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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. 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

  4. Vesicle uncoating regulated by SH3-SH3 domain-mediated complex formation between endophilin and intersectin at synapses

    PubMed Central

    Pechstein, Arndt; Gerth, Fabian; Milosevic, Ira; Jäpel, Maria; Eichhorn-Grünig, Marielle; Vorontsova, Olga; Bacetic, Jelena; Maritzen, Tanja; Shupliakov, Oleg; Freund, Christian; Haucke, Volker

    2015-01-01

    Neurotransmission involves the exo-endocytic cycling of synaptic vesicle (SV) membranes. Endocytic membrane retrieval and clathrin-mediated SV reformation require curvature-sensing and membrane-bending BAR domain proteins such as endophilin A. While their ability to sense and stabilize curved membranes facilitates membrane recruitment of BAR domain proteins, the precise mechanisms by which they are targeted to specific sites of SV recycling has remained unclear. Here, we demonstrate that the multi-domain scaffold intersectin 1 directly associates with endophilin A to facilitate vesicle uncoating at synapses. Knockout mice deficient in intersectin 1 accumulate clathrin-coated vesicles at synapses, a phenotype akin to loss of endophilin function. Intersectin 1/endophilin A1 complex formation is mediated by direct binding of the SH3B domain of intersectin to a non-canonical site on the SH3 domain of endophilin A1. Consistent with this, intersectin-binding defective mutant endophilin A1 fails to rescue clathrin accumulation at neuronal synapses derived from endophilin A1-3 triple knockout (TKO) mice. Our data support a model in which intersectin aids endophilin A recruitment to sites of clathrin-mediated SV recycling, thereby facilitating vesicle uncoating. PMID:25520322

  5. 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

  6. 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

  7. 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.

  8. 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

  9. The SH3 Domain Acts as a Scaffold for the N-Terminal Intrinsically Disordered Regions of c-Src.

    PubMed

    Maffei, Mariano; Arbesú, Miguel; Le Roux, Anabel-Lise; Amata, Irene; Roche, Serge; Pons, Miquel

    2015-05-01

    Regulation of c-Src activity by the intrinsically disordered Unique domain has recently been demonstrated. However, its connection with the classical regulatory mechanisms is still missing. Here we show that the Unique domain is part of a long loop closed by the interaction of the SH4 and SH3 domains. The conformational freedom of the Unique domain is further restricted through direct contacts with SH3 that are allosterically modulated by binding of a poly-proline ligand in the presence and in the absence of lipids. Our results highlight the scaffolding role of the SH3 domain for the c-Src N-terminal intrinsically disordered regions and suggest a connection between the regulatory mechanisms involving the SH3 and Unique domains.

  10. 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

  11. 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

  12. 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

  13. 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

  14. SETA: a novel SH3 domain-containing adapter molecule associated with malignancy in astrocytes.

    PubMed Central

    Bögler, O.; Furnari, F. B.; Kindler-Roehrborn, A.; Sykes, V. W.; Yung, R.; Huang, H. J.; Cavenee, W. K.

    2000-01-01

    Differential display polymerase chain reaction analysis was used to compare five differentiation states of the O-2A progenitor-like cell line CG4: progenitor cells and cells at 12 h or 4 days after the induction of differentiation into oligodendrocytes or astrocytes. This led to the identification of 52 sequence tags that were expressed differentially with cellular phenotype. One sequence was upregulated during differentiation of CG4 cells and represented a novel gene that we named SETA (SH3 domain-containing gene expressed in tumorigenic astrocytes). This gene encodes an SH3 domain-containing adapter protein with sequence similarity to the CD2AP (CD2 adapter protein) and CMS (Cas ligand with multiple Src homology) genes. SETA mRNA was expressed at high levels in the developing rat brain but was barely detectable in the normal adult rat or human brain. However, SETA mRNA was found in approximately one half of the human gliomas tested, including astrocytomas grades II, III, and IV, as well as oligodendrogliomas, mixed oligoastrocytomas, and human glioma-derived cell lines. A rat glioma generated by treatment with the alkylating carcinogen ethylnitrosourea on postnatal day 1 and a derived cell line also expressed SETA mRNA. Furthermore, in an in vitro model of astrocytoma progression based on p53-/- astrocytes, expression of SETA was restricted to cells that are tumorigenic. PMID:11302255

  15. Structural investigation of the binding of a herpesviral protein to the SH3 domain of tyrosine kinase Lck.

    PubMed

    Schweimer, Kristian; Hoffmann, Silke; Bauer, Finn; Friedrich, Ute; Kardinal, Christian; Feller, Stephan M; Biesinger, Brigitte; Sticht, Heinrich

    2002-04-23

    Herpesvirus saimiri codes for a tyrosine kinase interacting protein (Tip) that interacts with both the SH3 domain and the kinase domain of the T-cell-specific tyrosine kinase Lck via two separate motifs. The activation of Lck by Tip is considered as a key event in the transformation of human T-lymphocytes during herpesviral infection. We investigated the interaction of proline-rich Tip peptides with the LckSH3 domain starting with the structural characterization of the unbound interaction partners. The solution structure of the LckSH3 was determined by heteronuclear multidimensional nuclear magnetic resonance (NMR) spