dogs failed to demonstrate means-end understanding, an important form of relying on physical causal connection, when tested in a string-pulling task. Here, we tested dogs in an ‘on/off’ task using a novel approach. Thirty-two dogs were confronted with four different conditions in which they could choose between two boards one with a reward ‘on’ and another one with a reward ‘off’ (reward was placed next to the board). The dogs chose the correct board when 1) both rewards were placed at the same distance from the dog, when 2) the reward placed ‘on’ the board was closer to the dog, and 3) even when the reward placed ‘off’ the board was much closer to the dog and was food. Interestingly, in the latter case dogs did not perform above chance, if instead of a direct reward, the dogs had to retrieve an object placed on the board to get a food reward. In contrast to previous string pulling studies, our results show that dogs are able to solve a means-end task even if proximity of the unsupported reward is a confounding factor. PMID:21445577

  11. Are monkeys able to plan for future exchange?

    PubMed

    Bourjade, Marie; Thierry, Bernard; Call, Josep; Dufour, Valérie

    2012-09-01

    Whether or not non-human animals can plan for the future is a hotly debated issue. We investigate this question further and use a planning-to-exchange task to study future planning in the cooperative domain in two species of monkeys: the brown capuchin (Cebus apella) and the Tonkean macaque (Macaca tonkeana). The rationale required subjects to plan for a future opportunity to exchange tokens for food by collecting tokens several minutes in advance. Subjects who successfully planned for the exchange task were expected to select suitable tokens during a collection period (5/10 min), save them for a fixed period of time (20/30 min), then take them into an adjacent compartment and exchange them for food with an experimenter. Monkeys mostly failed to transport tokens when entering the testing compartment; hence, they do not seem able to plan for a future exchange with a human partner. Three subjects did however manage to solve the task several times, albeit at very low rates. They brought the correct version of three possible token types, but rarely transported more than one suitable token at a time. Given that the frequency of token manipulation predicted transport, success might have occurred by chance. This was not the case, however, since in most cases subjects were not already holding the token in their hands before they entered the testing compartment. Instead, these results may reflect subjects' strengths and weaknesses in their time-related comprehension of the task. PMID:22532073

  12. Ankyrin repeat domain 28 (ANKRD28), a novel binding partner of DOCK180, promotes cell migration by regulating focal adhesion formation.

    PubMed

    Tachibana, Mitsuhiro; Kiyokawa, Etsuko; Hara, Shigeo; Iemura, Shun-Ichiro; Natsume, Tohru; Manabe, Toshiaki; Matsuda, Michiyuki

    2009-03-10

    DOCK180 is a guanine exchange factor of Rac1 originally identified as a protein bound to an SH3 domain of the Crk adaptor protein. DOCK180 induces tyrosine phosphorylation of p130(Cas), and recruits the Crk-p130(Cas) complex to focal adhesions. To understand the role of DOCK180 in cell adhesion and migration, we searched for DOCK180-binding proteins with a nano-LC/MS/MS system, and identified ANKRD28, a protein that contains twenty-six ankyrin domain repeats. Knockdown of ANKRD28 by RNA interference reduced the velocity of migration of HeLa cells, suggesting that this protein plays a physiologic role in the DOCK180-Rac1 signaling pathway. Furthermore, knockdown of ANKRD28 was found to alter the distribution of focal adhesion proteins such as Crk, paxillin, and p130(Cas). On the other hand, expression of ANKRD28, p130(Cas), Crk, and DOCK180 induced hyper-phosphorylation of p130(Cas), and impaired detachment of the cell membrane during migration. Consequently, cells expressing ANKRD28 exhibited multiple long cellular processes. ANKRD28 associated with DOCK180 in an SH3-dependent manner and competed with ELMO, another protein bound to the SH3 domain of DOCK180. In striking contrast to ANKRD28, overexpression of ELMO induced extensive lamellipodial protrusion around the entire circumference. These data suggest that ANKRD28 specifies the localization and the activity of the DOCK180-Rac1 pathway. PMID:19118547

  13. IMPROVED COILED-COIL DESIGN ENHANCES INTERACTION WITH BCR-ABL AND INDUCES APOPTOSIS

    PubMed Central

    Dixon, Andrew S.; Miller, Geoffrey D.; Bruno, Benjamin J.; Constance, Jonathan E.; Woessner, David W.; Fidler, Trevor P.; Robertson, James C.; Cheatham, Thomas E.; Lim, Carol S.

    2012-01-01

    The oncoprotein Bcr-Abl drives aberrant downstream activity through trans-autophosphorylation of homo-oligomers in chronic myelogenous leukemia (CML).1,2 The formation of Bcr-Abl oligomers is achieved through the coiled-coil domain at the N-terminus of Bcr.3, 4 We have previously reported a modified version of this coiled-coil domain, CCmut2, which exhibits disruption of Bcr-Abl oligomeric complexes and results in decreased proliferation of CML cells and induction of apoptosis.5 A major contributing factor to these enhanced capabilities is the destabilization of the CCmut2 homo-dimers, increasing the availability to interact with and inhibit Bcr-Abl. Here, we included an additional mutation (K39E) that could in turn further destabilize the mutant homo-dimer. Incorporation of this modification into CCmut2 (C38A, S41R, L45D, E48R, Q60E) generated what we termed CCmut3, and resulted in further improvements in the binding properties with the wild-type coiled-coil domain representative of Bcr-Abl. A separate construct containing one revert mutation, CCmut4, did not demonstrate improved oligomeric properties and indicated the importance of the L45D mutation. CCmut3 demonstrated improved oligomerization via a two-hybrid assay as well as through colocalization studies, in addition to showing similar biologic activity as CCmut2. The improved binding between CCmut3 and the Bcr-Abl coiled-coil may be used to redirect Bcr-Abl to alternative subcellular locations with interesting therapeutic implications. PMID:22136227

  14. Structure of the ABL2/ARG kinase in complex with dasatinib

    PubMed Central

    Ha, Byung Hak; Simpson, Mark Adam; Koleske, Anthony J.; Boggon, Titus J.

    2015-01-01

    ABL2/ARG (ABL-related gene) belongs to the ABL (Abelson tyrosine-protein kinase) family of tyrosine kinases. ARG plays important roles in cell morphogenesis, motility, growth and survival, and many of these biological roles overlap with the cellular functions of the ABL kinase. Chronic myeloid leukemia (CML) is associated with constitutive ABL kinase activation resulting from fusion between parts of the breakpoint cluster region (BCR) and ABL1 genes. Similarly, fusion of the ETV6 (Tel) and ARG genes drives some forms of T-cell acute lymphoblastic leukemia (T-ALL) and acute myeloid leukemia (AML). Dasatinib is a tyrosine kinase inhibitor used for the treatment of CML by inhibiting ABL, and while it also inhibits ARG, there is currently no structure of ARG in complex with dasatinib. Here, the co-crystal structure of the mouse ARG catalytic domain with dasatinib at 2.5 Å resolution is reported. Dasatinib-bound ARG is found in the DFG-in conformation although it is nonphos­phorylated on the activation-loop tyrosine. In this structure the glycine-rich P-loop is found in a relatively open conformation compared with other known ABL family–inhibitor complex structures. PMID:25849507

  15. A role for FOXO1 in BCR-ABL1-independent tyrosine kinase inhibitor resistance in chronic myeloid leukemia.

    PubMed

    Wagle, M; Eiring, A M; Wongchenko, M; Lu, S; Guan, Y; Wang, Y; Lackner, M; Amler, L; Hampton, G; Deininger, M W; O'Hare, T; Yan, Y

    2016-07-01

    Chronic myeloid leukemia (CML) patients who relapse on imatinib due to acquired ABL1 kinase domain mutations are successfully treated with second-generation ABL1-tyrosine kinase inhibitors (ABL-TKIs) such as dasatinib, nilotinib or ponatinib. However, ~40% of relapsed patients have uncharacterized BCR-ABL1 kinase-independent mechanisms of resistance. To identify these mechanisms of resistance and potential treatment options, we generated ABL-TKI-resistant K562 cells through prolonged sequential exposure to imatinib and dasatinib. Dual-resistant K562 cells lacked BCR-ABL1 kinase domain mutations, but acquired other genomic aberrations that were characterized by next-generation sequencing and copy number analyses. Proteomics showed that dual-resistant cells had elevated levels of FOXO1, phospho-ERK and BCL-2, and that dasatinib no longer inhibited substrates of the PI3K/AKT pathway. In contrast to parental cells, resistant cells were sensitive to growth inhibition and apoptosis induced by the class I PI3K inhibitor, GDC-0941 (pictilisib), which also induced FOXO1 nuclear translocation. FOXO1 was elevated in a subset of primary specimens from relapsed CML patients lacking BCR-ABL1 kinase domain mutations, and these samples were responsive to GDC-0941 treatment ex vivo. We conclude that elevated FOXO1 contributes to BCR-ABL1 kinase-independent resistance experienced by these CML patients and that PI3K inhibition coupled with BCR-ABL1 inhibition may represent a novel therapeutic approach. PMID:27044711

  16. A role for FOXO1 in BCR–ABL1-independent tyrosine kinase inhibitor resistance in chronic myeloid leukemia

    PubMed Central

    Wagle, M; Eiring, A M; Wongchenko, M; Lu, S; Guan, Y; Wang, Y; Lackner, M; Amler, L; Hampton, G; Deininger, M W; O'Hare, T; Yan, Y

    2016-01-01

    Chronic myeloid leukemia (CML) patients who relapse on imatinib due to acquired ABL1 kinase domain mutations are successfully treated with second-generation ABL1-tyrosine kinase inhibitors (ABL-TKIs) such as dasatinib, nilotinib or ponatinib. However, ~40% of relapsed patients have uncharacterized BCR–ABL1 kinase-independent mechanisms of resistance. To identify these mechanisms of resistance and potential treatment options, we generated ABL-TKI-resistant K562 cells through prolonged sequential exposure to imatinib and dasatinib. Dual-resistant K562 cells lacked BCR–ABL1 kinase domain mutations, but acquired other genomic aberrations that were characterized by next-generation sequencing and copy number analyses. Proteomics showed that dual-resistant cells had elevated levels of FOXO1, phospho-ERK and BCL-2, and that dasatinib no longer inhibited substrates of the PI3K/AKT pathway. In contrast to parental cells, resistant cells were sensitive to growth inhibition and apoptosis induced by the class I PI3K inhibitor, GDC-0941 (pictilisib), which also induced FOXO1 nuclear translocation. FOXO1 was elevated in a subset of primary specimens from relapsed CML patients lacking BCR–ABL1 kinase domain mutations, and these samples were responsive to GDC-0941 treatment ex vivo. We conclude that elevated FOXO1 contributes to BCR–ABL1 kinase-independent resistance experienced by these CML patients and that PI3K inhibition coupled with BCR–ABL1 inhibition may represent a novel therapeutic approach. PMID:27044711

  17. Domain Engineering

    NASA Astrophysics Data System (ADS)

    Bjørner, Dines

    Before software can be designed we must know its requirements. Before requirements can be expressed we must understand the domain. So it follows, from our dogma, that we must first establish precise descriptions of domains; then, from such descriptions, “derive” at least domain and interface requirements; and from those and machine requirements design the software, or, more generally, the computing systems.

  18. Development of Alkyne-Containing Pyrazolopyrimidines To Overcome Drug Resistance of Bcr-Abl Kinase.

    PubMed

    Liu, Xu; Kung, Alvin; Malinoski, Brock; Prakash, G K Surya; Zhang, Chao

    2015-12-10

    Despite the success of imatinib at inhibiting Bcr-Abl and treating chronic myelogenous leukemia (CML), resistance to the therapy occurs over time in patients. In particular, the resistance to imatinib caused by the gatekeeper mutation T315I in Bcr-Abl remains a challenge in the clinic. Inspired by the successful development of ponatinib to curb drug resistance, we hypothesize that the incorporation of an alkyne linker in other heterocyclic scaffolds can also achieve potent inhibition of Bcr-Abl(T315I) by allowing for simultaneous occupancy of both the active site and the allosteric pocket in the Abl kinase domain. Herein, we describe the design, synthesis, and characterization of a series of alkyne-containing pyrazolopyrimidines as Bcr-Abl inhibitors. Our results demonstrate that some alkyne-containing pyrazolopyrimidines potently inhibit not only Abl(T315I) in vitro but also Bcr-Abl(T315I) in cells. These pyrazolopyrimidines can serve as lead compounds for future development of novel targeted therapy to overcome drug resistance of CML. PMID:26562217

  19. Effects of Character Education on the Self-Esteem of Intellectually Able and Less Able Elementary Students in Kuwait

    ERIC Educational Resources Information Center

    Tannir, Abir; Al-Hroub, Anies

    2013-01-01

    This research study investigates effects of character education activities on the self-esteem of intellectually able and less able students in the lower elementary level in Kuwait. The participants were 39 students in grade three with an average age of eight years old. Students were first divided into two ability subgroups (intellectually able vs.…

  20. Effective Teaching of Able Pupils in the Primary School: The Findings of the Oxfordshire Effective Teachers of Able Pupils Project.

    ERIC Educational Resources Information Center

    Eyre, Deborah; Coates, David; Fitzpatrick, Mary; Higgins, Chris; McClure, Lynne; Wilson, Helen; Chamberlin, Rosemary

    2002-01-01

    A review of British research on effective teaching of able students leads to a report on the Oxfordshire Effective Teachers of Able Pupils Project. This study found effective teachers shared similar beliefs about learning, had empathy with the needs of able children, created a secure classroom environment, held high expectations, used…

  1. Characterization of leukemias with ETV6-ABL1 fusion.

    PubMed

    Zaliova, Marketa; Moorman, Anthony V; Cazzaniga, Giovanni; Stanulla, Martin; Harvey, Richard C; Roberts, Kathryn G; Heatley, Sue L; Loh, Mignon L; Konopleva, Marina; Chen, I-Ming; Zimmermannova, Olga; Schwab, Claire; Smith, Owen; Mozziconacci, Marie-Joelle; Chabannon, Christian; Kim, Myungshin; Frederik Falkenburg, J H; Norton, Alice; Marshall, Karen; Haas, Oskar A; Starkova, Julia; Stuchly, Jan; Hunger, Stephen P; White, Deborah; Mullighan, Charles G; Willman, Cheryl L; Stary, Jan; Trka, Jan; Zuna, Jan

    2016-09-01

    To characterize the incidence, clinical features and genetics of ETV6-ABL1 leukemias, representing targetable kinase-activating lesions, we analyzed 44 new and published cases of ETV6-ABL1-positive hematologic malignancies [22 cases of acute lymphoblastic leukemia (13 children, 9 adults) and 22 myeloid malignancies (18 myeloproliferative neoplasms, 4 acute myeloid leukemias)]. The presence of the ETV6-ABL1 fusion was ascertained by cytogenetics, fluorescence in-situ hybridization, reverse transcriptase-polymerase chain reaction and RNA sequencing. Genomic and gene expression profiling was performed by single nucleotide polymorphism and expression arrays. Systematic screening of more than 4,500 cases revealed that in acute lymphoblastic leukemia ETV6-ABL1 is rare in childhood (0.17% cases) and slightly more common in adults (0.38%). There is no systematic screening of myeloproliferative neoplasms; however, the number of ETV6-ABL1-positive cases and the relative incidence of acute lymphoblastic leukemia and myeloproliferative neoplasms suggest that in adulthood ETV6-ABL1 is more common in BCR-ABL1-negative chronic myeloid leukemia-like myeloproliferations than in acute lymphoblastic leukemia. The genomic profile of ETV6-ABL1 acute lymphoblastic leukemia resembled that of BCR-ABL1 and BCR-ABL1-like cases with 80% of patients having concurrent CDKN2A/B and IKZF1 deletions. In the gene expression profiling all the ETV6-ABL1-positive samples clustered in close vicinity to BCR-ABL1 cases. All but one of the cases of ETV6-ABL1 acute lymphoblastic leukemia were classified as BCR-ABL1-like by a standardized assay. Over 60% of patients died, irrespectively of the disease or age subgroup examined. In conclusion, ETV6-ABL1 fusion occurs in both lymphoid and myeloid leukemias; the genomic profile and clinical behavior resemble BCR-ABL1-positive malignancies, including the unfavorable prognosis, particularly of acute leukemias. The poor outcome suggests that treatment with

  2. Targeting the SH2-Kinase Interface in Bcr-Abl Inhibits Leukemogenesis

    SciTech Connect

    Grebien, Florian; Hantschel, Oliver; Wojcik, John; Kaupe, Ines; Kovacic, Boris; Wyrzucki, Arkadiusz M.; Gish, Gerald D.; Cerny-Reiterer, Sabine; Koide, Akiko; Beug, Hartmut; Pawson, Tony; Valent, Peter; Koide, Shohei; Superti-Furga, Giulio

    2012-10-25

    Chronic myelogenous leukemia (CML) is caused by the constitutively active tyrosine kinase Bcr-Abl and treated with the tyrosine kinase inhibitor (TKI) imatinib. However, emerging TKI resistance prevents complete cure. Therefore, alternative strategies targeting regulatory modules of Bcr-Abl in addition to the kinase active site are strongly desirable. Here, we show that an intramolecular interaction between the SH2 and kinase domains in Bcr-Abl is both necessary and sufficient for high catalytic activity of the enzyme. Disruption of this interface led to inhibition of downstream events critical for CML signaling and, importantly, completely abolished leukemia formation in mice. Furthermore, disruption of the SH2-kinase interface increased sensitivity of imatinib-resistant Bcr-Abl mutants to TKI inhibition. An engineered Abl SH2-binding fibronectin type III monobody inhibited Bcr-Abl kinase activity both in vitro and in primary CML cells, where it induced apoptosis. This work validates the SH2-kinase interface as an allosteric target for therapeutic intervention.

  3. Single Point Mutation in Bin/Amphiphysin/Rvs (BAR) Sequence of Endophilin Impairs Dimerization, Membrane Shaping, and Src Homology 3 Domain-mediated Partnership*

    PubMed Central

    Gortat, Anna; San-Roman, Mabel Jouve; Vannier, Christian; Schmidt, Anne A.

    2012-01-01

    Bin/Amphiphysin/Rvs (BAR) domain-containing proteins are essential players in the dynamics of intracellular compartments. The BAR domain is an evolutionarily conserved dimeric module characterized by a crescent-shaped structure whose intrinsic curvature, flexibility, and ability to assemble into highly ordered oligomers contribute to inducing the curvature of target membranes. Endophilins, diverging into A and B subgroups, are BAR and SH3 domain-containing proteins. They exert activities in membrane dynamic processes such as endocytosis, autophagy, mitochondrial dynamics, and permeabilization during apoptosis. Here, we report on the involvement of the third α-helix of the endophilin A BAR sequence in dimerization and identify leucine 215 as a key residue within a network of hydrophobic interactions stabilizing the entire BAR dimer interface. With the combination of N-terminal truncation retaining the high dimerization capacity of the third α-helices of endophilin A and leucine 215 substitution by aspartate (L215D), we demonstrate the essential role of BAR sequence-mediated dimerization on SH3 domain partnership. In comparison with wild type, full-length endophilin A2 heterodimers with one protomer bearing the L215D substitution exhibit very significant changes in membrane binding and shaping activities as well as a dramatic decrease of SH3 domain partnership. This suggests that subtle changes in the conformation and/or rigidity of the BAR domain impact both the control of membrane curvature and downstream binding to effectors. Finally, we show that expression, in mammalian cells, of endophilin A2 bearing the L215D substitution impairs the endocytic recycling of transferrin receptors. PMID:22167186

  4. Expression of BCR-ABL1 oncogene relative to ABL1 gene changes overtime in chronic myeloid leukemia

    SciTech Connect

    Gupta, Manu; Milani, Lili; Hermansson, Monica; Simonsson, Bengt; Markevaern, Berit; Syvaenen, Ann Christine; Barbany, Gisela

    2008-02-15

    Using a quantitative single nucleotide polymorphism (SNP) assay we have investigated the changes in the expression of the BCR-ABL1 oncogene relative to the wild-type ABL1 and BCR alleles in cells from chronic myeloid leukemia (CML) patients not responding to therapy. The results show a progressive increase in the BCR-ABL1 oncogene expression at the expense of decreased expression of the ABL1 allele, not involved in the fusion. No relative changes in the expression of the two BCR alleles were found. These results demonstrate that allele-specific changes in gene expression, with selective, progressive silencing of the wild-type ABL1 allele in favor of the oncogenic BCR-ABL1 allele occur in CML patients with therapy-resistant disease.

  5. In Planta Mutagenesis of Src Homology 3 Domain-like Fold of NdhS, a Ferredoxin-binding Subunit of the Chloroplast NADH Dehydrogenase-like Complex in Arabidopsis

    PubMed Central

    Yamamoto, Hiroshi; Shikanai, Toshiharu

    2013-01-01

    Chloroplast NADH dehydrogenase-like (NDH) complex mediates cyclic electron transport around photosystem I and chlororespiration in angiosperms. The Src homology 3 domain (SH3)-like fold protein NdhS/CRR31 is an NDH subunit that is necessary for high affinity binding of ferredoxin, indicating that chloroplast NDH functions as a ferredoxin:plastoquinone oxidoreductase. However, the mechanism of the interaction between NdhS and ferredoxin is unclear. In this study, we analyzed their interaction in planta by using site-directed mutagenesis of NdhS. In general, binding of ferredoxin to its target proteins depends on electrostatic interaction. In silico analysis predicted the presence of a positively charged pocket in the SH3-like domain of NdhS, where nine charged residues are highly conserved among plants. Systematic alteration of these sites with neutral glutamine revealed that only arginine 193 was required for high NDH activity in vivo. Further replacement of arginine 193 with negatively charged aspartate or glutamate or hydrophobic alanine significantly decreased the efficiency of ferredoxin-dependent plastoquinone reduction by NDH in ruptured chloroplasts. Similar results were obtained in in vivo analyses of NDH activity and electron transport. From these results, we propose that the positive charge of arginine 193 in the SH3-like domain of NdhS is critical for electrostatic interaction with ferredoxin in vivo. PMID:24225949

  6. BCR-ABL/p62/SQSTM1: a cannibal embrace.

    PubMed

    Auberger, Patrick

    2012-10-25

    In this issue of Blood, Goussetis et al identify autophagy as a new pathway for the degradation of the oncoprotein BCR-ABL. They show that the therapeutic drug arsenic trioxide (AS(2)O(3)) targets BCR-ABL for autophagic degradation via a p62/SQSTM1-dependent mechanism that is critical for the antileukemic effect of the drug. PMID:23100300

  7. History Untold: Celebrating Ohio History through ABLE Students.

    ERIC Educational Resources Information Center

    Kent State Univ., OH. Ohio Literacy Resource Center.

    This document is a compilation of 25 pieces of writing presenting Ohio adult basic and literacy education (ABLE) students' perspectives of community and personal history. The items included in the compilation were written by ABLE students across Ohio. The compilation is organized in three sections as follows: (1) people (9 items, including a…

  8. Domain-mediated protein interaction prediction: From genome to network.

    PubMed

    Reimand, Jüri; Hui, Shirley; Jain, Shobhit; Law, Brian; Bader, Gary D

    2012-08-14

    Protein-protein interactions (PPIs), involved in many biological processes such as cellular signaling, are ultimately encoded in the genome. Solving the problem of predicting protein interactions from the genome sequence will lead to increased understanding of complex networks, evolution and human disease. We can learn the relationship between genomes and networks by focusing on an easily approachable subset of high-resolution protein interactions that are mediated by peptide recognition modules (PRMs) such as PDZ, WW and SH3 domains. This review focuses on computational prediction and analysis of PRM-mediated networks and discusses sequence- and structure-based interaction predictors, techniques and datasets for identifying physiologically relevant PPIs, and interpreting high-resolution interaction networks in the context of evolution and human disease. PMID:22561014

  9. Identification of novel inhibitors of BCR-ABL tyrosine kinase via virtual screening.

    PubMed

    Peng, Hui; Huang, Niu; Qi, Jing; Xie, Ping; Xu, Chen; Wang, Jianxiang; Yang, Chunzheng

    2003-11-01

    Inhibition of BCR-ABL tyrosine kinase activity has shown to be essential for the treatment of chronic myelogenous leukemia (CML). However, drug resistance has quickly arisen in recent clinical trials for STI571 (Gleevec), which is the first approved drug of CML by inhibiting ABL tyrosine kinase. It is desirable to develop new types of ABL tyrosine kinase inhibitors that may overcome this drug resistance problem. Here we present the discovery of novel inhibitors targeted at the catalytic domain of ABL tyrosine kinase by using three-dimensional database searching techniques. From a database containing 200,000 commercially available compounds, the top 1000 compounds with the best DOCK energy score were selected and subjected to structural diversity and drug likeness analysis, 15 compounds were submitted for biological assay. Eight out of the 15 showed inhibitory activity against K562 cells with IC(50) value ranging from 10 to 200 microM. Two promising compounds showed inhibition in further ABL tyrosine phosphorylation assay. It is anticipated that those two compounds can serve as lead compounds for further drug design and optimization. PMID:14552760

  10. Regulation of Neuronal Cell Death by c-Abl-Hippo/MST2 Signaling Pathway

    PubMed Central

    Xiao, Lei; Bai, Yujie; Qu, Aiqin; Zheng, Zheng; Yuan, Zengqiang

    2012-01-01

    Background Mammalian Ste20-like kinases (MSTs) are the mammalian homologue of Drosophila hippo and play critical roles in regulation of cell death, organ size control, proliferation and tumorigenesis. MSTs exert pro-apoptotic function through cleavage, autophosphorylation and in turn phosphorylation of downstream targets, such as Histone H2B and FOXO (Forkhead box O). Previously we reported that protein kinase c-Abl mediates oxidative stress-induced neuronal cell death through phosphorylating MST1 at Y433, which is not conserved among mammalian MST2, Drosophila Hippo and C.elegans cst-1/2. Methodology/Principal Findings Using immunoblotting, in vitro kinase and cell death assay, we demonstrate that c-Abl kinase phosphorylates MST2 at an evolutionarily conserved site, Y81, within the kinase domain. We further show that the phosphorylation of MST2 by c-Abl leads to the disruption of the interaction with Raf-1 proteins and the enhancement of homodimerization of MST2 proteins. It thereby enhances the MST2 activation and induces neuronal cell death. Conclusions/Significance The identification of the c-Abl tyrosine kinase as a novel upstream activator of MST2 suggests that the conserved c-Abl-MST signaling cascade plays an important role in oxidative stress-induced neuronal cell death. PMID:22590567

  11. Identification of alternative topological domains in chromatin

    PubMed Central

    2014-01-01

    Chromosome conformation capture experiments have led to the discovery of dense, contiguous, megabase-sized topological domains that are similar across cell types and conserved across species. These domains are strongly correlated with a number of chromatin markers and have since been included in a number of analyses. However, functionally-relevant domains may exist at multiple length scales. We introduce a new and efficient algorithm that is able to capture persistent domains across various resolutions by adjusting a single scale parameter. The ensemble of domains we identify allows us to quantify the degree to which the domain structure is hierarchical as opposed to overlapping, and our analysis reveals a pronounced hierarchical structure in which larger stable domains tend to completely contain smaller domains. The identified novel domains are substantially different from domains reported previously and are highly enriched for insulating factor CTCF binding and histone marks at the boundaries. PMID:24868242

  12. Formation of long and winding nuclear F-actin bundles by nuclear c-Abl tyrosine kinase

    SciTech Connect

    Aoyama, Kazumasa; Yuki, Ryuzaburo; Horiike, Yasuyoshi; Kubota, Sho; Yamaguchi, Noritaka; Morii, Mariko; Ishibashi, Kenichi; Nakayama, Yuji; Kuga, Takahisa; Hashimoto, Yuuki; Tomonaga, Takeshi; Yamaguchi, Naoto

    2013-12-10

    The non-receptor-type tyrosine kinase c-Abl is involved in actin dynamics in the cytoplasm. Having three nuclear localization signals (NLSs) and one nuclear export signal, c-Abl shuttles between the nucleus and the cytoplasm. Although monomeric actin and filamentous actin (F-actin) are present in the nucleus, little is known about the relationship between c-Abl and nuclear actin dynamics. Here, we show that nuclear-localized c-Abl induces nuclear F-actin formation. Adriamycin-induced DNA damage together with leptomycin B treatment accumulates c-Abl into the nucleus and increases the levels of nuclear F-actin. Treatment of c-Abl-knockdown cells with Adriamycin and leptomycin B barely increases the nuclear F-actin levels. Expression of nuclear-targeted c-Abl (NLS-c-Abl) increases the levels of nuclear F-actin even without Adriamycin, and the increased levels of nuclear F-actin are not inhibited by inactivation of Abl kinase activity. Intriguingly, expression of NLS-c-Abl induces the formation of long and winding bundles of F-actin within the nucleus in a c-Abl kinase activity-dependent manner. Furthermore, NLS-c-AblΔC, which lacks the actin-binding domain but has the full tyrosine kinase activity, is incapable of forming nuclear F-actin and in particular long and winding nuclear F-actin bundles. These results suggest that nuclear c-Abl plays critical roles in actin dynamics within the nucleus. - Highlights: • We show the involvement of c-Abl tyrosine kinase in nuclear actin dynamics. • Nuclear F-actin is formed by nuclear-localized c-Abl and its kinase-dead version. • The c-Abl actin-binding domain is prerequisite for nuclear F-actin formation. • Formation of long nuclear F-actin bundles requires nuclear c-Abl kinase activity. • We discuss a role for nuclear F-actin bundle formation in chromatin regulation.

  13. C-Abl as a modulator of p53

    SciTech Connect

    Levav-Cohen, Yaara; Goldberg, Zehavit; Zuckerman, Valentina; Grossman, Tamar; Haupt, Sue; Haupt, Ygal . E-mail: haupt@md.huji.ac.il

    2005-06-10

    P53 is renowned as a cellular tumor suppressor poised to instigate remedial responses to various stress insults that threaten DNA integrity. P53 levels and activities are kept under tight regulation involving a complex network of activators and inhibitors, which determine the type and extent of p53 growth inhibitory signaling. Within this complexity, the p53-Mdm2 negative auto-regulatory loop serves as a major route through which intra- and extra-cellular stress signals are channeled to appropriate p53 responses. Mdm2 inhibits p53 transcriptional activities and through its E3 ligase activity promotes p53 proteasomal degradation either within the nucleus or following nuclear export. Upon exposure to stress signals these actions of Mdm2 have to be moderated, or even interrupted, in order to allow sufficient p53 to accumulate in an active form. Multiple mechanisms involving a variety of factors have been demonstrated to mediate this interruption. C-Abl is a critical factor that under physiological conditions is required for the maximal and efficient accumulation of active p53 in response to DNA damage. C-Abl protects p53 by antagonizing the inhibitory effect of Mdm2, an action that requires a direct interplay between c-Abl and Mdm2. In addition, c-Abl protects p53 from other inhibitors of p53, such as the HPV-E6/E6AP complex, that inhibits and degrades p53 in HPV-infected cells. Surprisingly, the oncogenic form of c-Abl, the Bcr-Abl fusion protein in CML cells, also promotes the accumulation of wt p53. However, in contrast to the activation of p53 by c-Abl, its oncogenic form, Bcr-Abl, counteracts the growth inhibitory activities of p53 by modulating the p53-Mdm2 loop. Thus, it appears that by modulating the p53-Mdm2 loop, c-Abl and its oncogenic forms critically determine the type and extent of the cellular response to DNA damage.

  14. Organic acids and selected nitrogen species for ABLE-3

    NASA Technical Reports Server (NTRS)

    Talbot, Robert W.

    1991-01-01

    The NASA Global Tropospheric Experiment (GTE) executed airborne science missions aboard the NASA Wallops Electra (NA429) in the North American high latitude (greater than 45 deg North) atmosphere during Jul. to Aug. 1988 and Jul. to Aug. 1990. These missions were part of GTE's Atmospheric Boundary Layer Experiment (ABLE). The 1988 mission , ABLE-3A, examined the ecosystems of Alaska as a source and/or sink for important tropospheric gases and particles, and gained new information on the chemical composition of the Arctic atmosphere during the summertime. During 1990 the second high latitude mission, ABLE-3B, focused on the Hudson Bay Lowland and Labrador regions of Canada. Both of these missions provided benchmark data sets on atmosphere biosphere exchange and atmospheric chemistry over largely uninhabited regions of North America. In support of the GTE/ABLE-3A and -3B field missions, the University of New Hampshire flew instrumentation aboard the Wallops Electra research aircraft to provide measurements of the trace gases nitric (HNO3), formic (HCOOH), and acetic (CH3COOH) acid. In addition, measurements were conducted to determine the major water soluble ionic composition of the atmospheric aerosol. For ABLE-3B, groundbased measurements of the acidic trace gases were also performed from the NASA micrometerological tower situated at Schefferville, Laborador. These measurements were aimed at assessing dry deposition of acidic gases to the taiga ecosystem in the Laborador region of Canada.

  15. Introducing the advanced burn life support (ABLS) course in Italy.

    PubMed

    D'Asta, F; Homsi, J; Clark, P; Buffalo, M C; Melandri, D; Carboni, A; Pinzauti, E; Graziano, A; Masellis, A; Bussolin, L; Messineo, A

    2014-05-01

    Systematic education based on internationally standardized programs is a well-established practice in Italy, especially in the emergency health care system. However, until recently, a specific program to treat burns was not available to guide emergency physicians, nurses, or volunteers acting as first responders. In 2010, two national faculty members, acting as ABA observers, and one Italian course coordinator, trained and certified in the United States, conducted a week-long training program which fully certified 10 Italian instructors. Authorized ABLS provider courses were conducted in Italy between 2010 and 2012, including one organized prior to the 20th annual meeting of the Italian Society of Burns (SIUst). In order to increase the effectiveness and diffusion of the course in Italy, changes were approved by the ABA to accommodate societal differences, including the translation of the manual into Italian. The ABA has also approved the creation and publication of a bilingual ABLS Italian website for the purpose of promoting the ABLS course in Italy. In response to high demand, a second ABLS Instructor course was organized in 2012 and has been attended by physicians and nurses from several Italian burn centers. In the following discourse the experiences of the first 15 Italian ABLS courses will be discussed. PMID:23992873

  16. Src kinase activity and SH2 domain regulate the dynamics of Src association with lipid and protein targets

    PubMed Central

    Shvartsman, Dmitry E.; Donaldson, John C.; Diaz, Begoña; Gutman, Orit; Martin, G. Steven; Henis, Yoav I.

    2007-01-01

    Src functions depend on its association with the plasma membrane and with specific membrane-associated assemblies. Many aspects of these interactions are unclear. We investigated the functions of kinase, SH2, and SH3 domains in Src membrane interactions. We used FRAP beam-size analysis in live cells expressing a series of c-Src–GFP proteins with targeted mutations in specific domains together with biochemical experiments to determine whether the mutants can generate and bind to phosphotyrosyl proteins. Wild-type Src displays lipid-like membrane association, whereas constitutively active Src-Y527F interacts transiently with slower-diffusing membrane-associated proteins. These interactions require Src kinase activity and SH2 binding, but not SH3 binding. Furthermore, overexpression of paxillin, an Src substrate with a high cytoplasmic population, competes with membrane phosphotyrosyl protein targets for binding to activated Src. Our observations indicate that the interactions of Src with lipid and protein targets are dynamic and that the kinase and SH2 domain cooperate in the membrane targeting of Src. PMID:17698610

  17. Coordinated activation of the Rac-GAP β2-chimaerin by an atypical proline-rich domain and diacylglycerol.

    PubMed

    Gutierrez-Uzquiza, Alvaro; Colon-Gonzalez, Francheska; Leonard, Thomas A; Canagarajah, Bertram J; Wang, HongBin; Mayer, Bruce J; Hurley, James H; Kazanietz, Marcelo G

    2013-01-01

    Chimaerins, a family of GTPase activating proteins for the small G-protein Rac, have been implicated in development, neuritogenesis and cancer. These Rac-GTPase activating proteins are regulated by the lipid second messenger diacylglycerol generated by tyrosine kinases such as the epidermal growth factor receptor. Here we identify an atypical proline-rich motif in chimaerins that binds to the adaptor protein Nck1. Unlike most Nck1 partners, chimaerins bind to the third SH3 domain of Nck1. This association is mediated by electrostatic interactions of basic residues within the Pro-rich motif with acidic clusters in the SH3 domain. Epidermal growth factor promotes the binding of β2-chimaerin to Nck1 in the cell periphery in a diacylglycerol-dependent manner. Moreover, β2-chimaerin translocation to the plasma membrane and its peripheral association with Rac1 requires Nck1. Our studies underscore a coordinated mechanism for β2-chimaerin activation that involves lipid interactions via the C1 domain and protein-protein interactions via the N-terminal proline-rich region. PMID:23673634

  18. Structural basis for the inhibition of HIV-1 Nef by a high-affinity binding single-domain antibody

    PubMed Central

    2014-01-01

    Background The HIV-1 Nef protein is essential for AIDS pathogenesis by its interaction with host cell surface receptors and signaling factors. Despite its critical role as a virulence factor Nef is not targeted by current antiviral strategies. Results We have determined the crystal structure of the complex formed by a camelid single-domain antibody fragment, termed sdAb19, bound to HIV-1 Nef together with a stabilizing SH3 domain. sdAb19 forms a stoichiometric 1:1 complex with Nef and binds to a conformationally conserved surface at the C-terminus of Nef that overlaps with functionally important interaction sites involved in Nef-induced perturbations of signaling and trafficking pathways. The antibody fragment binds Nef with low nanomolar affinity, which could be attenuated to micromolar affinity range by site-directed mutagenesis of key interaction residues in sdAb19. Fusion of the SH3 domain to sdAb19, termed Neffin, leads to a significantly increased affinity for Nef and formation of a stoichiometric 2:2 Nef–Neffin complex. The 19 kDa Neffin protein inhibits all functions of Nef as CD4 and MHC-I downregulation, association with Pak2, and the increase in virus infectivity and replication. Conclusions Together, sdAb19 and Neffin thus represent efficient tools for the rational development of antiviral strategies against HIV-1 Nef. PMID:24620746

  19. Reproductive fitness advantage of BCR-ABL expressing leukemia cells.

    PubMed

    Traulsen, Arne; Pacheco, Jorge M; Dingli, David

    2010-08-01

    Mutations in oncogenes and tumor suppressor genes confer a fitness advantage to cells that can lead to cancer. The tumor phenotype normally results from the interaction of many mutant genes making it difficult to estimate the fitness advantage provided by any oncogene, except when tumors depend on one oncogene only. We utilize a model of chronic myeloid leukemia (CML), to quantitate the fitness advantage conferred by expression of BCR-ABL in hematopoietic cells from in vivo patient data. We show that BCR-ABL expression provides a high fitness advantage, which explains why this single mutation drives the chronic phase of CML. PMID:20153920

  20. Nilotinib treatment in mouse models of P190 Bcr/Abl lymphoblastic leukemia

    PubMed Central

    Kaur, Pavinder; Feldhahn, Niklas; Zhang, Bin; Trageser, Daniel; Müschen, Markus; Pertz, Veerle; Groffen, John; Heisterkamp, Nora

    2007-01-01

    Background Ph-positive leukemias are caused by the aberrant fusion of the BCR and ABL genes. Nilotinib is a selective Bcr/Abl tyrosine kinase inhibitor related to imatinib, which is widely used to treat chronic myelogenous leukemia. Because Ph-positive acute lymphoblastic leukemia only responds transiently to imatinib therapy, we have used mouse models to test the efficacy of nilotinib against lymphoblastic leukemia caused by the P190 form of Bcr/Abl. Results After transplant of 10,000 highly malignant leukemic cells into compatible recipients, untreated mice succumbed to leukemia within 21 days, whereas mice treated with 75 mg/kg nilotinib survived significantly longer. We examined cells from mice that developed leukemia while under treatment for Bcr/Abl kinase domain point mutations but these were not detected. In addition, culture of such cells ex vivo showed that they were as sensitive as the parental cell line to nilotinib but that the presence of stromal support allowed resistant cells to grow out. Nilotinib also exhibited impressive anti-leukemia activity in P190 Bcr/Abl transgenic mice that had developed overt leukemia/lymphoma masses and that otherwise would have been expected to die within 7 days. Visible lymphoma masses disappeared within six days of treatment and leukemic cell numbers in peripheral blood were significantly reduced. Treated mice survived more than 30 days. Conclusion These results show that nilotinib has very impressive anti-leukemia activity but that lymphoblastic leukemia cells can become unresponsive to it both in vitro and in vivo through mechanisms that appear to be Bcr/Abl independent. PMID:17958915

  1. The Inclusion of Differently Abled Students in the Regular Classroom.

    ERIC Educational Resources Information Center

    Lewis, Angela

    This study sought to evaluate the implementation of a program to foster the inclusion of differently abled students into a regular elementary school classroom. The report is based on interviews with eight regular and two special education teachers, as well as the school principal, along with classroom materials and information on inclusion…

  2. Classroom Activities for Able Students: In Fifth and Sixth Grades.

    ERIC Educational Resources Information Center

    Economopoulos, Marjorie P.

    1981-01-01

    Challenging problems and project ideas for mathematically able students in grades 5 and 6 are reviewed. Number patterns are viewed as a good tool to introduce inductive and deductive thinking. Applications of mathematics in sales, the stock market, opinion polls, weather prediction, and other areas are discussed. (MP)

  3. The Able Privilege Scale: A New Educational Tool

    ERIC Educational Resources Information Center

    Larson, Alan B.; Choate, Robert O.

    2011-01-01

    The Able Privilege Scale (APS) is a theoretically derived reflexive instrument that asks participants to assess their level of personal power and privilege in relation to society depending upon the presence, and/or type, of disability. The 23-items on the APS are derived form McIntosh's concept of white privilege (1988) and adapted to reflect…

  4. Imatinib may be ABL to improve anti-angiogenic therapy

    PubMed Central

    Raimondi, Claudio; Fantin, Alessandro; Ruhrberg, Christiana

    2015-01-01

    We recently reported that neuropilin 1 (NRP1) drives angiogenesis by promoting extracellular matrix signaling in endothelial cells via ABL1 kinase. Imatinib targets this pathway in pathological angiogenesis and may provide a novel opportunity for anti-angiogenic therapy of age-related macular degeneration, proliferative diabetic retinopathy, or solid tumor growth. PMID:27308396

  5. A Comprehensive Plan for Attracting Able Students to Teacher Education.

    ERIC Educational Resources Information Center

    Evans, Sally; And Others

    In response to the need to draw academically able students into the teaching profession, the College of Education and Human Services at Wright State University (Ohio) has initiated several new programs and policies that form part of a comprehensive plan to recruit, select, and retain potentially talented teachers. The special programs include: (1)…

  6. Tyrosine phosphorylation of RAS by ABL allosterically enhances effector binding

    PubMed Central

    Ting, Pamela Y.; Johnson, Christian W.; Fang, Cong; Cao, Xiaoqing; Graeber, Thomas G.; Mattos, Carla; Colicelli, John

    2015-01-01

    RAS proteins are signal transduction gatekeepers that mediate cell growth, survival, and differentiation through interactions with multiple effector proteins. The RAS effector RAS- and RAB-interacting protein 1 (RIN1) activates its own downstream effectors, the small GTPase RAB5 and the tyrosine kinase Abelson tyrosine-protein kinase (ABL), to modulate endocytosis and cytoskeleton remodeling. To identify ABL substrates downstream of RAS-to-RIN1 signaling, we examined human HEK293T cells overexpressing components of this pathway. Proteomic analysis revealed several novel phosphotyrosine peptides, including Harvey rat sarcoma oncogene (HRAS)-pTyr137. Here we report that ABL phosphorylates tyrosine 137 of H-, K-, and NRAS. Increased RIN1 levels enhanced HRAS-Tyr137 phosphorylation by nearly 5-fold, suggesting that RAS-stimulated RIN1 can drive ABL-mediated RAS modification in a feedback circuit. Tyr137 is well conserved among RAS orthologs and is part of a transprotein H-bond network. Crystal structures of HRASY137F and HRASY137E revealed conformation changes radiating from the mutated residue. Although consistent with Tyr137 participation in allosteric control of HRAS function, the mutations did not alter intrinsic GTP hydrolysis rates in vitro. HRAS-Tyr137 phosphorylation enhanced HRAS signaling capacity in cells, however, as reflected by a 4-fold increase in the association of phosphorylated HRASG12V with its effector protein RAF proto-oncogene serine/threonine protein kinase 1 (RAF1). These data suggest that RAS phosphorylation at Tyr137 allosterically alters protein conformation and effector binding, providing a mechanism for effector-initiated modulation of RAS signaling.—Ting, P. Y., Johnson, C. W., Fang, C., Cao, X., Graeber, T. G., Mattos, C., Colicelli, J. Tyrosine phosphorylation of RAS by ABL allosterically enhances effector binding. PMID:25999467

  7. Solution structure of the DNA binding domain of HIV-1 integrase.

    PubMed

    Lodi, P J; Ernst, J A; Kuszewski, J; Hickman, A B; Engelman, A; Craigie, R; Clore, G M; Gronenborn, A M

    1995-08-01

    The solution structure of the DNA binding domain of HIV-1 integrase (residues 220-270) has been determined by multidimensional NMR spectroscopy. The protein is a dimer in solution, and each subunit is composed of a five-stranded beta-barrel with a topology very similar to that of the SH3 domain. The dimer is formed by a stacked beta-interface comprising strands 2, 3, and 4, with the two triple-stranded antiparallel beta-sheets, one from each subunit, oriented antiparallel to each other. One surface of the dimer, bounded by the loop between strands beta 1 and beta 2, forms a saddle-shaped groove with dimensions of approximately 24 x 23 x 12 A in cross section. Lys264, which has been shown from mutational data to be involved in DNA binding, protrudes from this surface, implicating the saddle-shaped groove as the potential DNA binding site. PMID:7632683

  8. In planta mutagenesis of Src homology 3 domain-like fold of NdhS, a ferredoxin-binding subunit of the chloroplast NADH dehydrogenase-like complex in Arabidopsis: a conserved Arg-193 plays a critical role in ferredoxin binding.

    PubMed

    Yamamoto, Hiroshi; Shikanai, Toshiharu

    2013-12-20

    Chloroplast NADH dehydrogenase-like (NDH) complex mediates cyclic electron transport around photosystem I and chlororespiration in angiosperms. The Src homology 3 domain (SH3)-like fold protein NdhS/CRR31 is an NDH subunit that is necessary for high affinity binding of ferredoxin, indicating that chloroplast NDH functions as a ferredoxin:plastoquinone oxidoreductase. However, the mechanism of the interaction between NdhS and ferredoxin is unclear. In this study, we analyzed their interaction in planta by using site-directed mutagenesis of NdhS. In general, binding of ferredoxin to its target proteins depends on electrostatic interaction. In silico analysis predicted the presence of a positively charged pocket in the SH3-like domain of NdhS, where nine charged residues are highly conserved among plants. Systematic alteration of these sites with neutral glutamine revealed that only arginine 193 was required for high NDH activity in vivo. Further replacement of arginine 193 with negatively charged aspartate or glutamate or hydrophobic alanine significantly decreased the efficiency of ferredoxin-dependent plastoquinone reduction by NDH in ruptured chloroplasts. Similar results were obtained in in vivo analyses of NDH activity and electron transport. From these results, we propose that the positive charge of arginine 193 in the SH3-like domain of NdhS is critical for electrostatic interaction with ferredoxin in vivo. PMID:24225949

  9. Influence of entrainment and countergradient on the ABL diurnal development

    NASA Astrophysics Data System (ADS)

    Hernández-Ceballos, M. A.

    2009-09-01

    The representation of the diurnal evolution of the boundary layer (ABL) by NCAR-Penn State Mesoscale Model (MM5) and by the mesoscale model Weather Research Forecast (WRF) is compared. Special attention is paid to determine the role of processes that occur near and below the inversion zone: the positive correlation between the heat flux and the gradient (countergradient) and the role of entrainment of heat originating from the free troposphere. Both processes play a key role in the modelling of the diurnal variability of temperature, moisture and atmospheric compounds. A number of 13 simulations are carried out to determine the sensitivity of the model results to the formulation of the ABL height and countergradient heat flux in the Medium Range Forecast (MRF) ABL scheme. Model results are compared with experimental data obtained from the DOMINO (Diel Oxidant Mechanisms in relation to Nitrogen oxides) campaign. It was organized by Max Planck Institute for Atmospheric Chemistry (Germany) in collaboration with the National Institute for Aerospace Technology (Spain). The DOMINO campaign took place at the "Atmospheric Sounding Station - El Arenosillo", a platform dedicated to atmospheric measurements in the Southwest of Spain. All numerical experiments are grouped in four clusters, each focussing on the sensitivity of different relevant aspects. The following aspects of the formulation are analyzed: surface moisture availability (M), the countergradient term (γc) and the ABL height (h). This is done by modifying both the bulk critical Richardson number (Ric) at the inversion zone, and a coefficient of proportionality (b) that determines the excess temperature and countergradient. The importance of b is due to its direct relation in the definition of both, γc and h. The results got with MM5 model show that temperature and specific moisture temporal evolution is not very sensitive to changes in the soil moisture availability (M value from 0.6 to 0.1). Using the MRF

  10. Isolation of a Bacterial Strain Able To Degrade Branched Nonylphenol

    PubMed Central

    Tanghe, Tom; Dhooge, Willem; Verstraete, Willy

    1999-01-01

    Conventional enrichment of microorganisms on branched nonylphenol (NP) as only carbon and energy source yielded mixed cultures able to grow on the organic compound. However, plating yielded no single colonies capable, alone or in combination with other isolates, of degrading the NP in liquid culture. Therefore, a special approach was used, referred to as “serial dilution-plate resuspension,” to reduce culture complexity. In this way, one isolate, TTNP3, tentatively identified as a Sphingomonas sp., was found to be able to grow on NP in liquid culture. Remarkably, this isolate was able to be filtered through a 0.45-μm-pore-diameter filter. Moreover, isolate TTNP3 did not form visible colonies on mineral medium with NP, and it formed visible colonies on R2A agar only after a prolonged incubation of 1 week. High-performance liquid chromatography and gas chromatography-mass spectroscopy analysis of the culture media indicated that the strain starts the degradation of NP with a fission of the phenol ring and preferably uses the para isomer of NP and not the ortho isomer. No distinct accumulation of an intermediary product could be observed. PMID:9925611

  11. Site-selective Characterization of Src Homology 3 Domain Molecular Recognition with Cyanophenylalanine Infrared Probes

    PubMed Central

    Horness, Rachel E.; Basom, Edward J.; Thielges, Megan C.

    2015-01-01

    Local heterogeneity of microenvironments in proteins is important in biological function, but difficult to characterize experimentally. One approach is the combination of infrared (IR) spectroscopy and site-selective incorporation of probe moieties with spectrally resolved IR absorptions that enable characterization within inherently congested protein IR spectra. We employed this method to study molecular recognition of a Src homology 3 (SH3) domain from the yeast protein Sho1 for a peptide containing the proline-rich recognition sequence of its physiological binding partner Pbs2. Nitrile IR probes were introduced at four distinct sites in the protein by selective incorporation of p-cyanophenylalanine via the amber codon suppressor method and characterized by IR spectroscopy. Variation among the IR absorption bands reports on heterogeneity in local residue environments dictated by the protein structure, as well as on residue-dependent changes upon peptide binding. The study informs on the molecular recognition of SH3Sho1 and illustrates the speed and simplicity of this approach for characterization of select microenvironments within proteins. PMID:26491469

  12. ENHANCED AND SELECTIVE KILLING OF CHRONIC MYELOGENOUS LEUKEMIA CELLS WITH AN ENGINEERED BCR-ABL BINDING PROTEIN AND IMATINIB

    PubMed Central

    Constance, Jonathan E.; Woessner, David W.; Matissek, Karina J.; Mossalam, Mohanad; Lim, Carol S.

    2012-01-01

    The oncoprotein Bcr-Abl stimulates pro-survival pathways and suppresses apoptosis from its exclusively cytoplasmic locale, but when targeted to the mitochondrial compartment of leukemia cells, Bcr-Abl was potently cytotoxic. Therefore, we designed a protein construct to act as a mitochondrial chaperone to move Bcr-Abl to the mitochondria. The chaperone (i.e., the 43.6 kDa intracellular cryptic escort (iCE)) contains an EGFP tag and two previously characterized motifs: 1) An optimized Bcr-Abl binding motif that interacts with the coiled-coil domain of Bcr (ccmut3; 72 residues), and 2) A cryptic mitochondrial targeting signal (cMTS; 51 residues) that selectively targets the mitochondria in oxidatively stressed cells (i.e., Bcr-Abl positive leukemic cells) via phosphorylation at a key residue (T193) by protein kinase C. While the iCE colocalized with Bcr-Abl it did not re-localize to the mitochondria. However, the iCE was selectively toxic to Bcr-Abl positive K562 cells as compared to Bcr-Abl negative Cos-7 fibroblasts and 1471.1 murine breast cancer cells. The toxicity of the iCE to leukemic cells was equivalent to 10μM imatinib at 48 hours and the iCE combined with imatinib potentiated cell death beyond imatinib or the iCE alone. Substitution of either the ccmut3 or the cMTS with another Bcr-Abl binding domain (derived from Ras/Rab interaction protein 1 (RIN1; 295 residues)) or MTS (i.e., the canonical IMS derived from Smac/Diablo; 49 residues) did not match the cytotoxicity of the iCE. Additionally, a phosphorylation null mutant of the iCE also abolished the killing effect. The mitochondrial toxicity of Bcr-Abl and the iCE in Bcr-Abl positive K562 leukemia cells was confirmed by flow cytometric analysis of 7-AAD, TUNEL, and annexin-V staining. DNA segmentation and cell viability were assessed by microscopy. Subcellular localization of constructs was determined using confocal microscopy (including statistical colocalization analysis). Overall, the iCE was highly

  13. Isolation of Microorganisms Able To Metabolize Purified Natural Rubber

    PubMed Central

    Heisey, R. M.; Papadatos, S.

    1995-01-01

    Bacteria able to grow on purified natural rubber in the absence of other organic carbon sources were isolated from soil. Ten isolates reduced the weight of vulcanized rubber from latex gloves by >10% in 6 weeks. Scanning electron microscopy clearly revealed the ability of the microorganisms to colonize, penetrate, and dramatically alter the physical structure of the rubber. The rubber-metabolizing bacteria were identified on the basis of fatty acid profiles and cell wall characteristics. Seven isolates were strains of Streptomyces, two were strains of Amycolatopsis, and one was a strain of Nocardia. PMID:16535106

  14. Crystal Structure of the FERM Domain of Focal Adhesion Kinase

    SciTech Connect

    Ceccarelli,D.; Song, H.; Poy, F.; Schaller, M.; Eck, M.

    2006-01-01

    Focal adhesion kinase (FAK) is a non-receptor tyrosine kinase that localizes to focal adhesions in adherent cells. Through phosphorylation of proteins assembled at the cytoplasmic tails of integrins, FAK promotes signaling events that modulate cellular growth, survival, and migration. The amino-terminal region of FAK contains a region of sequence homology with band 4.1 and ezrin/radixin/moesin (ERM) proteins termed a FERM domain. FERM domains are found in a variety of signaling and cytoskeletal proteins and are thought to mediate intermolecular interactions with partner proteins and phospholipids at the plasma membrane and intramolecular regulatory interactions. Here we report two crystal structures of an NH2-terminal fragment of avian FAK containing the FERM domain and a portion of the regulatory linker that connects the FERM and kinase domains. The tertiary folds of the three subdomains (F1, F2, and F3) are similar to those of known FERM structures despite low sequence conservation. Differences in the sequence and relative orientation of the F3 subdomain alters the nature of the interdomain interface, and the phosphoinositide binding site found in ERM family FERM domains is not present in FAK. A putative protein interaction site on the F3 lobe is masked by the proximal region of the linker. Additionally, in one structure the adjacent Src SH3 and SH2 binding sites in the linker associate with the surfaces of the F3 and F1 lobes, respectively. These structural features suggest the possibility that protein interactions of the FAK FERM domain can be regulated by binding of Src kinases to the linker segment.

  15. Combined STAT3 and BCR-ABL1 inhibition induces synthetic lethality in therapy-resistant chronic myeloid leukemia.

    PubMed

    Eiring, A M; Page, B D G; Kraft, I L; Mason, C C; Vellore, N A; Resetca, D; Zabriskie, M S; Zhang, T Y; Khorashad, J S; Engar, A J; Reynolds, K R; Anderson, D J; Senina, A; Pomicter, A D; Arpin, C C; Ahmad, S; Heaton, W L; Tantravahi, S K; Todic, A; Colaguori, R; Moriggl, R; Wilson, D J; Baron, R; O'Hare, T; Gunning, P T; Deininger, M W

    2015-03-01

    Mutations in the BCR-ABL1 kinase domain are an established mechanism of tyrosine kinase inhibitor (TKI) resistance in Philadelphia chromosome-positive leukemia, but fail to explain many cases of clinical TKI failure. In contrast, it is largely unknown why some patients fail TKI therapy despite continued suppression of BCR-ABL1 kinase activity, a situation termed BCR-ABL1 kinase-independent TKI resistance. Here, we identified activation of signal transducer and activator of transcription 3 (STAT3) by extrinsic or intrinsic mechanisms as an essential feature of BCR-ABL1 kinase-independent TKI resistance. By combining synthetic chemistry, in vitro reporter assays, and molecular dynamics-guided rational inhibitor design and high-throughput screening, we discovered BP-5-087, a potent and selective STAT3 SH2 domain inhibitor that reduces STAT3 phosphorylation and nuclear transactivation. Computational simulations, fluorescence polarization assays and hydrogen-deuterium exchange assays establish direct engagement of STAT3 by BP-5-087 and provide a high-resolution view of the STAT3 SH2 domain/BP-5-087 interface. In primary cells from chronic myeloid leukemia (CML) patients with BCR-ABL1 kinase-independent TKI resistance, BP-5-087 (1.0 μM) restored TKI sensitivity to therapy-resistant CML progenitor cells, including leukemic stem cells. Our findings implicate STAT3 as a critical signaling node in BCR-ABL1 kinase-independent TKI resistance, and suggest that BP-5-087 has clinical utility for treating malignancies characterized by STAT3 activation. PMID:25134459

  16. Expressing gait-line symmetry in able-bodied gait

    PubMed Central

    Jeleń, Piotr; Wit, Andrzej; Dudziński, Krzysztof; Nolan, Lee

    2008-01-01

    Background Gait-lines, or the co-ordinates of the progression of the point of application of the vertical ground reaction force, are a commonly reported parameter in most in-sole measuring systems. However, little is known about what is considered a "normal" or "abnormal" gait-line pattern or level of asymmetry. Furthermore, no reference databases on healthy young populations are available for this parameter. Thus the aim of this study is to provide such reference data in order to allow this tool to be better used in gait analysis. Methods Vertical ground reaction force data during several continuous gait cycles were collected using a Computer Dyno Graphy in-sole system® for 77 healthy young able-bodied subjects. A curve (termed gait-line) was obtained from the co-ordinates of the progression of the point of application of the force. An Asymmetry Coefficient Curve (AsC) was calculated between the mean gait-lines for the left and right foot for each subject. AsC limits of ± 1.96 and 3 standard deviations (SD) from the mean were then calculated. Gait-line data from 5 individual subjects displaying pathological gait due to disorders relating to the discopathy of the lumbar spine (three with considerable plantarflexor weakness, two with considerable dorsiflexor weakness) were compared to the AsC results from the able-bodied group. Results The ± 1.96 SD limit suggested that non-pathological gait falls within 12–16% asymmetry for gait-lines. Those exhibiting pathological gait fell outside both the ± 1.96 and ± 3SD limits at several points during stance. The subjects exhibiting considerable plantarflexor weakness all fell outside the ± 1.96SD limit from 30–50% of foot length to toe-off while those exhibiting considerable dorsiflexor weakness fell outside the ± 1.96SD limit between initial contact to 25–40% of foot length, and then surpassed the ± 3SD limit after 55–80% of foot length. Conclusion This analysis of gait-line asymmetry provides a reference

  17. HS-438, a new inhibitor of imatinib-resistant BCR-ABL T315I mutation in chronic myeloid leukemia.

    PubMed

    Yun, Sun-Mi; Jung, Kyung Hee; Kim, Soo Jung; Fang, Zhenghuan; Son, Mi Kwon; Yan, Hong Hua; Lee, Hyunseung; Kim, JinHee; Shin, Sanghye; Hong, Sungwoo; Hong, Soon-Sun

    2014-06-28

    Imatinib is a selective breakpoint cluster region-Abelson (BCR-ABL) tyrosine kinase inhibitor (TKI) that has significantly improved the prognosis of patients with chronic myeloid leukemia (CML). However, T315I gene mutations of the BCR-ABL kinase domain have been shown to confer resistance to imatinib. In the present study, we synthesized a novel BCR-ABL inhibitor, HS-438, and identified its anti-leukemic effects in vitro and in vivo. We found that HS-438 strongly inhibited the expression of BCR-ABL signaling pathways in wild-type BCR-ABL (BaF3/WT) cells as well as T315I-mutated BCR-ABL (BaF3/T315I) cells with resistance to imatinib. HS-438 induced cell cycle arrest, particularly during the G0/G1 cell cycle phase, and induced apoptosis. In BaF3/T315I xenograft models, HS-438 significantly delayed tumor growth, unlike imatinib. In summary, we suggest that HS-438 may be a novel drug candidate with the therapeutic potential to target BCR-ABL and overcome imatinib resistance in patients with CML. PMID:24657654

  18. ABI-like transcription factor gene TaABL1 from wheat improves multiple abiotic stress tolerances in transgenic plants.

    PubMed

    Xu, Dong-Bei; Gao, Shi-Qing; Ma, You-Zhi; Xu, Zhao-Shi; Zhao, Chang-Ping; Tang, Yi-Miao; Li, Xue-Yin; Li, Lian-Cheng; Chen, Yao-Feng; Chen, Ming

    2014-12-01

    The phytohormone abscisic acid (ABA) plays crucial roles in adaptive responses of plants to abiotic stresses. ABA-responsive element binding proteins (AREBs) are basic leucine zipper transcription factors that regulate the expression of downstream genes containing ABA-responsive elements (ABREs) in promoter regions. A novel ABI-like (ABA-insensitive) transcription factor gene, named TaABL1, containing a conserved basic leucine zipper (bZIP) domain was cloned from wheat. Southern blotting showed that three copies were present in the wheat genome. Phylogenetic analyses indicated that TaABL1 belonged to the AREB subfamily of the bZIP transcription factor family and was most closely related to ZmABI5 in maize and OsAREB2 in rice. Expression of TaABL1 was highly induced in wheat roots, stems, and leaves by ABA, drought, high salt, and low temperature stresses. TaABL1 was localized inside the nuclei of transformed wheat mesophyll protoplast. Overexpression of TaABL1 enhanced responses of transgenic plants to ABA and hastened stomatal closure under stress, thereby improving tolerance to multiple abiotic stresses. Furthermore, overexpression of TaABL1 upregulated or downregulated the expression of some stress-related genes controlling stomatal closure in transgenic plants under ABA and drought stress conditions, suggesting that TaABL1 might be a valuable genetic resource for transgenic molecular breeding. PMID:25344442

  19. Endocrine disrupters: the new players able to affect the epigenome

    PubMed Central

    Casati, Lavinia; Sendra, Ramon; Sibilia, Valeria; Celotti, Fabio

    2015-01-01

    Epigenetics represents the way by which the environment is able to program the genome; there are three main levels of epigenetic control on genome: DNA methylation, post-translational histone modification and microRNA expression. The term Epigenetics has been widened by NIH to include “both heritable changes in gene activity and expression but also stable, long-term alterations in the transcriptional potential of a cell that are not necessarily heritable.” These changes might be produced mostly by the early life environment and might affect health influencing the susceptibility to develop diseases, from cancer to mental disorder, during the entire life span. The most studied environmental influences acting on epigenome are diet, infections, wasting, child care, smoking and environmental pollutants, in particular endocrine disrupters (EDs). These are environmental xenobiotics able to interfere with the normal development of the male and female reproductive systems of wildlife, of experimental animals and possibly of humans, disrupting the normal reproductive functions. Data from literature indicate that EDs can act at different levels of epigenetic control, in some cases transgenerationally, in particular when the exposure to these compounds occurs during the prenatal and earliest period of life. Some of the best characterized EDs will be considered in this review. Among the EDs, vinclozolin (VZ), and methoxychlor (MXC) promote epigenetic transgenerational effects. Polychlorinated biphenils (PCBs), the most widespread environmental EDs, affect histone post-translational modifications in a dimorphic way, possibly as the result of an alteration of gene expression of the enzymes involved in histone modification, as the demethylase Jarid1b, an enzyme also involved in regulating the interaction of androgens with their receptor. PMID:26151052

  20. Endocrine disrupters: the new players able to affect the epigenome.

    PubMed

    Casati, Lavinia; Sendra, Ramon; Sibilia, Valeria; Celotti, Fabio

    2015-01-01

    Epigenetics represents the way by which the environment is able to program the genome; there are three main levels of epigenetic control on genome: DNA methylation, post-translational histone modification and microRNA expression. The term Epigenetics has been widened by NIH to include "both heritable changes in gene activity and expression but also stable, long-term alterations in the transcriptional potential of a cell that are not necessarily heritable." These changes might be produced mostly by the early life environment and might affect health influencing the susceptibility to develop diseases, from cancer to mental disorder, during the entire life span. The most studied environmental influences acting on epigenome are diet, infections, wasting, child care, smoking and environmental pollutants, in particular endocrine disrupters (EDs). These are environmental xenobiotics able to interfere with the normal development of the male and female reproductive systems of wildlife, of experimental animals and possibly of humans, disrupting the normal reproductive functions. Data from literature indicate that EDs can act at different levels of epigenetic control, in some cases transgenerationally, in particular when the exposure to these compounds occurs during the prenatal and earliest period of life. Some of the best characterized EDs will be considered in this review. Among the EDs, vinclozolin (VZ), and methoxychlor (MXC) promote epigenetic transgenerational effects. Polychlorinated biphenils (PCBs), the most widespread environmental EDs, affect histone post-translational modifications in a dimorphic way, possibly as the result of an alteration of gene expression of the enzymes involved in histone modification, as the demethylase Jarid1b, an enzyme also involved in regulating the interaction of androgens with their receptor. PMID:26151052

  1. Discovery and Characterization of a Cell-Permeable, Small-Molecule c-Abl Kinase Activator that Binds to the Myristoyl Binding Site

    SciTech Connect

    Yang, Jingsong; Campobasso, Nino; Biju, Mangatt P.; Fisher, Kelly; Pan, Xiao-Qing; Cottom, Josh; Galbraith, Sarah; Ho, Thau; Zhang, Hong; Hong, Xuan; Ward, Paris; Hofmann, Glenn; Siegfried, Brett; Zappacosta, Francesca; Washio, Yoshiaki; Cao, Ping; Qu, Junya; Bertrand, Sophie; Wang, Da-Yuan; Head, Martha S.; Li, Hu; Moores, Sheri; Lai, Zhihong; Johanson, Kyung; Burton, George; Erickson-Miller, Connie; Simpson, Graham; Tummino, Peter; Copeland, Robert A.; Oliff, Allen

    2014-10-02

    c-Abl kinase activity is regulated by a unique mechanism involving the formation of an autoinhibited conformation in which the N-terminal myristoyl group binds intramolecularly to the myristoyl binding site on the kinase domain and induces the bending of the {alpha}I helix that creates a docking surface for the SH2 domain. Here, we report a small-molecule c-Abl activator, DPH, that displays potent enzymatic and cellular activity in stimulating c-Abl activation. Structural analyses indicate that DPH binds to the myristoyl binding site and prevents the formation of the bent conformation of the {alpha}I helix through steric hindrance, a mode of action distinct from the previously identified allosteric c-Abl inhibitor, GNF-2, that also binds to the myristoyl binding site. DPH represents the first cell-permeable, small-molecule tool compound for c-Abl activation.

  2. Cascade-able spin torque logic gates with input-output isolation

    NASA Astrophysics Data System (ADS)

    Nikonov, Dmitri E.; Manipatruni, Sasikanth; Young, Ian A.

    2015-06-01

    Spin torque majority gate (STMG) is one of the promising options for beyond-complementary metal-oxide-semiconductor non-volatile logic circuits for normally-off computing. Modeling of prior schemes demonstrated logic completeness using majority operation and nonlinear transfer characteristics. However significant problems arose with cascade-ability and input output isolation manifesting as domain walls (DWs) stopping, reflecting off ends of wires or propagating back to the inputs. We introduce a new scheme to enable cascade-ability and isolation based on (a) in-plane DW automotion in interconnects, (b) exchange coupling of magnetization between two FM layers, and (c) ‘round-about’ topology for the majority gate. We performed micro-magnetic simulations that demonstrate switching operation of this STMG scheme. These circuits were verified to enable isolation of inputs from output signals and to be cascade-able without limitations.

  3. Pharmacogenetics of BCR/ABL Inhibitors in Chronic Myeloid Leukemia.

    PubMed

    Polillo, Marialuisa; Galimberti, Sara; Baratè, Claudia; Petrini, Mario; Danesi, Romano; Di Paolo, Antonello

    2015-01-01

    Chronic myeloid leukemia was the first haematological neoplasia that benefited from a targeted therapy with imatinib nearly 15 years ago. Since then, several studies have investigated the role of genes, their variants (i.e., polymorphisms) and their encoded proteins in the pharmacokinetics and pharmacodynamics of BCR-ABL1 tyrosine kinase activity inhibitors (TKIs). Transmembrane transporters seem to influence in a significant manner the disposition of TKIs, especially that of imatinib at both cellular and systemic levels. In particular, members of the ATP-binding cassette (ABC) family (namely ABCB1 and ABCG2) together with solute carrier (SLC) transporters (i.e., SLC22A1) are responsible for the differences in drug pharmacokinetics. In the case of the newer TKIs, such as nilotinib and dasatinib, the substrate affinity of these drugs for transporters is variable but lower than that measured for imatinib. In this scenario, the investigation of genetic variants as possible predictive markers has led to some discordant results. With the partial exception of imatinib, these discrepancies seem to limit the application of discovered biomarkers in the clinical settings. In order to overcome these issues, larger prospective confirmative trials are needed. PMID:26402671

  4. Pharmacogenetics of BCR/ABL Inhibitors in Chronic Myeloid Leukemia

    PubMed Central

    Polillo, Marialuisa; Galimberti, Sara; Baratè, Claudia; Petrini, Mario; Danesi, Romano; Di Paolo, Antonello

    2015-01-01

    Chronic myeloid leukemia was the first haematological neoplasia that benefited from a targeted therapy with imatinib nearly 15 years ago. Since then, several studies have investigated the role of genes, their variants (i.e., polymorphisms) and their encoded proteins in the pharmacokinetics and pharmacodynamics of BCR-ABL1 tyrosine kinase activity inhibitors (TKIs). Transmembrane transporters seem to influence in a significant manner the disposition of TKIs, especially that of imatinib at both cellular and systemic levels. In particular, members of the ATP-binding cassette (ABC) family (namely ABCB1 and ABCG2) together with solute carrier (SLC) transporters (i.e., SLC22A1) are responsible for the differences in drug pharmacokinetics. In the case of the newer TKIs, such as nilotinib and dasatinib, the substrate affinity of these drugs for transporters is variable but lower than that measured for imatinib. In this scenario, the investigation of genetic variants as possible predictive markers has led to some discordant results. With the partial exception of imatinib, these discrepancies seem to limit the application of discovered biomarkers in the clinical settings. In order to overcome these issues, larger prospective confirmative trials are needed. PMID:26402671

  5. Combined STAT3 and BCR-ABL1 Inhibition Induces Synthetic Lethality in Therapy-Resistant Chronic Myeloid Leukemia

    PubMed Central

    Mason, Clinton C.; Vellore, Nadeem A.; Resetca, Diana; Zabriskie, Matthew S.; Zhang, Tian Y.; Khorashad, Jamshid S.; Engar, Alexander J.; Reynolds, Kimberly R.; Anderson, David J.; Senina, Anna; Pomicter, Anthony D.; Arpin, Carolynn C.; Ahmad, Shazia; Heaton, William L.; Tantravahi, Srinivas K.; Todic, Aleksandra; Moriggl, Richard; Wilson, Derek J.; Baron, Riccardo

    2014-01-01

    Mutations in the BCR-ABL1 kinase domain are an established mechanism of tyrosine kinase inhibitor (TKI) resistance in Philadelphia chromosome-positive leukemia, but fail to explain many cases of clinical TKI failure. In contrast, it is largely unknown why some patients fail TKI therapy despite continued suppression of BCR-ABL1 kinase activity, a situation termed BCRABL1 kinase-independent TKI resistance. Here, we identified activation of signal transducer and activator of transcription 3 (STAT3) by extrinsic or intrinsic mechanisms as an essential feature of BCR-ABL1 kinase-independent TKI resistance. By combining synthetic chemistry, in vitro reporter assays, and molecular dynamics-guided rational inhibitor design and high-throughput screening, we discovered BP-5-087, a potent and selective STAT3 SH2 domain inhibitor that reduces STAT3 phosphorylation and nuclear transactivation. Computational simulations, fluorescence polarization assays, and hydrogen-deuterium exchange assays establish direct engagement of STAT3 by BP-5-087 and provide a high-resolution view of the STAT3 SH2 domain/BP-5-087 interface. In primary cells from CML patients with BCR-ABL1 kinase-independent TKI resistance, BP-5-087 (1.0 μM) restored TKI sensitivity to therapy-resistant CML progenitor cells, including leukemic stem cells (LSCs). Our findings implicate STAT3 as a critical signaling node in BCR-ABL1 kinase-independent TKI resistance, and suggest that BP-5-087 has clinical utility for treating malignancies characterized by STAT3 activation. PMID:25134459

  6. Inhibition of isoprenylcysteine carboxylmethyltransferase augments BCR-ABL1 tyrosine kinase inhibition-induced apoptosis in chronic myeloid leukemia.

    PubMed

    Sun, Wen Tian; Xiang, Wei; Ng, Bee Ling; Asari, Kartini; Bunte, Ralph M; Casey, Patrick J; Wang, Mei; Chuah, Charles

    2016-03-01

    Despite the success of BCR-ABL1 tyrosine kinase inhibitors in patients with chronic myeloid leukemia (CML), resistance to tyrosine kinase inhibitors remains a therapeutic challenge. One strategy used to overcome resistance is combination of existing BCR-ABL1 tyrosine kinase inhibitors with agents that target alternative pathways. We report that inhibition of isoprenylcysteine carboxylmethyltransferase (Icmt), a key enzyme in the protein prenylation pathway, with the selective inhibitor cysmethynil enhances the effect of BCR-ABL1 tyrosine kinase inhibitors in killing CML cells. Cysmethynil augments tyrosine kinase inhibitor-induced apoptosis in both BCR-ABL1 wild type and BCR-ABL1 kinase domain mutant-expressing cell lines. Importantly, the enhanced apoptosis observed with the combination of cysmethynil and imatinib is significant only in primary CML CD34+ progenitor cells, not normal cord blood progenitor cells. The combination was also selective in inhibiting colony formation in CML CD34+ cells. The enhanced apoptosis appears to be due to combination of immediate and persistent inhibition of MAPK signaling. Consistent with in vitro studies, cysmethynil and imatinib, in combination, enhance the in vivo effects of either drug used alone. We found that simultaneous inhibition of BCR-ABL1 and Icmt may represent a potential therapeutic strategy for CML. PMID:26706195

  7. shRNA library screening identifies nucleocytoplasmic transport as a mediator of BCR-ABL1 kinase-independent resistance.

    PubMed

    Khorashad, Jamshid S; Eiring, Anna M; Mason, Clinton C; Gantz, Kevin C; Bowler, Amber D; Redwine, Hannah M; Yu, Fan; Kraft, Ira L; Pomicter, Anthony D; Reynolds, Kimberly R; Iovino, Anthony J; Zabriskie, Matthew S; Heaton, William L; Tantravahi, Srinivas K; Kauffman, Michael; Shacham, Sharon; Chenchik, Alex; Bonneau, Kyle; Ullman, Katharine S; O'Hare, Thomas; Deininger, Michael W

    2015-03-12

    The mechanisms underlying tyrosine kinase inhibitor (TKI) resistance in chronic myeloid leukemia (CML) patients lacking explanatory BCR-ABL1 kinase domain mutations are incompletely understood. To identify mechanisms of TKI resistance that are independent of BCR-ABL1 kinase activity, we introduced a lentiviral short hairpin RNA (shRNA) library targeting ∼5000 cell signaling genes into K562(R), a CML cell line with BCR-ABL1 kinase-independent TKI resistance expressing exclusively native BCR-ABL1. A customized algorithm identified genes whose shRNA-mediated knockdown markedly impaired growth of K562(R) cells compared with TKI-sensitive controls. Among the top candidates were 2 components of the nucleocytoplasmic transport complex, RAN and XPO1 (CRM1). shRNA-mediated RAN inhibition or treatment of cells with the XPO1 inhibitor, KPT-330 (Selinexor), increased the imatinib sensitivity of CML cell lines with kinase-independent TKI resistance. Inhibition of either RAN or XPO1 impaired colony formation of CD34(+) cells from newly diagnosed and TKI-resistant CML patients in the presence of imatinib, without effects on CD34(+) cells from normal cord blood or from a patient harboring the BCR-ABL1(T315I) mutant. These data implicate RAN in BCR-ABL1 kinase-independent imatinib resistance and show that shRNA library screens are useful to identify alternative pathways critical to drug resistance in CML. PMID:25573989

  8. Fusion protein based on Grb2-SH2 domain for cancer therapy

    SciTech Connect

    Saito, Yuriko; Furukawa, Takako; Arano, Yasushi; Fujibayashi, Yasuhisa; Saga, Tsuneo

    2010-08-20

    Research highlights: {yields} Grb2 mediates EGFR signaling through binding to phosphorylate EGFR with SH2 domain. {yields} We generated fusion proteins containing 1 or 2 SH2 domains of Grb2 added with TAT. {yields} The one with 2 SH2 domains (TSSF) interfered ERK phosphorylation. {yields} TSSF significantly delayed the growth of EGFR overexpressing tumor in a mouse model. -- Abstract: Epidermal growth factor receptor (EGFR) is one of the very attractive targets for cancer therapy. In this study, we generated fusion proteins containing one or two Src-homology 2 (SH2) domains of growth factor receptor bound protein 2 (Grb2), which bind to phosphorylated EGFR, added with HIV-1 transactivating transcription for cell membrane penetration (termed TSF and TSSF, respectively). We examined if they can interfere Grb2-mediated signaling pathway and suppress tumor growth as expected from the lack of SH3 domain, which is necessary to intermediate EGFR-Grb2 cell signaling, in the fusion proteins. The transduction efficiency of TSSF was similar to that of TSF, but the binding activity of TSSF to EGFR was higher than that of TSF. Treatment of EGFR-overexpressing cells showed that TSSF decreased p42-ERK phosphorylation, while TSF did not. Both the proteins delayed cell growth but did not induce cell death in culture. TSSF also significantly suppressed tumor growth in vivo under consecutive administration. In conclusion, TSSF showed an ability to inhibit EGFR-Grb2 signaling and could have a potential to treat EGFR-activated cancer.

  9. Cooperative interactions between paired domain and homeodomain.

    PubMed

    Jun, S; Desplan, C

    1996-09-01

    The Pax proteins are a family of transcriptional regulators involved in many developmental processes in all higher eukaryotes. They are characterized by the presence of a paired domain (PD), a bipartite DNA binding domain composed of two helix-turn-helix (HTH) motifs,the PAI and RED domains. The PD is also often associated with a homeodomain (HD) which is itself able to form homo- and hetero-dimers on DNA. Many of these proteins therefore contain three HTH motifs each able to recognize DNA. However, all PDs recognize highly related DNA sequences, and most HDs also recognize almost identical sites. We show here that different Pax proteins use multiple combinations of their HTHs to recognize several types of target sites. For instance, the Drosophila Paired protein can bind, in vitro, exclusively through its PAI domain, or through a dimer of its HD, or through cooperative interaction between PAI domain and HD. However, prd function in vivo requires the synergistic action of both the PAI domain and the HD. Pax proteins with only a PD appear to require both PAI and RED domains, while a Pax-6 isoform and a new Pax protein, Lune, may rely on the RED domain and HD. We propose a model by which Pax proteins recognize different target genes in vivo through various combinations of their DNA binding domains, thus expanding their recognition repertoire. PMID:8787739

  10. AP24534, a Pan-BCR-ABL Inhibitor for Chronic Myeloid Leukemia, Potently Inhibits the T315I Mutant and Overcomes Mutation-Based Resistance

    SciTech Connect

    O’Hare, Thomas; Shakespeare, William C.; Zhu, Xiaotian; Eide, Christopher A.; Rivera, Victor M.; Wang, Frank; Adrian, Lauren T.; Zhou, Tianjun; Huang, Wei-Sheng; Xu, Qihong; Metcalf, III, Chester A.; Tyner, Jeffrey W.; Loriaux, Marc M.; Corbin, Amie S.; Wardwell, Scott; Ning, Yaoyu; Keats, Jeffrey A.; Wang, Yihan; Sundaramoorthi, Raji; Thomas, Mathew; Zhou, Dong; Snodgrass, Joseph; Commodore, Lois; Sawyer, Tomi K.; Dalgarno, David C.; Deininger, Michael W.N.; Druker, Brian J.; Clackson, Tim

    2010-09-07

    Inhibition of BCR-ABL by imatinib induces durable responses in many patients with chronic myeloid leukemia (CML), but resistance attributable to kinase domain mutations can lead to relapse and a switch to second-line therapy with nilotinib or dasatinib. Despite three approved therapeutic options, the cross-resistant BCR-ABL{sup T315I} mutation and compound mutants selected on sequential inhibitor therapy remain major clinical challenges. We report design and preclinical evaluation of AP24534, a potent, orally available multitargeted kinase inhibitor active against T315I and other BCR-ABL mutants. AP24534 inhibited all tested BCR-ABL mutants in cellular and biochemical assays, suppressed BCR-ABL{sup T315I}-driven tumor growth in mice, and completely abrogated resistance in cell-based mutagenesis screens. Our work supports clinical evaluation of AP24534 as a pan-BCR-ABL inhibitor for treatment of CML.

  11. Domain adaptive boosting method and its applications

    NASA Astrophysics Data System (ADS)

    Geng, Jie; Miao, Zhenjiang

    2015-03-01

    Differences of data distributions widely exist among datasets, i.e., domains. For many pattern recognition, nature language processing, and content-based analysis systems, a decrease in performance caused by the domain differences between the training and testing datasets is still a notable problem. We propose a domain adaptation method called domain adaptive boosting (DAB). It is based on the AdaBoost approach with extensions to cover the domain differences between the source and target domains. Two main stages are contained in this approach: source-domain clustering and source-domain sample selection. By iteratively adding the selected training samples from the source domain, the discrimination model is able to achieve better domain adaptation performance based on a small validation set. The DAB algorithm is suitable for the domains with large scale samples and easy to extend for multisource adaptation. We implement this method on three computer vision systems: the skin detection model in single images, the video concept detection model, and the object classification model. In the experiments, we compare the performances of several commonly used methods and the proposed DAB. Under most situations, the DAB is superior.

  12. SGX393 inhibits the CML mutant Bcr-Abl[superscript T315I] and preempts in vitro resistance when combined with nilotinib or dasatinib

    SciTech Connect

    O'Hare, Thomas; Eide, Christopher A.; Tyner, Jeffrey W.; Corbin, Amie S.; Wong, Matthew J.; Buchanan, Sean; Holme, Kevin; Jessen, Katayoun A.; Tang, Crystal; Lewis, Hal A.; Romero, Richard D.; Burley, Stephen K.; Deininger, Michael W.

    2010-01-12

    Imatinib inhibits Bcr-Abl, the oncogenic tyrosine kinase that causes chronic myeloid leukemia. The second-line inhibitors nilotinib and dasatinib are effective in patients with imatinib resistance resulting from Bcr-Abl kinase domain mutations. Bcr-Abl{sup T315I}, however, is resistant to all Abl kinase inhibitors in clinical use and is emerging as the most frequent cause of salvage therapy failure. SGX393 is a potent inhibitor of native and T315I-mutant Bcr-Abl kinase that blocks the growth of leukemia cell lines and primary hematopoietic cells expressing Bcr-Abl{sup T315I}, with minimal toxicity against Bcr-Abl-negative cell lines or normal bone marrow. A screen for Bcr-Abl mutants emerging in the presence of SGX393 revealed concentration-dependent reduction in the number and range of mutations. Combining SGX393 with nilotinib or dasatinib preempted emergence of resistant subclones, including Bcr-Abl{sup T315I}. These findings suggest that combination of a T315I inhibitor with the current clinically used inhibitors may be useful for reduction of Bcr-Abl mutants in Philadelphia chromosome-positive leukemia.

  13. Misfolding, Aggregation, and Disordered Segments in c-Abl and p53 in Human Cancer

    PubMed Central

    de Oliveira, Guilherme A. P.; Rangel, Luciana P.; Costa, Danielly C.; Silva, Jerson L.

    2015-01-01

    The current understanding of the molecular mechanisms that lead to cancer is not sufficient to explain the loss or gain of function in proteins related to tumorigenic processes. Among them, more than 100 oncogenes, 20–30 tumor-suppressor genes, and hundreds of genes participating in DNA repair and replication have been found to play a role in the origins of cancer over the last 25 years. The phosphorylation of serine, threonine, or tyrosine residues is a critical step in cellular growth and development and is achieved through the tight regulation of protein kinases. Phosphorylation plays a major role in eukaryotic signaling as kinase domains are found in 2% of our genes. The deregulation of kinase control mechanisms has disastrous consequences, often leading to gains of function, cell transformation, and cancer. The c-Abl kinase protein is one of the most studied targets in the fight against cancer and is a hotspot for drug development because it participates in several solid tumors and is the hallmark of chronic myelogenous leukemia. Tumor suppressors have the opposite effects. Their fundamental role in the maintenance of genomic integrity has awarded them a role as the guardians of DNA. Among the tumor suppressors, p53 is the most studied. The p53 protein has been shown to be a transcription factor that recognizes and binds to specific DNA response elements and activates gene transcription. Stress triggered by ionizing radiation or other mutagenic events leads to p53 phosphorylation and cell-cycle arrest, senescence, or programed cell death. The p53 gene is the most frequently mutated gene in cancer. Mutations in the DNA-binding domain are classified as class I or class II depending on whether substitutions occur in the DNA contact sites or in the protein core, respectively. Tumor-associated p53 mutations often lead to the loss of protein function, but recent investigations have also indicated gain-of-function mutations. The prion-like aggregation of mutant p

  14. Tripartite motif protein 32 facilitates cell growth and migration via degradation of Abl-interactor 2.

    PubMed

    Kano, Satoshi; Miyajima, Naoto; Fukuda, Satoshi; Hatakeyama, Shigetsugu

    2008-07-15

    Tripartite motif protein 32 (TRIM32) mRNA has been reported to be highly expressed in human head and neck squamous cell carcinoma, but the involvement of TRIM32 in carcinogenesis has not been fully elucidated. In this study, we found by using yeast two-hybrid screening that TRIM32 binds to Abl-interactor 2 (Abi2), which is known as a tumor suppressor and a cell migration inhibitor, and we showed that TRIM32 mediates the ubiquitination of Abi2. Overexpression of TRIM32 promoted degradation of Abi2, resulting in enhancement of cell growth, transforming activity, and cell motility, whereas a dominant-negative mutant of TRIM32 lacking the RING domain inhibited the degradation of Abi2. In addition, we found that TRIM32 suppresses apoptosis induced by cis-diamminedichloroplatinum (II) in HEp2 cell lines. These findings suggest that TRIM32 is a novel oncogene that promotes tumor growth, metastasis, and resistance to anticancer drugs. PMID:18632609

  15. Leukemogenesis induced by wild-type and STI571-resistant BCR/ABL is potently suppressed by C/EBPα

    PubMed Central

    Ferrari-Amorotti, Giovanna; Keeshan, Karen; Zattoni, Michela; Guerzoni, Clara; Iotti, Giorgio; Cattelani, Sara; Donato, Nick J.; Calabretta, Bruno

    2006-01-01

    Chronic phase–to–blast crisis transition in chronic myelogenous leukemia (CML) is associated with differentiation arrest and down-regulation of C/EBPα, a transcription factor essential for granulocyte differentiation. Patients with CML in blast crisis (CML-BC) became rapidly resistant to therapy with the breakpoint cluster region–Abelson murine leukemia (BCR/ABL) kinase inhibitor imatinib (STI571) because of mutations in the kinase domain that interfere with drug binding. We show here that the restoration of C/EBPα activity in STI571-sensitive or -resistant 32D-BCR/ABL cells induced granulocyte differentiation, inhibited proliferation in vitro and in mice, and suppressed leukemogenesis. Moreover, activation of C/EBPα eradicated leukemia in 4 of 10 and in 6 of 7 mice injected with STI571-sensitive or -resistant 32D-BCR/ABL cells, respectively. Differentiation induction and proliferation inhibition were required for optimal suppression of leukemogenesis, as indicated by the effects of p42 C/EBPα, which were more potent than those of K298E C/EBPα, a mutant defective in DNA binding and transcription activation that failed to induce granulocyte differentiation. Activation of C/EBPα in blast cells from 4 patients with CML-BC, including one resistant to STI571 and BMS-354825 and carrying the T315I Abl kinase domain mutation, also induced granulocyte differentiation. Thus, these data indicate that C/EBPα has potent antileukemia effects even in cells resistant to ATP-binding competitive tyrosine kinase inhibitors, and they portend the development of anti-leukemia therapies that rely on C/EBPα activation. PMID:16670262

  16. In-silico identification of inhibitors against mutated BCR-ABL protein of chronic myeloid leukemia: a virtual screening and molecular dynamics simulation study.

    PubMed

    Kumar, Himansu; Raj, Utkarsh; Gupta, Saurabh; Varadwaj, Pritish Kumar

    2016-10-01

    Aberrant and proliferative expression of the oncogene BCR-ABL in the bone marrow cells had been proven as the prime cause of chronic myeloid leukemia (CML). It has been established that tyrosine kinase domain of BCR-ABL protein is a potential therapeutic target for the treatment of CML. Imatinib is considered as a first-generation drug that can inhibit the enzymatic action by inhibiting the ATP binding with BCR-ABL protein. Later on, insensitivity of CML cells towards Imatinib has been observed may be due to mutation in tyrosine kinase domain of the ABL receptor. Subsequently, some other second-generation drugs have also been reported viz. Baustinib, Nilotinib, Dasatinib, Ponatinib, Bafetinib, etc., which can able to combat against mutated domain of ABL tyrosine kinase protein. By taking into account of bioavailability and resistance developed, there is an utmost need to find some more inhibitors for the mutated ABL tyrosine kinase protein. For virtual screening, a data-set has been generated by collecting the all available drug like natural compounds from ZINC and Drug Bank databases. Comparative docking analysis was also carried out on the active site of ABL tyrosine kinase receptor with reported reference inhibitors. Molecular dynamics simulation of the best screened interacting complex was done for 50 ns to validate the stability of the system. These selected inhibitors were further validated and analyzed through pharmacokinetics properties and series of ADMET parameters by in silico methods. Considering the above said parameters proposed molecules are concluded as potential leads for drug designing pipeline against CML. PMID:26479578

  17. Detection of bcr/abl fusion and duplication of abl in a Ph-negative CML patient by the dual-color FISH method

    SciTech Connect

    Zhao, L.; Liang, J.C.

    1994-09-01

    We report here the use of the fluorescence in situ hybridization (FISH) method to detect duplication of the abl gene and fusion of bcr/abl in a Ph-negative CML patient. The patient was in clinical remission when studied. Conventional cytogenetic analysis revealed an apparent normal diploid karyotype. Using a metaphase-FISH method previously described for detecting residual leukemic cells in CML patients in clinical remission, we failed to detect any gross abnormality involving chromsomes 9 and 22. However, using the dual-color bcr and abl probes (Oncor), we found that in 55% of the interphases, fusion of these two genes was detectable. More interestingly, in those cells in which fusion of the bcr and abl genes occurred, we observed an extra abl signal. Further examination of bcr and abl hybridization signals on metaphases revealed that the bcr/abl fusion occurred on a chromosome that resembled chromosome 22. The two abl signals were on two chromosomes that resembled chromosome 9s and one bcr signal could be found on another chromosome 22. Thus the bcr and abl dual-color probes allow us to detect a rare form of gene fusion and duplication that is not detectable by karyotyping or chromosome painting.

  18. The Impact of Extra-Domain Structures and Post-Translational Modifications in the Folding/Misfolding Behaviour of the Third PDZ Domain of MAGUK Neuronal Protein PSD-95

    PubMed Central

    Cobos, Eva S.; Villegas, Sandra; Martinez, Jose C.

    2014-01-01

    The modulation of binding affinities and specificities by post-translational modifications located out from the binding pocket of the third PDZ domain of PSD-95 (PDZ3) has been reported recently. It is achieved through an intra-domain electrostatic network involving some charged residues in the β2–β3 loop (were a succinimide modification occurs), the α3 helix (an extra-structural element that links the PDZ3 domain with the following SH3 domain in PSD-95, and contains the phosphorylation target Tyr397), and the ligand peptide. Here, we have investigated the main structural and thermodynamic aspects that these structural elements and their related post-translational modifications display in the folding/misfolding pathway of PDZ3 by means of site-directed mutagenesis combined with calorimetry and spectroscopy. We have found that, although all the assayed mutations generate proteins more prone to aggregation than the wild-type PDZ3, those directly affecting the α3 helix, like the E401R substitution or the truncation of the whole α3 helix, increase the population of the DSC-detected intermediate state and the misfolding kinetics, by organizing the supramacromolecular structures at the expense of the two β-sheets present in the PDZ3 fold. However, those mutations affecting the β2–β3 loop, included into the prone-to-aggregation region composed by a single β-sheet comprising β2 to β4 chains, stabilize the trimeric intermediate previously shown in the wild-type PDZ3 and slow-down aggregation, also making it partly reversible. These results strongly suggest that the α3 helix protects to some extent the PDZ3 domain core from misfolding. This might well constitute the first example where an extra-element, intended to link the PDZ3 domain to the following SH3 in PSD-95 and in other members of the MAGUK family, not only regulates the binding abilities of this domain but it also protects PDZ3 from misfolding and aggregation. The influence of the post

  19. Activation of tyrosinase kinase and microfilament-binding functions of c-abl by bcr sequences in bcr/abl fusion proteins.

    PubMed Central

    McWhirter, J R; Wang, J Y

    1991-01-01

    Chronic myelogenous leukemia and one type of acute lymphoblastic leukemia are characterized by a 9;22 chronosome translocation in which 5' sequences of the bcr gene become fused to the c-abl proto-oncogene. The resulting chimeric genes encode bcr/abl fusion proteins which have deregulated tyrosine kinase activity and appear to play an important role in induction of these leukemias. A series of bcr/abl genes were constructed in which nested deletions of the bcr gene were fused to the c-abl gene. The fusion proteins encoded by these genes were assayed for autophosphorylation in vivo and for differences in subcellular localization. Our results demonstrate that bcr sequences activate two functions of c-abl; the tyrosine kinase activity and a previously undescribed microfilament-binding function. Two regions of bcr which activate these functions to different degrees have been mapped: amino acids 1 to 63 were strongly activating and amino acids 64 to 509 were weakly activating. The tyrosine kinase and microfilament-binding functions were not interdependent, as a kinase defective bcr/abl mutant still associated with actin filaments and a bcr/abl mutant lacking actin association still had deregulated kinase activity. Modification of actin filament functions by the bcr/abl tyrosine kinase may be an important event in leukemogenesis. Images PMID:1705008

  20. Identification of a novel contactin-associated transmembrane receptor with multiple domains implicated in protein-protein interactions.

    PubMed Central

    Peles, E; Nativ, M; Lustig, M; Grumet, M; Schilling, J; Martinez, R; Plowman, G D; Schlessinger, J

    1997-01-01

    Receptor protein tyrosine phosphatase beta (RPTPbeta) expressed on the surface of glial cells binds to the glycosylphosphatidylinositol (GPI)-anchored recognition molecule contactin on neuronal cells leading to neurite outgrowth. We describe the cloning of a novel contactin-associated transmembrane receptor (p190/Caspr) containing a mosaic of domains implicated in protein-protein interactions. The extracellular domain of Caspr contains a neurophilin/coagulation factor homology domain, a region related to fibrinogen beta/gamma, epidermal growth factor-like repeats, neurexin motifs as well as unique PGY repeats found in a molluscan adhesive protein. The cytoplasmic domain of Caspr contains a proline-rich sequence capable of binding to a subclass of SH3 domains of signaling molecules. Caspr and contactin exist as a complex in rat brain and are bound to each other by means of lateral (cis) interactions in the plasma membrane. We propose that Caspr may function as a signaling component of contactin, enabling recruitment and activation of intracellular signaling pathways in neurons. The binding of RPTPbeta to the contactin-Caspr complex could provide a mechanism for cell-cell communication between glial cells and neurons during development. PMID:9118959

  1. Synthetic Protein Scaffolds Based on Peptide Motifs and Cognate Adaptor Domains for Improving Metabolic Productivity.

    PubMed

    Horn, Anselm H C; Sticht, Heinrich

    2015-01-01

    The efficiency of many cellular processes relies on the defined interaction among different proteins within the same metabolic or signaling pathway. Consequently, a spatial colocalization of functionally interacting proteins has frequently emerged during evolution. This concept has been adapted within the synthetic biology community for the purpose of creating artificial scaffolds. A recent advancement of this concept is the use of peptide motifs and their cognate adaptor domains. SH2, SH3, GBD, and PDZ domains have been used most often in research studies to date. The approach has been successfully applied to the synthesis of a variety of target molecules including catechin, D-glucaric acid, H2, hydrochinone, resveratrol, butyrate, gamma-aminobutyric acid, and mevalonate. Increased production levels of up to 77-fold have been observed compared to non-scaffolded systems. A recent extension of this concept is the creation of a covalent linkage between peptide motifs and adaptor domains, which leads to a more stable association of the scaffolded systems and thus bears the potential to further enhance metabolic productivity. PMID:26636078

  2. Synthetic Protein Scaffolds Based on Peptide Motifs and Cognate Adaptor Domains for Improving Metabolic Productivity

    PubMed Central

    Horn, Anselm H. C.; Sticht, Heinrich

    2015-01-01

    The efficiency of many cellular processes relies on the defined interaction among different proteins within the same metabolic or signaling pathway. Consequently, a spatial colocalization of functionally interacting proteins has frequently emerged during evolution. This concept has been adapted within the synthetic biology community for the purpose of creating artificial scaffolds. A recent advancement of this concept is the use of peptide motifs and their cognate adaptor domains. SH2, SH3, GBD, and PDZ domains have been used most often in research studies to date. The approach has been successfully applied to the synthesis of a variety of target molecules including catechin, D-glucaric acid, H2, hydrochinone, resveratrol, butyrate, gamma-aminobutyric acid, and mevalonate. Increased production levels of up to 77-fold have been observed compared to non-scaffolded systems. A recent extension of this concept is the creation of a covalent linkage between peptide motifs and adaptor domains, which leads to a more stable association of the scaffolded systems and thus bears the potential to further enhance metabolic productivity. PMID:26636078

  3. The dynamin-binding domains of Dap160/intersectin affect bulk membrane retrieval in synapses

    PubMed Central

    Winther, Åsa M. E.; Jiao, Wei; Vorontsova, Olga; Rees, Kathryn A.; Koh, Tong-Wey; Sopova, Elena; Schulze, Karen L.; Bellen, Hugo J.; Shupliakov, Oleg

    2013-01-01

    Summary Dynamin-associated protein 160 kDa (Dap160)/intersectin interacts with several synaptic proteins and affects endocytosis and synapse development. The functional role of the different protein interaction domains is not well understood. Here we show that Drosophila Dap160 lacking the dynamin-binding SH3 domains does not affect the development of the neuromuscular junction but plays a key role in synaptic vesicle recycling. dap160 mutants lacking dynamin-interacting domains no longer accumulate dynamin properly at the periactive zone, and it becomes dispersed in the bouton during stimulation. This is accompanied by a reduction in uptake of the dye FM1-43 and an accumulation of large vesicles and membrane invaginations. However, we do not observe an increase in the number of clathrin-coated intermediates. We also note a depression in evoked excitatory junction potentials (EJPs) during high-rate stimulation, accompanied by aberrantly large miniature EJPs. The data reveal the important role of Dap160 in the targeting of dynamin to the periactive zone, where it is required to suppress bulk synaptic vesicle membrane retrieval during high-frequency activity. PMID:23321638

  4. A temperature sensitive p210 BCR-ABL mutant defines the primary consequences of BCR-ABL tyrosine kinase expression in growth factor dependent cells.

    PubMed Central

    Kabarowski, J H; Allen, P B; Wiedemann, L M

    1994-01-01

    The Philadelphia translocation commonly observed in chronic myeloid leukaemia (CML) and a proportion of cases of acute leukaemia results in the creation of a chimeric fusion protein, BCR-ABL. The fusion protein exhibits an elevated tyrosine kinase activity as compared to normal ABL. Using a temperature sensitive mutant of p210 BCR-ABL (ts-p210) we find that the primary effect of BCR-ABL expression in an IL-3 dependent cell line is to prolong survival following growth factor withdrawal; only a small proportion of cells remain viable and rapidly evolve to complete growth factor independence. During passage in the presence of IL-3 at the temperature permissive for kinase activity, ts-p210 expressing cultures become dominated by completely growth factor independent cells within 10-30 days. There is also a significant difference between BCR-ABL and IL-3 mediated signalling with respect to the MAP kinase pathway; in contrast to IL-3 stimulation or v-ABL expression, BCR-ABL does not signal ERK 2 (MAP 2 kinase) activation, underlining the apparent inability of BCR-ABL to deliver an immediate proliferative signal in Ba/F3 cells. Our data suggest that growth factor independence does not simply reflect the convergence of BCR-ABL and IL-3 mediated signalling pathways and its development, at least in Ba/F3 cells, requires prolonged exposure to BCR-ABL kinase activity. We suggest that the myeloid expansion characteristic of CML may result from the prolongation of survival of myeloid progenitor cells under conditions of limiting growth factor rather than their uncontrolled proliferation. Images PMID:7813429

  5. [Detection of bcr/abl fusion gene and its derivative chromosome 9 deletions in CML by using home-made bcr/abl extra-signal probe].

    PubMed

    Lai, Yue-Yun; Feng, Lin; Wang, Zheng; Lü, Shan; Dang, Hui; Shi, Yan; He, Qi; Huang, Xiao-Jun

    2010-02-01

    This study was aimed to verify the efficacy of home-made LSI bcr/abl ES probe for detection of bcr/abl fusion gene and derivative chromosome 9 deletions in chronic myeloid leukemia (CML). Fluorescence in situ hybridization (FISH) was carried out with dual color bcr/abl extra signal (ES) probe in 97 cases of CML based on morphology and cytogenetic karyotype and 129 cases of non-hematological malignancies/non-myeloproliferative diseases with normal cytogenetic karyotype. For the patients with signals of 1R1G1F indicating der(9) deletions, FISH were done using ASS DNA probe. The results showed that 91 cases with standard t(9;22) and 6 cases with variant translocation of t(9;22) were detected by conventional G banding technique. All of the 97 patients displayed bcr/abl fusion gene by ES-FISH, including 16 cases with signal patterns of 1R1G1F showing der(9) deletions. Among the 16 cases with der(9) deletions, 13 cases were detected to have deletions of ASS gene. Meanwhile, none of the 129 cases of negative control showed bcr/abl fusion gene by ES-FISH. It is concluded that home-made LSI bcr/abl ES probe is effective to identify the bcr/abl fusion gene and der(9) deletions in CML, and the ES-FISH results are consistent with conventional cytogenetic karyotype. PMID:20137147

  6. Expression of the ETS transcription factor GABPα is positively correlated to the BCR-ABL1/ABL1 ratio in CML patients and affects imatinib sensitivity in vitro.

    PubMed

    Manukjan, Georgi; Ripperger, Tim; Santer, Laura; von Neuhoff, Nils; Ganser, Arnold; Schambach, Axel; Schlegelberger, Brigitte; Steinemann, Doris

    2015-10-01

    In Philadelphia-positive chronic myeloid leukemia (CML), imatinib resistance frequently emerges because of point mutations in the ABL1 kinase domain, but may also be the consequence of uncontrolled upstream signaling. Recently, the heteromeric transcription factor GA-binding protein (GABP) was found to promote CML-like myeloproliferative disease in mice. In a cohort of 70 CML patients, we found that expression of the GABP α subunit (GABPα) is positively correlated to the BCR-ABL1/ABL1 ratio. Moreover, significantly higher GABPα expression was detected in blast crisis than in chronic phase CML after performing data mining on 91 CML patients. In functional studies, imatinib sensitivity is enhanced after GABPα knockdown in tyrosine kinase inhibitors (TKI)-sensitive K-562, as well as by overexpression of a deletion mutant in TKI-resistant NALM-1 cells. Moreover, in K-562 cells, GABP-dependent expression variations of PRKD2 and RAC2, relevant signaling mediators in CML, were observed. Notably, protein kinase D2 (Prkd2) was reported to be a GABP target gene in mice. In line with this, we detected a positive correlation between GABPA and PRKD2 expression in primary human CML, indicating that the effects of GABP are mediated by PRKD2. These findings illustrate an important role for GABP in disease development and imatinib sensitivity in human CML. PMID:26072332

  7. Functional domains of an ATP-dependent DNA ligase.

    PubMed

    Doherty, A J; Wigley, D B

    1999-01-01

    The crystal structure of an ATP-dependent DNA ligase from bacteriophage T7 revealed that the protein comprised two structural domains. In order to investigate the biochemical activities of these domains, we have overexpressed them separately and purified them to homogeneity. The larger N-terminal domain retains adenylation and ligase activities, though both at a reduced level. The adenylation activity of the large domain is stimulated by the presence of the smaller domain, suggesting that a conformational change is required for adenylation in the full length protein. The DNA binding properties of the two fragments have also been studied. The larger domain is able to band shift both single and double-stranded DNA, while the smaller fragment is only able to bind to double-stranded DNA. These data suggest that the specificity of DNA ligases for nick sites in DNA is produced by a combination of these different DNA binding activities in the intact enzyme. PMID:9878388

  8. Identification of Domains for Malaysian University Staff Happiness Index Development

    ERIC Educational Resources Information Center

    Yassin, Sulaiman Md.

    2014-01-01

    Without any doubt happiness among staff in any organization is pertinent to ensure continued growth and development. However, not many studies were carried out to determine the domains that will be able to measure the level of happiness among staff in universities. Thus, the aim of this study is to elicit the domains that explain the overall…

  9. Structural Basis for Polyproline Recognition by the FE65 WW Domain

    SciTech Connect

    Meiyappan,M.; Birrane, G.; Ladias, J.; Yaluris, G.; Roberie, T.; Amiridis, M.

    2007-01-01

    The neuronal protein FE65 functions in brain development and amyloid precursor protein (APP) signaling through its interaction with the mammalian enabled (Mena) protein and APP, respectively. The recognition of short polyproline sequences in Mena by the FE65 WW domain has a central role in axon guidance and neuronal positioning in the developing brain. We have determined the crystal structures of the human FE65 WW domain (residues 253-289) in the apo form and bound to the peptides PPPPPPLPP and PPPPPPPPPL, which correspond to human Mena residues 313-321 and 347-356, respectively. The FE65 WW domain contains two parallel ligand-binding grooves, XP (formed by residues Y269 and W280) and XP2 (formed by Y269 and W271). Both Mena peptides adopt a polyproline helical II conformation and bind to the WW domain in a forward (N-C) orientation through selection of the PPPPP motif by the XP and XP2 grooves. This mode of ligand recognition is strikingly similar to polyproline interaction with SH3 domains. Importantly, comparison of the FE65 WW structures in the apo and liganded forms shows that the XP2 groove is formed by an induced-fit mechanism that involves movements of the W271 and Y269 side-chains upon ligand binding. These structures elucidate the molecular determinants underlying polyproline ligand selection by the FE65 WW domain and provide a framework for the design of small molecules that would interfere with FE65 WW-ligand interaction and modulate neuronal development and APP signaling.

  10. Structural Mechanism of the Pan-BCR-ABL Inhibitor Ponatinib (AP24534): Lessons for Overcoming Kinase Inhibitor Resistance

    SciTech Connect

    Zhou, Tianjun; Commodore, Lois; Huang, Wei-Sheng; Wang, Yihan; Thomas, Mathew; Keats, Jeff; Xu, Qihong; Rivera, Victor M.; Shakespeare, William C.; Clackson, Tim; Dalgarno, David C.; Zhu, Xiaotian

    2012-01-20

    The BCR-ABL inhibitor imatinib has revolutionized the treatment of chronic myeloid leukemia. However, drug resistance caused by kinase domain mutations has necessitated the development of new mutation-resistant inhibitors, most recently against the T315I gatekeeper residue mutation. Ponatinib (AP24534) inhibits both native and mutant BCR-ABL, including T315I, acting as a pan-BCR-ABL inhibitor. Here, we undertook a combined crystallographic and structure-activity relationship analysis on ponatinib to understand this unique profile. While the ethynyl linker is a key inhibitor functionality that interacts with the gatekeeper, virtually all other components of ponatinib play an essential role in its T315I inhibitory activity. The extensive network of optimized molecular contacts found in the DFG-out binding mode leads to high potency and renders binding less susceptible to disruption by single point mutations. The inhibitory mechanism exemplified by ponatinib may have broad relevance to designing inhibitors against other kinases with mutated gatekeeper residues.

  11. Driving evaluation methods for able-bodied persons and individuals with lower extremity disabilities: a review of assessment modalities

    PubMed Central

    Greve, Julia Maria D'Andréa; Santos, Luciana; Alonso, Angelica Castilho; Tate, Denise G

    2015-01-01

    Assessing the driving abilities of individuals with disabilities is often a very challenging task because each medical condition is accompanied by physical impairments and because relative individual functional performance may vary depending on personal characteristics. We identified existing driving evaluation modalities for able-bodied and lower extremity-impaired subjects (spinal cord injury patients and amputees) and evaluated the potential relationships between driving performance and the motor component of driving. An extensive scoping review of the literature was conducted to identify driving assessment tools that are currently used for able-bodied individuals and for those with spinal cord injury or lower extremity amputation. The literature search focused on the assessment of the motor component of driving. References were electronically obtained via Medline from the PubMed, Ovid, Web of Science and Google Scholar databases. This article compares the current assessments of driving performance for those with lower extremity impairments with the assessments used for able-bodied persons. Very few articles were found concerning “Lower Extremity Disabilities,” thus confirming the need for further studies that can provide evidence and guidance for such assessments in the future. Little is known about the motor component of driving and its association with the other driving domains, such as vision and cognition. The available research demonstrates the need for a more evidenced-based understanding of how to best evaluate persons with lower extremity impairment. PMID:26375567

  12. Abl Kinases Regulate HGF/Met Signaling Required for Epithelial Cell Scattering, Tubulogenesis and Motility

    PubMed Central

    Li, Ran; Knight, Jennifer F.; Park, Morag; Pendergast, Ann Marie

    2015-01-01

    Tight regulation of receptor tyrosine kinases (RTKs) is crucial for normal development and homeostasis. Dysregulation of RTKs signaling is associated with diverse pathological conditions including cancer. The Met RTK is the receptor for hepatocyte growth factor (HGF) and is dysregulated in numerous human tumors. Here we show that Abl family of non-receptor tyrosine kinases, comprised of Abl (ABL1) and Arg (ABL2), are activated downstream of the Met receptor, and that inhibition of Abl kinases dramatically suppresses HGF-induced cell scattering and tubulogenesis. We uncover a critical role for Abl kinases in the regulation of HGF/Met-dependent RhoA activation and RhoA-mediated actomyosin contractility and actin cytoskeleton remodeling in epithelial cells. Moreover, treatment of breast cancer cells with Abl inhibitors markedly decreases Met-driven cell migration and invasion. Notably, expression of a transforming mutant of the Met receptor in the mouse mammary epithelium results in hyper-activation of both Abl and Arg kinases. Together these data demonstrate that Abl kinases link Met activation to Rho signaling and Abl kinases are required for Met-dependent cell scattering, tubulogenesis, migration, and invasion. Thus, inhibition of Abl kinases might be exploited for the treatment of cancers driven by hyperactivation of HGF/Met signaling. PMID:25946048

  13. Synaptic Clustering of PSD-95 Is Regulated by c-Abl through Tyrosine Phosphorylation

    PubMed Central

    de Arce, Karen Perez; Varela-Nallar, Lorena; Farias, Olivia; Cifuentes, Alejandra; Bull, Paulina; Couch, Brian A.; Koleske, Anthony J.; Inestrosa, Nibaldo C.; Alvarez, Alejandra R.

    2010-01-01

    The c-Abl tyrosine kinase is present in mouse brain synapses, but its precise synaptic function is unknown. We found that c-Abl levels in the rat hippocampus increase postnatally, with expression peaking at the first postnatal week. In 14 d in vitro hippocampal neuron cultures, c-Abl localizes primarily to the postsynaptic compartment, in which it colocalizes with the postsynaptic scaffold protein postsynaptic density protein-95 (PSD-95) in apposition to presynaptic markers. c-Abl associates with PSD-95, and chemical or genetic inhibition of c-Abl kinase activity reduces PSD-95 tyrosine phosphorylation, leading to reduced PSD-95 clustering and reduced synapses in treated neurons. c-Abl can phosphorylate PSD-95 on tyrosine 533, and mutation of this residue reduces the ability of PSD-95 to cluster at postsynaptic sites. Our results indicate that c-Abl regulates synapse formation by mediating tyrosine phosphorylation and clustering of PSD-95. PMID:20220006

  14. RICH-1 has a BIN/Amphiphysin/Rvsp domain responsible for binding to membrane lipids and tubulation of liposomes.

    PubMed

    Richnau, Ninna; Fransson, Asa; Farsad, Khashayar; Aspenström, Pontus

    2004-07-30

    RhoGAP interacting with CIP4 homologs-1 (RICH-1) was previously found in a yeast two-hybrid screen for proteins interacting with the SH3 domain of the Cdc42-interacting protein 4 (CIP4). RICH-1 was shown to be a RhoGAP for Cdc42 and Rac. In this study, we show that the BIN/Amphiphysin/Rvsp (BAR) domain in RICH-1 confers binding to membrane lipids, and has the potential to deform spherical liposomes into tubes. In accordance with previous findings for the BAR domains in endophilin and amphiphysin, RICH-1-induced tubes appeared striated. We propose that these striated structures are formed by oligomerization of RICH-1 through a putative coiled-coil region within the BAR domain. In support of this notion, we show that RICH-1 forms oligomers in the presence of the chemical cross-linker BS3. These results point to an involvement of RICH-1 in membrane deformation events. PMID:15240152

  15. T-cell immunity to the joining region of p210BCR-ABL protein.

    PubMed Central

    Chen, W; Peace, D J; Rovira, D K; You, S G; Cheever, M A

    1992-01-01

    The hallmark of chronic myelogenous leukemia is the translocation of the human c-abl protooncogene (ABL) from chromosome 9 to the specific breakpoint cluster region (bcr) of the BCR gene on chromosome 22. The t(9;22)(q34;q11) translocation results in the formation of a BCR-ABL fusion gene that encodes a 210-kDa chimeric protein with abnormal tyrosine kinase activity. The ABL and BCR genes are expressed by normal cells and thus the encoded proteins are presumably nonimmunogenic. However, the joining-region segment of the p210BCR-ABL chimeric protein is composed of unique sequences of ABL amino acids joined to BCR amino acids that are expressed only by malignant cells. The current study demonstrates that the joining region of BCR-ABL protein is immunogenic to murine T cells. Immunization of mice with synthetic peptides corresponding to the joining region elicited peptide-specific, CD4+, class II major histocompatibility complex-restricted T cells. The BCR-ABL peptide-specific T cells recognized only the combined sequence of BCR-ABL amino acids and not BCR or ABL amino acid sequences alone. Importantly, the BCR-ABL peptide-specific T cells could recognize and proliferate in response to p210BCR-ABL protein. The response of peptide-specific T cells to protein demonstrated that p210BCR-ABL can be processed by antigen-presenting cells so that the joining segment is bound to class II major histocompatibility complex molecules in a configuration similar to that of the immunizing peptide and in a concentration high enough to stimulate the antigen-specific T-cell receptor. Thus, BCR-ABL protein represents a potential tumor-specific antigen related to the transforming event and shared by many individuals with chronic myelogenous leukemia. PMID:1346932

  16. In vivo binding properties of SH2 domains from GTPase-activating protein and phosphatidylinositol 3-kinase.

    PubMed Central

    Cooper, J A; Kashishian, A

    1993-01-01

    We have used a transient expression system and mutant platelet-derived growth factor (PDGF) receptors to study the binding specificities of the Src homology 2 (SH2) regions of the Ras GTPase-activator protein (GAP) and the p85 alpha subunit of phosphatidylinositol 3-kinase (PI3 kinase). A number of fusion proteins, each tagged with an epitope allowing recognition by a monoclonal antibody, were expressed at levels comparable to those of endogenous GAP. Fusion proteins containing the central SH2-SH3-SH2 region of GAP or the C-terminal region of p85 alpha, which includes two SH2 domains, bound to PDGF receptors in response to PDGF stimulation. Both fusion proteins showed the same requirements for tyrosine phosphorylation sites in the PDGF receptor as the full-length proteins from which they were derived, i.e., binding of the GAP fusion protein was reduced by mutation of Tyr-771, and binding of the p85 fusion protein was reduced by mutation of Tyr-740, Tyr-751, or both residues. Fusion proteins containing single SH2 domains from either GAP or p85 alpha did not bind detectably to PDGF receptors in this system, suggesting that two SH2 domains in a single polypeptide cooperate to raise the affinity of binding. The sequence specificities of individual SH2 domains were deduced from the binding properties of fusion proteins containing one SH2 domain from GAP and another from p85. The results suggest that the C-terminal GAP SH2 domain specifies binding to Tyr-771, the C-terminal p85 alpha SH2 domain binds to either Tyr-740 or Tyr-751, and each protein's N-terminal SH2 domain binds to unidentified phosphorylation sites.(ABSTRACT TRUNCATED AT 250 WORDS) Images PMID:8382774

  17. Compendium of NASA data base for the Global Tropospheric Experiment's Arctic Boundary Layer Experiments ABLE-3A and ABLE-3B

    SciTech Connect

    Gregory, G.L.; Scott, A.D. Jr.

    1994-11-01

    The report provides a compendium of NASA aircraft data that are available from NASA's Global Tropospheric Experiment's (GTE) Arctic Boundary Layer Experiments (ABLE) conducted in July and August of 1988 (ABLE-3A) and 1990 (ABLE-3B). ABLE-3A flight experiments were based at Barrow and Bethel, Alaska, and included survey/transit flights to Thule, Greenland. ABLE-3B flight experiments were based at North Bay (Ontario) and Goose Bay, Canada, and included flights northward to Frobisher Bay, Canada. The primary purposes of the experiments were (1) the measurement of the flux of various trace gases from high-arctic ecosystems, (2) the elucidation of factors important to the production and destruction of ozone, and (3) the documentation of source and chemical signature of air common to and transported into the regions. The report provides a representation, in the form of selected data plots, of aircraft data that are available in archived format via NASA Langley's Distributed Active Archive Center. The archived data bases include data for other species measured on the aircraft as well as numerous supporting data, including meteorological observations/products, results from surface studies, satellite observations, and sondes releases.

  18. Compendium of NASA data base for the Global Tropospheric Experiment's Arctic Boundary Layer Experiments ABLE-3A and ABLE-3B

    NASA Technical Reports Server (NTRS)

    Gregory, Gerald L.; Scott, A. Donald, Jr.

    1994-01-01

    The report provides a compendium of NASA aircraft data that are available from NASA's Global Tropospheric Experiment's (GTE) Arctic Boundary Layer Experiments (ABLE) conducted in July and August of 1988 (ABLE-3A) and 1990 (ABLE-3B). ABLE-3A flight experiments were based at Barrow and Bethel, Alaska, and included survey/transit flights to Thule, Greenland. ABLE-3B flight experiments were based at North Bay (Ontario) and Goose Bay, Canada, and included flights northward to Frobisher Bay, Canada. The primary purposes of the experiments were (1) the measurement of the flux of various trace gases from high-arctic ecosystems, (2) the elucidation of factors important to the production and destruction of ozone, and (3) the documentation of source and chemical signature of air common to and transported into the regions. The report provides a representation, in the form of selected data plots, of aircraft data that are available in archived format via NASA Langley's Distributed Active Archive Center. The archived data bases include data for other species measured on the aircraft as well as numerous supporting data, including meteorological observations/products, results from surface studies, satellite observations, and sondes releases.

  19. Distinct Domains within PSD-95 Mediate Synaptic Incorporation, Stabilization and Activity-Dependent Trafficking

    PubMed Central

    Sturgill, James F.; Steiner, Pascal; Czervionke, Brian L.

    2009-01-01

    The postsynaptic density (PSD) consists of a lattice-like array of interacting proteins that organizes and stabilizes receptors, ion channels, structural, and signaling proteins necessary for synaptic function. To study the stabilization of proteins within this structure and the contribution of these proteins to the integrity of the PSD, we tagged synaptic proteins with photoactivatable GFP (PAGFP) and used combined 2-photon laser scanning microscopy (2PLSM) and 2-photon laser photoactivation (2PLP) to measure their rate of turnover in individual spines of rat CA1 pyramidal neurons. We find that PSD-95 is highly stable within the spine, more so than other PSD-associated proteins such as CaMKIIα, CaMKIIβ, GluR2 and Stargazin. Analysis of a series of PSD-95 mutants revealed that distinct domains stabilize PSD-95 within the PSD and contribute to PSD formation. Stabilization of PSD-95 within the PSD requires N-terminal palmitoylation and protein interactions mediated by the 1st and 2nd PDZ domains whereas formation of a stable lattice of PSD-95 molecules within the PSD additionally requires the C-terminal SH3 domain. Furthermore, in a PDZ domain 1 and 2 dependent manner, activation of NMDA receptors with a chemical LTD protocol rapidly destabilizes PSD-95 and causes a subset of the PSD-95 molecules previously anchored in the spine to be released. Thus, through the analysis of rates of exchange of synaptic PSD-95, we determine separate domains of PSD-95 that play specific roles in establishing a stable postsynaptic lattice, in allowing proteins to enter this lattice, and in reorganizing this structure in response to plasticity-inducing stimuli. PMID:19828799

  20. 124I-Iodopyridopyrimidinone for PET of Abl Kinase–Expressing Tumors In Vivo

    PubMed Central

    Doubrovin, Mikhail; Kochetkova, Tatiana; Santos, Elmer; Veach, Darren R.; Smith-Jones, Peter; Pillarsetty, Nagavarakishore; Balatoni, Julius; Bornmann, William; Gelovani, Juri; Larson, Steven M.

    2015-01-01

    Because of the recent development of an iodopyridopyrimidinone Abl protein kinase inhibitor (PKI), 124I-SKI-212230 (124I-SKI230), we investigated the feasibility of a PET-based molecular imaging method for the direct visualization of Abl kinase expression and PKI treatment. Methods In vitro pharmacokinetic properties, including specific and nonspecific binding of 124I-SKI230 to its Abl kinase target and interaction with other PKIs, were assessed in cell-free medium and chronic myelogenous leukemia (CML) cells overexpressing BCR-Abl (K562), in comparison with BT-474 cells that are low in Abl expression. In a xenograft tumor model, we assessed the in vivo pharmacokinetics of 124I-SKI230 using PET and postmortem tissue sampling. We also tested a paradigm of 124I-SKI230 PET after treatment of the animal with a dose of Abl-specific PKI for the monitoring of the tumor response. Results In vitro studies confirmed that SKI230 binds to Abl kinase with nanomolar affinity, that selective uptake occurs in cell lines known to express Abl kinase, that RNAi knock-down supports specificity of cellular uptake due to Abl kinase, and that imatinib, an archetype Abl PKI, completely displaces SKI230. With SKI230, we obtained successful in vivo PET of Abl-expressing human tumors in a nude rat. We were also able to demonstrate evidence of substrate inhibition of in vivo radiotracer uptake in the xenograft tumor after treatment of the animal as a model of PKI treatment monitoring. Conclusion These results support the hypothesis that molecular imaging using PET will be useful for the study of in vivo pharmacodynamics of Abl PKI molecular therapy in humans. PMID:20048131

  1. New alternative splicing BCR/ABL-OOF shows an oncogenic role by lack of inhibition of BCR GTPase activity and an increased of persistence of Rac activation in chronic myeloid leukemia.

    PubMed

    Panuzzo, Cristina; Volpe, Gisella; Cibrario Rocchietti, Elisa; Casnici, Claudia; Crotta, Katia; Crivellaro, Sabrina; Carrà, Giovanna; Lorenzatti, Roberta; Peracino, Barbara; Torti, Davide; Morotti, Alessandro; Camacho-Leal, Maria Pilar; Defilippi, Paola; Marelli, Ornella; Saglio, Giuseppe

    2015-01-01

    In Chronic Myeloid Leukemia 80% of patients present alternative splice variants involving BCR exons 1, 13 or 14 and ABL exon 4, with a consequent impairment in the reading frame of the ABL gene. Therefore BCR/ABL fusion proteins (BCR/ABL-OOF) are characterized by an in-frame BCR portion followed by an amino acids sequence arising from the out of frame (OOF) reading of the ABL gene. The product of this new transcript contains the characteristic BCR domains while lacking the COOH-terminal Rho GTPase GAP domain. The present work aims to characterize the protein functionality in terms of cytoskeleton (re-)modelling, adhesion and activation of canonical oncogenic signalling pathways. Here, we show that BCR/ABL-OOF has a peculiar endosomal localization which affects EGF receptor activation and turnover. Moreover, we demonstrate that BCR/ABL-OOF expression leads to aberrant cellular adhesion due to the activation of Rac GTPase, increase in cellular proliferation, migration and survival. When overexpressed in a BCR/ABL positive cell line, BCR/ABL-OOF induces hyperactivation of Rac signaling axis offering a therapeutic window for Rac-targeted therapy. Our data support a critical role of BCR/ABL-OOF in leukemogenesis and identify a subset of patients that may benefit from Rac-targeted therapies. PMID:26682280

  2. The interactome of a PTB domain-containing adapter protein, Odin, revealed by SILAC

    PubMed Central

    Zhong, Jun; Chaerkady, Raghothama; Kandasamy, Kumaran; Gucek, Marjan; Cole, Robert N.; Pandey, Akhilesh

    2011-01-01

    Signal transduction pathways are tightly controlled by positive and negative regulators. We have previously identified Odin (also known as ankyrin repeat and sterile alpha motif domain containing 1A; gene symbol AKNS1A) as a negative regulator of growth factor signaling; however, the mechanisms through which Odin regulates these pathways remain to be elucidated. To determine how Odin negatively regulates growth factor signaling, we undertook a proteomic approach to systematically identify proteins that interact with Odin using the SILAC strategy. In this study, we identified 18 molecules that were specifically associated in a protein complex with Odin. Our study established that the complete family of 14-3-3 proteins occur in a protein complex with Odin, which is also supported by earlier reports that identified a few members of the 14-3-3 family as Odin interactors. Among the novel protein interactors of Odin were CD2-associated protein, SH3 domain kinase binding protein 1 and DAB2 interacting protein. We confirmed 8 of the eighteen interactions identified in the Odin protein complex by co-immunoprecipitation experiments. Finally, a literature-based network analysis revealed that Odin interacting partners are involved in various cellular processes, some of which are key molecules in regulating receptor endocytosis. PMID:21081186

  3. Crystal structure of the Rasputin NTF2-like domain from Drosophila melanogaster

    SciTech Connect

    Vognsen, Tina; Kristensen, Ole

    2012-03-30

    Highlights: Black-Right-Pointing-Pointer The crystal structure of the NTF2-like domain of Rasputin protein is presented. Black-Right-Pointing-Pointer Differences to known ligand binding sites of nuclear transport factor 2 are discussed. Black-Right-Pointing-Pointer A new ligand binding site for the Rasputin and G3BP proteins is proposed. -- Abstract: The crystal structure of the NTF2-like domain of the Drosophila homolog of Ras GTPase SH3 Binding Protein (G3BP), Rasputin, was determined at 2.7 A resolution. The overall structure is highly similar to nuclear transport factor 2: It is a homodimer comprised of a {beta}-sheet and three {alpha}-helices forming a cone-like shape. However, known binding sites for RanGDP and FxFG containing peptides show electrostatic and steric differences compared to nuclear transport factor 2. A HEPES molecule bound in the structure suggests a new, and possibly physiologically relevant, ligand binding site.

  4. Targeting ABL1-mediated Oxidative Stress Adaptation in Fumarate Hydratase-Deficient Cancer

    PubMed Central

    Sourbier, Carole; Ricketts, Christopher J.; Matsumoto, Shingo; Crooks, Daniel R.; Liao, Pei-Jyun; Mannes, Philip Z.; Yang, Youfeng; Wei, Ming-Hui; Srivastava, Gaurav; Ghosh, Sanchari; Chen, Viola; Vocke, Cathy D.; Merino, Maria; Srinivasan, Ramaprasad; Krishna, Murali C.; Mitchell, James B.; Pendergast, Ann Marie; Rouault, Tracey A.; Neckers, Len; Linehan, W. Marston

    2014-01-01

    SUMMARY Patients with germline fumarate hydratase (FH) mutation are predisposed to develop aggressive kidney cancer with few treatment options and poor therapeutic outcomes. Activity of the proto-oncogene ABL1 is upregulated in FH-deficient kidney tumors and drives a metabolic and survival signaling network necessary to cope with impaired mitochondrial function and abnormal accumulation of intracellular fumarate. Excess fumarate indirectly stimulates ABL1 activity while restoration of wild-type FH abrogates both ABL1 activation and the cytotoxicity caused by ABL1 inhibition or knockdown. ABL1 upregulates aerobic glycolysis via the mTOR/HIF1α pathway and neutralizes fumarate-induced proteotoxic stress by promoting nuclear localization of the anti-oxidant response transcription factor NRF2. Our findings identify ABL1 as a pharmacologically tractable therapeutic target in glycolytically dependent, oxidatively stressed tumors. PMID:25490448

  5. Role of c-Abl in the DNA damage stress response.

    PubMed

    Shaul, Yosef; Ben-Yehoyada, Merav

    2005-01-01

    c-Abl has been implicated in many cellular processes including differentiation, division, adhesion, death, and stress response. c-Abl is a latent tyrosine kinase that becomes activated in response to numerous extra- and intra-cellular stimuli. Here we briefly review the current knowledge about c-Abl involvement in the DNA-damage stress response and its implication on cell physiology. PMID:15686624

  6. c-Abl Activates Janus Kinase 2 in Normal Hematopoietic Cells*

    PubMed Central

    Tao, Wenjing; Leng, Xiaohong; Chakraborty, Sandip N.; Ma, Helen; Arlinghaus, Ralph B.

    2014-01-01

    Jak2 is involved in cytokine growth factor-stimulated signal transduction, but the mechanism of its activation is largely unknown. Here, we investigated Jak2 activation in a normal hematopoietic cell line, 32D mouse myeloid cells. The bimolecular fluorescence complementation studies showed that c-Abl formed a stable complex with Jak2 in live cells. Co-immunoprecipitation results showed that c-Abl bound to the βc chain of IL-3/IL-5/GM-CSF receptors. The kinase activities of both c-Abl and Jak2 were stimulated by IL-3 in 32D cells. Decreasing c-Abl protein expression in 32D cells by inducible shRNA decreased Jak2 activity and resulted in the failure of Jak2 activation in response to IL-3. Treatment of IL-3 and serum-starved 32D cells with 1 μm imatinib mysylate inhibited IL-3 stimulated kinase activities of both c-Abl and Jak2. In addition, the kinase-deficient Bcr-Abl mutant (p210K1172R) was defective for activation of Jak2 in 32D cells and impaired IL-3 independent growth, which was rescued by overexpression of c-Abl (+Abl). IL-3 efficiently inhibited apoptosis of 32Dp210K/R+Abl cells induced by imatinib mysylate but not Jak2 kinase inhibitor TG101209. In summary, our findings provide evidence that the kinase function of c-Abl and its C-terminal CT4 region is crucial for its interaction with Jak2 and its activation. c-Abl kinase activity induced by IL-3 is required for IL-3-stimulated Jak2 and Jak1 activation. Our findings reveal a novel regulatory role of c-Abl in Jak2 activation induced by IL-3 cytokine growth factor in 32D hematopoietic cells. PMID:24923444

  7. Domains and Naive Theories

    PubMed Central

    Gelman, Susan A.; Noles, Nicholaus S.

    2013-01-01

    Human cognition entails domain-specific cognitive processes that influence memory, attention, categorization, problem-solving, reasoning, and knowledge organization. This review examines domain-specific causal theories, which are of particular interest for permitting an examination of how knowledge structures change over time. We first describe the properties of commonsense theories, and how commonsense theories differ from scientific theories, illustrating with children’s classification of biological and non-biological kinds. We next consider the implications of domain-specificity for broader issues regarding cognitive development and conceptual change. We then examine the extent to which domain-specific theories interact, and how people reconcile competing causal frameworks. Future directions for research include examining how different content domains interact, the nature of theory change, the role of context (including culture, language, and social interaction) in inducing different frameworks, and the neural bases for domain-specific reasoning. PMID:24187603

  8. Targeting Abl Kinases to Regulate Vascular Leak During Sepsis and ARDS

    PubMed Central

    Rizzo, Alicia N.; Aman, Jurjan; van Nieuw Amerongen, Geerten P.; Dudek, Steven M.

    2015-01-01

    The vascular endothelium separates circulating fluid and inflammatory cells from the surrounding tissues. Vascular leak occurs in response to wide-spread inflammatory processes, such as sepsis and Acute Respiratory Distress Syndrome (ARDS), due to the formation of gaps between endothelial cells (EC). Although these disorders are leading causes of mortality in the ICU, no medical therapies exist to restore EC barrier function. Recent evidence highlights a key role for the Abl family of non-receptor tyrosine kinases in regulating vascular barrier integrity. These kinases have well-described roles in cancer progression and neuronal morphogenesis, but their functions in the vasculature have remained enigmatic until recently. The Abl family kinases, c-Abl (Abl1) and Abl related gene (Arg, Abl2), phosphorylate several cytoskeletal effectors that mediate vascular permeability, including myosin light chain kinase, cortactin, vinculin, and β-catenin. They also regulate cell-cell and cell-matrix junction dynamics, and the formation of actin-based cellular protrusions in multiple cell types. Additionally, both c-Abl and Arg are activated by hyperoxia and contribute to oxidant-induced EC injury. These numerous roles of Abl kinases in EC and the current clinical usage of imatinib and other Abl kinase inhibitors have spurred recent interest in repurposing these drugs for the treatment of vascular barrier dysfunction. This review will describe the structure and function of Abl kinases with an emphasis on their roles in mediating vascular barrier integrity. We will also provide a critical evaluation of the potential for exploiting Abl kinase inhibition as a novel therapy for inflammatory vascular leak syndromes. PMID:25814671

  9. Learning and Domain Adaptation

    NASA Astrophysics Data System (ADS)

    Mansour, Yishay

    Domain adaptation is a fundamental learning problem where one wishes to use labeled data from one or several source domains to learn a hypothesis performing well on a different, yet related, domain for which no labeled data is available. This generalization across domains is a very significant challenge for many machine learning applications and arises in a variety of natural settings, including NLP tasks (document classification, sentiment analysis, etc.), speech recognition (speakers and noise or environment adaptation) and face recognition (different lighting conditions, different population composition).

  10. History Untold: Celebrating Ohio History Through ABLE Students. Ohio History Project.

    ERIC Educational Resources Information Center

    Kent State Univ., OH. Ohio Literacy Resource Center.

    This document is a compilation of 33 pieces of writing presenting Ohio adult basic and literacy education (ABLE) students' perspectives of community and personal history. The items included in the compilation were written by ABLE students across Ohio in celebration of Ohio History Day. The compilation is organized in five sections as follows: (1)…

  11. 7 CFR 273.24 - Time limit for able-bodied adults.

    Code of Federal Regulations, 2014 CFR

    2014-01-01

    ... processed in accord with 7 CFR part 15. (h) Adjustments. FNS will make adjustments as follows: (1) Caseload... 7 Agriculture 4 2014-01-01 2014-01-01 false Time limit for able-bodied adults. 273.24 Section 273... Program Alternatives § 273.24 Time limit for able-bodied adults. (a) Definitions. For purposes of the...

  12. 7 CFR 273.24 - Time limit for able-bodied adults.

    Code of Federal Regulations, 2011 CFR

    2011-01-01

    ... processed in accord with 7 CFR part 15. (h) Adjustments. FNS will make adjustments as follows: (1) Caseload... 7 Agriculture 4 2011-01-01 2011-01-01 false Time limit for able-bodied adults. 273.24 Section 273... Program Alternatives § 273.24 Time limit for able-bodied adults. (a) Definitions. For purposes of the...

  13. 7 CFR 273.24 - Time limit for able-bodied adults.

    Code of Federal Regulations, 2013 CFR

    2013-01-01

    ... processed in accord with 7 CFR part 15. (h) Adjustments. FNS will make adjustments as follows: (1) Caseload... 7 Agriculture 4 2013-01-01 2013-01-01 false Time limit for able-bodied adults. 273.24 Section 273... Program Alternatives § 273.24 Time limit for able-bodied adults. (a) Definitions. For purposes of the...

  14. 7 CFR 273.24 - Time limit for able-bodied adults.

    Code of Federal Regulations, 2012 CFR

    2012-01-01

    ... processed in accord with 7 CFR part 15. (h) Adjustments. FNS will make adjustments as follows: (1) Caseload... 7 Agriculture 4 2012-01-01 2012-01-01 false Time limit for able-bodied adults. 273.24 Section 273... Program Alternatives § 273.24 Time limit for able-bodied adults. (a) Definitions. For purposes of the...

  15. 20 CFR 604.3 - Able and available requirement-general principles.

    Code of Federal Regulations, 2012 CFR

    2012-04-01

    ... 20 Employees' Benefits 3 2012-04-01 2012-04-01 false Able and available requirement-general principles. 604.3 Section 604.3 Employees' Benefits EMPLOYMENT AND TRAINING ADMINISTRATION, DEPARTMENT OF LABOR REGULATIONS FOR ELIGIBILITY FOR UNEMPLOYMENT COMPENSATION § 604.3 Able and available...

  16. C/EBPβ promotes BCR-ABL-mediated myeloid expansion and leukemic stem cell exhaustion.

    PubMed

    Hayashi, Y; Hirai, H; Kamio, N; Yao, H; Yoshioka, S; Miura, Y; Ashihara, E; Fujiyama, Y; Tenen, D G; Maekawa, T

    2013-03-01

    The BCR-ABL fusion oncoprotein accelerates differentiation and proliferation of myeloid cells during the chronic phase of chronic myeloid leukemia (CP-CML). Here, the role of CCAAT/enhancer binding protein β (C/EBPβ), a regulator for 'emergency granulopoiesis,' in the pathogenesis of CP-CML was examined. C/EBPβ expression was upregulated in Lineage(-) CD34(+) CD38(-) hematopoietic stem cells (HSCs) and myeloid progenitors isolated from bone marrow of patients with CP-CML. In EML cells, a mouse HSC line, BCR-ABL upregulated C/EBPβ, at least in part, through the activation of STAT5. Myeloid differentiation and proliferation induced by BCR-ABL was significantly impaired in C/EBPβ-deficient bone marrow cells in vitro. Mice that were transplanted with BCR-ABL-transduced C/EBPβ knockout bone marrow cells survived longer than mice that received BCR-ABL-transduced wild-type (WT) bone marrow cells. Significantly higher levels of leukemic stem cells were maintained in BCR-ABL-transduced C/EBPβ-deficient cells than in BCR-ABL-transduced WT cells. These results suggest that C/EBPβ is involved in BCR-ABL-mediated myeloid expansion. Further elucidation of the molecular mechanisms underlying the C/EBPβ-mediated stem cell loss might reveal a novel therapeutic strategy for eradication of CML stem cells. PMID:22948537

  17. Design of substrate-based BCR-ABL kinase inhibitors using the cyclotide scaffold

    PubMed Central

    Huang, Yen-Hua; Henriques, Sónia T.; Wang, Conan K.; Thorstholm, Louise; Daly, Norelle L.; Kaas, Quentin; Craik, David J.

    2015-01-01

    The constitutively active tyrosine kinase BCR-ABL is the underlying cause of chronic myeloid leukemia (CML). Current CML treatments rely on the long-term use of tyrosine kinase inhibitors (TKIs), which target the ATP binding site of BCR-ABL. Over the course of treatment, 20–30% of CML patients develop TKI resistance, which is commonly attributed to point mutations in the drug-binding region. We design a new class of peptide inhibitors that target the substrate-binding site of BCR-ABL by grafting sequences derived from abltide, the optimal substrate of Abl kinase, onto a cell-penetrating cyclotide MCoTI-II. Three grafted cyclotides show significant Abl kinase inhibition in vitro in the low micromolar range using a novel kinase inhibition assay. Our work also demonstrates that a reengineered MCoTI-II with abltide sequences grafted in both loop 1 and 6 inhibits the activity of [T315I]Abl in vitro, a mutant Abl kinase harboring the “gatekeeper” mutation which is notorious for being multidrug resistant. Results from serum stability and cell internalization studies confirm that the MCoTI-II scaffold provides enzymatic stability and cell-penetrating properties to the lead molecules. Taken together, our study highlights that reengineered cyclotides incorporating abltide-derived sequences are promising substrate-competitive inhibitors for Abl kinase and the T315I mutant. PMID:26264857

  18. PROGRESS REPORT FOR 1963-64 DESCRIBING THE PROJECT ABLE PROGRAM.

    ERIC Educational Resources Information Center

    MARGULIS, JOHAN G.

    PROJECT ABLE IS AN EXPERIMENTAL PROGRAM DESIGNED TO IDENTIFY AND TO ENCOURAGE POTENTIALLY ABLE PUPILS FROM CULTURALLY DEPRIVED OR LOW SOCIOECONOMIC BACKGROUNDS TO COMPLETE APPROPRIATE PROGRAMS OF SECONDARY EDUCATION. STUDENTS FROM THE SEVENTH AND TWELFTH GRADES FROM CULTURALLY POOR OR UNHAPPY HOMES WERE SELECTED BY PRINCIPALS AND FACULTIES OF…

  19. Physiologic Responses of Able-Bodied and Paraplegic Males to Maximal Arm Ergometry.

    ERIC Educational Resources Information Center

    Israel, Richard G.; And Others

    A study compared physiologic responses of healthy paraplegic males to those of healthy, able-bodied males during maximal arm ergometry. Fifteen able-bodied, healthy adult males and 13 healthy adult male paraplegics followed an exercise program involving heart rate, increased exercise loads, and oxygen uptake. Results from an analysis of the data…

  20. Able to Play: Mobilizing Communities for Children of All Abilities - Framing the Issue. Seminar Series

    ERIC Educational Resources Information Center

    W. K. Kellogg Foundation, 2005

    2005-01-01

    "Able to Play: Mobilizing Communities for Children of All Abilities" is one of a series of six seminars that the W.K. Kellogg Foundation hosted during 2005 to celebrate its 75th Anniversary year. The seminar, held in Lansing, Michigan, June 8-10, shared the lessons learned from the Able to Play Project. Advocates for people with disabilities,…

  1. Beneficial effects of combining nilotinib and imatinib in preclinical models of BCR-ABL+ leukemias

    PubMed Central

    Weisberg, Ellen; Catley, Laurie; Wright, Renee D.; Moreno, Daisy; Banerji, Lolita; Ray, Arghya; Manley, Paul W.; Mestan, Juergen; Fabbro, Doriano; Jiang, Jingrui; Hall-Meyers, Elizabeth; Callahan, Linda; DellaGatta, Jamie L.; Kung, Andrew L.

    2007-01-01

    Drug resistance resulting from emergence of imatinib-resistant BCR-ABL point mutations is a significant problem in advanced-stage chronic myelogenous leukemia (CML). The BCR-ABL inhibitor, nilotinib (AMN107), is significantly more potent against BCR-ABL than imatinib, and is active against many imatinib-resistant BCR-ABL mutants. Phase 1/2 clinical trials show that nilotinib can induce remissions in patients who have previously failed imatinib, indicating that sequential therapy with these 2 agents has clinical value. However, simultaneous, rather than sequential, administration of 2 BCR-ABL kinase inhibitors is attractive for many reasons, including the theoretical possibility that this could reduce emergence of drug-resistant clones. Here, we show that exposure of a variety of BCR-ABL+ cell lines to imatinib and nilotinib results in additive or synergistic cytotoxicity, including testing of a large panel of cells expressing BCR-ABL point mutations causing resistance to imatinib in patients. Further, using a highly quantifiable bioluminescent in vivo model, drug combinations were at least additive in antileukemic activity, compared with each drug alone. These results suggest that despite binding to the same site in the same target kinase, the combination of imatinib and nilotinib is highly efficacious in these models, indicating that clinical testing of combinations of BCR-ABL kinase inhibitors is warranted. PMID:17068153

  2. Re-evaluating the role of BCR/ABL in chronic myelogenous leukemia

    PubMed Central

    Ross, Theodora S; Mgbemena, Victoria E

    2014-01-01

    Chronic myelogenous leukemia (CML) requires the BCR/ABL tyrosine kinase for disease onset and maintenance. As a result, CML can be successfully treated with tyrosine kinase inhibitors (TKIs) such as imatinib. Most patients are maintained in a disease-suppressed state on daily TKI therapy for several years and in many cases this treatment prevents progression to the blast phase. If the TKI is discontinued, CML redevelops in 95% of patients as a result of persisting leukemia initiating cells (LICs). There are several hypotheses that describe the potential mechanism(s) responsible for LIC persistence in CML, but supporting evidence is limited. Furthermore, of the few patients who discontinue TKI therapy and are “cured” (i.e., in treatment-free remission), most have residual BCR/ABL-expressing cells in their hematopoietic tissues. There are also healthy individuals without a CML diagnosis who express the BCR/ABL mutation in a fraction of their hematopoietic cells. Finally, mice that express BCR/ABL from the Bcr locus as a knockin mutation do not develop CML. These mice have lower BCR/ABL levels than retroviral or transgenic models of BCR/ABL that do develop CML. Understanding why mice with BCR/ABL expressed from the Bcr locus and some people that express BCR/ABL are not afflicted with CML will provide insights into therapies to prevent or cure this disease. PMID:27308345

  3. 20 CFR 604.3 - Able and available requirement-general principles.

    Code of Federal Regulations, 2010 CFR

    2010-04-01

    ... LABOR REGULATIONS FOR ELIGIBILITY FOR UNEMPLOYMENT COMPENSATION § 604.3 Able and available requirement... exist, only that, at a minimum, the type of services the individual is able and available to perform is... available for work applies only to the week of unemployment for which UC is claimed. It does not apply...

  4. Causal Learning Across Domains

    ERIC Educational Resources Information Center

    Schulz, Laura E.; Gopnik, Alison

    2004-01-01

    Five studies investigated (a) children's ability to use the dependent and independent probabilities of events to make causal inferences and (b) the interaction between such inferences and domain-specific knowledge. In Experiment 1, preschoolers used patterns of dependence and independence to make accurate causal inferences in the domains of…

  5. Modeling Protein Domain Function

    ERIC Educational Resources Information Center

    Baker, William P.; Jones, Carleton "Buck"; Hull, Elizabeth

    2007-01-01

    This simple but effective laboratory exercise helps students understand the concept of protein domain function. They use foam beads, Styrofoam craft balls, and pipe cleaners to explore how domains within protein active sites interact to form a functional protein. The activity allows students to gain content mastery and an understanding of the…

  6. Domain wall filters

    SciTech Connect

    Baer, Oliver; Narayanan, Rajamani; Neuberger, Herbert; Witzel, Oliver

    2007-03-15

    We propose using the extra dimension separating the domain walls carrying lattice quarks of opposite handedness to gradually filter out the ultraviolet fluctuations of the gauge fields that are felt by the fermionic excitations living in the bulk. This generalization of the homogeneous domain wall construction has some theoretical features that seem nontrivial.

  7. c-Abl Modulates Tumor Cell Sensitivity to Antibody-Dependent Cellular Cytotoxicity (ADCC)

    PubMed Central

    Murray, Joseph C.; Aldeghaither, Dalal; Wang, Shangzi; Nasto, Rochelle E.; Jablonski, Sandra A.; Tang, Yong; Weiner, Louis M.

    2014-01-01

    Monoclonal antibodies can modulate cancer cell signal transduction and recruit antitumor immune effector mechanisms – including antibody-dependent cellular cytotoxicity (ADCC). Although several clinically effective antibodies can promote ADCC, therapeutic resistance is common. We hypothesized that oncogenic signaling networks within tumor cells affect their sensitivity to ADCC. We developed a screening platform and targeted 60 genes derived from an EGFR gene network using RNA interference (RNAi) in an in vitro ADCC model system. Knockdown of GRB7, PRKCE, and ABL1 enhanced ADCC by primary and secondary screens. ABL1 knockdown also reduced cell proliferation, independent of its ADCC enhancement effects. c-Abl overexpression decreased ADCC sensitivity and rescued the effects of ABL1 knockdown. Imatinib inhibition of c-Abl kinase activity also enhanced ADCC – phenocopying ABL1 knockdown – against several EGFR-expressing head-and-neck squamous cell carcinoma (HNSCC) cell lines by ex vivo primary NK cells. Our findings suggest that combining c-Abl inhibition with ADCC-promoting antibodies, such as cetuximab, could translate into increased therapeutic efficacy of monoclonal antibodies. PMID:25300860

  8. Activation of tyrosine kinase c-Abl contributes to α-synuclein-induced neurodegeneration.

    PubMed

    Brahmachari, Saurav; Ge, Preston; Lee, Su Hyun; Kim, Donghoon; Karuppagounder, Senthilkumar S; Kumar, Manoj; Mao, Xiaobo; Shin, Joo Ho; Lee, Yunjong; Pletnikova, Olga; Troncoso, Juan C; Dawson, Valina L; Dawson, Ted M; Ko, Han Seok

    2016-08-01

    Aggregation of α-synuclein contributes to the formation of Lewy bodies and neurites, the pathologic hallmarks of Parkinson disease (PD) and α-synucleinopathies. Although a number of human mutations have been identified in familial PD, the mechanisms that promote α-synuclein accumulation and toxicity are poorly understood. Here, we report that hyperactivity of the nonreceptor tyrosine kinase c-Abl critically regulates α-synuclein-induced neuropathology. In mice expressing a human α-synucleinopathy-associated mutation (hA53Tα-syn mice), deletion of the gene encoding c-Abl reduced α-synuclein aggregation, neuropathology, and neurobehavioral deficits. Conversely, overexpression of constitutively active c-Abl in hA53Tα-syn mice accelerated α-synuclein aggregation, neuropathology, and neurobehavioral deficits. Moreover, c-Abl activation led to an age-dependent increase in phosphotyrosine 39 α-synuclein. In human postmortem samples, there was an accumulation of phosphotyrosine 39 α-synuclein in brain tissues and Lewy bodies of PD patients compared with age-matched controls. Furthermore, in vitro studies show that c-Abl phosphorylation of α-synuclein at tyrosine 39 enhances α-synuclein aggregation. Taken together, this work establishes a critical role for c-Abl in α-synuclein-induced neurodegeneration and demonstrates that selective inhibition of c-Abl may be neuroprotective. This study further indicates that phosphotyrosine 39 α-synuclein is a potential disease indicator for PD and related α-synucleinopathies. PMID:27348587

  9. BCR-ABL-positive acute myeloid leukemia: a new entity? Analysis of clinical and molecular features.

    PubMed

    Neuendorff, Nina Rosa; Burmeister, Thomas; Dörken, Bernd; Westermann, Jörg

    2016-08-01

    BCR-ABL-positive acute myeloid leukemia (AML) is a rare subtype of AML that is now included as a provisional entity in the 2016 revised WHO classification of myeloid malignancies. Since a clear distinction between de novo BCR-ABL+ AML and chronic myeloid leukemia (CML) blast crisis is challenging in many cases, the existence of de novo BCR-ABL+ AML has been a matter of debate for a long time. However, there is increasing evidence suggesting that BCR-ABL+ AML is in fact a distinct subgroup of AML. In this study, we analyzed all published cases since 1975 as well as cases from our institution in order to present common clinical and molecular features of this rare disease. Our analysis shows that BCR-ABL predominantly occurs in AML-NOS, CBF leukemia, and AML with myelodysplasia-related changes. The most common BCR-ABL transcripts (p190 and p210) are nearly equally distributed. Based on the analysis of published data, we provide a clinical algorithm for the initial differential diagnosis of BCR-ABL+ AML. The prognosis of BCR-ABL+ AML seems to depend on the cytogenetic and/or molecular background rather than on BCR-ABL itself. A therapy with tyrosine kinase inhibitors (TKIs) such as imatinib, dasatinib, or nilotinib is reasonable, but-due to a lack of systematic clinical data-their use cannot be routinely recommended in first-line therapy. Beyond first-line treatment of AML, the use of TKI remains an individual decision, both in combination with intensive chemotherapy and/or as a bridge to allogeneic stem cell transplantation. In each single case, potential benefits have to be weighed against potential risks. PMID:27297971

  10. Role of p21 RAS in p210 bcr-abl transformation of murine myeloid cells.

    PubMed

    Mandanas, R A; Leibowitz, D S; Gharehbaghi, K; Tauchi, T; Burgess, G S; Miyazawa, K; Jayaram, H N; Boswell, H S

    1993-09-15

    The p21 RAS product has been implicated as part of the downstream signaling of certain nonreceptor tyrosine kinase oncogenes and several growth factor receptor-ligand interactions. We have reported that the chronic myelogenous leukemia oncogene p210 bcr-abl transforms a growth-factor-dependent myeloid cell line NFS/N1.H7 to interleukin-3 (IL-3) independence. In these p210 bcr-abl-transformed cells (H7 bcr-abl.A54) and in two other murine myeloid cell lines transformed to IL-3 independence by p210 bcr-abl, endogenous p21 RAS is activated as determined by an elevated ratio of associated guanosine triphosphate (GTP)/guanosine diphosphate (GDP), assayed by thin-layer chromatography of the nucleotides eluted from p21 RAS after immunoprecipitation with the Y13-259 antibody. Treatment of p210 bcr-abl-transformed cells with a specific tyrosine kinase inhibitor herbimycin A resulted in diminished tyrosine phosphorylation of p210 bcr-abl and associated proteins, without major reduction in expression of the p210 bcr-abl protein itself. Inhibition of p210 bcr-abl-dependent tyrosine phosphorylation resulted in a reduction of active p21RAS-GTP complexes in the transformed cells, in diminished expression of the nuclear early response genes c-jun and c-fos, and in lower cellular proliferation rate. To further implicate p21 RAS in these functional events downstream of p210 bcr-abl tyrosine phosphorylation, we targeted G-protein function directly by limiting the availability of GTP with the inosine monophosphate dehydrogenase inhibitor, tiazofurin (TR). In p210 bcr-abl-transformed cells treated for 4 hours with TR, in which the levels of GTP were reduced by 50%, but GDP, guanosine monophosphate, and adenosine triphosphate (ATP) were unaffected, p210 bcr-abl tyrosine phosphorylation was at control levels. However, expression of c-fos and c-jun nuclear proto-oncogenes were strongly inhibited and p21 RAS activity was downregulated. These findings show that p210 bcr-abl transduces

  11. Structural and biophysical investigation of the interaction of a mutant Grb2 SH2 domain (W121G) with its cognate phosphopeptide.

    PubMed

    Papaioannou, Danai; Geibel, Sebastian; Kunze, Micha B A; Kay, Christopher W M; Waksman, Gabriel

    2016-03-01

    The adaptor protein Grb2 is a key element of mitogenetically important signaling pathways. With its SH2 domain it binds to upstream targets while its SH3 domains bind to downstream proteins thereby relaying signals from the cell membranes to the nucleus. The Grb2 SH2 domain binds to its targets by recognizing a phosphotyrosine (pY) in a pYxNx peptide motif, requiring an Asn at the +2 position C-terminal to the pY with the residue either side of this Asn being hydrophobic. Structural analysis of the Grb2 SH2 domain in complex with its cognate peptide has shown that the peptide adopts a unique β-turn conformation, unlike the extended conformation that phosphopeptides adopt when bound to other SH2 domains. TrpEF1 (W121) is believed to force the peptide into this unusual conformation conferring this unique specificity to the Grb2 SH2 domain. Using X-ray crystallography, electron paramagnetic resonance (EPR) spectroscopy, and isothermal titration calorimetry (ITC), we describe here a series of experiments that explore the role of TrpEF1 in determining the specificity of the Grb2 SH2 domain. Our results demonstrate that the ligand does not adopt a pre-organized structure before binding to the SH2 domain, rather it is the interaction between the two that imposes the hairpin loop to the peptide. Furthermore, we find that the peptide adopts a similar structure when bound to both the wild-type Grb2 SH2 domain and a TrpEF1Gly mutant. This suggests that TrpEF1 is not the determining factor for the conformation of the phosphopeptide. PMID:26645482

  12. A Proposed System for Differentiating Elementary Mathematics for Exceptionally Able Students.

    ERIC Educational Resources Information Center

    Sirr, Palma M.

    1984-01-01

    A pilot mathematics project for one exceptionally able elementary student expanded to include other students and schools. Project activities included a needs assessment and development of a learning center approach and materials to differentiate the core elementary mathematics curriculum. (CL)

  13. Knee Replacement Patients May Be Able to Hit the Shower Sooner

    MedlinePlus

    ... page: https://medlineplus.gov/news/fullstory_159055.html Knee Replacement Patients May Be Able to Hit the Shower ... effect on surgical-site wound infection risk for [knee replacement surgery]," the researchers wrote. The findings were published ...

  14. Academic Attitudes of High Achieving and Low Achieving Academically Able Black Male Adolescents.

    ERIC Educational Resources Information Center

    Trotter, John Rhodes

    1981-01-01

    In order to identify the relationships between the attitudes and perceptions of peer pressure and the academic achievement of academically able male adolescents, this study compares the school attitudes of high achieving and low achieving Black male youth. (EF)

  15. A peptide biosensor for detecting intracellular Abl kinase activity using MALDI-TOF MS

    PubMed Central

    Placzek, Ekaterina A.; Plebanek, Michael P.; Lipchik, Andrew M.; Kidd, Stephanie R.; Parker, Laurie L.

    2009-01-01

    Many cancers are characterized by changes in protein phosphorylation as a result of kinase dysregulation. Disruption of Abl kinase signaling through the Philadelphia chromosome (causing the Bcr-Abl mutation) in chronic myeloid leukemia (CML) has provided a paradigm for development of kinase inhibitor drugs such as the specific inhibitor imatinib (also known as STI571 or Gleevec). However, since patients are treated indefinitely with this drug to maintain remission, resistance is increasingly becoming an issue. While there are many ways to detect kinase activity, most lack the ability to ‘multiplex’ the analysis (to detect more than one substrate simultaneously). Here we report a novel biosensor for detecting Abl kinase activity and sensitivity to inhibitor in live, intact cells overexpressing a CML model Abl kinase construct. This straightforward methodology could eventually provide a new tool for detecting kinase activity and inhibitor drug response in cancer cells that overexpress oncogenic kinases. PMID:19818327

  16. Rearrangements of c-myc and c-abl genes in tumour cells in Burkitt's lymphoma.

    PubMed Central

    Casares, S; Rodríguez, J M; Martin, A; Parrado, A

    1993-01-01

    Rearrangements of oncogenes c-myc and c-abl were detected by non-radioactive hybridisation in a case of Burkitt's lymphoma/leukaemia. The surface phenotype of Burkitt's cells were positive for CD19, CD20, HLA-DR, CD14, CD33 and surface immunoglobulin markers. Although cytogenetic analysis was not performed, the c-myc and heavy immunoglobulin genes had the same 14.2 kilobase EcoRI molecular size fragment, suggesting a possible t(8;14) translocation which is a common marker of this malignancy. The c-abl oncogene was also rearranged in DNA digested BamHI and EcoRI. The physiopathological implications of the rearranged c-abl gene are unknown, this being the first case, as for as is known, of Burkitt's lymphoma/leukaemia with a rearranged c-abl gene. Images PMID:8408711

  17. Combination of the ABL kinase inhibitor imatinib with the Janus kinase 2 inhibitor TG101348 for targeting residual BCR-ABL-positive cells

    PubMed Central

    2014-01-01

    Background The ABL kinase inhibitor imatinib is highly effective in treating most, but not all, patients with chronic myeloid leukemia (CML). This is because residual CML cells are generally present in the bone marrow microenvironment and are refractory to imatinib. Hematopoietic cytokine receptor signaling is mediated by Janus kinases (JAKs) and their downstream transcription factor, signal transducer and activator of transcription (STAT). TG101348 (SAR302503) is an oral inhibitor of JAK2. Methods We investigated the efficacy of imatinib and TG101348 using the break point cluster region-c-Abelson (BCR-ABL)-positive cell line and primary CML samples wherein leukemia cells were protected by a feeder cell line (HS-5). Results Imatinib treatment resulted in partial inhibition of cell growth in HS-5-conditioned medium. Furthermore, combined treatment with imatinib and TG101348 abrogated the protective effects of HS-5-conditioned medium on K562 cells. Phosphorylation of Crk-L, a BCR-ABL substrate, decreased considerably, while apoptosis increased. In addition, the combined treatment of CD34-positive primary samples resulted in considerably increased cytotoxicity, decreased Crk-L phosphorylation, and increased apoptosis. We also investigated TG101348 activity against feeder cells and observed that STAT5 phosphorylation, granulocyte macrophage colony-stimulating factor, and interleukin 6 levels decreased, indicating reduced cytokine production in HS-5 cells treated with TG101348. Conclusions These results showed that JAK inhibitors may enhance the cytotoxic effect of imatinib against residual CML cells and that a combined approach may be a powerful strategy against the stroma-associated drug resistance of Philadelphia chromosome-positive cells. PMID:24775308

  18. Optical Frequency Domain Imaging

    NASA Astrophysics Data System (ADS)

    Bouma, Brett E.; Tearney, Guillermo J.; Vakoc, Benjamin; Yun, Seok Hyun

    In this chapter, we discuss a frequency-domain approach, optical frequency-domain imaging (OFDI), which is based on optical frequency-domain reflectometry and uses a wavelength-swept laser and standard single-element photodetectors. The chapter begins with an overview of the fundamental aspects of the technology, including the detected signal, sensitivity, depth range, and resolution, and then goes on to discuss specific component technologies including the light source, interferometer and acquisition electronics, and image processing. The final section of the chapter provides a brief glimpse at some of the biomedical applications that most directly take advantage of the improved speed and sensitivity of OFDI.

  19. Evaluation of deoxyhypusine synthase inhibitors targeting BCR-ABL positive leukemias.

    PubMed

    Ziegler, Patrick; Chahoud, Tuhama; Wilhelm, Thomas; Pällman, Nora; Braig, Melanie; Wiehle, Valeska; Ziegler, Susanne; Schröder, Marcus; Meier, Chris; Kolodzik, Adrian; Rarey, Matthias; Panse, Jens; Hauber, Joachim; Balabanov, Stefan; Brümmendorf, Tim H

    2012-12-01

    Effective inhibition of BCR-ABL tyrosine kinase activity with Imatinib represents a breakthrough in the treatment of patients with chronic myeloid leukemia (CML). However, more than 30 % of patients with CML in chronic phase do not respond adequately to Imatinib and the drug seems not to affect the quiescent pool of BCR-ABL positive leukemic stem and progenitor cells. Therefore, despite encouraging clinical results, Imatinib can still not be considered a curative treatment option in CML. We recently reported downregulation of eukaryotic initiation factor 5A (eIF5A) in Imatinib treated K562 cells. Furthermore, the inhibition of eIF5A by siRNA in combination with Imatinib has been shown to exert synergistic cytotoxic effects on BCR-ABL positive cell lines. Based on the structure of known deoxyhypusine synthase (DHS) inhibitors such as CNI-1493, a drug design approach was applied to develop potential compounds targeting DHS. Here we report the biological evaluation of selected novel (DHSI-15) as compared to established (CNI-1493, deoxyspergualin) DHS inhibitors. We show that upon the compounds tested, DHSI-15 and deoxyspergualin exert strongest antiproliferative effects on BCR-ABL cells including Imatinib resistant mutants. However, this effect did not seem to be restricted to BCR-ABL positive cell lines or primary cells. Both compounds are able to induce apoptosis/necrosis during long term incubation of BCR-ABL positive BA/F3 derivates. Pharmacological synergism can be observed for deoxyspergualin and Imatinib, but not for DHSI-15 and Imatinib. Finally we show that deoxyspergualin is able to inhibit proliferation of CD34+ progenitor cells from CML patients. We conclude that inhibition of deoxyhypusine synthase (DHS) can be supportive for the anti-proliferative treatment of leukemia and merits further investigation including other cancers. PMID:22415796

  20. miRNA143 Induces K562 Cell Apoptosis Through Downregulating BCR-ABL

    PubMed Central

    Liang, Bing; Song, Yanbin; Zheng, Wenling; Ma, Wenli

    2016-01-01

    Background Leukemia seriously threats human health and life. MicroRNA regulates cell growth, proliferation, apoptosis, and cell cycle. Whether microRNA could be treated as a target for leukemia is still unclear and the mechanism by which microRNA143 regulates K562 cells needs further investigation. Material/Methods miRNA143 and its scramble miRNA were synthesized and transfected to K562 cells. MTT assay was used to detect K562 cell proliferation. Flow cytometry and a caspase-3 activity detection kit were used to test K562 cell apoptosis. Western blot analysis was performed to determine breakpoint cluster region-Abelson (BCR-ABL) expression. BCR-ABL overexpression and siRNA were used to change BCR-ABL level, and cell apoptosis was detected again after lipofection transfection. Results miRNA143 transfection inhibited K562 cell growth and induced its apoptosis. miRNA143 transfection decreased BCR-ABL expression. BCR-ABL overexpression suppressed miRNA143-induced K562 cell apoptosis, while its reduction enhanced miRNA143-induced apoptosis. Conclusions miRNA143 induced K562 cell apoptosis through downregulating BCR-ABL. miRNA143 might be a target for a new leukemia therapy. PMID:27492780

  1. Current status of ABL tyrosine kinase inhibitors stop studies for chronic myeloid leukemia

    PubMed Central

    2016-01-01

    ABL tyrosine kinase inhibitors (TKIs) dramatically improves chronic myeloid leukemia (CML) prognosis and most CML patients are now able to lead lives that are equivalent to those of healthy individuals. However, high cost to CML patients of long-term treatment and adverse effects (AEs) remain problems. At the setout, a clinical study involving the discontinuation of imatinib was conducted in France. Then, several stop studies of first-generation (imatinib) and second-generation ABL TKIs (dasatinib, nilotinib), which induce earlier response than imatinib, have also been started. These studies revealed that almost half of CML patients who are treated with ABL TKIs and achieve a certain period of sustained deep molecular response can stop ABL TKIs safely and obtain treatment free remission (TFR). AEs of ABL TKIs withdrawal and predicting factors for successful discontinuation including immunity are becoming clear gradually through these studies. It is important to conduct a comprehensive examination of the results of studies with a wide variety of protocols in order to determine which discontinuation method results in the highest probability of TFR in clinical settings. PMID:27583255

  2. ABL kinase mutation and relapse in 4 pediatric Philadelphia chromosome-positive acute lymphoblastic leukemia cases.

    PubMed

    Aoe, Michinori; Shimada, Akira; Muraoka, Michiko; Washio, Kana; Nakamura, Yoshimi; Takahashi, Takahide; Imada, Masahide; Watanabe, Toshiyuki; Okada, Ken; Nishiuchi, Ritsuo; Miyamura, Takako; Chayama, Kosuke; Shibakura, Misako; Oda, Megumi; Morishima, Tsuneo

    2014-01-01

    The tyrosine kinase inhibitor (TKI) imatinib mesylate (IM) revolutionized the treatment of Philadelphia chromosome-positive acute lymphoblastic leukemia (Ph-ALL), which had showed poor prognosis before the dawn of IM treatment. However, if Ph-ALL patients showed IM resistance due to ABL kinase mutation, second-generation TKI, dasatinib or nilotinib, was recommended. We treated 4 pediatric Ph-ALL patients with both IM and bone marrow transplantation (BMT); however, 3 relapsed. We retrospectively examined the existence of ABL kinase mutation using PCR and direct sequencing methods, but there was no such mutation in all 4 diagnostic samples. Interestingly, two relapsed samples from patients who were not treated with IM before relapse did not show ABL kinase mutation and IM was still effective even after relapse. On the other hand, one patient who showed resistance to 3 TKI acquired dual ABL kinase mutations, F359C at the IM-resistant phase and F317I at the dasatinib-resistant phase, simultaneously. In summary, Ph-ALL patients relapsed with or without ABL kinase mutation. Furthermore, ABL kinase mutation was only found after IM treatment, so an IM-resistant clone might have been selected during the IM treatment and intensive chemotherapy. The appropriate combination of TKI and BMT must be discussed to cure Ph-ALL patients. PMID:24652384

  3. Hybrid pyrimidine alkynyls inhibit the clinically resistance related Bcr-Abl(T315I) mutant.

    PubMed

    Lu, Xiaoyun; Zhang, Zhang; Ren, Xiaomei; Pan, Xiaofeng; Wang, Deping; Zhuang, Xiaoxi; Luo, Jingfeng; Yu, Rongmin; Ding, Ke

    2015-09-01

    A series of pyrimidine alkynyl derivatives were designed and synthesized as new Bcr-Abl inhibitors by hybriding the structural moieties from GNF-7, ponatinib and nilotinib. One of the most potent compounds 4e strongly suppresses Bcr-Abl(WT) and Bcr-Abl(T315I) kinase with IC50 values of 5.0 and 9.0 nM, and inhibits the proliferation of K562 and murine Ba/F3 cells ectopically expressing Bcr-Abl(T315I) cells with IC50 values of 2 and 50 nM, respectively. It also displays good pharmacokinetics properties with an oral bioavailability of 35.3% and T(1/2) value of 48.7 h, and demonstrates significantly suppression on tumor growth in xenografted mice of K562 and Ba/F3 cells expressing Bcr-Abl(T315I). These inhibitors may serve as lead compounds for further developing new anticancer drugs overcoming the clinically acquired resistance against current Bcr-Abl inhibitors. PMID:26195136

  4. Current status of ABL tyrosine kinase inhibitors stop studies for chronic myeloid leukemia.

    PubMed

    Kimura, Shinya

    2016-01-01

    ABL tyrosine kinase inhibitors (TKIs) dramatically improves chronic myeloid leukemia (CML) prognosis and most CML patients are now able to lead lives that are equivalent to those of healthy individuals. However, high cost to CML patients of long-term treatment and adverse effects (AEs) remain problems. At the setout, a clinical study involving the discontinuation of imatinib was conducted in France. Then, several stop studies of first-generation (imatinib) and second-generation ABL TKIs (dasatinib, nilotinib), which induce earlier response than imatinib, have also been started. These studies revealed that almost half of CML patients who are treated with ABL TKIs and achieve a certain period of sustained deep molecular response can stop ABL TKIs safely and obtain treatment free remission (TFR). AEs of ABL TKIs withdrawal and predicting factors for successful discontinuation including immunity are becoming clear gradually through these studies. It is important to conduct a comprehensive examination of the results of studies with a wide variety of protocols in order to determine which discontinuation method results in the highest probability of TFR in clinical settings. PMID:27583255

  5. Step Frequency and Step Length of 200-m Sprint in Able-bodied and Amputee Sprinters.

    PubMed

    Hobara, H; Sano, Y; Kobayashi, Y; Heldoorn, T A; Mochimaru, M

    2016-02-01

    The goal of this study was to examine the hypothesis that the difference in the 200-m sprint performance of amputee and able-bodied sprinters is due to a shorter step length rather than a lower step frequency. Men's elite-level 200-m races with a total of 16 able-bodied, 13 unilateral transtibial, 5 bilateral transtibial, and 16 unilateral transfemoral amputee sprinters were analyzed from publicly available internet broadcasts. For each run, the average forward velocity, step frequency, and step length over the entire 200-m distance were analyzed for each sprinter. The average forward velocity of able-bodied sprinters was faster than that of the other 3 groups, but there was no significant difference in average step frequency between able-bodied and transtibial amputee sprinters. However, the average step length of able-bodied sprinters was significantly longer than that of the transtibial amputee sprinters. In contrast, the step frequency and step length of transfemoral amputees were significantly lower and shorter than those of the other 3 groups. These results suggest that the differences in 200-m sprint performance between able-bodied and amputee sprinters are dependent on amputation level. PMID:26509370

  6. Effective Moment Feature Vectors for Protein Domain Structures

    PubMed Central

    Shi, Jian-Yu; Yiu, Siu-Ming; Zhang, Yan-Ning; Chin, Francis Yuk-Lun

    2013-01-01

    Imaging processing techniques have been shown to be useful in studying protein domain structures. The idea is to represent the pairwise distances of any two residues of the structure in a 2D distance matrix (DM). Features and/or submatrices are extracted from this DM to represent a domain. Existing approaches, however, may involve a large number of features (100–400) or complicated mathematical operations. Finding fewer but more effective features is always desirable. In this paper, based on some key observations on DMs, we are able to decompose a DM image into four basic binary images, each representing the structural characteristics of a fundamental secondary structure element (SSE) or a motif in the domain. Using the concept of moments in image processing, we further derive 45 structural features based on the four binary images. Together with 4 features extracted from the basic images, we represent the structure of a domain using 49 features. We show that our feature vectors can represent domain structures effectively in terms of the following. (1) We show a higher accuracy for domain classification. (2) We show a clear and consistent distribution of domains using our proposed structural vector space. (3) We are able to cluster the domains according to our moment features and demonstrate a relationship between structural variation and functional diversity. PMID:24391828

  7. The Promise of Domain Adaptation

    NASA Astrophysics Data System (ADS)

    Mahabal, Ashish A.; Li, Jingling; Vaijanapurkar, Samarth; Bue, Brian; Miller, Adam; Donalek, Ciro; Djorgovski, Stanislav G.; Drake, Andrew J.; Graham, Matthew; CRTS, iPTF

    2016-01-01

    Most new surveys spend an appreciable time in collecting data on which to train classifiers before they can be used on future observations from the same dataset. The result generating phase can start much earlier if the training could incorporate data accumulated from older surveys enhanced with a small set from the new survey. This is exactly what Domain Adaptation (DA) allows us to do. The main idea behind DAs can be summarized thus: if we have two classes of separable objects in some feature space of a Source survey (S), we can define a hyperplane to separate the two types. In a second Target survey (T), for the same features the hyperplane would be inclined differently. DA methods get the mapping between the two hyperplanes using a small fraction of data from the Target (T) survey and can then be used to predict the classes of the remaining majority of data in T. We discuss the parameters that need to be tuned, the difficulties involved, and ways to improve the results. As we move towards bigger, and deeper surveys, being able to use existing labelled information to conduct classification in future surveys will be more cost-effective and promote time efficiency as well. Starting with the light curve data of 50,000 periodic objects from Catalina Real-Time Transient Survey (CRTS), we have applied domain adaptation techniques such as Geodesic Flow Kernel (GFK) with Random forest classifier and Co-training for domain adaptation (CODA) to the CRTS data which has 35,000 points overlapping with Palomar Transient Factory (PTF), and 12,000 with Lincoln Near-Earth Asteroid Research (LINEAR). The results suggest that domain adaptation is an area worth exploring as the knowledge between these surveys is transferable and the approaches to find the mappings between these surveys can be applied to the remaining data as well as for near future surveys such as CRTS-II, Zwicky Transient Facility (ZTF) and the Large Synoptic Survey Telescope (LSST) to name a few at the optical

  8. Visualizing Knowledge Domains.

    ERIC Educational Resources Information Center

    Borner, Katy; Chen, Chaomei; Boyack, Kevin W.

    2003-01-01

    Reviews visualization techniques for scientific disciplines and information retrieval and classification. Highlights include historical background of scientometrics, bibliometrics, and citation analysis; map generation; process flow of visualizing knowledge domains; measures and similarity calculations; vector space model; factor analysis;…

  9. Oscillons and domain walls

    SciTech Connect

    Hindmarsh, Mark; Salmi, Petja

    2008-05-15

    Oscillons, extremely long-lived localized oscillations of a scalar field, are shown to be produced by evolving domain wall networks in {phi}{sup 4} theory in two spatial dimensions. We study the oscillons in frequency space using the classical spectral function at zero momentum, and obtain that the velocity distribution is suppressed as {gamma}{sup -2} at large Lorentz factor {gamma}, with oscillons produced up to at least {gamma}{approx}10. This leads us to speculate that oscillons are produced at cusps, regions of the domain wall travelling near the speed of light. In order to gain some insight onto the dilute oscillon 'gas' produced by the domain walls, we prepare a denser gas by filling the simulation volume with oscillons boosted in random directions. We finish the study by revisiting collisions between oscillons and between an oscillon and a domain wall, showing that in the latter case they can pass straight through with minimal distortion.

  10. Disrupting BCR-ABL in Combination with Secondary Leukemia-Specific Pathways in CML Cells Leads to Enhanced Apoptosis and Decreased Proliferation

    PubMed Central

    Woessner, David W.; Lim, Carol S.

    2013-01-01

    Chronic myeloid leukemia (CML) is a myeloproliferative disorder caused by expression of the fusion gene BCR-ABL following a chromosomal translocation in the hematopoietic stem cell.1 Therapeutic management of CML uses tyrosine kinase inhibitors (TKIs), which blocks ABL-signaling and effectively kill peripheral cells with BCR-ABL. However, TKIs are not curative, and chronic use of is required in order to treat CML. The primary failure for TKIs is through development of a resistant population due to mutations in the TKI binding regions.2, 3 This led us to develop the mutant coiled-coil, CCmut2, an alternative method for BCR-ABL signaling inhibition by targeting the N-terminal oligomerization domain of BCR, necessary for ABL activation.4 In this report we explore additional pathways which are important for leukemic stem cell survival in K562 cells. Using a candidate-based approach we test the combination of CCmut2 and inhibitors of unique secondary pathways in leukemic cells. Transformative potential was reduced following silencing of the leukemic stem cell factor Alox55 by RNA interference. Furthermore, blockade of the oncogenic protein MUC-16 by the novel peptide GO-201 yielded reductions in proliferation and increased cell death. Finally, we found that inhibiting macroautophagy7 using chloroquine in addition to blocking BCR-ABL signaling with the CCmut2 was most effective in limiting cell survival and proliferation. This study has elucidated possible combination therapies for CML using novel blockade of BCR-ABL and secondary leukemia-specific pathways. PMID:23211037

  11. Tandem BRCT Domains

    PubMed Central

    Mesquita, Rafael D.; Woods, Nicholas T.; Seabra-Junior, Eloy S.; Monteiro, Alvaro N.A.

    2010-01-01

    The cell’s ability to sense and respond to specific stimuli is a complex system derived from precisely regulated protein-protein interactions. Some of these protein-protein interactions are mediated by the recognition of linear peptide motifs by protein modular domains. BRCT (BRCA1 C-terminal) domains and their linear motif counterparts, which contain phosphoserines, are one such pair-wise interaction system that seems to have evolved to serve as a surveillance system to monitor threats to the cell’s genetic integrity. Evidence indicates that BRCT domains found in tandem can cooperate to provide sequence-specific binding of phosphorylated peptides as is the case for the breast and ovarian cancer susceptibility gene BRCA1 and the PAX transcription factor–interacting protein PAXIP1. Particular interest has been paid to tandem BRCT domains as “readers” of signaling events in the form of phosphorylated serine moieties induced by the activation of DNA damage response kinases ATM, ATR, and DNA-PK. However, given the diversity of tandem BRCT-containing proteins, questions remain as to the origin and evolution of this domain. Here, we discuss emerging views of the origin and evolving roles of tandem BRCT domain repeats in the DNA damage response. PMID:21533002

  12. Phosphorylation by the c-Abl protein tyrosine kinase inhibits parkin's ubiquitination and protective function

    PubMed Central

    Ko, Han Seok; Lee, Yunjong; Shin, Joo-Ho; Karuppagounder, Senthilkumar S.; Gadad, Bharathi Shrikanth; Koleske, Anthony J.; Pletnikova, Olga; Troncoso, Juan C.; Dawson, Valina L.; Dawson, Ted M.

    2010-01-01

    Mutations in PARK2/Parkin, which encodes a ubiquitin E3 ligase, cause autosomal recessive Parkinson disease (PD). Here we show that the nonreceptor tyrosine kinase c-Abl phosphorylates tyrosine 143 of parkin, inhibiting parkin's ubiquitin E3 ligase activity and protective function. c-Abl is activated by dopaminergic stress and by dopaminergic neurotoxins, 1-methyl-4-phenylpyridinium (MPP+) in vitro and in vivo by 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP), leading to parkin inactivation, accumulation of the parkin substrates aminoacyl-tRNA synthetase-interacting multifunctional protein type 2 (AIMP2) (p38/JTV-1) and fuse-binding protein 1 (FBP1), and cell death. STI-571, a c-Abl-family kinase inhibitor, prevents the phosphorylation of parkin, maintaining parkin in a catalytically active and protective state. STI-571’s protective effects require parkin, as shRNA knockdown of parkin prevents STI-571 protection. Conditional knockout of c-Abl in the nervous system also prevents the phosphorylation of parkin, the accumulation of its substrates, and subsequent neurotoxicity in response to MPTP intoxication. In human postmortem PD brain, c-Abl is active, parkin is tyrosine-phosphorylated, and AIMP2 and FBP1 accumulate in the substantia nigra and striatum. Thus, tyrosine phosphorylation of parkin by c-Abl is a major posttranslational modification that inhibits parkin function, possibly contributing to pathogenesis of sporadic PD. Moreover, inhibition of c-Abl may be a neuroprotective approach in the treatment of PD. PMID:20823226

  13. Inhibition of c-Abl Kinase Activity Renders Cancer Cells Highly Sensitive to Mitoxantrone

    PubMed Central

    Tuomela, Johanna; Sandholm, Jouko; Aittokallio, Kaappo; Siljamäki, Elina; Kallio, Marko; Kähäri, Veli-Matti; Hietanen, Sakari

    2014-01-01

    Although c-Abl has increasingly emerged as a key player in the DNA damage response, its role in this context is far from clear. We studied the effect of inhibition of c-Abl kinase activity by imatinib with chemotherapy drugs and found a striking difference in cell survival after combined mitoxantrone (MX) and imatinib treatment compared to a panel of other chemotherapy drugs. The combinatory treatment induced apoptosis in HeLa cells and other cancer cell lines but not in primary fibroblasts. The difference in MX and doxorubicin was related to significant augmentation of DNA damage. Transcriptionally active p53 accumulated in cells in which human papillomavirus E6 normally degrades p53. The combination treatment resulted in caspase activation and apoptosis, but this effect did not depend on either p53 or p73 activity. Despite increased p53 activity, the cells arrested in G2 phase became defective in this checkpoint, allowing cell cycle progression. The effect after MX treatment depended partially on c-Abl: Short interfering RNA knockdown of c-Abl rendered HeLa cells less sensitive to MX. The effect of imatinib was decreased by c-Abl siRNA suggesting a role for catalytically inactive c-Abl in the death cascade. These findings indicate that MX has a unique cytotoxic effect when the kinase activity of c-Abl is inhibited. The treatment results in increased DNA damage and c-Abl–dependent apoptosis, which may offer new possibilities for potentiation of cancer chemotherapy. PMID:25148385

  14. Quality Control Methods for Optimal BCR-ABL1 Clinical Testing in Human Whole Blood Samples

    PubMed Central

    Stanoszek, Lauren M.; Crawford, Erin L.; Blomquist, Thomas M.; Warns, Jessica A.; Willey, Paige F.S.; Willey, James C.

    2014-01-01

    Reliable breakpoint cluster region (BCR)–Abelson (ABL) 1 measurement is essential for optimal management of chronic myelogenous leukemia. There is a need to optimize quality control, sensitivity, and reliability of methods used to measure a major molecular response and/or treatment failure. The effects of room temperature storage time, different primers, and RNA input in the reverse transcription (RT) reaction on BCR-ABL1 and β-glucuronidase (GUSB) cDNA yield were assessed in whole blood samples mixed with K562 cells. BCR-ABL1 was measured relative to GUSB to control for sample loading, and each gene was measured relative to known numbers of respective internal standard molecules to control for variation in quality and quantity of reagents, thermal cycler conditions, and presence of PCR inhibitors. Clinical sample and reference material measurements with this test were concordant with results reported by other laboratories. BCR-ABL1 per 103 GUSB values were significantly reduced (P = 0.004) after 48-hour storage. Gene-specific primers yielded more BCR-ABL1 cDNA than random hexamers at each RNA input. In addition, increasing RNA inhibited the RT reaction with random hexamers but not with gene-specific primers. Consequently, the yield of BCR-ABL1 was higher with gene-specific RT primers at all RNA inputs tested, increasing to as much as 158-fold. We conclude that optimal measurement of BCR-ABL1 per 103 GUSB in whole blood is obtained when gene-specific primers are used in RT and samples are analyzed within 24 hours after blood collection. PMID:23541592

  15. An alpha-helical extension of the ELMO1 pleckstrin homology domain mediates direct interaction to DOCK180 and is critical in Rac signaling.

    PubMed

    Komander, David; Patel, Manishha; Laurin, Mélanie; Fradet, Nadine; Pelletier, Ariane; Barford, David; Côté, Jean-François

    2008-11-01

    The mammalian DOCK180 protein belongs to an evolutionarily conserved protein family, which together with ELMO proteins, is essential for activation of Rac GTPase-dependent biological processes. Here, we have analyzed the DOCK180-ELMO1 interaction, and map direct interaction interfaces to the N-terminal 200 amino acids of DOCK180, and to the C-terminal 200 amino acids of ELMO1, comprising the ELMO1 PH domain. Structural and biochemical analysis of this PH domain reveals that it is incapable of phospholipid binding, but instead structurally resembles FERM domains. Moreover, the structure revealed an N-terminal amphiphatic alpha-helix, and point mutants of invariant hydrophobic residues in this helix disrupt ELMO1-DOCK180 complex formation. A secondary interaction between ELMO1 and DOCK180 is conferred by the DOCK180 SH3 domain and proline-rich motifs at the ELMO1 C-terminus. Mutation of both DOCK180-interaction sites on ELMO1 is required to disrupt the DOCK180-ELMO1 complex. Significantly, although this does not affect DOCK180 GEF activity toward Rac in vivo, Rac signaling is impaired, implying additional roles for ELMO in mediating intracellular Rac signaling. PMID:18768751

  16. Functional Characterization of a Newly Identified Group B Streptococcus Pullulanase Eliciting Antibodies Able to Prevent Alpha-Glucans Degradation

    PubMed Central

    Bosello, Mattia; Berti, Francesco; Mariani, Massimo; Telford, John L.; Grandi, Guido; Soriani, Marco

    2008-01-01

    Streptococcal pullulanases have been recently proposed as key components of the metabolic machinery involved in bacterial adaptation to host niches. By sequence analysis of the Group B Streptococcus (GBS) genome we found a novel putative surface exposed protein with pullulanase activity. We named such a protein SAP. The sap gene is highly conserved among GBS strains and homologous genes, such as PulA and SpuA, have been described in other pathogenic streptococci. The SAP protein contains two N-terminal carbohydrate-binding motifs, followed by a catalytic domain and a C-terminal LPXTG cell wall-anchoring domain. In vitro analysis revealed that the recombinant form of SAP is able to degrade α-glucan polysaccharides, such as pullulan, glycogen and starch. Moreover, NMR analysis showed that SAP acts as a type I pullulanase. Studies performed on whole bacteria indicated that the presence of α-glucan polysaccharides in culture medium up-regulated the expression of SAP on bacterial surface as confirmed by FACS analysis and confocal imaging. Deletion of the sap gene resulted in a reduced capacity of bacteria to grow in medium containing pullulan or glycogen, but not glucose or maltose, confirming the pivotal role of SAP in GBS metabolism of α-glucans. As reported for other streptococcal pullulanases, we found specific anti-SAP antibodies in human sera from healthy volunteers. Investigation of the functional role of anti-SAP antibodies revealed that incubation of GBS in the presence of sera from animals immunized with SAP reduced the capacity of the bacterium to degrade pullulan. Of interest, anti-SAP sera, although to a lower extent, also inhibited Group A Streptococcus pullulanase activity. These data open new perspectives on the possibility to use SAP as a potential vaccine component inducing functional cross-reacting antibodies interfering with streptococcal infections. PMID:19023424

  17. Efficacy of ponatinib against ABL tyrosine kinase inhibitor-resistant leukemia cells

    SciTech Connect

    Okabe, Seiichi Tauchi, Tetsuzo; Tanaka, Yuko; Ohyashiki, Kazuma

    2013-06-07

    Highlights: •Efficacy of ponatinib against ABL tyrosine kinase inhibitor-resistant leukemia cells okabe et al. •Imatinib or nilotinib resistance was involved Src family kinase. •The BCR-ABL point mutation (E334V) was highly resistant to imatinib or nilotinib. •Ponatinib was a powerful strategy against imatinib or nilotinib resistant Ph-positive cells. -- Abstract: Because a substantial number of patients with chronic myeloid leukemia acquire resistance to ABL tyrosine kinase inhibitors (TKIs), their management remains a challenge. Ponatinib, also known as AP24534, is an oral multi-targeted TKI. Ponatinib is currently being investigated in a pivotal phase 2 clinical trial. In the present study, we analyzed the molecular and functional consequences of ponatinib against imatinib- or nilotinib-resistant (R) K562 and Ba/F3 cells. The proliferation of imatinib- or nilotinib-resistant K562 cells did not decrease after treatment with imatinib or nilotinib. Src family kinase Lyn was activated. Point mutation Ba/F3 cells (E334 V) were also highly resistant to imatinib and nilotinib. Treatment with ponatinib for 72 h inhibited the growth of imatinib- and nilotinib-resistant cells. The phosphorylation of BCR-ABL, Lyn, and Crk-L was reduced. This study demonstrates that ponatinib has an anti-leukemia effect by reducing ABL and Lyn kinase activity and this information may be of therapeutic relevance.

  18. BCR-ABL1(+) acute myeloid leukemia: clonal selection of a BCR-ABL1(-) subclone as a cause of refractory disease with nilotinib treatment.

    PubMed

    Neuendorff, Nina Rosa; Schwarz, Michaela; Hemmati, Philipp; Türkmen, Seval; Bommer, Christiane; Burmeister, Thomas; Dörken, Bernd; le Coutre, Philipp; Arnold, Renate; Westermann, Jörg

    2015-01-01

    The presence of a Philadelphia chromosome with a corresponding BCR-ABL1 rearrangement is the hallmark of chronic myeloid leukemia, but is considered a very rare event in de novo acute myeloid leukemia (AML). Here, we report the first case in which a dominant Philadelphia chromosome-positive subclone was detected upon relapse in a formerly Philadelphia chromosome-negative MLL-AF6(+) AML. Due to refractory disease under salvage chemotherapy, the patient was started on nilotinib treatment. As a result, the Philadelphia chromosome-positive subclone was eradicated within 1 month; however, disease progressed and was again dominated by the Philadelphia chromosome-negative founding clone, demonstrating rapid clonal expansion under nilotinib-induced selection pressure. PMID:25401297

  19. Domains in Ferroelectric Nanostructures

    NASA Astrophysics Data System (ADS)

    Gregg, Marty

    2010-03-01

    Ferroelectric materials have great potential in influencing the future of small scale electronics. At a basic level, this is because ferroelectric surfaces are charged, and so interact strongly with charge-carrying metals and semiconductors - the building blocks for all electronic systems. Since the electrical polarity of the ferroelectric can be reversed, surfaces can both attract and repel charges in nearby materials, and can thereby exert complete control over both charge distribution and movement. It should be no surprise, therefore, that microelectronics industries have already looked very seriously at harnessing ferroelectric materials in a variety of applications, from solid state memory chips (FeRAMs) to field effect transistors (FeFETs). In all such applications, switching the direction of the polarity of the ferroelectric is a key aspect of functional behavior. The mechanism for switching involves the field-induced nucleation and growth of domains. Domain coarsening, through domain wall propagation, eventually causes the entire ferroelectric to switch its polar direction. It is thus the existence and behavior of domains that determine the switching response, and ultimately the performance of the ferroelectric device. A major issue, associated with the integration of ferroelectrics into microelectronic devices, has been that the fundamental properties associated with ferroelectrics, when in bulk form, appear to change quite dramatically and unpredictably when at the nanoscale: new modes of behaviour, and different functional characteristics from those seen in bulk appear. For domains, in particular, the proximity of surfaces and boundaries have a dramatic effect: surface tension and depolarizing fields both serve to increase the equilibrium density of domains, such that minor changes in scale or morphology can have major ramifications for domain redistribution. Given the importance of domains in dictating the overall switching characteristics of a device

  20. Essential role for telomerase in chronic myeloid leukemia induced by BCR-ABL in mice

    PubMed Central

    Vicente-Dueñas, Carolina; Barajas-Diego, Marcos; Romero-Camarero, Isabel; González-Herrero, Inés; Flores, Teresa; Sánchez-García, Isidro

    2012-01-01

    The telomerase protein is constitutively activated in malignant cells from many patients with cancer, including the chronic myeloid leukemia (CML), but whether telomerase is essential for the pathogenesis of this disease is not known. Here, we used telomerase deficient mice to determine the requirement for telomerase in CML induced by BCR-ABL in mouse models of CML. Loss of one telomerase allele or complete deletion of telomerase prevented the development of leukemia induced by BCR-ABL. However, BCR-ABL was expressed and active in telomerase heterozygous and null leukemic hematopoietic stem cells. These results demonstrate that telomerase is essential for oncogene-induced reprogramming of hematopoietic stem cells in CML development and validate telomerase and the genes it regulates as targets for therapy in CML. PMID:22408137

  1. A Cross-Sectional Study of Dermatological Problems Among Differently-Abled Children

    PubMed Central

    Rao, Chythra; Rao, Raghavendra

    2012-01-01

    Background: Differently-abled children may be particularly susceptible to skin disorders either as a direct consequence of their disability or due of lack awareness regarding personal and environmental hygiene. Aim: To study the prevalence of skin problems in differently-abled children. Materials and Methods: A total of 122 differently-abled children in two special schools were examined for this study. Details regarding the disabilities were obtained from the school records. Results: More than half (53.2%) of the children had skin problems. Infections and infestations, acne, and dermatitis were the common dermatoses encountered in the present study. Conclusion: Periodic skin examination is of vital importance for all people with disabilities to reach a proper diagnosis and to offer treatment. Health education for teachers and parents on maintenance of sound personal hygiene and a clean environment is also recommended. PMID:22470206

  2. Monitoring Aerosol Optical Properties in the ABL, Using Lidar System and Sunphotometer in Buenos Aires, Argentina

    NASA Astrophysics Data System (ADS)

    Pallotta, J.; Pawelko, E.; Otero, L.; Ristori, P.; D'Elia, R.; Gonzalez, F.; Dworniczak, J.; Vilar, O.; Quel, E.

    2009-03-01

    At the Lasers and Applications Research Center (CEILAP, CITEFA-CONICET, (34°33' S, 58°30' W), located in an industrial suburb of the metropolitan area (Villa Martelli, Buenos Aires, Argentina), operates a multiwavelength lidar, based on a Nd:Yag laser (Continuum Surelite III P-IV). This system emits in 1064, 532 and 355 nm simultaneously (10 Hz, 600 mJ @ 1064 nm) and allows the monitoring of the optical aerosols properties in the atmospheric boundary layer (ABL). On the same experimental site, an AERONET sunphotometer provides the AOT value. An analysis of boundary layer behaviour in some relevant days of March, from the years 2004 to 2006 is presented. On the days analyzed, no aerosols events and clouds were registered over the ABL. Evolutions of some characteristics of the ABL are presented, such as the height of the boundary layer, height of entrainment zone (EZ) and the entrainment flux ratio.

  3. Aerosol Property Comparison Within and Above the ABL at the ARM Program SGP Site

    SciTech Connect

    Delle Monache, L

    2002-05-01

    This thesis determines what, if any, measurements of aerosol properties made at the Earth surface are representative of those within the entire air column. Data from the Atmospheric Radiation Measurement site at the Southern Great Plains, the only location in the world where ground-based and in situ airborne measurements are routinely made. Flight legs during the one-year period from March 2000 were categorized as either within or above the atmospheric boundary layer (ABL) by use of an objective mixing height determination technique. Correlations between aerosol properties measured at the surface and those within and above the ABL were computed. Aerosol extensive and intensive properties measured at the surface were found representative of values within the ABL, but not of within the free atmosphere.

  4. A Convenient Cell Culture Model for CML Acquired Resistance Through BCR-ABL Mutations.

    PubMed

    Wang, Zhiqiang; Chen, WenYong

    2016-01-01

    Tyrosine kinase inhibitors (TKIs) are the effective treatments for chronic myeloid leukemia (CML). However, clinical resistance to TKIs that leads to patient relapse remains a challenge. Acquisition of BCR-ABL mutations is crucial in the resistance but the underlying molecular mechanisms are poorly understood. Here we describe a cell culture model for CML acquired resistance in which blast crisis CML cells undergo initial apoptosis upon treatment with therapeutically effective doses of TKIs, but the cells regrow quickly with development of resistance through BCR-ABL mutations. This model mimics the clinical process of acquisition of BCR-ABL mutations and will be an important tool to dissect molecular mechanisms of CML drug resistance and to explore strategies to overcome resistance. PMID:27581146

  5. Biologically based risk estimation for radiation-induced CML. Inferences from BCR and ABL geometric distributions.

    PubMed

    Radivoyevitch, T; Kozubek, S; Sachs, R K

    2001-03-01

    Chronic myeloid leukemia (CML) invites biologically based radiation risk modeling because CML is simultaneously well-understood, homogeneous and prevalent. CML is known to be caused by a translocation involving the ABL and BCR genes, almost all CML patients have the BCR-ABL translocation, and CML is prevalent enough that its induction is unequivocally detected among Hiroshima A-bomb survivors. In a previous paper, a linear-quadratic-exponential (LQE) dose-response model was used to estimate the lifetime excess risk of CML in the limit of low doses of gamma-rays, R gamma. This estimate assumed that BCR-ABL translocation dose-response curves in stem cells for both neutrons and gamma-rays, differ only by a common proportionality constant from dicentric aberration dose-response curves in lymphocytes. In the present paper we challenge this assumption by predicting the BCR-ABL dose response. The predictions are based on the biophysical theory of dual radiation action (TDRA) as it applies to recent BCR-to-ABL distance data in G0 human lymphocytes; this data shows BCR and ABL geometric distributions that are not uniform and not independent, with close association of the two genes in some cells. The analysis speaks against the previous proportionality assumption. We compute 11 plausible LQE estimates of R gamma, 2 based on the proportionality assumption and 9 based on TDRA predictions. For each estimate of R gamma we also compute an associated estimate of the number of CML target cells, N; the biological basis of the LQE model allows us to form such estimates. Consistency between N and hematological considerations provides a plausibility check of the risk estimates. Within the group of estimates investigated, the most plausible lifetime excess risk estimates tend to lie near R gamma = 0.01 Gy-1, substantially higher than risk estimates based on the proportionality assumption. PMID:11357705

  6. Synthesis and biological evaluation of analogues of the kinase inhibitor nilotinib as Abl and Kit inhibitors

    PubMed Central

    Duveau, Damien Y.; Hu, Xin; Walsh, Martin J.; Shukla, Suneet; Skoumbourdis, Amanda P.; Boxer, Matthew B.; Ambudkar, Suresh V.; Shen, Min; Thomas, Craig J.

    2013-01-01

    The importance of the trifluoromethyl group in the polypharmacological profile of nilotinib was investigated. Molecular editing of nilotinib led to the design, synthesis and biological evaluation of analogues where the trifluoromethyl group was replaced by a proton, fluorine and a methyl group. While these analogues were less active than nilotinib toward Abl, their activity toward Kit was comparable, with the monofluorinated analogue being the most active. Docking of nilotinib and of analogues 2a–c to the binding pocket of Abl and of Kit showed that the lack of shape complementarity in Kit is compensated by the stabilizing effect from its juxtamembrane region. PMID:23273517

  7. Being Able to Learn: Researching the History of a Therapeutic Community

    PubMed Central

    Boyling, Elaine

    2011-01-01

    This article uses grounded theory methods to research the history of a therapeutic community and highlights the possibilities of ‘being able to learn’ as an aim for history of medicine research. For this case study, processes of research and learning allow consideration of the ‘dilemma of paternalism’ and the interaction between history, policy and practice. How can students enter into discourses about learning? How could this discourse help to make university systems of administration and assessment provide a more appropriate environment for staff and students to ‘be able to learn’?

  8. Axion domain wall baryogenesis

    SciTech Connect

    Daido, Ryuji; Kitajima, Naoya; Takahashi, Fuminobu

    2015-07-28

    We propose a new scenario of baryogenesis, in which annihilation of axion domain walls generates a sizable baryon asymmetry. Successful baryogenesis is possible for a wide range of the axion mass and decay constant, m≃10{sup 8}–10{sup 13} GeV and f≃10{sup 13}–10{sup 16} GeV. Baryonic isocurvature perturbations are significantly suppressed in our model, in contrast to various spontaneous baryogenesis scenarios in the slow-roll regime. In particular, the axion domain wall baryogenesis is consistent with high-scale inflation which generates a large tensor-to-scalar ratio within the reach of future CMB B-mode experiments. We also discuss the gravitational waves produced by the domain wall annihilation and its implications for the future gravitational wave experiments.

  9. Why Preferences in College Admissions May Yield a More-Able Student Body

    ERIC Educational Resources Information Center

    Li, Dong; Weisman, Dennis L.

    2011-01-01

    Critics of affirmative action policies contend that the elimination of racial preferences in college admissions would lead to a "more-able" student body. We develop a simple model comprised of three classes of college admissions--merit, race and legacy--to show that it is possible that a change in admissions policy that reduces racial preferences…

  10. The Value-Able Child: Teaching Values at Home and School. Grades K-3.

    ERIC Educational Resources Information Center

    Bostrom, Kathleen Long

    Noting that parents and educators need to place greater emphasis on teaching children values, this book shows parents, teachers, and group leaders how to work as a team to teach the values young children need to lead happy, "value-able" lives. The book's introduction defines values and presents a rationale for teaching values to young children in…

  11. Navajo-ABLE: Replication Model Navajo Assistive Technology Loan Program. Final Program Evaluation.

    ERIC Educational Resources Information Center

    Norton, Katie Jebb

    This final report discusses the activities and outcomes of the Navajo Assistive Bank of Loanable Equipment (Navajo-ABLE), a federally funded program designed to provide assistive technology (AT) devices, services, technical information, funding information, and training for Navajo children and youth with disabilities. The program was operated and…

  12. High School Physical Educators' Beliefs about Teaching Differently Abled Students in an Urban Public School District

    ERIC Educational Resources Information Center

    Hersman, Bethany L.; Hodge, Samuel R.

    2010-01-01

    The purpose of this study was to examine general physical education (GPE) teachers' beliefs about teaching differently abled students in inclusive classes.The participants were 5 GPE teachers from a large urban school district. The research method was explanatory multiple-case study situated in planned behavior theory. Data were gathered using a…

  13. What Should Teacher Educators Know and Be Able to Do? Perspectives from Practicing Teacher Educators

    ERIC Educational Resources Information Center

    Goodwin, A. Lin; Smith, Laura; Souto-Manning, Mariana; Cheruvu, Ranita; Tan, Mei Ying; Reed, Rebecca; Taveras, Lauren

    2014-01-01

    Commonsense reasoning says that quality teacher education relies on quality teacher educators. Yet, there is minimal attention to what teacher educators should know and be able to do. Unquestionably, teacher educators cannot teach what they do not know; but what "should" they know, and should they be prepared? This study of 293 teacher…

  14. Able Linguist Days--A Collaborative Approach between a University and Schools

    ERIC Educational Resources Information Center

    Riordan, Tanya

    2006-01-01

    This is a challenging time for sectors traditionally associated with modem foreign languages (MFL) education. MFL departments in secondary schools are striving to encourage their able linguists to continue with their language studies at Key Stage 4 (KS4); further education colleges are trying to make their numbers viable to run AS and A2 courses;…

  15. Literacy through Play: How Families with Able Children Support Their Literacy Development

    ERIC Educational Resources Information Center

    Williams, Mary; Rask, Hilma

    2003-01-01

    This article reports on findings from a research study that set out to identify factors that enable children considered to be able by their teachers to extend and develop their literacy. The data substantiated much that is already known about the significance of preschool home influences on the emergence of literacy. It underlined the importance…

  16. Strategies for the 21st Century: Integrating Technology into the ABLE Environment.

    ERIC Educational Resources Information Center

    Mingle, Mary E. H.

    Integrating technology into the Adult Basic Literacy Education (ABLE) classroom can be very helpful to students and teachers, but it requires a shift in the teacher's role. The idea of "delivering" instruction--teacher-centered classes or tutor-directed lessons--should be replaced with student-centered, self-paced learning. Although the first…

  17. Modelling c-Abl Signalling in Activated Neutrophils: the Anti-inflammatory Effect of Seliciclib.

    PubMed

    Jackson, Robert C; Radivoyevitch, Tomas

    2013-03-01

    When mammalian tissues are infected by bacteria or fungi, inflammatory cytokines are released that cause circulating neutrophils to invade the infected tissue. The cytosolic tyrosine kinase, c-Abl, in these tissue neutrophils is activated by TNFα. c-Abl then phosphorylates STAT transcription factors, which results in production of the antiapoptotic protein Mcl-1. The normally short-lived tissue neutrophils are then unable to enter apoptosis. c-Abl also causes release of reactive oxygen species (ROS) from the mitochondria of the activated neutrophils. These ROS, and ROS generated by NADPH oxidase, are bactericidal agents of the innate immune system. In some inflammatory diseases, such as chronic obstructive pulmonary disease (COPD), the invading neutrophils become permanently activated, and the resulting ROS overproduction causes severe tissue damage. The cyclin-dependent kinase inhibitor, seliciclib, blocks transcription through inhibition of cdk9. This results in a relatively rapid decline of antiapoptotic Mcl-1 transcripts in activated neutrophils, an increase in neutrophil apoptosis, and less ROS leakage and oxidative damage. We present here a model of neutrophil kinetics that simulates the principal pathways of c-Abl signalling and use it to explore possible treatment options for inflammatory lung disease. PMID:24765523

  18. Development of resistance to dasatinib in Bcr/Abl-positive acute lymphoblastic leukemia

    PubMed Central

    Fei, Fei; Stoddart, Sonia; Müschen, Markus; Kim, Yong-mi; Groffen, John; Heisterkamp, Nora

    2010-01-01

    Dasatinib is a potent dual Abl/Src inhibitor approved for treatment of Ph-positive leukemias. At a once-daily dose and a relatively short half-life of 3-5 hours, tyrosine kinase inhibition is not sustained. However, transient inhibition of K562 leukemia cells with a high-dose pulse of dasatinib or long-term treatment with a lower dose was reported to irreversibly induce apoptosis. Here, the effect of dasatinib on treatment of Bcr/Abl-positive acute lymphoblastic leukemia (ALL) cells was evaluated in the presence of stromal support. Dasatinib eradicated Bcr/Abl ALL cells, caused significant apoptosis and eliminated tyrosine phosphorylation on Bcr/Abl, Src, Crkl and Stat-5. However, treatment of mouse ALL cells with lower doses of dasatinib over an extended period of time allowed the emergence of viable drug-resistant cells. Interestingly, dasatinib treatment increased cell surface expression of CXCR4, which is important for survival of B-lineage cells, but this did not promote survival. Combined treatment of cells with dasatinib and a CXCR4 inhibitor resulted in enhanced cell death. These results do not support the concept that long-term treatment with low dose dasatinib monotherapy will be effective in causing irreversible apoptosis in Ph-positive ALL, but suggest that combined treatment with dasatinib and drugs such as AMD3100 may be effective. PMID:20111071

  19. 46 CFR 12.403 - Service or training requirements for able seaman (A/B) endorsements.

    Code of Federal Regulations, 2014 CFR

    2014-10-01

    ... 46 Shipping 1 2014-10-01 2014-10-01 false Service or training requirements for able seaman (A/B) endorsements. 12.403 Section 12.403 Shipping COAST GUARD, DEPARTMENT OF HOMELAND SECURITY MERCHANT MARINE OFFICERS AND SEAMEN REQUIREMENTS FOR RATING ENDORSEMENTS Requirements for National Deck Rating Endorsements § 12.403 Service or...

  20. High Self-Perceived Stress and Poor Coping in Intellectually Able Adults with Autism Spectrum Disorder

    ERIC Educational Resources Information Center

    Hirvikoski, Tatja; Blomqvist, My

    2015-01-01

    Despite average intellectual capacity, autistic traits may complicate performance in many everyday situations, thus leading to stress. This study focuses on stress in everyday life in intellectually able adults with autism spectrum disorders. In total, 53 adults (25 with autism spectrum disorder and 28 typical adults from the general population)…

  1. Project Final Report Regional Forest-ABL Coupling: Influence on CO2 and Climate

    SciTech Connect

    Kenneth J. Davis; Chuixiang Yi

    2003-04-02

    Ecosystem CO{sub 2} exchange and atmospheric boundary layer (ABL) mixing are correlated diurnally and seasonally. Tracer transport models predict that these covariance signals produce a meridional gradient of annual mean CO{sub 2} concentration in the marine boundary layer that is half as strong as the signal produced by fossil fuel emissions. This rectifier effect has been predicted by many inversion models. However, observations to constrain the strength of the rectifier effect in nature are lacking. The fundamental objective of this project was to measure the strength of these covariance signals between ecosystem CO{sub 2} flux and ABL dynamics by employing ABL profiling systems at eddy flux tower sites. We found that (1) the observed diurnal and seasonal covariance between ecosystem CO{sub 2} fluxes and ABL turbulent mixing are strong; (2) the inversion model underestimates the diurnal and seasonal covariance; (3) the rectifier effect in the model appears to be too weak. However, these results are subject to significant uncertainties associated with the use of a point measurement to represent an area, fair weather bias among the data and instruments, and nonlinear transport processes between continental and marine boundary layers.

  2. RT-PCR is a more accurate diagnostic tool for detection of BCR-ABL rearrangement

    SciTech Connect

    Zehnbauer, B.A.; Allen, A.P.; McGrath, S.D.

    1994-09-01

    Detection of the Philadelphia chromosome (Ph1) or genomic Southern hybridization for clonal gene rearrangement (GSH-R) has provided very specific identification of BCR-ABL gene rearrangement. Reverse transcriptase-polymerase chain reaction (RT-PCR) is diagnostic for patterns of BCR-ABL expression which are undetected by GSH-R and/or Ph1 and provides increased sensitivity both at diagnosis and in detection of minimal residual leukemia. Fifty-three specimens (of 150 tested from 119 consecutive leukemia patients) were RT-PCR positive for BCR-ABL gene expression confirmed by hybridization of PCR products with b{sub 3}a{sub 2}, b{sub 2}a{sub 2}, or e{sub 1}a{sub 2} junction-specific oligonucleotides. In 6 cases of CML with GSH-R{sup {minus}}at diagnosis, RT-PCR provided specific BCR-ABL identification. Deletion of BCR regions, low mitotic index, or e{sub 1}a{sub 2} expression caused failure to detect GSH-R or Ph1 translocation.

  3. Classification of Gaia16abl and Gaia16abn as type Ia supernovae

    NASA Astrophysics Data System (ADS)

    Piascik, A. S.; Steele, I. A.

    2016-02-01

    We conducted a spectroscopic observation of transient Gaia16abl at 2016-02-07T06:04:31 UT. This transient was observed by the Gaia Photometric Science survey on 2016-01-28T15:49:02 at position RA=3:15:22.38 DEC=+52:21:23.9 near galaxy GALEXASC J031526.23+522128.2.

  4. Predicting domain-domain interactions using a parsimony approach

    PubMed Central

    Guimarães, Katia S; Jothi, Raja; Zotenko, Elena; Przytycka, Teresa M

    2006-01-01

    We propose a novel approach to predict domain-domain interactions from a protein-protein interaction network. In our method we apply a parsimony-driven explanation of the network, where the domain interactions are inferred using linear programming optimization, and false positives in the protein network are handled by a probabilistic construction. This method outperforms previous approaches by a considerable margin. The results indicate that the parsimony principle provides a correct approach for detecting domain-domain contacts. PMID:17094802

  5. Evidence that Autophosphorylation of the Major Sporulation Kinase in Bacillus subtilis Is Able To Occur in trans

    PubMed Central

    Devi, Seram Nganbiton; Kiehler, Brittany; Haggett, Lindsey

    2015-01-01

    ABSTRACT Entry into sporulation in Bacillus subtilis is governed by a multicomponent phosphorelay, a complex version of a two-component system which includes at least three histidine kinases (KinA to KinC), two phosphotransferases (Spo0F and Spo0B), and a response regulator (Spo0A). Among the three histidine kinases, KinA is known as the major sporulation kinase; it is autophosphorylated with ATP upon starvation and then transfers a phosphoryl group to the downstream components in a His-Asp-His-Asp signaling pathway. Our recent study demonstrated that KinA forms a homotetramer, not a dimer, mediated by the N-terminal domain, as a functional unit. Furthermore, when the N-terminal domain was overexpressed in the starving wild-type strain, sporulation was impaired. We hypothesized that this impairment of sporulation could be explained by the formation of a nonfunctional heterotetramer of KinA, resulting in the reduced level of phosphorylated Spo0A (Spo0A∼P), and thus, autophosphorylation of KinA could occur in trans. To test this hypothesis, we generated a series of B. subtilis strains expressing homo- or heterogeneous KinA protein complexes consisting of various combinations of the phosphoryl-accepting histidine point mutant protein and the catalytic ATP-binding domain point mutant protein. We found that the ATP-binding-deficient protein was phosphorylated when the phosphorylation-deficient protein was present in a 1:1 stoichiometry in the tetramer complex, while each of the mutant homocomplexes was not phosphorylated. These results suggest that ATP initially binds to one protomer within the tetramer complex and then the γ-phosphoryl group is transmitted to another in a trans fashion. We further found that the sporulation defect of each of the mutant proteins is complemented when the proteins are coexpressed in vivo. Taken together, these in vitro and in vivo results reinforce the evidence that KinA autophosphorylation is able to occur in a trans fashion

  6. Beyond the "Textbook ABL": Numerical Simulations and Experimental Implications of Baroclinicity and Unsteadiness

    NASA Astrophysics Data System (ADS)

    Bou-Zeid, E.; Momen, M.

    2015-12-01

    Understanding and predicting the flow of air, and how it transports heat and trace gases, in the atmospheric boundary layer are increasingly becoming critical to a wide range of applications including wind and solar energy, urban design, agriculture, and assessment of climate change impacts and adaptations. These applications all require a level of sophistication and detail in our ability to probe and model the ABL and its interaction with the earth surface that manifestly exceeds our current capabilities. Previous work largely focused on the "textbook ABL", which is barotropic, in (quasi) steady-state, and interacts with a horizontal and homogeneous earth surface; it is evident that the "real-world ABL", even over flat terrain, rarely meets these simplifying conditions. In this talk we overview two complicating features that have been largely overlooked thus far despite their ubiquity: baroclinicity and unsteadiness. Large-eddy simulations of ABL flow with a time-varying (unsteady) or height-varying (baroclinic) pressure forcings are analyzed to understand how they modulate the bulk structure (mean fields) and turbulence (higher order moments). Our results indicate that for the unsteady ABL, the dynamics are primarily controlled by the relative magnitudes of three times scales: the inertial time scale (~ 12 hours in mid latitude), the turbulent time scale (~ 0.5 hours), and the forcing variability time scale (varies depending on meso and synoptic scale dynamics). For the baroclinic simulations, the strength and more importantly the direction of the baroclinicity can result in profiles that are vastly different from the classic barotropic case, with for example peaks in the turbulent kinetic energy that are in the middle of the layer. Both features also results in first and second order moments that, if interpreted to results from a steady barotropic case, can be highly misleading when experimental results are analyzed.

  7. Frequency of BCR-ABL Transcript Types in Syrian CML Patients

    PubMed Central

    Farhat-Maghribi, Sulaf; Habbal, Wafa; Monem, Fawza

    2016-01-01

    Background. In Syria, CML patients are started on tyrosine kinase inhibitors (TKIs) and monitored until complete molecular response is achieved. BCR-ABL mRNA transcript type is not routinely identified, contrary to the recommendations. In this study we aimed to identify the frequency of different BCR-ABL transcripts in Syrian CML patients and highlight their significance on monitoring and treatment protocols. Methods. CML patients positive for BCR-ABL transcripts by quantitative RT-PCR were enrolled. BCR-ABL transcript types were investigated using a home-made PCR method that was adapted from published protocols and optimized. The transcript types were then confirmed using a commercially available research kit. Results. Twenty-four transcripts were found in 21 patients. The most common was b2a2, followed by b3a2, b3a3, and e1a3 present solely in 12 (57.1%), 3 (14.3%), 2 (9.5%), and 1 (4.8%), respectively. Three samples (14.3%) contained dual transcripts. While b3a2 transcript was apparently associated with warning molecular response to imatinib treatment, b2a2, b3a3, and e1a3 transcripts collectively proved otherwise (P = 0.047). Conclusion. It might be advisable to identify the BCR-ABL transcript type in CML patients at diagnosis, using an empirically verified method, in order to link the detected transcript with the clinical findings, possible resistance to treatment, and appropriate monitoring methods. PMID:27313614

  8. Activity of the Aurora Kinase inhibitor VX-680 against Bcr/Abl positive acute lymphoblastic leukemias

    PubMed Central

    Fei, Fei; Stoddart, Sonia; Groffen, John; Heisterkamp, Nora

    2010-01-01

    The emergence of resistance to tyrosine kinase inhibitors due to point mutations in Bcr/Abl is a challenging problem for Philadelphia-chromosome positive (Ph-positive) acute lymphoblastic leukemia (ALL) patients, especially for those with the T315I mutation, against which neither nilotinib or dasatinib shows significant activity. VX-680 is a pan-Aurora kinase inhibitor active against all Bcr/Abl proteins but has not been extensively examined in preclinical models of Ph-positive ALL. Here, we have tested VX-680 for treatment of Bcr/Abl positive ALL when leukemic cells are protected by the presence of stroma. Under these conditions, VX-680 showed significant effects on primary human Ph-positive ALL cells both with and without the T315I mutation, including ablation of tyrosine phosphorylation downstream of Bcr/Abl, decreased viability and induction of apoptosis. However, drug treatment of human Ph-positive ALL cells for 3 days followed by drug removal allowed the outgrowth of abnormal cells 21 days later, and upon culture of mouse Bcr/Abl ALL cells on stroma with lower concentrations of VX-680, drug-resistant cells emerged. Combined treatment of human ALL cells lacking the T315I mutation with both VX-680 and dasatinib caused significantly more cytotoxicity than each drug alone. We suggest that use of VX-680 together with a second effective drug as first-line treatment for Ph-positive ALL is likely to be safer and more useful than second-line treatment with VX-680 as monotherapy for drug-resistant T315I Ph-positive ALL. PMID:20388735

  9. Nonrandom chromosomal change (trisomy 11) in murine plasmacytomas induced by an ABL-MYC retrovirus.

    PubMed

    Wiener, F; Coleman, A; Mock, B A; Potter, M

    1995-03-01

    Trisomy of chromosome 11 (Ts11) is the second most frequent nonrandom chromosomal change in murine plasmacytomas (PCTs). The frequency of Ts11 is significantly higher in PCTs induced in pristane-conditioned mice infected by Abelson-murine leukemia virus (52%) compared to those induced by pristane alone (8.1%). Although the significance of Ts11 in mouse plasmacytomagenesis is not clearly understood it is hypothesized that a gene or genes located on chromosome (Chr) 11 may specifically promote the development of PCTs in which both oncogenes, c-myc and v-abl, are abundantly expressed. To test this assumption we induced PCTs by three highly effective plasmacytomagenic retroviruses: ABL-MYC, J3V1, and RIM. Nearly 90% of PCTs that arose in BALB/c, (BALB/c x DBA/2N)F1, BALB/c-nu/nu, and 5-month-old SCID mice infected with ABL-MYC virus were trisomic for Chr 11. In contrast, < 10% of PCTs induced by J3V1 or RIM retroviral constructs encompassing either v-myc and v-raf or c-myc and v-Ha-ras oncogenes, respectively, contained Ts11. We have also investigated whether the entire Chr 11 or any particular subregion is preferentially duplicated in the process of ABL-MYC plasmacytomagenesis. By inducing PCTs in F1 heterozygous mice that are carriers of reciprocal translocations involving Chr 11 we found that the duplicated chromosomal region is located distal to the T4Dn breakpoint (11B5 band) on the telomeric segment of Chr 11. The regular duplication of this chromosomal segment strongly suggests the presence of a gene or genes whose amplification is of critical importance for v-abl associated murine plasmacytomagenesis. PMID:7867005

  10. Increased magnetic damping of a single domain wall and adjacent magnetic domains detected by spin torque diode in a nanostripe

    NASA Astrophysics Data System (ADS)

    Lequeux, Steven; Sampaio, Joao; Bortolotti, Paolo; Devolder, Thibaut; Matsumoto, Rie; Yakushiji, Kay; Kubota, Hitoshi; Fukushima, Akio; Yuasa, Shinji; Nishimura, Kazumasa; Nagamine, Yoshinori; Tsunekawa, Koji; Cros, Vincent; Grollier, Julie

    2015-11-01

    Spin torque resonance has been used to simultaneously probe the dynamics of a magnetic domain wall and of magnetic domains in a nanostripe magnetic tunnel junction. Due to the large associated resistance variations, we are able to analyze quantitatively the resonant properties of these single nanoscale magnetic objects. In particular, we find that the magnetic damping of both the domains and the domain wall is doubled compared to the damping value of the host magnetic layer. We estimate the contributions to the damping arising from the dipolar couplings between the different layers in the junction and from the intralayer spin pumping effect, and find that they cannot explain the large damping enhancement that we observe. We conclude that the measured increased damping is intrinsic to large amplitudes excitations of spatially localized modes or solitons such as vibrating or propagating domain walls.

  11. Increased magnetic damping of a single domain wall and adjacent magnetic domains detected by spin torque diode in a nanostripe

    SciTech Connect

    Lequeux, Steven; Sampaio, Joao; Bortolotti, Paolo; Cros, Vincent; Grollier, Julie; Matsumoto, Rie; Yakushiji, Kay; Kubota, Hitoshi; Fukushima, Akio; Yuasa, Shinji; Nishimura, Kazumasa; Nagamine, Yoshinori; Tsunekawa, Koji

    2015-11-02

    Spin torque resonance has been used to simultaneously probe the dynamics of a magnetic domain wall and of magnetic domains in a nanostripe magnetic tunnel junction. Due to the large associated resistance variations, we are able to analyze quantitatively the resonant properties of these single nanoscale magnetic objects. In particular, we find that the magnetic damping of both the domains and the domain wall is doubled compared to the damping value of the host magnetic layer. We estimate the contributions to the damping arising from the dipolar couplings between the different layers in the junction and from the intralayer spin pumping effect, and find that they cannot explain the large damping enhancement that we observe. We conclude that the measured increased damping is intrinsic to large amplitudes excitations of spatially localized modes or solitons such as vibrating or propagating domain walls.

  12. Quantifying intramolecular binding in multivalent interactions: a structure-based synergistic study on Grb2-Sos1 complex.

    PubMed

    Sethi, Anurag; Goldstein, Byron; Gnanakaran, S

    2011-10-01

    Numerous signaling proteins use multivalent binding to increase the specificity and affinity of their interactions within the cell. Enhancement arises because the effective binding constant for multivalent binding is larger than the binding constants for each individual interaction. We seek to gain both qualitative and quantitative understanding of the multivalent interactions of an adaptor protein, growth factor receptor bound protein-2 (Grb2), containing two SH3 domains interacting with the nucleotide exchange factor son-of-sevenless 1 (Sos1) containing multiple polyproline motifs separated by flexible unstructured regions. Grb2 mediates the recruitment of Sos1 from the cytosol to the plasma membrane where it activates Ras by inducing the exchange of GDP for GTP. First, using a combination of evolutionary information and binding energy calculations, we predict an additional polyproline motif in Sos1 that binds to the SH3 domains of Grb2. This gives rise to a total of five polyproline motifs in Sos1 that are capable of binding to the two SH3 domains of Grb2. Then, using a hybrid method combining molecular dynamics simulations and polymer models, we estimate the enhancement in local concentration of a polyproline motif on Sos1 near an unbound SH3 domain of Grb2 when its other SH3 domain is bound to a different polyproline motif on Sos1. We show that the local concentration of the Sos1 motifs that a Grb2 SH3 domain experiences is approximately 1000 times greater than the cellular concentration of Sos1. Finally, we calculate the intramolecular equilibrium constants for the crosslinking of Grb2 on Sos1 and use thermodynamic modeling to calculate the stoichiometry. With these equilibrium constants, we are able to predict the distribution of complexes that form at physiological concentrations. We believe this is the first systematic analysis that combines sequence, structure, and thermodynamic analyses to determine the stoichiometry of the complexes that are dominant in

  13. An English language interface for constrained domains

    NASA Technical Reports Server (NTRS)

    Page, Brenda J.

    1989-01-01

    The Multi-Satellite Operations Control Center (MSOCC) Jargon Interpreter (MJI) demonstrates an English language interface for a constrained domain. A constrained domain is defined as one with a small and well delineated set of actions and objects. The set of actions chosen for the MJI is from the domain of MSOCC Applications Executive (MAE) Systems Test and Operations Language (STOL) directives and contains directives for signing a cathode ray tube (CRT) on or off, calling up or clearing a display page, starting or stopping a procedure, and controlling history recording. The set of objects chosen consists of CRTs, display pages, STOL procedures, and history files. Translation from English sentences to STOL directives is done in two phases. In the first phase, an augmented transition net (ATN) parser and dictionary are used for determining grammatically correct parsings of input sentences. In the second phase, grammatically typed sentences are submitted to a forward-chaining rule-based system for interpretation and translation into equivalent MAE STOL directives. Tests of the MJI show that it is able to translate individual clearly stated sentences into the subset of directives selected for the prototype. This approach to an English language interface may be used for similarly constrained situations by modifying the MJI's dictionary and rules to reflect the change of domain.

  14. Lattice gas simulations of replicating domains

    SciTech Connect

    Dawson, S.P.; Hasslacher, B.; Pearson, J.E.

    1993-12-31

    We use the lattice gas cellular automation (LGCA) developed to simulate a process of pattern-formation recently observed in reaction-diffusion systems. We study the reaction mechanism, which is an extension of the Selkov model for glycolytic oscillations. We are able to reproduce the self-replicating domains observed in this work. We use the LGCA simulation to estimate the smallest length-scale on which this process can occur under conditions encountered in the cell. These estimates are similar to those obtained for Turing patterns in the same setting.

  15. Time-domain imaging

    NASA Technical Reports Server (NTRS)

    Tolliver, C. L.

    1989-01-01

    The quest for the highest resolution microwave imaging and principle of time-domain imaging has been the primary motivation for recent developments in time-domain techniques. With the present technology, fast time varying signals can now be measured and recorded both in magnitude and in-phase. It has also enhanced our ability to extract relevant details concerning the scattering object. In the past, the interface of object geometry or shape for scattered signals has received substantial attention in radar technology. Various scattering theories were proposed to develop analytical solutions to this problem. Furthermore, the random inversion, frequency swept holography, and the synthetic radar imaging, have two things in common: (1) the physical optic far-field approximation, and (2) the utilization of channels as an extra physical dimension, were also advanced. Despite the inherent vectorial nature of electromagnetic waves, these scalar treatments have brought forth some promising results in practice with notable examples in subsurface and structure sounding. The development of time-domain techniques are studied through the theoretical aspects as well as experimental verification. The use of time-domain imaging for space robotic vision applications has been suggested.

  16. Cellulose binding domain proteins

    DOEpatents

    Shoseyov, O.; Shpiegl, I.; Goldstein, M.; Doi, R.

    1998-11-17

    A cellulose binding domain (CBD) having a high affinity for crystalline cellulose and chitin is disclosed, along with methods for the molecular cloning and recombinant production. Fusion products comprising the CBD and a second protein are likewise described. A wide range of applications are contemplated for both the CBD and the fusion products, including drug delivery, affinity separations, and diagnostic techniques. 16 figs.

  17. Cellulose binding domain proteins

    DOEpatents

    Shoseyov, Oded; Shpiegl, Itai; Goldstein, Marc; Doi, Roy

    1998-01-01

    A cellulose binding domain (CBD) having a high affinity for crystalline cellulose and chitin is disclosed, along with methods for the molecular cloning and recombinant production thereof. Fusion products comprising the CBD and a second protein are likewise described. A wide range of applications are contemplated for both the CBD and the fusion products, including drug delivery, affinity separations, and diagnostic techniques.

  18. WAP domain proteins as modulators of mucosal immunity.

    PubMed

    Wilkinson, Thomas S; Roghanian, Ali; Simpson, Alexander John; Sallenave, Jean-Michel

    2011-10-01

    WAP (whey acidic protein) is an important whey protein present in milk of mammals. This protein has characteristic domains, rich in cysteine residues, called 4-DSC (four-disulfide core domain). Other proteins, mainly present at mucosal surfaces, have been shown to also possess these characteristic WAP-4-DSC domains. The present review will focus on two WAP-4-DSC containing proteins, namely SLPI (secretory leucocyte protease inhibitor) and trappin-2/elafin. Although first described as antiproteases able to inhibit in particular host neutrophil proteases [NE (neutrophil elastase), cathepsin-G and proteinase-3] and as such, able to limit maladaptive tissue damage during inflammation, it has become apparent that these molecules have a variety of other functions (direct antimicrobial activity, bacterial opsonization, induction of adaptive immune responses, promotion of tissue repair, etc.). After providing information about the 'classical' antiproteasic role of these molecules, we will discuss the evidence pertaining to their pleiotropic functions in inflammation and immunity. PMID:21936824

  19. Cytoprotective effect of imatinib mesylate in non-BCR-ABL-expressing cells along with autophagosome formation

    SciTech Connect

    Ohtomo, Tadashi; Miyazawa, Keisuke; Naito, Munekazu; Moriya, Shota; Kuroda, Masahiko; Itoh, Masahiro; Tomoda, Akio

    2010-01-01

    Treatment with imatinib mesylate (IM) results in an increased viable cell number of non-BCR-ABL-expressing cell lines by inhibiting spontaneous apoptosis. Electron microscopy revealed an increase of autophagosomes in response to IM. IM attenuated the cytotoxic effect of cytosine arabinoside, as well as inhibiting cell death with serum-deprived culture. Cytoprotection with autophagosome formation by IM was observed in various leukemia and cancer cell lines as well as normal murine embryonic fibroblasts (MEFs). Complete inhibition of autophagy by knockdown of atg5 in the Tet-off atg5{sup -/-} MEF system attenuated the cytoprotective effect of IM, indicating that the effect is partially dependent on autophagy. However, cytoprotection by IM was not mediated through suppression of ROS production via mitophagy, ER stress via ribophagy, or proapoptotic function of ABL kinase. Although the target tyrosine kinase(s) of IM remains unclear, our data provide novel therapeutic possibilities of using IM for cytoprotection.

  20. High self-perceived stress and poor coping in intellectually able adults with autism spectrum disorder.

    PubMed

    Hirvikoski, Tatja; Blomqvist, My

    2015-08-01

    Despite average intellectual capacity, autistic traits may complicate performance in many everyday situations, thus leading to stress. This study focuses on stress in everyday life in intellectually able adults with autism spectrum disorders. In total, 53 adults (25 with autism spectrum disorder and 28 typical adults from the general population) completed the Perceived Stress Scale. Autistic traits were assessed using the Autism Spectrum Quotient. Adults with autism spectrum disorder reported significantly higher subjective stress and poorer ability to cope with stress in everyday life, as compared to typical adults. Autistic traits were associated with both subjective stress/distress and coping in this cross-sectional series. The long-term consequences of chronic stress in everyday life, as well as treatment intervention focusing on stress and coping, should be addressed in future research as well as in the clinical management of intellectually able adults with autism spectrum disorder. PMID:25073750

  1. Comparison of Aerosol Properties Within and Above the ABL at the ARM Program's SGP Site

    SciTech Connect

    Delle Monache, L

    2002-05-01

    The goal of this thesis is to determine under what conditions, if any, measurements of aerosol properties made at the Earth's surface are representative of aerosol properties within the column of air above the surface. This thesis will use data from the Atmospheric Radiation Measurement (ARM) site at the Southern Great Plains (SGP) which is the only location in the world where ground-based and in situ airborne measurements are made on a routine basis. All flight legs in the one-year period from March 2000-March 2001 were categorized as either within or above the atmospheric boundary layer using an objective mixing height determination technique. The correlations between the aerosol properties measured at the surface and the measured within and above the ABL were then computed. The conclusion of this comparison is that the aerosol extensive and intensive properties measured at the surface are representative of values within the ABL, but not within the free atmosphere.

  2. Smart adaptable system for older adults' Daily Life Activities Management - The ABLE platform.

    PubMed

    Giokas, Kostas; Anastasiou, Athanasios; Tsirmpas, Charalampos; Koutsouri, Georgia; Koutsouris, Dimitris; Iliopoulou, Dimitra

    2014-01-01

    In this paper we propose a system (ABLE) that will act as the main platform for a number of low-cost, mature technologies that will be integrated in order to create a dynamically adaptive Daily Life Activities Management environment in order to facilitate the everyday life of senior (but not exclusively) citizens at home. While the main target group of ABLE's users is the ageing population its use can be extended to all people that are vulnerable or atypical in body, intellect or emotions and are categorized by society as disabled. The classes of assistive products that are well defined in the international standard, ISO9999 such as assistive products for personal medical treatment, personal care and protection, communication, information and reaction and for personal mobility, will be easily incorporated in our proposed platform. Furthermore, our platform could integrate and implement the above classes under several service models that will be analyzed further. PMID:25571318

  3. Phosphates sensing: two polyamino-phenolic zinc receptors able to discriminate and signal phosphates in water.

    PubMed

    Ambrosi, Gianluca; Formica, Mauro; Fusi, Vieri; Giorgi, Luca; Guerri, Annalisa; Macedi, Eleonora; Micheloni, Mauro; Paoli, Paola; Pontellini, Roberto; Rossi, Patrizia

    2009-07-01

    Two Zn(II)-dinuclear systems were studied as receptors for phosphates; they were obtained by using the two polyamino-phenolic ligands 3,3'-bis[N,N-bis(2-aminoethyl)aminomethyl]-2,2'-dihydroxybiphenyl (L1) and 2,6-bis[N,N-bis(2-aminoethyl)aminomethyl]phenol (L2) in which the difference lies in the spacers between the two dien units, biphenol or phenol in L1 and L2, respectively. The metallo-receptors obtained are able to selectively discriminate phosphate (Pi) from pyrophosphate (PPi) and vice versa in aqueous solution in a wide range of pH (6 < pH < 10). The L1 receptor system shows selectivity toward PPi over Pi, and on the contrary the L2 system exhibits opposite selectivity. This different selectivity is ascribed to the different Zn(II)-Zn(II) distances between the two metal centers which, showing a similar coordination requirement and binding phosphate in a bridge disposition, fit in a different way with the different guests. Furthermore, NMR studies supported the model of interaction proposed between guests and receptors, highlighting that they are also able to bind biological phosphates such as G6P and ATP at physiological pH. Fluorescence studies showed that the receptor system based on L1 is able to signal the presence in solution of Pi and PPi at physiological pH; the presence of Pi is detected by a quenching of the emission, that of PPi by an enhancement of it. With the aid of an external colored sensor (PCV), the receptors were then used to produce simple signaling systems for phosphates based on the displacement method; the two chemosensors obtained are able to signal and quantify these anions at physiological pH, preserving the selectivity between phosphate and pyrophosphate and extending it to G6P and ATP. PMID:19432470

  4. Immunomodulatory properties of carbon nanotubes are able to compensate immune function dysregulation caused by microgravity conditions.

    PubMed

    Crescio, Claudia; Orecchioni, Marco; Ménard-Moyon, Cécilia; Sgarrella, Francesco; Pippia, Proto; Manetti, Roberto; Bianco, Alberto; Delogu, Lucia Gemma

    2014-08-21

    Spaceflights lead to dysregulation of the immune cell functionality affecting the expression of activation markers and cytokine production. Short oxidized multi-walled carbon nanotubes functionalized by 1,3-dipolar cycloaddition have been reported to activate immune cells. In this Communication we have performed surface marker assays and multiplex ELISA on primary monocytes and T cells under microgravity. We have discovered that carbon nanotubes, through their immunostimulatory properties, are able to fight spaceflight immune system dysregulations. PMID:25029354

  5. Evaluation of a modified Fitts law BCI target acquisition task in able and motor disabled individuals

    PubMed Central

    Felton, E A; Radwin, R G; Wilson, J A; Williams, J C

    2013-01-01

    A brain-computer interface (BCI) is a communication system that takes recorded brain signals and translates them into real-time actions, in this case movement of a cursor on a computer screen. This work applied Fitts’ law to the evaluation of performance on a target acquisition task during sensorimotor rhythm-based BCI training. Fitts’ law, which has been used as a predictor of movement time in studies of human movement, was used here to determine the information transfer rate, which was based on target acquisition time and target difficulty. The information transfer rate was used to make comparisons between control modalities and subject groups on the same task. Data were analyzed from eight able-bodied and five motor disabled participants who wore an electrode cap that recorded and translated their electroencephalogram (EEG) signals into computer cursor movements. Direct comparisons were made between able-bodied and disabled subjects and between EEG and joystick cursor control in able-bodied subjects. Fitts’ law aptly described the relationship between movement time and index of difficulty for each task movement direction when evaluated separately and averaged together. This study showed that Fitts’ law can be successfully applied to computer cursor movement controlled by neural signals. PMID:19700814

  6. Prune-able fuzzy ART neural architecture for robot map learning and navigation in dynamic environments.

    PubMed

    Araújo, Rui

    2006-09-01

    Mobile robots must be able to build their own maps to navigate in unknown worlds. Expanding a previously proposed method based on the fuzzy ART neural architecture (FARTNA), this paper introduces a new online method for learning maps of unknown dynamic worlds. For this purpose the new Prune-able fuzzy adaptive resonance theory neural architecture (PAFARTNA) is introduced. It extends the FARTNA self-organizing neural network with novel mechanisms that provide important dynamic adaptation capabilities. Relevant PAFARTNA properties are formulated and demonstrated. A method is proposed for the perception of object removals, and then integrated with PAFARTNA. The proposed methods are integrated into a navigation architecture. With the new navigation architecture the mobile robot is able to navigate in changing worlds, and a degree of optimality is maintained, associated to a shortest path planning approach implemented in real-time over the underlying global world model. Experimental results obtained with a Nomad 200 robot are presented demonstrating the feasibility and effectiveness of the proposed methods. PMID:17001984

  7. Association of HLA antigens and BCR-ABL transcripts in leukemia patients with the Philadelphia chromosome

    PubMed Central

    de Carvalho, Daiana Landenberger; Barbosa, Cristian Dias; de Carvalho, André Luiz; Beck, Sandra Trevisan

    2012-01-01

    Objective This study aimed to verify the association between human leukocyte antigens and the bcr-abl fusion protein resulting from t(9;22)(q34;q11) in chronic leukemia myeloid and acute lymphoblastic leukemia patients. Methods Forty-seven bcr-abl positive individuals were evaluated. Typing was performed bymicrolymphocytotoxicity and molecular biological methods (human leukocyte antigens Class I and Class II). A control group was obtained from the data of potential bone marrow donors registered in the Brazilian Bone Marrow Donor Registry (REDOME). Results Positive associations with HLA-A25 and HLA-B18 were found for the b2a2 transcript, as well as a tendency towards a positive association with HLA-B40 and a negative association with HLA-A68. The b3a2 transcript showed positive associations with HLA-B40 and HLA-DRB1*3. Conclusion The negative association between human leukocyte antigens and the BCR-ABL transcript suggests that binding and presentation of peptides derived from the chimeric protein are effective to increase a cytotoxic T lymphocyte response appropriate for the destruction of leukemic cells. PMID:23049441

  8. Comparison of Aerosol Properties within and above the ABL at the ARM Program's SGP Site

    SciTech Connect

    Monache, L.D.; Perry, K.D.; Cederwall, R.T.

    2002-02-26

    The goal of this study was to determine under what conditions, if any, measurements of aerosol properties made at the Earth's surface are representative of the aerosol properties within the column of air above the surface. This project used data from the Atmospheric Radiation Measurement (ARM) site at the Southern Great Plains (SGP) site (Stokes and Schwartz 1994), which is one of the only locations in the world where ground-based and in situ airborne measurements of atmospheric aerosol are made on a routine basis. All flight legs in the one-year period from March 2000 to March 2001 were categorized as either within or above the atmospheric boundary layer (ABL) using an objective mixing height determination technique. The correlations between the aerosol properties measured at the surface and those measured within and above the ABL were then computed. The conclusion of this comparison is that the aerosol extensive properties (those that depend upon the amount of aerosol that is present in the atmosphere, i.e., either the number or mass concentrations), and intensive properties (those that do not depend upon the amount of aerosol present) measured at the surface are representative of values within the ABL, but not within the free atmosphere.

  9. Explaining why Gleevec is a specific and potent inhibitor of Abl kinase

    PubMed Central

    Lin, Yen-Lin; Meng, Yilin; Jiang, Wei; Roux, Benoît

    2013-01-01

    Tyrosine kinases present attractive drug targets for specific types of cancers. Gleevec, a well-known therapeutic agent against chronic myelogenous leukemia, is an effective inhibitor of Abl tyrosine kinase. However, Gleevec fails to inhibit closely homologous tyrosine kinases, such as c-Src. Because many structural features of the binding site are conserved, the molecular determinants responsible for binding specificity are not immediately apparent. Some have attributed the difference in binding specificity of Gleevec to subtle variations in ligand–protein interactions (binding affinity control), whereas others have proposed that it is the conformation of the DFG motif, in which ligand binding is only accessible to Abl and not to c-Src (conformational selection control). To address this issue, the absolute binding free energy was computed using all-atom molecular dynamics simulations with explicit solvent. The results of the free energy simulations are in good agreement with experiments, thereby enabling a meaningful decomposition of the binding free energy to elucidate the factors controlling Gleevec’s binding specificity. The latter is shown to be controlled by a conformational selection mechanism and also by differences in key van der Waals interactions responsible for the stabilization of Gleevec in the binding pocket of Abl. PMID:23319661

  10. Using the ABLE facility to observe urbanization effects on planetary boundary layer processes

    SciTech Connect

    Coulter, R.L.; Klazura, J.; Lesht, B.M.; Shannon, J.D.; Sisterson, D.L.; Wesely, M.L.

    1998-12-31

    The Argonne Boundary Layer Experiments (ABLE) facility, located in south central Kansas, east of Wichita, is devoted primarily to investigations of and within the planetary boundary layer (PBL), including the dynamics of the mixed layer during both day and night; effects of varying land use and landform; the interactive role of precipitation, runoff, and soil moisture; storm development; and energy budgets on scales of 10 to 100 km. With an expected lifetime of 10--15 years, the facility is well situated to observe the effects of gradual urbanization on PBL dynamics and structure as the Wichita urban area expands to the east and several small municipalities located within the study area expand. Combining the continuous measurements of ABLE with (1) ancillary continuous measurements of, for example, the Atmospheric Radiation Measurement (ARM) program and the Global Energy Water Cycle Experiment (GEWEX) programs and with (2) shorter, more intensive studies within ABLE, such as the Cooperative Atmosphere Surface Exchange Studies (CASES) Program, allows hypothesized features of urbanization, including heat island effects, precipitation enhancement, and modification of the surface energy budget partitioning, to be studied.

  11. GNF-2 Inhibits Dengue Virus by Targeting Abl Kinases and the Viral E Protein.

    PubMed

    Clark, Margaret J; Miduturu, Chandra; Schmidt, Aaron G; Zhu, Xuling; Pitts, Jared D; Wang, Jinhua; Potisopon, Supanee; Zhang, Jianming; Wojciechowski, Amy; Hann Chu, Justin Jang; Gray, Nathanael S; Yang, Priscilla L

    2016-04-21

    Dengue virus infects more than 300 million people annually, yet there is no widely protective vaccine or drugs against the virus. Efforts to develop antivirals against classical targets such as the viral protease and polymerase have not yielded drugs that have advanced to the clinic. Here, we show that the allosteric Abl kinase inhibitor GNF-2 interferes with dengue virus replication via activity mediated by cellular Abl kinases but additionally blocks viral entry via an Abl-independent mechanism. To characterize this newly discovered antiviral activity, we developed disubstituted pyrimidines that block dengue virus entry with structure-activity relationships distinct from those driving kinase inhibition. We demonstrate that biotin- and fluorophore-conjugated derivatives of GNF-2 interact with the dengue glycoprotein, E, in the pre-fusion conformation that exists on the virion surface, and that this interaction inhibits viral entry. This study establishes GNF-2 as an antiviral compound with polypharmacological activity and provides "lead" compounds for further optimization efforts. PMID:27105280

  12. The Endocytic Fate of the Transferrin Receptor Is Regulated by c-Abl Kinase.

    PubMed

    Cao, Hong; Schroeder, Barbara; Chen, Jing; Schott, Micah B; McNiven, Mark A

    2016-08-01

    Clathrin-mediated endocytosis of transferrin (Tf) and its cognate receptor (TfR1) is a central pathway supporting the uptake of trophic iron. It has generally been assumed that this is a constitutive process. However, we have reported that the non-receptor tyrosine kinase, Src, is activated by Tf to facilitate the internalization of the Tf-TfR1 ligand-receptor complex. As an extension of these findings, we have tested whether subsequent trafficking steps might be regulated by additional kinase-dependent cascades, and we observed a significant endocytic block by inhibiting c-Abl kinase by a variety of methods. Importantly, Tf internalization was reduced significantly in all of these cell models and could be restored by re-expression of WT c-Abl. Surprisingly, this attenuated Tf-TfR1 endocytosis was due to a substantial drop in both the surface and total cellular receptor levels. Additional studies with the LDL receptor showed a similar effect. Surprisingly, immunofluorescence microscopy of imatinib-treated cells revealed a marked colocalization of internalized TfR1 with late endosomes/lysosomes, whereas attenuating the lysosome function with several inhibitors reduced this receptor loss. Importantly, inhibition of c-Abl resulted in a striking redistribution of the chaperone Hsc70 from a diffuse cytosolic localization to an association with the TfR1 at the late endosome-lysosome. Pharmacological inhibition of Hsc70 ATPase activity in cultured cells by the drug VER155008 prevents this chaperone-receptor interaction, resulting in an accumulation of the TfR1 in the early endosome. Thus, inhibition of c-Abl minimizes receptor recycling pathways and results in chaperone-dependent trafficking of the TfR1 to the lysosome for degradation. These findings implicate a novel role for c-Abl and Hsc70 as an unexpected regulator of Hsc70-mediated transport of trophic receptor cargo between the early and late endosomal compartments. PMID:27226592

  13. Compound mutations in BCR-ABL1 are not major drivers of primary or secondary resistance to ponatinib in CP-CML patients

    PubMed Central

    Hodgson, J. Graeme; Shah, Neil P.; Cortes, Jorge E.; Kim, Dong-Wook; Nicolini, Franck E.; Talpaz, Moshe; Baccarani, Michele; Müller, Martin C.; Li, Jin; Parker, Wendy T.; Lustgarten, Stephanie; Clackson, Tim; Haluska, Frank G.; Guilhot, Francois; Kantarjian, Hagop M.; Soverini, Simona; Hochhaus, Andreas; Hughes, Timothy P.; Rivera, Victor M.; Branford, Susan

    2016-01-01

    BCR-ABL1 kinase domain mutations can confer resistance to first- and second-generation tyrosine kinase inhibitors (TKIs) in chronic myeloid leukemia (CML). In preclinical studies, clinically achievable concentrations of the third-generation BCR-ABL1 TKI ponatinib inhibit T315I and all other single BCR-ABL1 mutants except T315M, which generates a single amino acid exchange, but requires 2 sequential nucleotide exchanges. In addition, certain compound mutants (containing ≥2 mutations in cis) confer resistance. Initial analyses based largely on conventional Sanger sequencing (SS) have suggested that the preclinical relationship between BCR-ABL1 mutation status and ponatinib efficacy is generally recapitulated in patients receiving therapy. Thus far, however, such analyses have been limited by the inability of SS to definitively identify compound mutations or mutations representing less than ∼20% of total alleles (referred to as “low-level mutations”), as well as limited patient follow-up. Here we used next-generation sequencing (NGS) to define the baseline BCR-ABL1 mutation status of 267 heavily pretreated chronic phase (CP)-CML patients from the PACE trial, and used SS to identify clonally dominant mutants that may have developed on ponatinib therapy (30.1 months median follow-up). Durable cytogenetic and molecular responses were observed irrespective of baseline mutation status and included patients with compound mutations. No single or compound mutation was identified that consistently conferred primary and/or secondary resistance to ponatinib in CP-CML patients. Ponatinib is effective in CP-CML irrespective of baseline mutation status. PMID:26603839

  14. Photodynamic treatment (ALA-PDT) suppresses the expression of the oncogenic Bcr-Abl kinase and affects the cytoskeleton organization in K562 cells.

    PubMed

    Pluskalová, Michaela; Peslová, Gabriela; Grebenová, Dana; Halada, Petr; Hrkal, Zbynek

    2006-06-01

    K562 is the chronic myelogenous leukemia (CML)-derived cell line that expresses high levels of chimeric oncoprotein Bcr-Abl. The deregulated (permanent) kinase activity of Bcr-Abl leads to continuous proliferation of K562 cells and their resistance to the apoptosis promotion by conventional drugs. The photodynamic treatment (PDT) based on the application of 5-aminolevulinic acid (ALA) and irradiation with blue light (ALA-PDT) resulted in the suppression of K562 cells proliferation. It was followed by a necrosis-like cell death [K. Kuzelová, D. Grebenová, M. Pluskalová, I. Marinov, Z. Hrkal, J. Photochem. Photobiol. B 73 (2004) 67-78]. ALA-PDT led to the perturbation of the Hsp90/p23 multichaperone complex of which the Bcr-Abl is the client protein. Bcr-Abl protein was suppressed whereas the bcr-abl mRNA level was not affected. Further on, we observed several changes in the cytoskeleton organization. We detected ALA-PDT-mediated disruption of filamental actin structure using FITC-Phalloidin staining. In connection with this we uncovered certain cytoskeleton organizing proteins involved in the cell response to the treatment. Among these proteins, Septin2, which plays a role in maintaining actin bundles, was suppressed. Another one, PDZ-LIM domain protein 1 (CLP36) was altered. This protein acts as an adaptor molecule for LIM-kinase which phosphorylates and thus inactivates cofilin. Cofilin was indeed dephosphorylated and could thus be activated and operate as an actin-depolymerizing factor. We propose the scheme of molecular response of K562 cells to ALA-PDT. PMID:16495075

  15. Simplified Parallel Domain Traversal

    SciTech Connect

    Erickson III, David J

    2011-01-01

    Many data-intensive scientific analysis techniques require global domain traversal, which over the years has been a bottleneck for efficient parallelization across distributed-memory architectures. Inspired by MapReduce and other simplified parallel programming approaches, we have designed DStep, a flexible system that greatly simplifies efficient parallelization of domain traversal techniques at scale. In order to deliver both simplicity to users as well as scalability on HPC platforms, we introduce a novel two-tiered communication architecture for managing and exploiting asynchronous communication loads. We also integrate our design with advanced parallel I/O techniques that operate directly on native simulation output. We demonstrate DStep by performing teleconnection analysis across ensemble runs of terascale atmospheric CO{sub 2} and climate data, and we show scalability results on up to 65,536 IBM BlueGene/P cores.

  16. Magnetic bubble domain memories

    NASA Technical Reports Server (NTRS)

    Ypma, J. E.

    1974-01-01

    Some attractive features of Bubble Domain Memory and its relation to existing technologies are discussed. Two promising applications are block access mass memory and tape recorder replacement. The required chip capabilities for these uses are listed, and the specifications for a block access mass memory designed to fit between core and HPT disk are presented. A feasibility model for a tape recorder replacement is introduced.

  17. LRIG1 extracellular domain: structure and function analysis.

    PubMed

    Xu, Yibin; Soo, Priscilla; Walker, Francesca; Zhang, Hui Hua; Redpath, Nicholas; Tan, Chin Wee; Nicola, Nicos A; Adams, Timothy E; Garrett, Thomas P; Zhang, Jian-Guo; Burgess, Antony W

    2015-05-22

    We have expressed and purified three soluble fragments of the human LRIG1-ECD (extracellular domain): the LRIG1-LRR (leucine-rich repeat) domain, the LRIG1-3Ig (immunoglobulin-like) domain, and the LRIG1-LRR-1Ig fragment using baculovirus vectors in insect cells. The two LRIG1 domains crystallised so that we have been able to determine the three-dimensional structures at 2.3Å resolution. We developed a three-dimensional structure for the LRIG1-ECD using homology modelling based on the LINGO-1 structure. The LRIG1-LRR domain and the LRIG1-LRR-1Ig fragment are monomers in solution, whereas the LRIG1-3Ig domain appears to be dimeric. We could not detect any binding of the LRIG1 domains or the LRIG1-LRR-1Ig fragment to the EGF receptor (EGFR), either in solution using biosensor analysis or when the EGFR was expressed on the cell surface. The FLAG-tagged LRIG1-LRR-1Ig fragment binds weakly to colon cancer cells regardless of the presence of EGFRs. Similarly, neither the soluble LRIG1-LRR nor the LRIG1-3Ig domains nor the full-length LRIG1 co-expressed in HEK293 cells inhibited ligand-stimulated activation of cell-surface EGFR. PMID:25765764

  18. The impact of multiple low-level BCR-ABL1 mutations on response to ponatinib.

    PubMed

    Parker, Wendy T; Yeung, David T O; Yeoman, Alexandra L; Altamura, Haley K; Jamison, Bronte A; Field, Chani R; Hodgson, J Graeme; Lustgarten, Stephanie; Rivera, Victor M; Hughes, Timothy P; Branford, Susan

    2016-04-14

    The third-generation tyrosine kinase inhibitor (TKI) ponatinib shows activity against all common BCR-ABL1 single mutants, including the highly resistant BCR-ABL1-T315I mutant, improving outcome for patients with refractory chronic myeloid leukemia (CML). However, responses are variable, and causal baseline factors have not been well-studied. The type and number of low-level BCR-ABL1 mutations present after imatinib resistance has prognostic significance for subsequent treatment with nilotinib or dasatinib as second-line therapy. We therefore investigated the impact of low-level mutations detected by sensitive mass-spectrometry before ponatinib initiation (baseline) on treatment response in 363 TKI-resistant patients enrolled in the PONATINIB for Chronic Myeloid Leukemia Evaluation and Ph(+)Acute Lymphoblastic Leukemia trial, including 231 patients in chronic phase (CP-CML). Low-level mutations were detected in 53 patients (15%, including low-level T315I in 14 patients); most, however, did not undergo clonal expansion during ponatinib treatment and, moreover, no specific individual mutations were associated with inferior outcome. We demonstrate however, that the number of mutations detectable by mass spectrometry after TKI resistance is associated with response to ponatinib treatment and could be used to refine the therapeutic approach. Although CP-CML patients with T315I (63/231, 27%) had superior responses overall, those with multiple mutations detectable by mass spectrometry (20, 32%) had substantially inferior responses compared with those with T315I as the sole mutation detected (43, 68%). In contrast, for CP-CML patients without T315I, the inferior responses previously observed with nilotinib/dasatinib therapy for imatinib-resistant patients with multiple mutations were not seen with ponatinib treatment, suggesting that ponatinib may prove to be particularly advantageous for patients with multiple mutations detectable by mass spectrometry after TKI resistance

  19. The impact of multiple low-level BCR-ABL1 mutations on response to ponatinib

    PubMed Central

    Yeung, David T. O.; Yeoman, Alexandra L.; Altamura, Haley K.; Jamison, Bronte A.; Field, Chani R.; Hodgson, J. Graeme; Lustgarten, Stephanie; Rivera, Victor M.; Hughes, Timothy P.; Branford, Susan

    2016-01-01

    The third-generation tyrosine kinase inhibitor (TKI) ponatinib shows activity against all common BCR-ABL1 single mutants, including the highly resistant BCR-ABL1-T315I mutant, improving outcome for patients with refractory chronic myeloid leukemia (CML). However, responses are variable, and causal baseline factors have not been well-studied. The type and number of low-level BCR-ABL1 mutations present after imatinib resistance has prognostic significance for subsequent treatment with nilotinib or dasatinib as second-line therapy. We therefore investigated the impact of low-level mutations detected by sensitive mass-spectrometry before ponatinib initiation (baseline) on treatment response in 363 TKI-resistant patients enrolled in the PONATINIB for Chronic Myeloid Leukemia Evaluation and Ph+ Acute Lymphoblastic Leukemia trial, including 231 patients in chronic phase (CP-CML). Low-level mutations were detected in 53 patients (15%, including low-level T315I in 14 patients); most, however, did not undergo clonal expansion during ponatinib treatment and, moreover, no specific individual mutations were associated with inferior outcome. We demonstrate however, that the number of mutations detectable by mass spectrometry after TKI resistance is associated with response to ponatinib treatment and could be used to refine the therapeutic approach. Although CP-CML patients with T315I (63/231, 27%) had superior responses overall, those with multiple mutations detectable by mass spectrometry (20, 32%) had substantially inferior responses compared with those with T315I as the sole mutation detected (43, 68%). In contrast, for CP-CML patients without T315I, the inferior responses previously observed with nilotinib/dasatinib therapy for imatinib-resistant patients with multiple mutations were not seen with ponatinib treatment, suggesting that ponatinib may prove to be particularly advantageous for patients with multiple mutations detectable by mass spectrometry after TKI resistance

  20. Domain Specific vs Domain General: Implications for Dynamic Assessment

    ERIC Educational Resources Information Center

    Kaniel, Shlomo

    2010-01-01

    The article responds to the need for evidence-based dynamic assessment. The article is divided into two sections: In Part 1 we examine the scientific answer to the question of how far human mental activities and capabilities are domain general (DG) / domain specific (DS). A highly complex answer emerges from the literature review of domains such…

  1. The Amazon Boundary-Layer Experiment (ABLE 2B) - A meteorological perspective

    NASA Technical Reports Server (NTRS)

    Garstang, Michael; Greco, Steven; Scala, John; Swap, Robert; Ulanski, Stanley; Fitzjarrald, David; Martin, David; Browell, Edward; Shipman, Mark; Connors, Vickie

    1990-01-01

    The Amazon Boundary-Layer Experiments (ABLE) 2A and 2B, which were performed near Manaus, Brazil in July-August, 1985, and April-May, 1987 are discussed. The experiments were performed to study the sources, sinks, concentrations, and transports of trace gases and aerosols in rain forest soils, wetlands, and vegetation. Consideration is given the design and preliminary results of the experiment, focusing on the relationships between meteorological scales of motion and the flux, transports, and reactions of chemical species and aerosols embedded in the atmospheric fluid. Meteorological results are presented and the role of the meteorological results in the atmospheric chemistry experiment is examined.

  2. Are Caribbean reef sharks, Carcharhinus perezi, able to perceive human body orientation?

    PubMed

    Ritter, Erich K; Amin, Raid

    2014-05-01

    The present study examines the potential capability of Caribbean reef sharks to perceive human body orientation, as well as discussing the sharks' swimming patterns in a person's vicinity. A standardized video method was used to record the scenario of single SCUBA divers kneeling in the sand and the approach patterns of sharks, combined with a control group of two divers kneeling back-to-back. When approaching a single test-subject, significantly more sharks preferred to swim outside the person's field of vision. The results suggest that these sharks are able to identify human body orientation, but the mechanisms used and factors affecting nearest distance of approach remain unclear. PMID:24305995

  3. [Dynamics of mortality of able-bodied population in CIS countries].

    PubMed

    Maksimova, T M; Belov, V B; Rogovina, A G

    2006-01-01

    Comparative analysis founded on WHO European database was carried out concerning mortality in progress as compared between Russia and CIS countries. It is testified that the Russian Federation is distinguished by excelling rate of reduction of able-bodied population of middle age group. It is suggested that in this process significant role is played by health care system defects along with life-style factors. Motivation for enhancing activity and attention of medical workers as related to this age group, especially males is needed. PMID:17004373

  4. Spread spectrum time domain reflectometry

    NASA Astrophysics Data System (ADS)

    Smith, Paul Samuel

    For many years, wiring has been treated as a system that could be installed and expected to work for the life of the aircraft. As aircraft age far beyond their original expected life span, this attitude is rapidly changing. Wiring problems have recently been identified as the cause of several tragic mishaps and hundreds of thousands of lost mission hours. Intermittent wiring faults have been and continue to be difficult to resolve. Test methods that pinpoint faults on the ground can miss intermittent failures. New test methods involving spread spectrum signals are investigated that could be used in flight to locate intermittent failures, including open circuits, short circuits, and arcs. Spread spectrum time domain reflectometry (SSTDR) and sequence time domain reflectometry (STDR) are analyzed in light of the signals commonly present on aircraft wiring. Pseudo noise codes used for the generation of STDR and SSTDR signals are analyzed for application in a STDR/SSTDR test system in the presence of noise. The effects of Mil-Std 1553 and white noise on the STDR and SSTDR signals are discussed analytically, through simulations, and with the use of test hardware. A test system using STDR and SSTDR is designed, built, and used to collect STDR and SSTDR test data. The data collected with the STDR/SSTDR test hardware is analyzed and compared to the theoretical results. Experimental data for open and short circuits collected using SSTDR and a curve fitting algorithm shows a maximum range estimation error of +/-0.2 ft for 75O coaxial cable up to 100ft, and +/-0.6ft for a sample 32.5ft non-controlled impedance aircraft cable. Mil-Std 1553 is specified to operate reliably with a signal-to-noise ratio of 17.5dB, and the SSTDR test system was able to locate an open circuit on a cable also carrying simulated Mil-Std 1553 data where the SSTDR signal was 50dB below the Mil-Std 1553 signal. STDR and SSTDR are shown to be effective in detecting and locating dry and wet arcs on wires.

  5. TE-domestication and horizontal transfer in a putative Nef-AP1mu mimic of HLA-A cytoplasmic domain re-trafficking.

    PubMed

    Murray, Joseph S; Murray, Elaina H

    2016-01-01

    Loc-103275158 provisional protein conserved the nominal MHC-I CD tyrosine phosphorylation site, and it has an N-terminal SH3 domain that we docked in one conformation to its internal Nef-like domain. Here, we suggest that phosphorylation of the protein's CD-loop signals an exchange between the internal Nef-like domain and a lentiviral-Nef for binding the N-terminal SH3 domain - freeing the Nef-like domain to bind MHC-I CD. Since the 5'-tigger sequence encodes part of the pseudo α1/α2 MHC-I domain, and the 3'-tigger part of the Nef-like domain, we speculate that transposition proceeded phylogenetically disparate horizontal transfers, involving adjacent 5'- and 3'- parasitic footprints, which we also found in the Loc-103275158 orf. PMID:27511291

  6. BPS pion domain walls in the supersymmetric chiral Lagrangian

    NASA Astrophysics Data System (ADS)

    Gudnason, Sven Bjarke; Nitta, Muneto; Sasaki, Shin

    2016-07-01

    We construct exact solutions of BPS pion domain walls in the four-dimensional N =1 supersymmetric S U (N ) chiral Lagrangian with pion masses introduced via linear and quadratic superpotentials. The model admits N discrete vacua in the center of S U (N ) for the linear superpotential. In addition to the latter, new vacua appear for the quadratic superpotential. We find that the domain wall solutions of pions (Nambu-Goldstone bosons) that interpolate between a pair of (pion) vacua preserve half of supersymmetry. Contrary to our expectations, we have not been able to find domain walls involving the quasi-Nambu-Goldstone bosons present in the theory, which in turn has the consequence that not all vacua of the theory are connected by a BPS domain wall solution.

  7. Motivation for everyday social participation in cognitively able individuals with autism spectrum disorder

    PubMed Central

    Chen, Yu-Wei; Bundy, Anita C; Cordier, Reinie; Chien, Yi-Ling; Einfeld, Stewart L

    2015-01-01

    Objective The purpose of the present study was to examine motivation for the contextual nature of motivations for social participation in cognitively able adolescents and adults with autism spectrum disorder, using self-determination theory as a theoretical framework. Methods Fourteen Australians and 16 Taiwanese (aged 16–45 years) with Asperger’s syndrome and high functioning autism were asked to carry a device which prompted them seven times/day for 7 days, to record what they were doing, with whom, perceived difficulty and social reciprocity, and the reasons for engaging in a situation, which were then coded into degree of self-determination. Results Multilevel analyses showed that participants were more likely to be self-determined while engaging in “solitary/parallel leisure” and “social activities” than in other types of activities. Interactions with “family members” and “casual/intimate friends” were also positively associated with self-determined motivation. Further, participants were more likely to perceive higher levels of being listened to during interaction with casual/intimate friends than in interaction with other people. Global social anxiety served as a moderator for their perceptions of difficulty and social reciprocity during social engagement. Conclusion The findings highlight the context-dependent motivations for social engagement of cognitively able individuals with autism spectrum disorder. PMID:26508865

  8. Intracellular Retention of ABL Kinase Inhibitors Determines Commitment to Apoptosis in CML Cells

    PubMed Central

    Dziadosz, Marek; Schnöder, Tina; Heidel, Florian; Schemionek, Mirle; Melo, Junia V.; Kindler, Thomas; Müller-Tidow, Carsten; Koschmieder, Steffen; Fischer, Thomas

    2012-01-01

    Clinical development of imatinib in CML established continuous target inhibition as a paradigm for successful tyrosine kinase inhibitor (TKI) therapy. However, recent reports suggested that transient potent target inhibition of BCR-ABL by high-dose TKI (HD-TKI) pulse-exposure is sufficient to irreversibly commit cells to apoptosis. Here, we report a novel mechanism of prolonged intracellular TKI activity upon HD-TKI pulse-exposure (imatinib, dasatinib) in BCR-ABL-positive cells. Comprehensive mechanistic exploration revealed dramatic intracellular accumulation of TKIs which closely correlated with induction of apoptosis. Cells were rescued from apoptosis upon HD-TKI pulse either by repetitive drug wash-out or by overexpression of ABC-family drug transporters. Inhibition of ABCB1 restored sensitivity to HD-TKI pulse-exposure. Thus, our data provide evidence that intracellular drug retention crucially determines biological activity of imatinib and dasatinib. These studies may refine our current thinking on critical requirements of TKI dose and duration of target inhibition for biological activity of TKIs. PMID:22815843

  9. Identification of Aspergillus species in Central Europe able to produce G-type aflatoxins.

    PubMed

    Baranyi, Nikolett; Despot, Daniela Jakšić; Palágyi, Andrea; Kiss, Noémi; Kocsubé, Sándor; Szekeres, András; Kecskeméti, Anita; Bencsik, Ottó; Vágvölgyi, Csaba; Klarić, Maja Šegvić; Varga, János

    2015-09-01

    The occurrence of potential aflatoxin producing fungi was examined in various agricultural products and indoor air in Central European countries including Hungary, Serbia and Croatia. For species identification, both morphological and sequence based methods were applied. Aspergillus flavus was detected in several samples including maize, cheese, nuts, spices and indoor air, and several isolates were able to produce aflatoxins. Besides, three other species of Aspergillus section Flavi, A. nomius, A. pseudonomius and A. parasiticus were also isolated from cheese, maize and indoor air, respectively. This is the first report on the occurrence of A. nomius and A. pseudonomius in Central Europe. All A. nomius, A. pseudonomius and A. parasiticus isolates were able to produce aflatoxins B1, B2, G1 and G2. The A. nomius isolate came from cheese produced very high amounts of aflatoxins (above 1 mg ml⁻¹). All A. nomius, A. pseudonomius and A. parasiticus isolates produced much higher amounts of aflatoxin G1 then aflatoxin B1. Further studies are in progress to examine the occurrence of producers of these highly carcinogenic mycotoxins in agricultural products and indoor air in Central Europe. PMID:26344029

  10. Glyphosate Inhibits PPAR Gamma Induction and Differentiation of Preadipocytes and is able to Induce Oxidative Stress.

    PubMed

    Martini, Claudia N; Gabrielli, Matías; Brandani, Javier N; Vila, María Del C

    2016-08-01

    Glyphosate-based herbicides (GF) are extensively used for weed control. Thus, it is important to investigate their putative toxic effects. We have reported that GF at subagriculture concentrations inhibits proliferation and differentiation to adipocytes of 3T3-L1 fibroblasts. In this investigation, we evaluated the effect of GF on genes upregulated during adipogenesis. GF was able to inhibit the induction of PPAR gamma, the master gene in adipogenesis but not C/EBP beta, which precedes PPAR gamma activation. GF also inhibited differentiation and proliferation of another model of preadipocyte: mouse embryonic fibroblasts. In exponentially growing 3T3-L1 cells, GF increased lipid peroxidation and the activity of the antioxidant enzyme, superoxide dismutase. We also found that proliferation was inhibited with lower concentrations of GF when time of exposure was extended. Thus, GF was able to inhibit proliferation and differentiation of preadipocytes and to induce oxidative stress, which is indicative of its ability to alter cellular physiology. PMID:27044015

  11. A Cell-Based Assay for Measuring Endogenous BcrAbl Kinase Activity and Inhibitor Resistance.

    PubMed

    Ouellette, Steven B; Noel, Brett M; Parker, Laurie L

    2016-01-01

    Kinase enzymes are an important class of drug targets, particularly in cancer. Cell-based kinase assays are needed to understand how potential kinase inhibitors act on their targets in a physiologically relevant context. Current cell-based kinase assays rely on antibody-based detection of endogenous substrates, inaccurate disease models, or indirect measurements of drug action. Here we expand on previous work from our lab to introduce a 96-well plate compatible approach for measuring cell-based kinase activity in disease-relevant human chronic myeloid leukemia cell lines using an exogenously added, multi-functional peptide substrate. Our cellular models natively express the BcrAbl oncogene and are either sensitive or have acquired resistance to well-characterized BcrAbl tyrosine kinase inhibitors. This approach measures IC50 values comparable to established methods of assessing drug potency, and its robustness indicates that it can be employed in drug discovery applications. This medium-throughput assay could bridge the gap between single target focused, high-throughput in vitro assays and lower-throughput cell-based follow-up experiments. PMID:27598410

  12. Plasmacytomagenesis in Eμ-v-abl transgenic mice is accelerated when apoptosis is restrained

    PubMed Central

    Vandenberg, Cassandra J.; Waring, Paul; Strasser, Andreas

    2014-01-01

    Mice susceptible to plasma cell tumors provide a useful model for human multiple myeloma. We previously showed that mice expressing an Eµ-v-abl oncogene solely develop plasmacytomas. Here we show that loss of the proapoptotic BH3-only protein Bim or, to a lesser extent, overexpression of antiapoptotic Bcl-2 or Mcl-1, significantly accelerated the development of plasmacytomas and increased their incidence. Disease was preceded by an increased abundance of plasma cells, presumably reflecting their enhanced survival capacity in vivo. Plasmacytomas of each genotype expressed high levels of v-abl and frequently harbored a rearranged c-myc gene, probably as a result of chromosome translocation. As in human multiple myelomas, elevated expression of cyclin D genes was common, and p53 deregulation was rare. Our results for plasmacytomas highlight the significance of antiapoptotic changes in multiple myeloma, which include elevated expression of Mcl-1 and, less frequently, Bcl-2, and suggest that closer attention to defects in Bim expression is warranted. PMID:24986687

  13. Inverse regulation of bridging integrator 1 and BCR-ABL1 in chronic myeloid leukemia.

    PubMed

    Trino, Stefania; De Luca, Luciana; Simeon, Vittorio; Laurenzana, Ilaria; Morano, Annalisa; Caivano, Antonella; La Rocca, Francesco; Pietrantuono, Giuseppe; Bianchino, Gabriella; Grieco, Vitina; Signorino, Elisabetta; Fragasso, Alberto; Bochicchio, Maria Teresa; Venturi, Claudia; Rosti, Gianantonio; Martinelli, Giovanni; Del Vecchio, Luigi; Cilloni, Daniela; Musto, Pellegrino

    2016-01-01

    Endocytosis is the major regulator process of tyrosine kinase receptor (RTK) functional activities. Bridging integrator 1 (BIN1) is a key protein involved in RTK intracellular trafficking. Here, we report, by studying 34 patients with chronic myeloid leukemia (CML) at diagnosis, that BIN1 gene is downregulated in CML as compared to healthy controls, suggesting an altered endocytosis of RTKs. Rab interactor 1 (RIN1), an activator of BIN1, displayed a similar behavior. Treatment of 57 patients by tyrosine kinase inhibitors caused, along with BCR-ABL1 inactivation, an increase of BIN1 and RIN1 expression, potentially restoring endocytosis. There was a significant inverse correlation between BIN1-RIN1 and BCR-ABL1 expression. In vitro experiments on both CML and nontumorigenic cell lines treated with Imatinib confirmed these results. In order to provide another proof in favor of BIN1 and RIN1 endocytosis function in CML, we demonstrated that Imatinib induced, in K562 cell line, BIN1-RIN1 upregulation accompanied by a parallel AXL receptor internalization into cytoplasmic compartment. This study shows a novel deregulated mechanism in CML patients, indicating BIN1 and RIN1 as players in the maintenance of the abnormal RTK signaling in this hematological disease. PMID:26194865

  14. Tolerance to silver of an Aspergillus fumigatus strain able to grow on cyanide containing wastes.

    PubMed

    Sabatini, L; Battistelli, M; Giorgi, L; Iacobucci, M; Gobbi, L; Andreozzi, E; Pianetti, A; Franchi, R; Bruscolini, F

    2016-04-01

    We studied the strategy of an Aspergillus fumigatus strain able to grow on metal cyanide wastes to cope with silver. The tolerance test revealed that the Minimum Inhibitory Concentration of Ag(I) was 6mM. In 1mM AgNO3 aqueous solution the fungus was able to reduce and sequestrate silver into the cell in the form of nanoparticles as evidenced by the change in color of the biomass and Electron Microscopy observations. Extracellular silver nanoparticle production also occurred in the filtrate solution after previous incubation of the fungus in sterile, double-distilled water for 72h, therefore evidencing that culture conditions may influence nanoparticle formation. The nanoparticles were characterized by UV-vis spectrometry, X-ray diffraction and Energy Dispersion X-ray analysis. Atomic absorption spectrometry revealed that the optimum culture conditions for silver absorption were at pH 8.5.The research is part of a polyphasic study concerning the behavior of the fungal strain in presence of metal cyanides; the results provide better understanding for further research targeted at a rationale use of the microorganism in bioremediation plans, also in view of possible metal recovery. Studies will be performed to verify if the fungus maintains its ability to produce nanoparticles using KAg(CN)2. PMID:26705888

  15. Towards identification of finger flexions using single channel surface electromyography – able bodied and amputee subjects

    PubMed Central

    2013-01-01

    Background This research has established a method for using single channel surface electromyogram (sEMG) recorded from the forearm to identify individual finger flexion. The technique uses the volume conduction properties of the tissues and uses the magnitude and density of the singularities in the signal as a measure of strength of the muscle activity. Methods SEMG was recorded from the flexor digitorum superficialis muscle during four different finger flexions. Based on the volume conduction properties of the tissues, sEMG was decomposed into wavelet maxima and grouped into four groups based on their magnitude. The mean magnitude and the density of each group were the inputs to the twin support vector machines (TSVM). The algorithm was tested on 11 able-bodied and one trans-radial amputated volunteer to determine the accuracy, sensitivity and specificity. The system was also tested to determine inter-experimental variations and variations due to difference in the electrode location. Results Accuracy and sensitivity of identification of finger actions from single channel sEMG signal was 93% and 94% for able-bodied and 81% and 84% for trans-radial amputated respectively, and there was only a small inter-experimental variation. Conclusions Volume conduction properties based sEMG analysis provides a suitable basis for identifying finger flexions from single channel sEMG. The reported system requires supervised training and automatic classification. PMID:23758881

  16. Immunomodulatory properties of carbon nanotubes are able to compensate immune function dysregulation caused by microgravity conditions

    NASA Astrophysics Data System (ADS)

    Crescio, Claudia; Orecchioni, Marco; Ménard-Moyon, Cécilia; Sgarrella, Francesco; Pippia, Proto; Manetti, Roberto; Bianco, Alberto; Delogu, Lucia Gemma

    2014-07-01

    Spaceflights lead to dysregulation of the immune cell functionality affecting the expression of activation markers and cytokine production. Short oxidized multi-walled carbon nanotubes functionalized by 1,3-dipolar cycloaddition have been reported to activate immune cells. In this Communication we have performed surface marker assays and multiplex ELISA on primary monocytes and T cells under microgravity. We have discovered that carbon nanotubes, through their immunostimulatory properties, are able to fight spaceflight immune system dysregulations.Spaceflights lead to dysregulation of the immune cell functionality affecting the expression of activation markers and cytokine production. Short oxidized multi-walled carbon nanotubes functionalized by 1,3-dipolar cycloaddition have been reported to activate immune cells. In this Communication we have performed surface marker assays and multiplex ELISA on primary monocytes and T cells under microgravity. We have discovered that carbon nanotubes, through their immunostimulatory properties, are able to fight spaceflight immune system dysregulations. Electronic supplementary information (ESI) available: Experimental section, structures of f-MWCNTs and uptake by human primary immune cells. See DOI: 10.1039/c4nr02711f

  17. The chimeric ubiquitin ligase SH2-U-box inhibits the growth of imatinib-sensitive and resistant CML by targeting the native and T315I-mutant BCR-ABL

    PubMed Central

    Ru, Yi; Wang, Qinhao; Liu, Xiping; Zhang, Mei; Zhong, Daixing; Ye, Mingxiang; Li, Yuanchun; Han, Hua; Yao, Libo; Li, Xia

    2016-01-01

    Chronic myeloid leukemia (CML) is characterized by constitutively active fusion protein tyrosine kinase BCR-ABL. Although the tyrosine kinase inhibitor (TKI) against BCR-ABL, imatinib, is the first-line therapy for CML, acquired resistance almost inevitably emerges. The underlying mechanism are point mutations within the BCR-ABL gene, among which T315I is notorious because it resists to almost all currently available inhibitors. Here we took use of a previously generated chimeric ubiquitin ligase, SH2-U-box, in which SH2 from the adaptor protein Grb2 acts as a binding domain for activated BCR-ABL, while U-box from CHIP functions as an E3 ubiquitin ligase domain, so as to target the ubiquitination and degradation of both native and T315I-mutant BCR-ABL. As such, SH2-U-box significantly inhibited proliferation and induced apoptosis in CML cells harboring either the wild-type or T315I-mutant BCR-ABL (K562 or K562R), with BCR-ABL-dependent signaling pathways being repressed. Moreover, SH2-U-box worked in concert with imatinib in K562 cells. Importantly, SH2-U-box-carrying lentivirus could markedly suppress the growth of K562-xenografts in nude mice or K562R-xenografts in SCID mice, as well as that of primary CML cells. Collectively, by degrading the native and T315I-mutant BCR-ABL, the chimeric ubiquitin ligase SH2-U-box may serve as a potential therapy for both imatinib-sensitive and resistant CML. PMID:27329306

  18. The chimeric ubiquitin ligase SH2-U-box inhibits the growth of imatinib-sensitive and resistant CML by targeting the native and T315I-mutant BCR-ABL.

    PubMed

    Ru, Yi; Wang, Qinhao; Liu, Xiping; Zhang, Mei; Zhong, Daixing; Ye, Mingxiang; Li, Yuanchun; Han, Hua; Yao, Libo; Li, Xia

    2016-01-01

    Chronic myeloid leukemia (CML) is characterized by constitutively active fusion protein tyrosine kinase BCR-ABL. Although the tyrosine kinase inhibitor (TKI) against BCR-ABL, imatinib, is the first-line therapy for CML, acquired resistance almost inevitably emerges. The underlying mechanism are point mutations within the BCR-ABL gene, among which T315I is notorious because it resists to almost all currently available inhibitors. Here we took use of a previously generated chimeric ubiquitin ligase, SH2-U-box, in which SH2 from the adaptor protein Grb2 acts as a binding domain for activated BCR-ABL, while U-box from CHIP functions as an E3 ubiquitin ligase domain, so as to target the ubiquitination and degradation of both native and T315I-mutant BCR-ABL. As such, SH2-U-box significantly inhibited proliferation and induced apoptosis in CML cells harboring either the wild-type or T315I-mutant BCR-ABL (K562 or K562R), with BCR-ABL-dependent signaling pathways being repressed. Moreover, SH2-U-box worked in concert with imatinib in K562 cells. Importantly, SH2-U-box-carrying lentivirus could markedly suppress the growth of K562-xenografts in nude mice or K562R-xenografts in SCID mice, as well as that of primary CML cells. Collectively, by degrading the native and T315I-mutant BCR-ABL, the chimeric ubiquitin ligase SH2-U-box may serve as a potential therapy for both imatinib-sensitive and resistant CML. PMID:27329306

  19. Kernel Manifold Alignment for Domain Adaptation.

    PubMed

    Tuia, Devis; Camps-Valls, Gustau

    2016-01-01

    The wealth of sensory data coming from different modalities has opened numerous opportunities for data analysis. The data are of increasing volume, complexity and dimensionality, thus calling for new methodological innovations towards multimodal data processing. However, multimodal architectures must rely on models able to adapt to changes in the data distribution. Differences in the density functions can be due to changes in acquisition conditions (pose, illumination), sensors characteristics (number of channels, resolution) or different views (e.g. street level vs. aerial views of a same building). We call these different acquisition modes domains, and refer to the adaptation problem as domain adaptation. In this paper, instead of adapting the trained models themselves, we alternatively focus on finding mappings of the data sources into a common, semantically meaningful, representation domain. This field of manifold alignment extends traditional techniques in statistics such as canonical correlation analysis (CCA) to deal with nonlinear adaptation and possibly non-corresponding data pairs between the domains. We introduce a kernel method for manifold alignment (KEMA) that can match an arbitrary number of data sources without needing corresponding pairs, just few labeled examples in all domains. KEMA has interesting properties: 1) it generalizes other manifold alignment methods, 2) it can align manifolds of very different complexities, performing a discriminative alignment preserving each manifold inner structure, 3) it can define a domain-specific metric to cope with multimodal specificities, 4) it can align data spaces of different dimensionality, 5) it is robust to strong nonlinear feature deformations, and 6) it is closed-form invertible, which allows transfer across-domains and data synthesis. To authors' knowledge this is the first method addressing all these important issues at once. We also present a reduced-rank version of KEMA for computational

  20. Kernel Manifold Alignment for Domain Adaptation

    PubMed Central

    Tuia, Devis; Camps-Valls, Gustau

    2016-01-01

    The wealth of sensory data coming from different modalities has opened numerous opportunities for data analysis. The data are of increasing volume, complexity and dimensionality, thus calling for new methodological innovations towards multimodal data processing. However, multimodal architectures must rely on models able to adapt to changes in the data distribution. Differences in the density functions can be due to changes in acquisition conditions (pose, illumination), sensors characteristics (number of channels, resolution) or different views (e.g. street level vs. aerial views of a same building). We call these different acquisition modes domains, and refer to the adaptation problem as domain adaptation. In this paper, instead of adapting the trained models themselves, we alternatively focus on finding mappings of the data sources into a common, semantically meaningful, representation domain. This field of manifold alignment extends traditional techniques in statistics such as canonical correlation analysis (CCA) to deal with nonlinear adaptation and possibly non-corresponding data pairs between the domains. We introduce a kernel method for manifold alignment (KEMA) that can match an arbitrary number of data sources without needing corresponding pairs, just few labeled examples in all domains. KEMA has interesting properties: 1) it generalizes other manifold alignment methods, 2) it can align manifolds of very different complexities, performing a discriminative alignment preserving each manifold inner structure, 3) it can define a domain-specific metric to cope with multimodal specificities, 4) it can align data spaces of different dimensionality, 5) it is robust to strong nonlinear feature deformations, and 6) it is closed-form invertible, which allows transfer across-domains and data synthesis. To authors’ knowledge this is the first method addressing all these important issues at once. We also present a reduced-rank version of KEMA for computational

  1. Frequency domain nonlinear optics

    NASA Astrophysics Data System (ADS)

    Legare, Francois

    2016-05-01

    The universal dilemma of gain narrowing occurring in fs amplifiers prevents ultra-high power lasers from delivering few-cycle pulses. This problem is overcome by a new amplification concept: Frequency domain Optical Parametric Amplification - FOPA. It enables simultaneous up-scaling of peak power and amplified spectral bandwidth and can be performed at any wavelength range of conventional amplification schemes, however, with the capability to amplify single cycles of light. The key idea for amplification of octave-spanning spectra without loss of spectral bandwidth is to amplify the broad spectrum ``slice by slice'' in the frequency domain, i.e. in the Fourier plane of a 4f-setup. The striking advantages of this scheme, are its capability to amplify (more than) one octave of bandwidth without shorting the corresponding pulse duration. This is because ultrabroadband phase matching is not defined by the properties of the nonlinear crystal employed but the number of crystals employed. In the same manner, to increase the output energy one simply has to increase the spectral extension in the Fourier plane and to add one more crystal. Thus, increasing pulse energy and shortening its duration accompany each other. A proof of principle experiment was carried out at ALLS on the sub-two cycle IR beam line and yielded record breaking performance in the field of few-cycle IR lasers. 100 μJ two-cycle pulses from a hollow core fibre compression setup were amplified to 1.43mJ without distorting spatial or temporal properties. Pulse duration at the input of FOPA and after FOPA remains the same. Recently, we have started upgrading this system to be pumped by 250 mJ to reach 40 mJ two-cycle IR few-cycle pulses and latest results will be presented at the conference. Furthermore, the extension of the concept of FOPA to other nonlinear optical processes will be discussed. Frequency domain nonlinear optics.

  2. On Probability Domains III

    NASA Astrophysics Data System (ADS)

    Frič, Roman; Papčo, Martin

    2015-12-01

    Domains of generalized probability have been introduced in order to provide a general construction of random events, observables and states. It is based on the notion of a cogenerator and the properties of product. We continue our previous study and show how some other quantum structures fit our categorical approach. We discuss how various epireflections implicitly used in the classical probability theory are related to the transition to fuzzy probability theory and describe the latter probability theory as a genuine categorical extension of the former. We show that the IF-probability can be studied via the fuzzy probability theory. We outline a "tensor modification" of the fuzzy probability theory.

  3. Transfer of high domain knowledge to a similar domain.

    PubMed

    Jessup, Ryan K

    2009-01-01

    Researchers have widely examined domain knowledge yet rarely investigate the transfer of knowledge from one domain to another. This study sought to fill in the literature gap concerning the impact of domain knowledge on memory in a similar situation. Specifically, this study examined whether high knowledge of baseball could enhance memory for the similar yet unknown domain of cricket, using a 2 (knowledge) x 2 (prime) design. An interaction occurred, indicating that when primed, baseball knowledge improves memory for cricket events in participants with high baseball knowledge but reduces memory in their low-knowledge counterparts. These results suggest that extensive knowledge in one domain allows it to serve as an organizational framework for incoming information in a similar domain; conversely, priming poorly understood domain knowledge results in negative transfer. PMID:19353932

  4. BCL6 enables Ph+ acute lymphoblastic leukemia cells to survive BCR-ABL1 kinase inhibition

    PubMed Central

    Duy, Cihangir; Hurtz, Christian; Shojaee, Seyedmehdi; Cerchietti, Leandro; Geng, Huimin; Swaminathan, Srividya; Klemm, Lars; Kweon, Soo-mi; Nahar, Rahul; Braig, Melanie; Park, Eugene; Kim, Yong-mi; Hofmann, Wolf-Karsten; Herzog, Sebastian; Jumaa, Hassan; Koeffler, H Phillip; Yu, J. Jessica; Heisterkamp, Nora; Graeber, Thomas G.; Wu, Hong; Ye, B. Hilda; Melnick, Ari; Müschen, Markus

    2011-01-01

    Tyrosine kinase inhibitors (TKI) are widely used to treat patients with leukemia driven by BCR-ABL11 and other oncogenic tyrosine kinases2,3. Recent efforts focused on the development of more potent TKI that also inhibit mutant tyrosine kinases4,5. However, even effective TKI typically fail to eradicate leukemia-initiating cells6–8, which often cause recurrence of leukemia after initially successful treatment. Here we report on the discovery of a novel mechanism of drug-resistance, which is based on protective feedback signaling of leukemia cells in response to TKI-treatment. We identified BCL6 as a central component of this drug-resistance pathway and demonstrate that targeted inhibition of BCL6 leads to eradication of drug-resistant and leukemia-initiating subclones. BCL6 is a known proto-oncogene that is often translocated in diffuse large B cell lymphoma (DLBCL)9. In response to TKI-treatment, BCR-ABL1 acute lymphoblastic leukemia (ALL) cells upregulate BCL6 protein levels by ~90-fold, i.e. to similar levels as in DLBCL (Fig. 1a). Upregulation of BCL6 in response to TKI-treatment represents a novel defense mechanism, which enables leukemia cells to survive TKI-treatment: Previous work suggested that TKI-mediated cell death is largely p53-independent. Here we demonstrate that BCL6 upregulation upon TKI-treatment leads to transcriptional inactivation of the p53 pathway. BCL6-deficient leukemia cells fail to inactivate p53 and are particularly sensitive to TKI-treatment. BCL6−/− leukemia cells are poised to undergo cellular senescence and fail to initiate leukemia in serial transplant recipients. A combination of TKI-treatment and a novel BCL6 peptide inhibitor markedly increased survival of NOD/SCID mice xenografted with patient-derived BCR-ABL1 ALL cells. We propose that dual targeting of oncogenic tyrosine kinases and BCL6-dependent feedback (Supplementary Fig. 1) represents a novel strategy to eradicate drug-resistant and leukemia-initiating subclones in

  5. Gads (Grb2-related adaptor downstream of Shc) is required for BCR-ABL-mediated lymphoid leukemia

    PubMed Central

    Gillis, LC; Berry, DM; Minden, MD; McGlade, CJ; Barber, DL

    2016-01-01

    Philadelphia chromosome-positive leukemias, including chronic myeloid leukemia and B-cell acute lymphoblastic leukemia (B-ALL), are driven by the oncogenic BCR-ABL fusion protein. Animal modeling experiments utilizing retroviral transduction and subsequent bone marrow transplantation have demonstrated that BCR-ABL generates both myeloid and lymphoid disease in mice receiving whole bone marrow transduced with BCR-ABL. Y177 of BCR-ABL is critical to the development of myeloid disease, and phosphorylation of Y177 has been shown to induce GRB2 binding to BCR-ABL, followed by activation of the Ras and phosphoinositide 3 kinase signaling pathways. We show that the GRB2-related adapter protein, GADS, also associates with BCR-ABL, specifically through Y177 and demonstrate that BCR-ABL-driven lymphoid disease requires Gads. BCR-ABL transduction of Gads(−/−) bone marrow results in short latency myeloid disease within 3–4 weeks of transplant, while wild-type mice succumb to both a longer latency lymphoid and myeloid diseases. We report that GADS mediates a unique BCR-ABL complex with SLP-76 in BCR-ABL-positive cell lines and B-ALL patient samples. These data suggest that GADS mediates lymphoid disease downstream of BCR-ABL through the recruitment of specific signaling intermediates. PMID:23399893

  6. Inhibition of STAT5: A therapeutic option in BCR-ABL1-driven leukemia

    PubMed Central

    Berger, Angelika; Sexl, Veronika; Valent, Peter; Moriggl, Richard

    2014-01-01

    The two transcription factors STAT5A and STAT5B are central signaling molecules in leukemias driven by Abelson fusion tyrosine kinases and they fulfill all criteria of drug targets. STAT5A and STAT5B display unique nuclear shuttling mechanisms and they have a key role in resistance of leukemic cells against treatment with tyrosine kinase inhibitors (TKI). Moreover, STAT5A and STAT5B promote survival of leukemic stem cells. We here discuss the possibility of targeting up-stream kinases with TKI, direct STAT5 inhibition via SH2 domain obstruction and blocking nuclear translocation of STAT5. All discussed options will result in a stop of STAT5 transport to the nucleus to block STAT5-mediated transcriptional activity. In summary, recently described shuttling functions of STAT5 are discussed as potentially druggable pathways in leukemias. PMID:25333255

  7. Inhibition of Aurora Kinase B Is Important for Biologic Activity of the Dual Inhibitors of BCR-ABL and Aurora Kinases R763/AS703569 and PHA-739358 in BCR-ABL Transformed Cells

    PubMed Central

    Illert, Anna L.; Seitz, Anna K.; Rummelt, Christoph; Kreutmair, Stefanie; Engh, Richard A.; Goodstal, Samantha; Peschel, Christian; Duyster, Justus; von Bubnoff, Nikolas

    2014-01-01

    ABL tyrosine kinase inhibitors (TKI) like Imatinib, Dasatinib and Nilotinib are the gold standard in conventional treatment of CML. However, the emergence of resistance remains a major problem. Alternative therapeutic strategies of ABL TKI-resistant CML are urgently needed. We asked whether dual inhibition of BCR-ABL and Aurora kinases A-C could overcome resistance mediated by ABL kinase mutations. We therefore tested the dual ABL and Aurora kinase inhibitors PHA-739358 and R763/AS703569 in Ba/F3- cells ectopically expressing wild type (wt) or TKI-resistant BCR-ABL mutants. We show that both compounds exhibited strong anti-proliferative and pro-apoptotic activity in ABL TKI resistant cell lines including cells expressing the strongly resistant T315I mutation. Cell cycle analysis indicated polyploidisation, a consequence of continued cell cycle progression in the absence of cell division by Aurora kinase inhibition. Experiments using drug resistant variants of Aurora B indicated that PHA-739358 acts on both, BCR-ABL and Aurora Kinase B, whereas Aurora kinase B inhibition might be sufficient for the anti-proliferative activity observed with R763/AS703569. Taken together, our data demonstrate that dual ABL and Aurora kinase inhibition might be used to overcome ABL TKI resistant CML. PMID:25426931

  8. Rainfall and surface kinematic conditions over central amazonia during ABLE 2B

    NASA Technical Reports Server (NTRS)

    Greco, Steven; Swap, Robert; Garstang, Michael; Ulanski, Stanley; Shipham, Mark

    1990-01-01

    Rainfall, rainfall systems, and surface kinematics of the central Amazon basin wet season are investigated using meteorological and chemical data collected during the wet season Amazon Boundary Layer Experiment (ABLE) near Manaus, Brazil. Through analysis of (GOES-West) imagery, it is determined that, based on location of the initial development, there are three main types of convective systems which influence a mesoscale network near Manaus, namely the Coastal Occurring Systems (COS), the Basin Occurring Systems (BOS), and the Locally Occurring Systems (LOS). Chemical analysis of rainwater delivered by these systems shows significant differences in concentrations of formate, acetate, pyruvate, sulfate, and hydrogen ion, and measurements of aerosol concentrations near Manaus show large influxes of aerosols into central Amazonia after passage of BOS and COS. Results of satellite based classification of the rain-producing systems are discussed.

  9. Links between social understanding and social behavior in verbally able children with autism.

    PubMed

    Travis, L; Sigman, M; Ruskin, E

    2001-04-01

    This study investigated the relations between various measures of social understanding and social interaction competence in verbally able children with autism. Measures of social understanding included measures of verbalizable knowledge (false belief understanding, affective perspective taking), as well as measures of more intuitive forms of social responsiveness (empathy, concern to distress, and initiating joint attention). Two measures of social interaction competence were employed: level of engagement with peers on the playground, and prosocial behavior in a structured laboratory task. For children with autism, initiating joint attention and empathy were strongly related to both measures of social interaction competence. No understanding-behavior links were identified for a language-age matched comparison sample of developmentally delayed children. Several accounts of these understanding-behavior links are considered, including the possibility that for children with autism, more impaired forms of understanding are more closely linked to behavior because they serve as limits on competence. PMID:11450811

  10. Selection of filamentous fungi of the Beauveria genus able to metabolize quercetin like mammalian cells

    PubMed Central

    de M. B. Costa, Eula Maria; Pimenta, Fabiana Cristina; Luz, Wolf Christian; de Oliveira, Valéria

    2008-01-01

    Microbial biotransformations constitute an important alternative as models for drug metabolism study in mammalians and have been used for the industrial synthesis of chemicals with pharmaceutical purposes. Several microorganisms with unique biotransformation ability have been found by intensive screening and put in commercial applications. Ten isolates of Beauveria sp genus filamentous fungi, isolated from soil in the central Brazil, and Beauveria bassiana ATCC 7159 were evaluated for their capability of quercetin biotransformation. Biotransformation processes were carried out for 24 up to 96 hours and monitored by mass spectrometry analyses of the culture broth. All strains were able to metabolize quercetin, forming mammalian metabolites. The results were different from those presented by other microorganisms previously utilized, attrackting attention because of the great diversity of reactions. Methylated, sulphated, monoglucuronidated, and glucuronidated conjugated metabolites were simultaneously detected. PMID:24031237

  11. JNK pathway activation is able to synchronize neuronal death and glial phagocytosis in Drosophila

    PubMed Central

    Shklover, J; Mishnaevski, K; Levy-Adam, F; Kurant, E

    2015-01-01

    Glial phagocytosis of superfluous neurons and damaged or aberrant neuronal material is crucial for normal development and maintenance of the CNS. However, the molecular mechanisms underlying the relationship between neuronal death and glial phagocytosis are poorly understood. We describe a novel mechanism that is able to synchronize neuronal cell death and glial phagocytosis of dying neurons in the Drosophila embryonic CNS. This mechanism involves c-Jun N-terminal kinase (JNK) signaling, which is required for developmental apoptosis of specific neurons during embryogenesis. We demonstrate that the dJNK pathway gain-of-function in neurons leads to dJNK signaling in glia, which results in upregulation of glial phagocytosis. Importantly, this promotion of phagocytosis is not mediated by upregulation of the glial phagocytic receptors SIMU and DRPR, but by increasing glial capacity to degrade apoptotic particles inside phagosomes. The proposed mechanism may be important for removal of damaged neurons in the developing and mature CNS. PMID:25695602

  12. The Learning Benefits of Being Willing and Able to Engage in Scientific Argumentation

    NASA Astrophysics Data System (ADS)

    Bathgate, Meghan; Crowell, Amanda; Schunn, Christian; Cannady, Mac; Dorph, Rena

    2015-07-01

    Engaging in science as an argumentative practice can promote students' critical thinking, reflection, and evaluation of evidence. However, many do not approach science in this way. Furthermore, the presumed confrontational nature of argumentation may run against cultural norms particularly during the sensitive time of early adolescence. This paper explores whether middle-school students' ability to engage in critical components of argumentation in science impacts science classroom learning. It also examines whether students' willingness to do so attenuates or moderates that benefit. In other words, does one need to be both willing and able to engage critically with the discursive nature of science to receive benefits to learning? This study of middle-school students participating in four months of inquiry science shows a positive impact of argumentative sensemaking ability on learning, as well as instances of a moderating effect of one's willingness to engage in argumentative discourse. Possible mechanisms and the potential impacts to educational practices are discussed.

  13. Cationic Two-Photon Lanthanide Bioprobes Able to Accumulate in Live Cells.

    PubMed

    Bui, Anh Thy; Beyler, Maryline; Liao, Yuan-Yuan; Grichine, Alexei; Duperray, Alain; Mulatier, Jean-Christophe; Guennic, Boris Le; Andraud, Chantal; Maury, Olivier; Tripier, Raphaël

    2016-07-18

    An original cationic water-soluble cyclen-based Eu(III) complex [EuL(1)](+) featuring a chromophore-functionalized antenna to increase the two-photon (2P) absorption properties was synthesized. The photophysical properties were thoroughly studied in various solvents and rationalized with the help of theoretical calculations. The complex exhibits an optimized 2P absorption cross section. Finally, 2P microscopy imaging experiments on living T24 human cancer cells highlighted the spontaneous internalization and the biological stability of this 2P bioprobe in vitro. Macrocyclic-based antennas open new perspectives for future optimization of the photophysical properties and allows envisaging the design of Eu, Tb, Yb, and Sm bioprobes. This result also opens the way for the design of functional two-photon Ln complexes able to monitor intracellular physicochemical parameters. PMID:27367598

  14. Trypanosoma cruzi Epimastigotes Are Able to Manage Internal Cholesterol Levels under Nutritional Lipid Stress Conditions

    PubMed Central

    Pereira, Miria Gomes; Visbal, Gonzalo; Salgado, Leonardo T.; Vidal, Juliana Cunha; Godinho, Joseane L. P.; De Cicco, Nuccia N. T.; Atella, Geórgia C.; de Souza, Wanderley; Cunha-e-Silva, Narcisa

    2015-01-01

    Trypanosoma cruzi epimastigotes store high amounts of cholesterol and cholesteryl esters in reservosomes. These unique organelles are responsible for cellular digestion by providing substrates for homeostasis and parasite differentiation. Here we demonstrate that under nutritional lipid stress, epimastigotes preferentially mobilized reservosome lipid stocks, instead of lipid bodies, leading to the consumption of parasite cholesterol reservoirs and production of ergosterol. Starved epimastigotes acquired more LDL-NBD-cholesterol by endocytosis and distributed the exogenous cholesterol to their membranes faster than control parasites. Moreover, the parasites were able to manage internal cholesterol levels, alternating between consumption and accumulation. With normal lipid availability, parasites esterified cholesterol exhibiting an ACAT-like activity that was sensitive to Avasimibe in a dose-dependent manner. This result also implies that exogenous cholesterol has a role in lipid reservoirs in epimastigotes. PMID:26068009

  15. Trypanosoma cruzi Epimastigotes Are Able to Manage Internal Cholesterol Levels under Nutritional Lipid Stress Conditions.

    PubMed

    Pereira, Miria Gomes; Visbal, Gonzalo; Salgado, Leonardo T; Vidal, Juliana Cunha; Godinho, Joseane L P; De Cicco, Nuccia N T; Atella, Geórgia C; de Souza, Wanderley; Cunha-e-Silva, Narcisa

    2015-01-01

    Trypanosoma cruzi epimastigotes store high amounts of cholesterol and cholesteryl esters in reservosomes. These unique organelles are responsible for cellular digestion by providing substrates for homeostasis and parasite differentiation. Here we demonstrate that under nutritional lipid stress, epimastigotes preferentially mobilized reservosome lipid stocks, instead of lipid bodies, leading to the consumption of parasite cholesterol reservoirs and production of ergosterol. Starved epimastigotes acquired more LDL-NBD-cholesterol by endocytosis and distributed the exogenous cholesterol to their membranes faster than control parasites. Moreover, the parasites were able to manage internal cholesterol levels, alternating between consumption and accumulation. With normal lipid availability, parasites esterified cholesterol exhibiting an ACAT-like activity that was sensitive to Avasimibe in a dose-dependent manner. This result also implies that exogenous cholesterol has a role in lipid reservoirs in epimastigotes. PMID:26068009

  16. Towards a Density Functional Theory Exchange-Correlation Functional able to describe localization/delocalization

    NASA Astrophysics Data System (ADS)

    Mattsson, Ann E.; Wills, John M.

    2013-03-01

    The inability to computationally describe the physics governing the properties of actinides and their alloys is the poster child of failure of existing Density Functional Theory exchange-correlation functionals. The intricate competition between localization and delocalization of the electrons, present in these materials, exposes the limitations of functionals only designed to properly describe one or the other situation. We will discuss the manifestation of this competition in real materials and propositions on how to construct a functional able to accurately describe properties of these materials. I addition we will discuss both the importance of using the Dirac equation to describe the relativistic effects in these materials, and the connection to the physics of transition metal oxides. Sandia National Laboratories is a multi-program laboratory managed and operated by Sandia Corporation, a wholly owned subsidiary of Lockheed Martin Corporation, for the U.S. Department of Energy's National Nuclear Security Administration under contract DE-AC04-94AL85000.

  17. Machine-able Yttria Stabilized Zirconia Composites for Thermal Insulation in Nuclear Reactors

    NASA Astrophysics Data System (ADS)

    Lo, J.; Zhang, R.; Santos, R.

    2016-02-01

    Ceramics are a promising insulating material for high temperature environment. To qualify for in-core use in nuclear reactors, there are many other materials requirements to be met, such as neutron irradiation resistance, corrosion resistance, low thermal conductivity, high coefficient of thermal expansion, high strength, high fracture toughness, ease of fabricability, etc. And among the promising ceramics meeting most of the requirements, with the exception of fabricability, is yttria-stabilized zirconia (YSZ). Like all ceramics, YSZ is hard, brittle and difficult to machine. At CanmetMATERIALS, YSZ-based composites for in-core insulation that are machine-able and capable of being formed into complex shapes have been developed. In this paper, the focus is geared towards the fabrication and property evaluation of such composites. In addition, the machinability aspect of the YSZ composites was addressed with a demonstration of a machined component.

  18. A mathematical model for a didactic device able to simulate a 2D Newtonian gravitational field

    NASA Astrophysics Data System (ADS)

    De Marchi, Fabrizio

    2015-01-01

    In this paper we propose a mathematical model to describe a theoretical device able to simulate an inverse-square force on a test mass moving on a horizontal plane. We use two pulleys, a counterweight, a wire and a smooth rail, in addition to the test mass. The tension of the wire (i.e. the attractive force on the test mass) is determined by the position of a counterweight free to move on a rail placed under the plane. The profile of the rail is calculated in order to obtain the required Newtonian force. Details of this calculation are reported in the paper, and numerical simulations are provided in order to investigate the stability of the orbits under the effect of the main friction forces and other perturbative effects. This work points out that there are some criticalities intrinsic to the apparatus and gives some suggestions about how to minimize their impact.

  19. A derivative of epigallocatechin-3-gallate induces apoptosis via SHP-1-mediated suppression of BCR-ABL and STAT3 signalling in chronic myelogenous leukaemia

    PubMed Central

    Jung, Ji Hoon; Yun, Miyong; Choo, Eun-Jeong; Kim, Sun-Hee; Jeong, Myoung-Seok; Jung, Deok-Beom; Lee, Hyemin; Kim, Eun-Ok; Kato, Nobuo; Kim, Bonglee; Srivastava, Sanjay K; Kaihatsu, Kunihiro; Kim, Sung-Hoon

    2015-01-01

    Background and Purpose Epigallocatechin-3-gallate (EGCG) is a component of green tea known to have chemo-preventative effects on several cancers. However, EGCG has limited clinical application, which necessitates the development of a more effective EGCG prodrug as an anticancer agent. Experimental Approach Derivatives of EGCG were evaluated for their stability and anti-tumour activity in human chronic myeloid leukaemia (CML) K562 and KBM5 cells. Key Results EGCG-mono-palmitate (EGCG-MP) showed most prolonged stability compared with other EGCG derivatives. EGCG-MP exerted greater cytotoxicity and apoptosis in K562 and KBM5 cells than the other EGCG derivatives. EGCG-MP induced Src-homology 2 domain-containing tyrosine phosphatase 1 (SHP-1) leading decreased oncogenic protein BCR-ABL and STAT3 phosphorylation in CML cells, compared with treatment with EGCG. Furthermore, EGCG-MP reduced phosphorylation of STAT3 and survival genes in K562 cells, compared with EGCG. Conversely, depletion of SHP-1 or application of the tyrosine phosphatase inhibitor pervanadate blocked the ability of EGCG-MP to suppress phosphorylation of BCR-ABL and STAT3, and the expression of survival genes downstream of STAT3. In addition, EGCG-MP treatment more effectively suppressed tumour growth in BALB/c athymic nude mice compared with untreated controls or EGCG treatment. Immunohistochemistry revealed increased caspase 3 and SHP-1 activity and decreased phosphorylation of BCR-ABL in the EGCG-MP-treated group relative to that in the EGCG-treated group. Conclusions and Implications EGCG-MP induced SHP-1-mediated inhibition of BCR-ABL and STAT3 signalling in vitro and in vivo more effectively than EGCG. This derivative may be a potent chemotherapeutic agent for CML treatment. PMID:25825203

  20. The Arctic Boundary Layer Expedition (ABLE-3B): July - August 1990

    NASA Technical Reports Server (NTRS)

    Harriss, R. C.; Wofsy, S. C.; Hoell, J. M., Jr.; Bendura, R. J.; Drewry, J. W.; Mcneal, R. J.; Pierce, D.; Rabine, V.; Snell, R. L.

    1994-01-01

    The Arctic Boundary Layer Expedition (ABLE) 3B used data from ground-based, aircraft, and satellite platforms to characterize the chemistry and dynamics of the troposphere in subarctic and Arctic regions of midcontinent and eastern Canada during July - August 1990. This paper reports the experimental design for ABLE 3B and a brief overview of results. The detailed results are presented in a series of papers in this issue. The chemical composition of the atmospheric mixed layer over remote tundra, boreal wetland, and forested environments was influenced by emissions of CH4 and nonmethane hydrocarbons from biogenic sources, emissions of gases and aerosols from local biomass burning, and transport of pollutants into the study areas from urban/industrial sources. Minimum concentrations of both trace gas and aerosol species in boundary layer air were associated with Arctic source areas. In the free troposphere the biospheric influence was undetectable, and major sources of chemical variability were related to long-range transport of pollutants into the study areas from biomass burning and industrial sources in Alaska and the Great Lakes regions, respectively. Minimum concentrations of both trace gas and aerosol species in the free troposphere were associated with a persistent, widespread air mass which both chemistry and air mass trajectory analyses suggested had originated in the tropical Pacific. Subsidence of air from the upper troposphere and lower stratosphere frequently enhanced ozone and influenced other trace gas and aerosol species at midtropospheric altitudes. The North American Arctic is a complex dynamical and chemical environment with considerable spatial and temporal variability in aerosol and trace gas concentrations. The use of atmospheric chemical indicators for climate change detection will require a much more comprehensive Arctic monitoring program than currently exists.

  1. Fit women are not able to use the whole aerobic capacity during aerobic dance.

    PubMed

    Edvardsen, Elisabeth; Ingjer, Frank; Bø, Kari

    2011-12-01

    Edvardsen, E, Ingjer, F, and Bø, K. Fit women are not able to use the whole aerobic capacity during aerobic dance. J Strength Cond Res 25(12): 3479-3485, 2011-This study compared the aerobic capacity during maximal aerobic dance and treadmill running in fit women. Thirteen well-trained female aerobic dance instructors aged 30 ± 8.17 years (mean ± SD) exercised to exhaustion by running on a treadmill for measurement of maximal oxygen uptake (VO(2)max) and peak heart rate (HRpeak). Additionally, all subjects performed aerobic dancing until exhaustion after a choreographed videotaped routine trying to reach the same HRpeak as during maximal running. The p value for statistical significance between running and aerobic dance was set to ≤0.05. The results (mean ± SD) showed a lower VO(2)max in aerobic dance (52.2 ± 4.02 ml·kg·min) compared with treadmill running (55.9 ± 5.03 ml·kg·min) (p = 0.0003). Further, the mean ± SD HRpeak was 182 ± 9.15 b·min in aerobic dance and 192 ± 9.62 b·min in treadmill running, giving no difference in oxygen pulse between the 2 exercise forms (p = 0.32). There was no difference in peak ventilation (aerobic dance: 108 ± 10.81 L·min vs. running: 113 ± 11.49 L·min). In conclusion, aerobic dance does not seem to be able to use the whole aerobic capacity as in running. For well endurance-trained women, this may result in a lower total workload at maximal intensities. Aerobic dance may therefore not be as suitable as running during maximal intensities in well-trained females. PMID:22080322

  2. The Intimin periplasmic domain mediates dimerisation and binding to peptidoglycan.

    PubMed

    Leo, Jack C; Oberhettinger, Philipp; Chaubey, Manish; Schütz, Monika; Kühner, Daniel; Bertsche, Ute; Schwarz, Heinz; Götz, Friedrich; Autenrieth, Ingo B; Coles, Murray; Linke, Dirk

    2015-01-01

    Intimin and Invasin are prototypical inverse (Type Ve) autotransporters and important virulence factors of enteropathogenic Escherichia coli and Yersinia spp. respectively. In addition to a C-terminal extracellular domain and a β-barrel transmembrane domain, both proteins also contain a short N-terminal periplasmic domain that, in Intimin, includes a lysin motif (LysM), which is thought to mediate binding to peptidoglycan. We show that the periplasmic domain of Intimin does bind to peptidoglycan both in vitro and in vivo, but only under acidic conditions. We were able to determine a dissociation constant of 0.8 μM for this interaction, whereas the Invasin periplasmic domain, which lacks a LysM, bound only weakly in vitro and failed to bind peptidoglycan in vivo. We present the solution structure of the Intimin LysM, which has an additional α-helix conserved within inverse autotransporter LysMs but lacking in others. In contrast to previous reports, we demonstrate that the periplasmic domain of Intimin mediates dimerisation. We further show that dimerisation and peptidoglycan binding are general features of LysM-containing inverse autotransporters. Peptidoglycan binding by the periplasmic domain in the infection process may aid in resisting mechanical and chemical stress during transit through the gastrointestinal tract. PMID:25353290

  3. Feature-level sentiment analysis by using comparative domain corpora

    NASA Astrophysics Data System (ADS)

    Quan, Changqin; Ren, Fuji

    2016-06-01

    Feature-level sentiment analysis (SA) is able to provide more fine-grained SA on certain opinion targets and has a wider range of applications on E-business. This study proposes an approach based on comparative domain corpora for feature-level SA. The proposed approach makes use of word associations for domain-specific feature extraction. First, we assign a similarity score for each candidate feature to denote its similarity extent to a domain. Then we identify domain features based on their similarity scores on different comparative domain corpora. After that, dependency grammar and a general sentiment lexicon are applied to extract and expand feature-oriented opinion words. Lastly, the semantic orientation of a domain-specific feature is determined based on the feature-oriented opinion lexicons. In evaluation, we compare the proposed method with several state-of-the-art methods (including unsupervised and semi-supervised) using a standard product review test collection. The experimental results demonstrate the effectiveness of using comparative domain corpora.

  4. STAS Domain Structure and Function

    PubMed Central

    Sharma, Alok K.; Rigby, Alan C.; Alper, Seth L.

    2011-01-01

    Pendrin shares with nearly all SLC26/SulP anion transporters a carboxy-terminal cytoplasmic segment organized around a Sulfate Transporter and Anti-Sigma factor antagonist (STAS) domain. STAS domains of divergent amino acid sequence exhibit a conserved fold of 4 β strands interspersed among 5 α helices. The first STAS domain proteins studied were single-domain anti-sigma factor antagonists (anti-anti-σ). These anti-anti-σ indirectly stimulate bacterial RNA polymerase by inactivating inhibitory anti-σ kinases, liberating σ factors to direct specific transcription of target genes or operons. Some STAS domains are nucleotide-binding phosphoproteins or nucleotidases. Others are interaction/transduction modules within multidomain sensors of light, oxygen and other gasotransmitters, cyclic nucleotides, inositol phosphates, and G proteins. Additional multidomain STAS protein sequences suggest functions in sensing, metabolism, or transport of nutrients such as sugars, amino acids, lipids, anions, vitamins, or hydrocarbons. Still other multidomain STAS polypeptides include histidine and serine/threonine kinase domains and ligand-activated transcription factor domains. SulP/SLC26 STAS domains and adjacent sequences interact with other transporters, cytoskeletal scaffolds, and with enzymes metabolizing transported anion substrates, forming putative metabolons. STAS domains are central to membrane targeting of many SulP/SLC26 anion transporters, and STAS domain mutations are associated with at least three human recessive diseases. This review summarizes STAS domain structure and function. PMID:22116355

  5. 1-o-acetylbritannilactone (ABL) inhibits angiogenesis and lung cancer cell growth through regulating VEGF-Src-FAK signaling

    SciTech Connect

    Zhengfu, He; Hu, Zhang; Huiwen, Miao; Zhijun, Li; Jiaojie, Zhou; Xiaoyi, Yan; Xiujun, Cai

    2015-08-21

    The search for safe, effective and affordable therapeutics against non-small cell lung cancer (NSCLC) and other lung cancers is important. Here we explored the potential effect of 1-o-acetylbritannilactone (ABL), a novel extract from Inula britannica-F, on angiogenesis and lung cancer cell growth. We demonstrated that ABL dose-dependently inhibited vascular endothelial growth factor (VEGF)-induced proliferation, migration, and capillary structure formation of cultured human umbilical vascular endothelial cells (HUVECs). In vivo, ABL administration suppressed VEGF-induced new vasculature formation in Matrigel plugs. For the mechanism investigations, we found that ABL largely inhibited VEGF-mediated activation of Src kinase and focal adhesion kinase (FAK) in HUVECs. Furthermore, treatment of A549 NSCLC cells with ABL resulted in cell growth inhibition and Src-FAK in-activation. Significantly, administration of a single dose of ABL (12 mg/kg/day) remarkably suppressed growth of A549 xenografts in nude mice. In vivo microvessels formation and Src activation were also significantly inhibited in ABL-treated xenograft tumors. Taken together, our findings suggest that ABL suppresses angiogenesis and lung cancer cell growth possibly via regulating the VEGFR-Src-FAK signaling. - Highlights: • 1-o-acetylbritannilactone (ABL) inhibits VEGF-induced angiogenesis in vivo. • ABL inhibits VEGF-induced HUVEC migration, proliferation, capillary tube formation. • ABL inhibits VEGF-mediated activation of Src and FAK in HUVECs. • ABL inhibits growth and Src-FAK activation in A549 cells. • ABL administration inhibits A549 tumor angiogenesis and growth in nude mice.

  6. Imatinib binding to human c-Src is coupled to inter-domain allostery and suggests a novel kinase inhibition strategy.

    PubMed

    Tsutsui, Yuko; Deredge, Daniel; Wintrode, Patrick L; Hays, Franklin A

    2016-01-01

    Imatinib (Gleevec), a non-receptor tyrosine kinase inhibitor (nRTKI), is one of the most successful anti-neoplastic drugs in clinical use. However, imatinib-resistant mutations are increasingly prevalent in patient tissues and driving development of novel imatinib analogs. We present a detailed study of the conformational dynamics, in the presence and absence of bound imatinib, for full-length human c-Src using hydrogen-deuterium exchange and mass spectrometry. Our results demonstrate that imatinib binding to the kinase domain effects dynamics of proline-rich or phosphorylated peptide ligand binding sites in distal c-Src SH3 and SH2 domains. These dynamic changes in functional regulatory sites, distal to the imatinib binding pocket, show similarities to structural transitions involved in kinase activation. These data also identify imatinib-sensitive, and imatinib-resistant, mutation sites. Thus, the current study identifies novel c-Src allosteric sites associated with imatinib binding and kinase activation and provide a framework for follow-on development of TKI binding modulators. PMID:27480221

  7. Imatinib binding to human c-Src is coupled to inter-domain allostery and suggests a novel kinase inhibition strategy

    PubMed Central

    Tsutsui, Yuko; Deredge, Daniel; Wintrode, Patrick L.; Hays, Franklin A.

    2016-01-01

    Imatinib (Gleevec), a non-receptor tyrosine kinase inhibitor (nRTKI), is one of the most successful anti-neoplastic drugs in clinical use. However, imatinib-resistant mutations are increasingly prevalent in patient tissues and driving development of novel imatinib analogs. We present a detailed study of the conformational dynamics, in the presence and absence of bound imatinib, for full-length human c-Src using hydrogen-deuterium exchange and mass spectrometry. Our results demonstrate that imatinib binding to the kinase domain effects dynamics of proline-rich or phosphorylated peptide ligand binding sites in distal c-Src SH3 and SH2 domains. These dynamic changes in functional regulatory sites, distal to the imatinib binding pocket, show similarities to structural transitions involved in kinase activation. These data also identify imatinib-sensitive, and imatinib-resistant, mutation sites. Thus, the current study identifies novel c-Src allosteric sites associated with imatinib binding and kinase activation and provide a framework for follow-on development of TKI binding modulators. PMID:27480221

  8. Beyond the Number Domain

    PubMed Central

    Cantlon, Jessica F.; Platt, Michael L.; Brannon, Elizabeth M.

    2009-01-01

    In a world without numbers, we would be unable to build a skyscraper, hold a national election, plan a wedding, or pay for a chicken at the market. The numerical symbols used in all these behaviors build on the approximate number system (ANS) which represents the number of discrete objects or events as a continuous mental magnitude. In this review, we first discuss evidence that the ANS bears a set of behavioral and brain signatures that are universally displayed across animal species, human cultures, and development. We then turn to the question of whether the ANS constitutes a specialized cognitive and neural domain--a question central to understanding how this system works, the nature of its evolutionary and developmental trajectory, and its physical instantiation in the brain. PMID:19131268

  9. Crystallization of PTP Domains.

    PubMed

    Levy, Colin; Adams, James; Tabernero, Lydia

    2016-01-01

    Protein crystallography is the most powerful method to obtain atomic resolution information on the three-dimensional structure of proteins. An essential step towards determining the crystallographic structure of a protein is to produce good quality crystals from a concentrated sample of purified protein. These crystals are then used to obtain X-ray diffraction data necessary to determine the 3D structure by direct phasing or molecular replacement if the model of a homologous protein is available. Here, we describe the main approaches and techniques to obtain suitable crystals for X-ray diffraction. We include tools and guidance on how to evaluate and design the protein construct, how to prepare Se-methionine derivatized protein, how to assess the stability and quality of the sample, and how to crystallize and prepare crystals for diffraction experiments. While general strategies for protein crystallization are summarized, specific examples of the application of these strategies to the crystallization of PTP domains are discussed. PMID:27514806

  10. Final Report [The c-Abl signaling network in the radioadaptive response

    SciTech Connect

    Chi-Min, Yuan

    2014-01-28

    The radioadaptive response, or radiation hormesis, i.e. a low dose of radiation can protect cells and organisms from the effects of a subsequent higher dose, is a widely recognized phenomenon. Mechanisms underlying such radiation hormesis, however, remain largely unclear. Preliminary studies indicate an important role of c-Abl signaling in mediating the radioadaptive response. We propose to investigate how c-Abl regulates the crosstalk between p53 and NFκB in response to low doses irradiation. We found in our recent study that low dose IR induces a reciprocal p53 suppression and NFκB activation, which induces HIF-a and subsequently a metabolic reprogramming resulting in a transition from oxidative phosphorylation to glycolysis. Of importance is that this glycolytic switch is essential for the radioadaptive response. This low-dose radiationinduced HIF1α activation was in sharp contrast with the high-dose IR-induced p53 activation and HIF1α inhibition. HIF1α and p53 seem to play distinct roles in mediating the radiation dose-dependent metabolic response. The induction of HIF1α-mediated glycolysis is restricted to a low dose range of radiation, which may have important implications in assessing the level of radiation exposure and its potential health risk. Our results support a dose-dependent metabolic response to IR. When IR doses are below the threshold of causing detectable DNA damage (<0.2Gy) and thus little p53 activation, HIF1α is induced resulting in induction of glycolysis and increased radiation resistance. When the radiation dose reaches levels eliciting DNA damage, p53 is activated and diminishes the activity of HIF1α and glycolysis, leading to the induction of cell death. Our work challenges the LNT model of radiation exposure risk and provides a metabolic mechanism of radioadaptive response. The study supports a need for determining the p53 and HIF1α activity as a potential reliable biological readout of radiation exposure in humans. The

  11. Washing-resistant surfactant coated surface is able to inhibit pathogenic bacteria adhesion

    NASA Astrophysics Data System (ADS)

    Treter, Janine; Bonatto, Fernando; Krug, Cristiano; Soares, Gabriel Vieira; Baumvol, Israel Jacob Rabin; Macedo, Alexandre José

    2014-06-01

    Surface-active substances, which are able to organize themselves spontaneously on surfaces, triggering changes in the nature of the solid-liquid interface, are likely to influence microorganism adhesion and biofilm formation. Therefore, this study aimed to evaluate chemical non-ionic surfactants activity against pathogenic microbial biofilms and to cover biomaterial surfaces in order to obtain an anti-infective surface. After testing 11 different surfactants, Pluronic F127 was selected for further studies due to its non-biocidal properties and capability to inhibit up to 90% of biofilm formation of Gram-positive pathogen and its clinical isolates. The coating technique using direct impregnation on the surface showed important antibiofilm formation characteristics, even after extensive washes. Surface roughness and bacterial surface polarity does not influence the adhesion of Staphylococcus epidermidis, however, the material coated surface became extremely hydrophilic. The phenotype of S. epidermidis does not seem to have been affected by the contact with surfactant, reinforcing the evidence that a physical phenomenon is responsible for the activity. This paper presents a simple method of surface coating employing a synthetic surfactant to prevent S. epidermidis biofilm formation.

  12. Are reproductive health NGOs in Uganda able to engage in the health SWAp?

    PubMed

    Mugisha, Frank; Birungi, Harriet; Askew, Ian

    2005-01-01

    This paper explores the ability for reproductive health (RH) non-governmental organizations (NGO) in Uganda to survive in the context of SWAp and decentralization. The authors argue that, contrary to the perceptions that this context may increase NGO's financial vulnerability, a SWAp and a decentralized system may provide an opportunity that should be embraced by NGOs to enhance their sustainability and effectiveness by reducing their current dependency on donor funding. The paper discusses the systemic weaknesses of many NGOs that currently make them vulnerable, and observes that unless these weaknesses are addressed, such NGOs will lose their space in the SWAp and decentralization arena. The authors suggest that NGOs need to recognize the opportunities that participating in public-private partnerships through a SWAp can offer them for long-term and significant funding. They need also to develop their capacity to pro-actively participate in a SWAp and decentralized context by becoming more entrepreneurial in nature, through re-orienting their organizational philosophies and strategic planning and budgeting so as to be able to partner effectively with the public sector in accessing funds made available through health sector reform. PMID:16138736

  13. Fuzzy logic system able to detect interesting areas of a video sequence

    NASA Astrophysics Data System (ADS)

    De Vleeschouwer, Christophe; Marichal, Xavier; Delmot, Thierry; Macq, Benoit M. M.

    1997-06-01

    This paper introduces an automatic tool able to analyze the picture according to the semantic interest an observer attributes to its content. Its aim is to give a 'level of interest' to the distinct areas of the picture extracted by any segmentation tool. For the purpose of dealing with semantic interpretation of images, a single criterion is clearly insufficient because the human brain, due to its a priori knowledge and its huge memory of real-world concrete scenes, combines different subjective criteria in order to assess its final decision. The developed method permits such combination through a model using assumptions to express some general subjective criteria. Fuzzy logic enables the user to encode knowledge in a form that is very close the way experts think about the decision process. This fuzzy modeling is also well suited to represent multiple collaborating or even conflicting experts opinions. Actually, the assumptions are verified through a non-hierarchical strategy that considers them in a random order, each partial result contributing to the final one. Presented results prove that the tool is effective for a wide range of natural pictures. It is versatile and flexible in that it can be used stand-alone or can take into account any a priori knowledge about the scene.

  14. Off-Target Effects of BCR-ABL and JAK2 Inhibitors.

    PubMed

    Green, Myke R; Newton, Michael D; Fancher, Karen M

    2016-02-01

    The advent of targeted oncolytic agents has created a revolution in the treatment of malignancies. Perhaps best exemplified in myeloproliferative neoplasms (MPN), the tyrosine kinase inhibitors, including inhibitors of BCR-ABL tyrosine kinase and JAK2, have dramatically changed outcomes in persons with MPN. However, clinically relevant dosing of these adenosine triphosphate-mimetic agents in humans leads to inhibition of numerous tyrosine kinases beyond those touted by drug manufacturers and studied in landmark clinical trials. These so-called off-target effects have been linked to both clinical efficacy and toxicity. Rational drug development and serendipitous discovery of drug molecules allows the clinician to select targeted oncolytic agents to treat a specific clinical diagnosis and/or avoid exacerbation of concomitant disease states due to effects upon signaling pathways. Understanding the off-target binding and effects upon signaling pathway of the agents approved for the treatment of MPN will empower the clinician to adroitly select pharmacotherapy, predict toxicities, and utilize these agents in clinical practice for indications beyond MPN. PMID:24351780

  15. Klebsiella pneumoniae Is Able to Trigger Epithelial-Mesenchymal Transition Process in Cultured Airway Epithelial Cells

    PubMed Central

    Leone, Laura; Mazzetta, Francesca; Martinelli, Daniela; Valente, Sabatino; Alimandi, Maurizio; Raffa, Salvatore; Santino, Iolanda

    2016-01-01

    The ability of some bacterial pathogens to activate Epithelial-Mesenchymal Transition normally is a consequence of the persistence of a local chronic inflammatory response or depends on a direct interaction of the pathogens with the host epithelial cells. In this study we monitored the abilities of the K. pneumoniae to activate the expression of genes related to EMT-like processes and the occurrence of phenotypic changes in airway epithelial cells during the early steps of cell infection. We describe changes in the production of intracellular reactive oxygen species and increased HIF-1α mRNA expression in cells exposed to K. pneumoniae infection. We also describe the upregulation of a set of transcription factors implicated in the EMT processes, such as Twist, Snail and ZEB, indicating that the morphological changes of epithelial cells already appreciable after few hours from the K. pneumoniae infection are tightly regulated by the activation of transcriptional pathways, driving epithelial cells to EMT. These effects appear to be effectively counteracted by resveratrol, an antioxidant that is able to exert a sustained scavenging of the intracellular ROS. This is the first report indicating that strains of K. pneumoniae may promote EMT-like programs through direct interaction with epithelial cells without the involvement of inflammatory cells. PMID:26812644

  16. Bosutinib: a dual SRC/ABL kinase inhibitor for the treatment of chronic myeloid leukemia.

    PubMed

    Keller, Gunhild; Schafhausen, Philippe; Brummendorf, Tim H

    2009-10-01

    The tyrosine kinase inhibitor imatinib mesylate (IM) set new standards in the treatment of chronic myeloid leukemia (CML). However, emergence of resistance to IM became a major therapeutic challenge. Bosutinib (SKI-606), a 7-alkoxy-3-quinolinecarbonitrile, functions as a dual inhibitor of SRC and ABL kinases, and preclinical studies demonstrated a high antiproliferative activity in human and murine CML cell lines. In ongoing Phase I/II clinical trials, bosutinib yielded promising results revealing high clinical efficacy, good tolerability and reduced toxicity in IM-resistant or -intolerant CML patients. In this article, we provide an overview on the mechanism of action, and the preclinical and currently available clinical data for bosutinib. Owing to its favorable toxicity profile and its high antileukemic activity, bosutinib is a promising novel treatment option for patients with CML. A recently initiated, randomized open-label Phase III clinical study will clarify its role in first-line therapy of Philadelphia chromosome-positive chronic-phase CML. PMID:21083014

  17. Discrimination against differently abled children among rural communities in India: Need for action

    PubMed Central

    Janardhana, N.; Muralidhar, D.; Naidu, D. M.; Raghevendra, Guru

    2015-01-01

    Background: Persons with disabilities comprise at least 4 to 8 percent of the Indian population. Children with disabilities in India are subject to multiple deprivations and limited opportunities in several dimensions of their lives. Their families and caregivers also go through lot of stress and challenges in having a person with disability at home which ultimately leads to grave discriminatory practices towards these children. Materials and Methods: The article attempts to analyze and describe the common discriminatory grounds that children with disabilities commonly face from their immediate families and from the larger community through analyzing the filed visit reports of the Basic Needs India Staff providing on job training (handholding support) for the community based rehabilitation workers. Results: The case studies describes the various ugly forms of the discriminatory practices seen in the community towards differently abled children, same been categorized as denial of disability, physical restraints, social boycott, denial of property rights, decreased marital life prospects due to disabled member in family, implications on sexuality of people with disability, women with disability, discrepancies in state welfare programs, and problems in measuring disabilities. Conclusion: During the last two decades, there has been a growing realization that institutional care for the disabled is not entirely suitable for their individual needs, dignity and independence. A movement towards community based rehabilitation has picked up pace and contribute toward greater independence and self sustainability of the disabled. PMID:25810627

  18. Dietary constituents are able to play a beneficial role in canine epidermal barrier function.

    PubMed

    Watson, Adrian L; Fray, Tim R; Bailey, Julie; Baker, Claire B; Beyer, Sally A; Markwell, Peter J

    2006-01-01

    Epidermal barrier function is a critical attribute of mammalian skin. The barrier is responsible for preventing skin-associated pathologies through controlling egress of water and preventing ingress of environmental agents. Maintaining the quality and integrity of the epidermal barrier is therefore of considerable importance. Structurally, the barrier is composed of two main parts, the corneocytes and the intercellular lamellar lipid. The epidermal lamellar lipid comprises mainly ceramides, sterols and fatty acids. Twenty-seven nutritional components were screened for their ability to upregulate epidermal lipid synthesis. Seven of the 27 nutritional components (pantothenate, choline, nicotinamide, histidine, proline, pyridoxine and inositol) were subsequently retested using an in vitro transepidermal diffusion experimental model, providing a functional assessment of barrier properties. Ultimately, the best performing five nutrients were fed to dogs at supplemented concentrations in a 12-week feeding study. Barrier function was measured using transepidermal water loss (TEWL). It was found that a combination of pantothenate, choline, nicotinamide, histidine and inositol, when fed at supplemented concentrations, was able to significantly reduce TEWL in dogs after 9 weeks. PMID:16364034

  19. Activated mast cells release biological activities able to support eosinophil production from mouse hemopoietic precursors.

    PubMed

    Oskéritzian, C; Milon, G; Braquet, P; Mencia-Huerta, J M; David, B

    1996-02-01

    Mouse bone marrow cells cultured for 6 days in the presence of recombinant murine IL-3 and granulocyte-macrophage colony-stimulating factor (GM-CSF) were used as a source of precursors responsive to eosinopoietins. They were further cultured for 7 days in the presence of either a combination of recombinant cytokines or supernatants of bone marrow-derived mast cells (BMMC) activated with either immunological or nonimmunological stimuli. Cytosmears of collected cells were analyzed for eosinophil contents and allowed to demonstrate that supernatants of passively sensitized BMMC support both total cell proliferation and eosinophil production, after various periods of incubation with monoclonal rat anti-mouse IgE antibodies (the 6HD5 mAbs). In contrast, a stimulation with 100 ng/ml dinitrophenylated bovine serum albumin (DNP-BSA) did not generate supernatants displaying such bioactivities. Low doses of methyl ester of L (but not D)-leucine or of the calcium ionophore A23187 also allowed the release of eosinopoietic bioactivities. In addition, immunoreactive IL-5, GM-CSF, and IL-3 were quantified in the BMMC supernatants. These results demonstrate that activated BMMC are able to effect eosinophil production. PMID:8603429

  20. Synthesis of a pH dependent covalent imprinted polymer able to recognize organotin species.

    PubMed

    Gallego-Gallegos, Mercedes; Muñoz-Olivas, Riansares; Cámara, C; Mancheño, María J; Sierra, Miguel A

    2006-01-01

    The covalent imprinting approach has for the first time been successfully applied for the synthesis of an imprinted polymer able to recognize organotin species. The synthesis has been accomplished by co-polymerization of the complex Bu(2)SnO-m-vinylbenzoin as the imprinting template plus co-monomer sodium methacrylate, and ethylene glycol dimethacrylate as cross-linker. The imprinting effect has been evidenced within the narrow pH range 2.5< pH< 3.5. At lower pH values, the imprinting effect is prevented by the exclusive existence of non-specific interactions, whereas pH>3.5 provokes a strong rebind of the template in both imprinted and non-imprinted polymers. This pH dependency can be explained as a selective chemical modification which reduces bind diversity following a model based on enolization by protonation of the specific cavities. Characterization of the adsorption isotherms showed good agreement with the Langmuir-Freundlich (LF) model, presenting quite homogeneous binding sites for a bulk material and high capacity in the imprinting pH range. In addition, the affinity spectrum (AS) method has been represented showing the typical profiles of LF isotherm for both sub-saturation and saturation levels, being in general agreement with the encountered values for fitting coefficients. The covalent molecular imprinted polymer has been successfully evaluated in a SPE process for further OTC determination in the certified mussel tissue (CRM 477). PMID:16365669

  1. Azithromycin is able to control Toxoplasma gondii infection in human villous explants

    PubMed Central

    2014-01-01

    Background Although Toxoplasma gondii infection is normally asymptomatic, severe cases of toxoplasmosis may occur in immunosuppressed patients or congenitally infected newborns. When a fetal infection is established, the recommended treatment is a combination of pyrimethamine, sulfadiazine and folinic acid (PSA). The aim of the present study was to evaluate the efficacy of azithromycin to control T. gondii infection in human villous explants. Methods Cultures of third trimester human villous explants were infected with T. gondii and simultaneously treated with either PSA or azithromycin. Proliferation of T. gondii, as well as production of cytokines and hormones by chorionic villous explants, was analyzed. Results Treatment with either azithromycin or PSA was able to control T. gondii infection in villous explants. After azithromycin or PSA treatment, TNF-α, IL-17A or TGF-β1 levels secreted by infected villous explants did not present significant differences. However, PSA-treated villous explants had decreased levels of IL-10 and increased IL-12 levels, while treatment with azithromycin increased production of IL-6. Additionally, T. gondii-infected villous explants increased secretion of estradiol, progesterone and HCG + β, while treatments with azithromycin or PSA reduced secretion of these hormones concurrently with decrease of parasite load. Conclusions In conclusion, these results suggest that azithromycin may be defined as an effective alternative drug to control T. gondii infection at the fetal-maternal interface. PMID:24885122

  2. Meteorological overview of the Arctic Boundary Layer Expedition (ABLE 3A) flight series

    NASA Technical Reports Server (NTRS)

    Shipham, Mark C.; Bachmeier, A. S.; Cahoon, Donald R., Jr.; Browell, Edward V.

    1992-01-01

    A meteorological overview of the Arctic Boundary Layer Expedition (ABLE 3A) flight series is presented. Synoptic analyses of mid-tropospheric circulation patterns are combined with isentropic back trajectory calculations to describe the long-range (400-3000 km) atmospheric transport mechanisms and pathways of air masses to the Arctic and sub-Arctic regions of North America during July and August 1988. Siberia and the northern Pacific Ocean were found to be the two most likely source areas for 3-day transport to the study areas in Alaska. Transport to the Barrow region was frequently influenced by polar vortices and associated short-wave troughs over the Arctic Ocean, while the Bethel area was most often affected by lows migrating across the Bering Sea and the Gulf of Alaska, as well as ridges of high pressure which built into interior Alaska. July 1988 was warmer and dryer than normal over much of Alaska. As a result, the 1988 Alaska fire season was one of the most active of the past decade. Airborne lidar measurements verified the presence of biomass burning plumes on many flights, often trapped in thin subsidence layer temperature inversions. Several cases of stratosphere/troposphere exchange were noted, based upon potential vorticity analyses and aircraft lidar data, especially in the Barrow region and during transit flights to and from Alaska.

  3. Nicotinamide: a vitamin able to shift macrophage differentiation toward macrophages with restricted inflammatory features.

    PubMed

    Weiss, Ronald; Schilling, Erik; Grahnert, Anja; Kölling, Valeen; Dorow, Juliane; Ceglarek, Uta; Sack, Ulrich; Hauschildt, Sunna

    2015-11-01

    The differentiation of human monocytes into macrophages is influenced by environmental signals. Here we asked in how far nicotinamide (NAM), a vitamin B3 derivative known to play a major role in nicotinamide adenine dinucleotide (NAD)-mediated signaling events, is able to modulate monocyte differentiation into macrophages developed in the presence of granulocyte macrophage colony-stimulating factor (GM-MØ) or macrophage colony-stimulating factor (M-MØ). We found that GM-MØ undergo biochemical, morphological and functional modifications in response to NAM, whereas M-MØ were hardly affected. GM-MØ exposed to NAM acquired an M-MØ-like structure while the LPS-induced production of pro-inflammatory cytokines and COX-derived eicosanoids were down-regulated. In contrast, NAM had no effect on the production of IL-10 or the cytochrome P450-derived eicosanoids. Administration of NAM enhanced intracellular NAD concentrations; however, it did not prevent the LPS-mediated drain on NAD pools. In search of intracellular molecular targets of NAM known to be involved in LPS-induced cytokine and eicosanoid synthesis, we found NF-κB activity to be diminished. In conclusion, our data show that vitamin B3, when present during the differentiation of monocytes into GM-MØ, interferes with biochemical pathways resulting in strongly reduced pro-inflammatory features. PMID:26385774

  4. Is dynamometry able to infer the risk of muscle mass loss in patients with COPD?

    PubMed Central

    Ramos, Dionei; Bertolini, Giovana Navarro; Leite, Marceli Rocha; Carvalho Junior, Luiz Carlos Soares; da Silva Pestana, Paula Roberta; dos Santos, Vanessa Ribeiro; Fortaleza, Ana Claudia de Souza; Rodrigues, Fernanda Maria Machado; Ramos, Ercy Mara Cipulo

    2015-01-01

    Introduction Sarcopenia is characterized by a progressive and generalized decrease of strength and muscle mass. Muscle mass loss is prevalent in patients with chronic obstructive pulmonary disease (COPD) as a result of both the disease and aging. Some methods have been proposed to assess body composition (and therefore identify muscle mass loss) in this population. Despite the high accuracy of some methods, they require sophisticated and costly equipment. Aim The purpose of this study was to infer the occurrence of muscle mass loss measured by a sophisticated method (dual energy X-ray absorptiometry [DEXA]) using a more simple and affordable equipment (dynamometer). Methods Fifty-seven stable subjects with COPD were evaluated for anthropometric characteristics, lung function, functional exercise capacity, body composition, and peripheral muscle strength. A binary logistic regression model verified whether knee-extension strength (measured by dynamometry) could infer muscle mass loss (from DEXA). Results Patients with decreased knee-extension strength were 5.93 times more likely to have muscle mass loss, regardless of sex, disease stage, and functional exercise capacity (P=0.045). Conclusion Knee-extension dynamometry was able to infer muscle mass loss in patients with COPD. PMID:26229459

  5. Activation of a novel Bcr/Abl destruction pathway by WP1130 induces apoptosis of chronic myelogenous leukemia cells

    PubMed Central

    Bartholomeusz, Geoffrey A.; Talpaz, Moshe; Kapuria, Vaibhav; Kong, Ling Yuan; Wang, Shimei; Estrov, Zeev; Priebe, Waldemar; Wu, Ji

    2007-01-01

    Imatinib mesylate (Gleevec) is effective therapy against Philadelphia chromosome–positive leukemia, but resistance develops in all phases of the disease. Bcr/Abl point mutations and other alterations reduce the kinase inhibitory activity of imatinib mesylate; thus, agents that target Bcr/Abl through unique mechanisms may be needed. Here we describe the activity of WP1130, a small molecule that specifically and rapidly down-regulates both wild-type and mutant Bcr/Abl protein without affecting bcr/abl gene expression in chronic myelogenous leukemia (CML) cells. Loss of Bcr/Abl protein correlated with the onset of apoptosis and reduced phosphorylation of Bcr/Abl substrates. WP1130 did not affect Hsp90/Hsp70 ratios within the cells and did not require the participation of the proteasomal pathway for loss of Bcr/Abl protein. WP1130 was more effective in reducing leukemic versus normal hematopoietic colony formation and strongly inhibited colony formation of cells derived from patients with T315I mutant Bcr/Abl–expressing CML in blast crisis. WP1130 suppressed the growth of K562 heterotransplanted tumors as well as both wild-type Bcr/Abl and T315I mutant Bcr/Abl–expressing BaF/3 cells transplanted into nude mice. Collectively, our results demonstrate that WP1130 reduces wild-type and T315I mutant Bcr/Abl protein levels in CML cells through a unique mechanism and may be useful in treating CML. PMID:17202319

  6. Multifunctionalities driven by ferroic domains

    SciTech Connect

    Yang, J. C.; Huang, Y. L.; Chu, Y. H.; He, Q.

    2014-08-14

    Considerable attention has been paid to ferroic systems in pursuit of advanced applications in past decades. Most recently, the emergence and development of multiferroics, which exhibit the coexistence of different ferroic natures, has offered a new route to create functionalities in the system. In this manuscript, we step from domain engineering to explore a roadmap for discovering intriguing phenomena and multifunctionalities driven by periodic domain patters. As-grown periodic domains, offering exotic order parameters, periodic local perturbations and the capability of tailoring local spin, charge, orbital and lattice degrees of freedom, are introduced as modeling templates for fundamental studies and novel applications. We discuss related significant findings on ferroic domain, nanoscopic domain walls, and conjunct heterostructures based on the well-organized domain patterns, and end with future prospects and challenges in the field.

  7. Dynamical domain wall and localization

    NASA Astrophysics Data System (ADS)

    Toyozato, Yuta; Higuchi, Masafumi; Nojiri, Shin'ichi

    2016-03-01

    Based on the previous works (Toyozato et al., 2013 [24]; Higuchi and Nojiri, 2014 [25]), we investigate the localization of the fields on the dynamical domain wall, where the four-dimensional FRW universe is realized on the domain wall in the five-dimensional space-time. Especially we show that the chiral spinor can localize on the domain wall, which has not been succeeded in the past works as the seminal work in George et al. (2009) [23].

  8. Using SDSS & GalaxyZoo Databases to Ask Research-able Questions in Learning Astronomy

    NASA Astrophysics Data System (ADS)

    Slater, Timothy F.

    2010-01-01

    Using Galaxy Zoo1 at: http://zoo1.galaxyzoo.org/ accessing SDSS and a multi-phase backwards faded scaffolding approach, we first ask students to classify 30 galaxies and consider proposed conclusion: "most galaxies are elliptical” based on the evidence collected. Here, student attention is isolated from generating a question or even a data collection protocol, but focused on the issue of "does the evidence match the conclusion?” The next phase focuses on generating conclusions from evidence, whereas the previous phase was focused on evaluating conclusions. Students explain their reasoning provide evidence in response to "What conclusions and generalizations can you make from the following data collected by a student in terms of do spiral galaxies generally spin clockwise or anticlockwise given that one observes 36 spirals spinning clockwise, 21 spirals spinning anticlockwise, and 16 appearing to be edge-on or unclear.” Next, students are asked to consider what evidence needs to be collected in order to complete a scientific inquiry related to a given question. Students propose what evidence is needed in order to pursue, "What fraction of galaxies observed appear to be in the process of merging with other galaxies?” Note students are explicitly asked not to actually gather data as it detracts from developing an understanding of how data collection needs to be tightly aligned with the question. And, in practice, students can intellectually engage with a data collection plan that is simply too ominous to actually collect. By this point, students have extended experience with inquiry in this domain. Students are now ready to wrestle with creating a fruitful question. Students are tasked to design an answerable research question, propose a plan to pursue evidence, collect data using the present astronomical data base and create an evidence-based conclusion about the nature and or frequency of galaxies.

  9. A tyrosine-phosphorylated carboxy-terminal peptide of the fibroblast growth factor receptor (Flg) is a binding site for the SH2 domain of phospholipase C-gamma 1.

    PubMed Central

    Mohammadi, M; Honegger, A M; Rotin, D; Fischer, R; Bellot, F; Li, W; Dionne, C A; Jaye, M; Rubinstein, M; Schlessinger, J

    1991-01-01

    Phospholipase C-gamma (PLC-gamma) is a substrate of the fibroblast growth factor receptor (FGFR; encoded by the flg gene) and other receptors with tyrosine kinase activity. It has been demonstrated that the src homology region 2 (SH2 domain) of PLC-gamma and of other signalling molecules such as GTPase-activating protein and phosphatidylinositol 3-kinase-associated p85 direct their binding toward tyrosine-autophosphorylated regions of the epidermal growth factor or platelet-derived growth factor receptor. In this report, we describe the identification of Tyr-766 as an autophosphorylation site of flg-encoded FGFR by direct sequencing of a tyrosine-phosphorylated tryptic peptide isolated from the cytoplasmic domain of FGFR expressed in Escherichia coli. The same phosphopeptide was found in wild-type FGFR phosphorylated either in vitro or in living cells. Like other growth factor receptors, tyrosine-phosphorylated wild-type FGFR or its cytoplasmic domain becomes associated with intact PLC-gamma or with a fusion protein containing the SH2 domain of PLC-gamma. To delineate the site of association, we have examined the capacity of a 28-amino-acid tryptic peptide containing phosphorylated Tyr-766 to bind to various constructs containing SH2 and other domains of PLC-gamma. It is demonstrated that the tyrosine-phosphorylated peptide binds specifically to the SH2 domain but not to the SH3 domain or other regions of PLC-gamma. Hence, Tyr-766 and its flanking sequences represent a major binding site in FGFR for PLC-gamma. Alignment of the amino acid sequences surrounding Tyr-766 with corresponding regions of other FGFRs revealed conserved tyrosine residues in all known members of the FGFR family. We propose that homologous tyrosine-phosphorylated regions in other FGFRs also function as binding sites for PLC-gamma and therefore are involved in coupling to phosphatidylinositol breakdown. Images PMID:1656221

  10. Domain transfer multiple kernel learning.

    PubMed

    Duan, Lixin; Tsang, Ivor W; Xu, Dong

    2012-03-01

    Cross-domain learning methods have shown promising results by leveraging labeled patterns from the auxiliary domain to learn a robust classifier for the target domain which has only a limited number of labeled samples. To cope with the considerable change between feature distributions of different domains, we propose a new cross-domain kernel learning framework into which many existing kernel methods can be readily incorporated. Our framework, referred to as Domain Transfer Multiple Kernel Learning (DTMKL), simultaneously learns a kernel function and a robust classifier by minimizing both the structural risk functional and the distribution mismatch between the labeled and unlabeled samples from the auxiliary and target domains. Under the DTMKL framework, we also propose two novel methods by using SVM and prelearned classifiers, respectively. Comprehensive experiments on three domain adaptation data sets (i.e., TRECVID, 20 Newsgroups, and email spam data sets) demonstrate that DTMKL-based methods outperform existing cross-domain learning and multiple kernel learning methods. PMID:21646679

  11. Hydrophilic Domains Enhance Nanobubble Stability.

    PubMed

    Nishiyama, Takashi; Takahashi, Koji; Ikuta, Tatsuya; Yamada, Yutaka; Takata, Yasuyuki

    2016-05-18

    Highly stable nanoscale gas states at solid/liquid interfaces, referred to as nanobubbles, have been widely studied for over a decade. In this study, nanobubbles generated on a hydrophobic Teflon amorphous fluoroplastic thin film in the presence and absence of hydrophilic carbon domains are investigated by peak force quantitative nanomechanics. On the hydrophobic surface without hydrophilic domains, a small number of nanobubbles are generated and then rapidly decrease in size. On the hydrophobic surface with hydrophilic domains, the hydrophilic domains have a significant effect on the generation and stability of nanobubbles, with bubbles remaining on the surface for up to three days. PMID:26864857

  12. Characterization of an epithelial, nearly diploid liver cell strain, from Chinese hamster, able to activate promutagens.

    PubMed

    Turchi, G; Carluccio, M A; Oesch, F; Gemperlein, I; Glatt, H R

    1987-03-01

    Epithelial liver cells of the Chinese hamster (CHEL cells) were propagated in culture for 35 passages. At favourable cell densities, the population doubling time in normal medium, was 20 h. L-Tyrosine amino transferase activity was retained at a measurable level, but its enhancement by dexamethasone was detected solely in cells of early passages. Pyruvate kinase was strongly activated by fructose-1,6-biphosphate at low substrate concentrations. These enzymatic properties suggest that the CHEL cells are derived from a sub-population of parenchymal hepatocytes or from cells closely related to parenchymal hepatocytes. With a lag period of a few hours, CHEL cultures metabolized benzo[a]pyrene. In cell homogenates the various monooxygenase activities investigated were below the detection limits. However, other xenobiotic-metabolizing activities, such as cytochrome P-450 reductase, glutathione transferase and UDP-glucuronosyl-transferase were high, with levels comparable to those observed in freshly isolated rat parenchymal cells. Epoxide hydrolase activity was also detected, but was lower than in the liver. The CHEL cells were able to activate benzo[a]pyrene, 7,12-dimethylbenz[a]anthracene and aflatoxin B1 to mutagens, as shown in a co-culture assay with V79 cells, in which acquisition of resistance to 6-thioguanine was studied. At early passages, the CHEL cells had a near diploid set of chromosomes. Then, gradually the frequency of cells with slight changes in the number of chromosomes and the frequency of tetraploids were increased. During the observation period (up to passage 20) the modal number of chromosomes shifted from 22 to 23. No gross morphological changes in the cultures were noticed during the 20 passages.(ABSTRACT TRUNCATED AT 250 WORDS) PMID:2901026

  13. A Novel Blasted and Grooved Low Profile Pedicle Screw Able to Resist High Compression Bending Loads

    PubMed Central

    Kim, Young-Sung; Choi, Hong-June; Kim, Kyung-Hyun; Park, Jeong-Yoon; Jeong, Hyun-Yong; Chin, Dong-Kyu; Kim, Keun-Su; Yoon, Young-Sul; Lee, Yoon-Chul; Cho, Yong-Eun

    2012-01-01

    Objective Polyaxial pedicle screws are a safe, useful adjunct to transpedicular fixation. However, the large screw head size can cause soft tissue irritation, high rod positioning, and facet joint injury. However, the mechanical resistance provided by small and low profile pedicle screws is very limited. We therefore developed a novel, low profile pedicle screw using grooving and blasting treatment that is able to resist a high compression bending load. Methods We evaluated the compression bending force to displacement and yield loads for seven different screw head types that differed with regard to their groove intervals and whether or not they had been blasted. Results The rank order of screw types that had the greatest compression bending force to displacement was as follows: (1) universal polyaxial, (2) low polyaxial with 0.1mm grooves and blasting, (3) low polyaxial with blasting, (4) low polyaxial with 0.15mm grooves and blasting, (5) low polyaxial with 0.05mm grooves and blasting, (6) low polyaxial with 0.05mm grooves, (7) and low polyaxial. Low polyaxial screws with 0.1mm grooves and blasting had the maximum yield load and highest compression bending force to displacement of all seven polyaxial screw head systems evaluated. Conclusion Blasting and grooving treatment of pedicle screw heads resulted in screw heads with a high yield load and compression bending force relative to displacement because of increased friction. Low polyaxial pedicle screws with 0.1 mm grooves treated by blasting have mechanical characteristics similar to those of universal polyaxial pedicle screws. PMID:25983790

  14. Microbial biofilms are able to destroy hydroxyapatite in the absence of host immunity in vitro

    PubMed Central

    Junka, Adam Feliks; Szymczyk, Patrycja; Smutnicka, Danuta; Kos, Marcin; Smolina, Iryna; Bartoszewicz, Marzenna; Chlebus, Edward; Turniak, Michal; Sedghizadeh, Parish P.

    2014-01-01

    Introduction It is widely thought that inflammation and osteoclastogenesis result in hydroxyapatite (HA) resorption and sequestra formation during osseous infections, and microbial biofilm pathogens induce the inflammatory destruction of HA. We hypothesized that biofilms associated with infectious bone disease can directly resorb HA in the absence of host inflammation or osteoclastogenesis. Therefore, we developed an in vitro model to test this hypothesis. Materials and Methods Customized HA discs were manufactured as a substrate for growing clinically relevant biofilm pathogens. Single-species biofilms of S.mutans, S.aureus, P.aeruginosa and C.albicans, and mixed-species biofilms of C.albicans + S.mutans were incubated on HA discs for 72 hours to grow mature biofilms. Three different non-biofilm control groups were also established for testing. HA discs were then evaluated by means of scanning electron microscopy, micro-CT metrotomography, x-ray spectroscopy and confocal microscopy with planimetric analysis. Additionally, quantitative cultures and pH assessment were performed. ANOVA was used to test for significance between treatment and control groups. Results All investigated biofilms were able to cause significant (P<0.05) and morphologically characteristic alterations in HA structure as compared to controls. The highest number of alterations observed was caused by mixed biofilms of C.albicans + S.mutans. S. mutans biofilm incubated in medium with additional sucrose content was the most detrimental to HA surfaces among single-species biofilms. Conclusion These findings suggest that direct microbial resorption of bone is possible in addition to immune-mediated destruction, which has important translational implications for the pathogenesis of chronic bone infections and for targeted antimicrobial therapeutics. PMID:25544303

  15. A Cognitive Neural Architecture Able to Learn and Communicate through Natural Language

    PubMed Central

    Golosio, Bruno; Cangelosi, Angelo; Gamotina, Olesya; Masala, Giovanni Luca

    2015-01-01

    Communicative interactions involve a kind of procedural knowledge that is used by the human brain for processing verbal and nonverbal inputs and for language production. Although considerable work has been done on modeling human language abilities, it has been difficult to bring them together to a comprehensive tabula rasa system compatible with current knowledge of how verbal information is processed in the brain. This work presents a cognitive system, entirely based on a large-scale neural architecture, which was developed to shed light on the procedural knowledge involved in language elaboration. The main component of this system is the central executive, which is a supervising system that coordinates the other components of the working memory. In our model, the central executive is a neural network that takes as input the neural activation states of the short-term memory and yields as output mental actions, which control the flow of information among the working memory components through neural gating mechanisms. The proposed system is capable of learning to communicate through natural language starting from tabula rasa, without any a priori knowledge of the structure of phrases, meaning of words, role of the different classes of words, only by interacting with a human through a text-based interface, using an open-ended incremental learning process. It is able to learn nouns, verbs, adjectives, pronouns and other word classes, and to use them in expressive language. The model was validated on a corpus of 1587 input sentences, based on literature on early language assessment, at the level of about 4-years old child, and produced 521 output sentences, expressing a broad range of language processing functionalities. PMID:26560154

  16. Environment-mediated drug resistance in Bcr/Abl-positive acute lymphoblastic leukemia

    PubMed Central

    Feldhahn, Niklas; Arutyunyan, Anna; Stoddart, Sonia; Zhang, Bin; Schmidhuber, Sabine; Yi, Sun-Ju; Kim, Yong-mi; Groffen, John; Heisterkamp, Nora

    2012-01-01

    Although cure rates for acute lymphoblastic leukemia (ALL) have increased, development of resistance to drugs and patient relapse are common. The environment in which the leukemia cells are present during the drug treatment is known to provide significant survival benefit. Here, we have modeled this process by culturing murine Bcr/Abl-positive acute lymphoblastic leukemia cells in the presence of stroma while treating them with a moderate dose of two unrelated drugs, the farnesyltransferase inhibitor lonafarnib and the tyrosine kinase inhibitor nilotinib. This results in an initial large reduction in cell viability of the culture and inhibition of cell proliferation. However, after a number of days, cell death ceases and the culture becomes drug-tolerant, enabling cell division to resume. Using gene expression profiling, we found that the development of drug resistance was accompanied by massive transcriptional upregulation of genes that are associated with general inflammatory responses such as the metalloproteinase MMP9. MMP9 protein levels and enzymatic activity were also increased in ALL cells that had become nilotinib-tolerant. Activation of p38, Akt and Erk correlated with the development of environment-mediated drug resistance (EMDR), and inhibitors of Akt and Erk in combination with nilotinib reduced the ability of the cells to develop resistance. However, inhibition of p38 promoted increased resistance to nilotinib. We conclude that development of EMDR by ALL cells involves changes in numerous intracellular pathways. Development of tolerance to drugs such as nilotinib may therefore be circumvented by simultaneous treatment with other drugs having divergent targets. PMID:22934254

  17. Delayed riluzole treatment is able to rescue injured rat spinal motoneurons.

    PubMed

    Nógrádi, A; Szabó, A; Pintér, S; Vrbová, G

    2007-01-19

    The effect of delayed 2-amino-6-trifluoromethoxy-benzothiazole (riluzole) treatment on injured motoneurons was studied. The L4 ventral root of adult rats was avulsed and reimplanted into the spinal cord. Immediately after the operation or with a delay of 5, 10, 14 or 16 days animals were treated with riluzole (n=5 in each group) while another four animals remained untreated. Three months after the operation the fluorescent dye Fast Blue was applied to the proximal end of the cut ventral ramus of the L4 spinal nerve to retrogradely label reinnervating neurons. Three days later the spinal cords were processed for counting the retrogradely labeled cells and choline acetyltransferase immunohistochemistry was performed to reveal the cholinergic cells in the spinal cords. In untreated animals there were 20.4+/-1.6 (+/-S.E.M.) retrogradely labeled neurons while in animals treated with riluzole immediately or 5 and 10 days after ventral root avulsion the number of labeled motoneurons ranged between 763+/-36 and 815+/-50 (S.E.M.). Riluzole treatment starting at 14 and 16 days after injury resulted in significantly lower number of reinnervating motoneurons (67+/-4 and 52+/-3 S.E.M., respectively). Thus, riluzole dramatically enhanced the survival and reinnervating capacity of injured motoneurons not only when treatment started immediately after injury but also in cases when riluzole treatment was delayed for up to 10 days. These results suggest that motoneurons destined to die after ventral root avulsion are programmed to survive for some time after injury and riluzole is able to rescue them during this period of time. PMID:17084537

  18. Is allicin able to reduce Campylobacter jejuni colonization in broilers when added to drinking water?

    PubMed

    Robyn, J; Rasschaert, G; Hermans, D; Pasmans, F; Heyndrickx, M

    2013-05-01

    Reducing Campylobacter shedding on the farm could result in a reduction of the number of human campylobacteriosis cases. In this study, we first investigated if allicin, allyl disulfide, and garlic oil extract were able to either prevent C. jejuni growth or kill C. jejuni in vitro. Allyl disulfide and garlic oil extract reduced C. jejuni numbers in vitro below a detectable level at a concentration of 50 mg/kg (no lower concentrations were tested), whereas allicin reduced C. jejuni numbers below a detectable level at a concentration as low as 7.5 mg/kg. In further experiments we screened for the anti-C. jejuni activity of allicin in a fermentation system closely mimicking the broiler cecal environment using cecal microbiota and mucus isolated from C. jejuni-free broilers. During these fermentation experiments, allicin reduced C. jejuni numbers below a detectable level after 24 h at a concentration of 50 mg/kg. In contrast, 25 mg/kg of allicin killed C. jejuni in the first 28 h of incubation, but anti-C. jejuni activity was lost after 48 h of incubation, probably due to the presence of mucin in the growth medium. This had been confirmed in fermentation experiments in the presence of broiler cecal mucus. Based on these results, we performed an in vivo experiment to assess the prevention or reduction of cecal C. jejuni colonization in broiler chickens when allicin was added to drinking water. We demonstrated that allicin in drinking water did not have a statistically significant effect on cecal C. jejuni colonization in broilers. It was assumed, based on in vitro experiments, that the activity of allicin was thwarted by the presence of mucin-containing mucus. Despite promising in vitro results, allicin was not capable of statistically influencing C. jejuni colonization in a broiler flock, although a trend toward lower cecal C. jejuni numbers in allicin-treated broilers was observed. PMID:23571353

  19. Theory of Mind and Executive Function in Preschoolers with Typical Development versus Intellectually Able Preschoolers with Autism Spectrum Disorder

    ERIC Educational Resources Information Center

    Kimhi, Yael; Shoam-Kugelmas, Dana; Agam Ben-Artzi, Galit; Ben-Moshe, Inbal; Bauminger-Zviely, Nirit

    2014-01-01

    Children with autism spectrum disorder (ASD) have difficulties in theory of mind (ToM) and executive function (EF), which may be linked because one domain (EF) affects the other (ToM). Group differences (ASD vs. typical development) were examined in both cognitive domains, as well as EF's associations and regressions with ToM. Participants…

  20. Mapping the hemagglutination domain of rotaviruses.

    PubMed Central

    Fuentes-Pananá, E M; López, S; Gorziglia, M; Arias, C F

    1995-01-01

    Most strains of animal rotaviruses are able to agglutinate erythrocytes, and the surface protein VP4 is the virus hemagglutinin. To map the hemagglutination domain on VP4 while preserving the conformation of the protein, we constructed full-length chimeras between the VP4 genes of hemagglutinating (YM) and nonhemagglutinating (KU) rotavirus strains. The parental and chimeric genes were expressed in insect cells, and the recombinant VP4 proteins were evaluated for their capacity to agglutinate human type O erythrocytes. Three chimeric genes, encoding amino acids 1 to 208 (QKU), 93 to 208 (QC), and 93 to 776 (QYM) of the YM VP4 protein in a KU VP4 background, were constructed. YM VP4 and chimeras QKU and QC were shown to specifically hemagglutinate, indicating that the region between amino acids 93 and 208 of YM VP4 is sufficient to determine the hemagglutination activity of the protein. PMID:7884915

  1. Mapping the Moral Domain

    PubMed Central

    Graham, Jesse; Nosek, Brian A.; Haidt, Jonathan; Iyer, Ravi; Koleva, Spassena; Ditto, Peter H.

    2010-01-01

    The moral domain is broader than the empathy and justice concerns assessed by existing measures of moral competence, and it is not just a subset of the values assessed by value inventories. To fill the need for reliable and theoretically-grounded measurement of the full range of moral concerns, we developed the Moral Foundations Questionnaire (MFQ) based on a theoretical model of five universally available (but variably developed) sets of moral intuitions: Harm/care, Fairness/reciprocity, Ingroup/loyalty, Authority/respect, and Purity/sanctity. We present evidence for the internal and external validity of the scale and the model, and in doing so present new findings about morality: 1. Comparative model fitting of confirmatory factor analyses provides empirical justification for a five-factor structure of moral concerns. 2. Convergent/discriminant validity evidence suggests that moral concerns predict personality features and social group attitudes not previously considered morally relevant. 3. We establish pragmatic validity of the measure in providing new knowledge and research opportunities concerning demographic and cultural differences in moral intuitions. These analyses provide evidence for the usefulness of Moral Foundations Theory in simultaneously increasing the scope and sharpening the resolution of psychological views of morality. PMID:21244182

  2. Development of an AAV9 coding for a 3XFLAG-TALEfrat#8-VP64 able to increase in vivo the human frataxin in YG8R mice.

    PubMed

    Chapdelaine, P; Gérard, C; Sanchez, N; Cherif, K; Rousseau, J; Ouellet, D L; Jauvin, D; Tremblay, J P

    2016-07-01

    Artificially designed transcription activator-like effector (TALE) proteins fused to a transcription activation domain (TAD), such as VP64, are able to activate specific eukaryotic promoters. They thus provide a good tool for targeted gene regulation as a therapy. However, the efficacy of such an agent in vivo remains to be demonstrated as the majority of studies have been carried out in cell culture. We produced an adeno-associated virus 9 (AAV9) coding for a TALEfrat#8 containing 13 repeat variable diresidues able to bind to the proximal promoter of human frataxin (FXN) gene. This TALEfrat#8 was fused with a 3XFLAG at its N terminal and a VP64 TAD at its C terminal, and driven by a CAG promoter. This AAV9_3XFLAG-TALEfrat#8-VP64 was injected intraperitoneally to 9-day-old and 4-month-old YG8R mice. After 1 month, the heart, muscle and liver were removed and their FXN mRNA and FXN protein were analyzed. The results show that the AAV9_3XFLAG-TALEfrat#8-VP64 increased the FXN mRNA and FXN protein in the three organs studied. These results corroborate our previous in vitro studies in the FRDA human fibroblasts. Our study indicates that an AAV coding for a TALE protein coupled with a TAD may be used to increase gene expression in vivo as a possible treatment not only for FRDA but also for other haploinsufficiency diseases. PMID:27082765

  3. MRP1 and P-glycoprotein expression assays would be useful in the additional detection of treatment non-responders in CML patients without ABL1 mutation.

    PubMed

    Park, Sang Hyuk; Park, Chan-Jeoung; Kim, Dae-Young; Lee, Bo-Ra; Kim, Young Jin; Cho, Young-Uk; Jang, Seongsoo

    2015-10-01

    We evaluated the ability of the rhodamine-123 efflux assay, multidrug resistance-associated protein-1 (MRP1) expression assay and P-glycoprotein (Pgp) expression assay to discriminate chronic myelogenous leukemia (CML) patients who had failed treatment or were at risk of failure. Each assay was performed in blood samples from CML patients (n=224) treated with tyrosine kinase inhibitors, taken at diagnosis (n=14) and follow-up (n=210). Patient samples were categorized as optimal response (n=120), suboptimal response (n=54), and treatment failure (n=36). Treatment-failed patients had a significantly higher MRP1 expression (5.24% vs. 3.54%, P=0.006) and Pgp expression (5.25% vs. 3.48%, P=0.005) than responders. Both MRP1 (%) and Pgp (%) were highly specific (95.2% and 94.5%) and relatively accurate (83.0% and 82.5%) in the detection of treatment non-responders. Of treatment-failed patients, 41.2% had a positive result in at least one assay and of these patients without ABL1 kinase domain mutation, 51.9% were positive in at least one assay. However, the rhodamine-123 efflux assay failed to discriminate two patient groups. Thus, both MRP1 and Pgp expression assays could be useful for additional identification of treatment non-responders in CML patients without ABL1 mutation. PMID:26248945

  4. Development of an AAV9 coding for a 3XFLAG-TALEfrat#8-VP64 able to increase in vivo the human frataxin in YG8R mice

    PubMed Central

    Chapdelaine, P; Gérard, C; Sanchez, N; Cherif, K; Rousseau, J; Ouellet, D L; Jauvin, D; Tremblay, J P

    2016-01-01

    Artificially designed transcription activator-like effector (TALE) proteins fused to a transcription activation domain (TAD), such as VP64, are able to activate specific eukaryotic promoters. They thus provide a good tool for targeted gene regulation as a therapy. However, the efficacy of such an agent in vivo remains to be demonstrated as the majority of studies have been carried out in cell culture. We produced an adeno-associated virus 9 (AAV9) coding for a TALEfrat#8 containing 13 repeat variable diresidues able to bind to the proximal promoter of human frataxin (FXN) gene. This TALEfrat#8 was fused with a 3XFLAG at its N terminal and a VP64 TAD at its C terminal, and driven by a CAG promoter. This AAV9_3XFLAG-TALEfrat#8-VP64 was injected intraperitoneally to 9-day-old and 4-month-old YG8R mice. After 1 month, the heart, muscle and liver were removed and their FXN mRNA and FXN protein were analyzed. The results show that the AAV9_3XFLAG-TALEfrat#8-VP64 increased the FXN mRNA and FXN protein in the three organs studied. These results corroborate our previous in vitro studies in the FRDA human fibroblasts. Our study indicates that an AAV coding for a TALE protein coupled with a TAD may be used to increase gene expression in vivo as a possible treatment not only for FRDA but also for other haploinsufficiency diseases. PMID:27082765

  5. Evolving Catalytic Properties of the MLL Family SET Domain

    PubMed Central

    Zhang, Ying; Mittal, Anshumali; Reid, James; Reich, Stephanie; Gamblin, Steven J.; Wilson, Jon R.

    2015-01-01

    Summary Methylation of histone H3 lysine-4 is a hallmark of chromatin associated with active gene expression. The activity of H3K4-specific modification enzymes, in higher eukaryotes the MLL (or KMT2) family, is tightly regulated. The MLL family has six members, each with a specialized function. All contain a catalytic SET domain that associates with a core multiprotein complex for activation. These SET domains segregate into three classes that correlate with the arrangement of targeting domains that populate the rest of the protein. Here we show that, unlike MLL1, the MLL4 SET domain retains significant activity without the core complex. We also present the crystal structure of an inactive MLL4-tagged SET domain construct and describe conformational changes that account for MLL4 intrinsic activity. Finally, our structure explains how the MLL SET domains are able to add multiple methyl groups to the target lysine, despite having the sequence characteristics of a classical monomethylase. PMID:26320581

  6. Screening retinal transplants with Fourier-domain OCT

    NASA Astrophysics Data System (ADS)

    Rao, Bin

    2009-02-01

    Transplant technologies have been studied for the recovery of vision loss from retinitis pigmentosa (RP) and age-related macular degeneration (AMD). In several rodent retinal degeneration models and in patients, retinal progenitor cells transplanted as layers to the subretinal space have been shown to restore or preserve vision. The methods for evaluation of transplants are expensive considering the large amount of animals. Alternatively, time-domain Stratus OCT was previously shown to be able to image the morphological structure of transplants to some extent, but could not clearly identify laminated transplants. The efficacy of screening retinal transplants with Fourier-domain OCT was studied on 37 S334ter line 3 rats with retinal degeneration 6-67 days after transplant surgery. The transplants were morphologically categorized as no transplant, detachment, rosettes, small laminated area and larger laminated area with both Fourier-domain OCT and histology. The efficacy of Fourier-domain OCT in screening retinal transplants was evaluated by comparing the categorization results with OCT and histology. Additionally, 4 rats were randomly selected for multiple OCT examinations (1, 5, 9, 14 and 21days post surgery) in order to determine the earliest image time of OCT examination since the transplanted tissue may need some time to show its tendency of growing. Finally, we demonstrated the efficacy of Fourier-domain OCT in screening retinal transplants in early stages and determined the earliest imaging time for OCT. Fourier-domain OCT makes itself valuable in saving resource spent on animals with unsuccessful transplants.

  7. 46 CFR 12.05-11 - General provisions respecting merchant mariner's document endorsed for service as able seamen.

    Code of Federal Regulations, 2011 CFR

    2011-10-01

    ... 46 Shipping 1 2011-10-01 2011-10-01 false General provisions respecting merchant mariner's... HOMELAND SECURITY MERCHANT MARINE OFFICERS AND SEAMEN REQUIREMENTS FOR RATING ENDORSEMENTS Able Seamen § 12.05-11 General provisions respecting merchant mariner's document endorsed for service as able...

  8. 46 CFR 12.05-11 - General provisions respecting merchant mariner's document endorsed for service as able seamen.

    Code of Federal Regulations, 2010 CFR

    2010-10-01

    ... 46 Shipping 1 2010-10-01 2010-10-01 false General provisions respecting merchant mariner's... HOMELAND SECURITY MERCHANT MARINE OFFICERS AND SEAMEN REQUIREMENTS FOR RATING ENDORSEMENTS Able Seamen § 12.05-11 General provisions respecting merchant mariner's document endorsed for service as able...

  9. Optimization of novel combi-molecules: identification of balanced and mixed bcr-abl/DNA targeting properties.

    PubMed

    Rachid, Zakaria; Katsoulas, Athanasia; Williams, Christopher; Larroque, Anne-Laure; McNamee, James; Jean-Claude, Bertrand J

    2007-08-01

    Steps toward the identification of combi-molecules with strong abl tyrosine kinase (TK) inhibitory property and significant DNA damaging potential are described. The optimized combi-molecule 13a was shown to induce approximately twofold stronger abl TK inhibitory activity than Gleevec and high levels of DNA damage in chronic myelogenous leukemic cells. PMID:17572088

  10. Meeting the Needs of Your Most Able Pupils: Art. Gifted and Talented Series [with CD-ROM

    ERIC Educational Resources Information Center

    Earle, Kim

    2006-01-01

    "Meeting the Needs of your Most Able Pupils in Art" provides specific guidance on: (1) Recognising High Ability and Multiple Intelligences Planning, (2) Differentiation and Extension; (3) Enrichment in Art Teacher Questioning Skills; (4) Support for More Able Pupils with Learning Difficulties (dyslexics, ADHD, sensory impairment); (5) Homework…

  11. Ontology development for Sufism domain

    NASA Astrophysics Data System (ADS)

    Iqbal, Rizwan

    2012-01-01

    Domain ontology is a descriptive representation of any particular domain which in detail describes the concepts in a domain, the relationships among those concepts and organizes them in a hierarchal manner. It is also defined as a structure of knowledge, used as a means of knowledge sharing to the community. An Important aspect of using ontologies is to make information retrieval more accurate and efficient. Thousands of domain ontologies from all around the world are available online on ontology repositories. Ontology repositories like SWOOGLE currently have over 1000 ontologies covering a wide range of domains. It was found that up to date there was no ontology available covering the domain of "Sufism". This unavailability of "Sufism" domain ontology became a motivation factor for this research. This research came up with a working "Sufism" domain ontology as well a framework, design of the proposed framework focuses on the resolution to problems which were experienced while creating the "Sufism" ontology. The development and working of the "Sufism" domain ontology are covered in detail in this research. The word "Sufism" is a term which refers to Islamic mysticism. One of the reasons to choose "Sufism" for ontology creation is its global curiosity. This research has also managed to create some individuals which inherit the concepts from the "Sufism" ontology. The creation of individuals helps to demonstrate the efficient and precise retrieval of data from the "Sufism" domain ontology. The experiment of creating the "Sufism" domain ontology was carried out on a tool called Protégé. Protégé is a tool which is used for ontology creation, editing and it is open source.

  12. Ontology development for Sufism domain

    NASA Astrophysics Data System (ADS)

    Iqbal, Rizwan

    2011-12-01

    Domain ontology is a descriptive representation of any particular domain which in detail describes the concepts in a domain, the relationships among those concepts and organizes them in a hierarchal manner. It is also defined as a structure of knowledge, used as a means of knowledge sharing to the community. An Important aspect of using ontologies is to make information retrieval more accurate and efficient. Thousands of domain ontologies from all around the world are available online on ontology repositories. Ontology repositories like SWOOGLE currently have over 1000 ontologies covering a wide range of domains. It was found that up to date there was no ontology available covering the domain of "Sufism". This unavailability of "Sufism" domain ontology became a motivation factor for this research. This research came up with a working "Sufism" domain ontology as well a framework, design of the proposed framework focuses on the resolution to problems which were experienced while creating the "Sufism" ontology. The development and working of the "Sufism" domain ontology are covered in detail in this research. The word "Sufism" is a term which refers to Islamic mysticism. One of the reasons to choose "Sufism" for ontology creation is its global curiosity. This research has also managed to create some individuals which inherit the concepts from the "Sufism" ontology. The creation of individuals helps to demonstrate the efficient and precise retrieval of data from the "Sufism" domain ontology. The experiment of creating the "Sufism" domain ontology was carried out on a tool called Protégé. Protégé is a tool which is used for ontology creation, editing and it is open source.

  13. 1958 NASA/USAF Space Probes (Able-1). Volume 1; Summary

    NASA Technical Reports Server (NTRS)

    1959-01-01

    Early in calendar year 1958 Space Technology Laboratories, Inc. (STL) (then Space Technology Laboratories, a division of the Ramo-Wooldridge Corp.) developed for the Air Force Ballistic Missile Division (AFBMD) an Advanced Re-entry Test Vehicle (ARTV) for the purpose of testing ballistic missile nose cones at the full range of 5500 nautical miles. The two-stage ARTV utilized the Thor ballistic missile and the second stage propulsion system developed for the Vanguard program. In late 1957 and early 1958, STL/AFBMD prepared studies of various missile combinations which could be utilized for space testing. The Thor, in combination with the Vanguard second and third stages, was one of the vehicles considered which offered a very early capability of placing a reasonable payload in a lunar orbit. These STL/AFBMD studies were presented to various appropriate groups including the Killian, Millikan, H. J . Stewart Committees; Headquarters, Air Research and Development Command, and ARDC Centers. Subsequently the Advanced Research Projects Agency (ARPA) contacted STL relative to the availability of hardware for an early lunar shot. By utilizing existing spares already purchased for the ARTV, and by making use of the ARTV contractors already in being, it appeared feasible to launch by the third quarter of calendar year 1958 a payload which would be captured by the moon's gravitational force. On 27 March 1958, ARPA directed STL to proceed with a program of three lunar shots. As much as possible, these shots were to utilize existing ARTV spare hardware and impose no interference with the ballistic missile programs. In September this program was transferred to the direction of the National Aeronautics and Space Administration (NASA). On 17 August 1958 the first launching of the Able-1 vehicle was attempted, but the flight was terminated by a propulsion failure of the first stage. Subsequent launchings were attempted on 13 October and 8 November 1958. Of these launchirigs the

  14. Erythrobacter atlanticus sp. nov., a bacterium from ocean sediment able to degrade polycyclic aromatic hydrocarbons.

    PubMed

    Zhuang, Lingping; Liu, Yang; Wang, Lin; Wang, Wanpeng; Shao, Zongze

    2015-10-01

    A Gram-stain-negative, motile, rod-shaped, orange-pigmented bacterium able to degrade polycyclic aromatic hydrocarbons was isolated from deep-sea sediment of the Atlantic Ocean and subjected to a polyphasic taxonomic study. The strain, designated s21-N3T, could grow at 4–37 °C (optimum 28 °C), at pH 5–10 (optimum pH 7–8) and with 1–7 % (w/v) NaCl (optimum 2–3 %). Strain s21-N3T was positive for nitrate reduction, denitrification, aesculin hydrolysis, oxidase and catalase, but negative for indole production and urease. Phylogenetic analyses based on 16S rRNA gene sequences showed that strain s21-N3T formed a distinct branch within the genus Erythrobacter, sharing high similarities with three closely related strains, Erythrobacter marinus HWDM-33T (98.67 %), ‘Erythrobacter luteus’ KA37 (97.80 %) and Erythrobacter gangjinensis K7-2T (97.59 %). The similarities between strain s21-N3T and other type strains of recognized species within the genus Erythrobacter ranged from 95.00 to 96.47 %. The digital DNA–DNA hybridization values and average nucleotide identity (ANI) values between strain s21-N3T and the three closely related strains Erythrobacter marinus HWDM-33T, ‘Erythrobacter luteus’ KA37 and Erythrobacter gangjinensis K7-2T were 18.60, 18.00 and 18.50 % and 74.24, 72.49 and 72.54 %, respectively. The principal fatty acids were summed feature 8 (C18 : 1ω7c/ω6c) and summed feature 3 (C16 : 1ω7c/ω6c). The respiratory lipoquinone was identified as Q-10. The major polar lipids comprised sphingoglycolipid, phosphatidylethanolamine, phosphatidylglycerol, phosphatidylcholine and diphosphatidylglycerol. The G+C content of the chromosomal DNA was determined to be 58.18 mol%. The combined genotypic and phenotypic distinctiveness demonstrated that strain s21-N3T represents a novel species of the genus Erythrobacter, for which the name Erythrobacter atlanticus sp. nov. is proposed, with the type strain s21-N3T (

  15. Expression of Interferon Consensus Sequence Binding Protein (ICSBP) Is Downregulated in Bcr-Abl-Induced Murine Chronic Myelogenous Leukemia-Like Disease, and Forced Coexpression of ICSBP Inhibits Bcr-Abl-Induced Myeloproliferative Disorder

    PubMed Central

    Hao, Sheryl X.; Ren, Ruibao

    2000-01-01

    Chronic myelogenous leukemia (CML) is a clonal myeloproliferative disorder resulting from the neoplastic transformation of a hematopoietic stem cell. The majority of cases of CML are associated with the (9;22) chromosome translocation that generates the bcr-abl chimeric gene. Alpha interferon (IFN-α) treatment induces hematological remission and prolongs life in 75% of CML patients in the chronic phase. It has been shown that mice deficient in interferon consensus sequence binding protein (ICSBP), a member of the interferon regulatory factor family, manifest a CML-like syndrome. We have shown that expression of Bcr-Abl in bone marrow (BM) cells from 5-fluorouracil (5-FU)-treated mice by retroviral transduction efficiently induces a myeloproliferative disease in mice resembling human CML. To directly test whether icsbp can function as a tumor suppressor gene, we examined the effect of ICSBP on Bcr-Abl-induced CML-like disease using this murine model for CML. We found that expression of the ICSBP protein was significantly decreased in Bcr-Abl-induced CML-like disease. Forced coexpression of ICSBP inhibited the Bcr-Abl-induced colony formation of BM cells from 5-FU-treated mice in vitro and Bcr-Abl-induced CML-like disease in vivo. Interestingly, coexpression of ICSBP and Bcr-Abl induced a transient B-lymphoproliferative disorder in the murine model of Bcr-Abl-induced CML-like disease. Overexpression of ICSBP consistently promotes rather than inhibits Bcr-Abl-induced B lymphoproliferation in a murine model where BM cells from non-5-FU-treated donors were used, indicating that ICSBP has a specific antitumor activity toward myeloid neoplasms. We also found that overexpression of ICSBP negatively regulated normal hematopoiesis. These data provide direct evidence that ICSBP can act as a tumor suppressor that regulates normal and neoplastic proliferation of hematopoietic cells. PMID:10648600

  16. Thundercloud: Domain specific information security training for the smart grid

    NASA Astrophysics Data System (ADS)

    Stites, Joseph

    In this paper, we describe a cloud-based virtual smart grid test bed: ThunderCloud, which is intended to be used for domain-specific security training applicable to the smart grid environment. The test bed consists of virtual machines connected using a virtual internal network. ThunderCloud is remotely accessible, allowing students to undergo educational exercises online. We also describe a series of practical exercises that we have developed for providing the domain-specific training using ThunderCloud. The training exercises and attacks are designed to be realistic and to reflect known vulnerabilities and attacks reported in the smart grid environment. We were able to use ThunderCloud to offer practical domain-specific security training for smart grid environment to computer science students at little or no cost to the department and no risk to any real networks or systems.

  17. The Caenorhabditis elegans gene unc-89, required fpr muscle M-line assembly, encodes a giant modular protein composed of Ig and signal transduction domains

    PubMed Central

    1996-01-01

    Mutations in the Caenorhabditis elegans gene unc-89 result in nematodes having disorganized muscle structure in which thick filaments are not organized into A-bands, and there are no M-lines. Beginning with a partial cDNA from the C. elegans sequencing project, we have cloned and sequenced the unc-89 gene. An unc-89 allele, st515, was found to contain an 84-bp deletion and a 10-bp duplication, resulting in an in- frame stop codon within predicted unc-89 coding sequence. Analysis of the complete coding sequence for unc-89 predicts a novel 6,632 amino acid polypeptide consisting of sequence motifs which have been implicated in protein-protein interactions. UNC-89 begins with 67 residues of unique sequences, SH3, dbl/CDC24, and PH domains, 7 immunoglobulins (Ig) domains, a putative KSP-containing multiphosphorylation domain, and ends with 46 Ig domains. A polyclonal antiserum raised to a portion of unc-89 encoded sequence reacts to a twitchin-sized polypeptide from wild type, but truncated polypeptides from st515 and from the amber allele e2338. By immunofluorescent microscopy, this antiserum localizes to the middle of A-bands, consistent with UNC-89 being a structural component of the M-line. Previous studies indicate that myofilament lattice assembly begins with positional cues laid down in the basement membrane and muscle cell membrane. We propose that the intracellular protein UNC-89 responds to these signals, localizes, and then participates in assembling an M-line. PMID:8603916

  18. Fractional diffusion on bounded domains

    DOE PAGESBeta

    Defterli, Ozlem; D'Elia, Marta; Du, Qiang; Gunzburger, Max Donald; Lehoucq, Richard B.; Meerschaert, Mark M.

    2015-03-13

    We found that the mathematically correct specification of a fractional differential equation on a bounded domain requires specification of appropriate boundary conditions, or their fractional analogue. In this paper we discuss the application of nonlocal diffusion theory to specify well-posed fractional diffusion equations on bounded domains.

  19. Polarized domains of myelinated axons.

    PubMed

    Salzer, James L

    2003-10-01

    The entire length of myelinated axons is organized into a series of polarized domains that center around nodes of Ranvier. These domains, which are crucial for normal saltatory conduction, consist of distinct multiprotein complexes of cell adhesion molecules, ion channels, and scaffolding molecules; they also differ in their diameter, organelle content, and rates of axonal transport. Juxtacrine signals from myelinating glia direct their sequential assembly. The composition, mechanisms of assembly, and function of these molecular domains will be reviewed. I also discuss similarities of this domain organization to that of polarized epithelia and present emerging evidence that disorders of domain organization and function contribute to the axonopathies of myelin and other neurologic disorders. PMID:14556710

  20. Structural domains of vault proteins: a role for the coiled coil domain in vault assembly.

    PubMed

    van Zon, Arend; Mossink, Marieke H; Schoester, Martijn; Scheffer, George L; Scheper, Rik J; Sonneveld, Pieter; Wiemer, Erik A C

    2002-03-01

    Vaults consist of multiple copies of three proteins (MVP, VPARP, and TEP1) and several untranslated RNAs. The function of vaults is unknown but the typical and evolutionary conserved structure indicates a role in intracellular transport. Although all vault components have been identified and characterized, not much is known about vault protein assembly. In this study we identified and analyzed structural domains involved in vault assembly with emphasis on protein-protein interactions. Using a yeast two-hybrid system, we demonstrate within MVP an intramolecular binding site and show that MVP molecules interact with each other via their coiled coil domain. We show that purified MVP is able to bind calcium, most likely at calcium-binding EF-hands. No interactions could be detected between TEP1 and other vault proteins. However, the N-terminal half of MVP binds to a specific domain in the C-terminus of VPARP. Furthermore, VPARP contains amino acid stretches mediating intramolecular binding. PMID:11855821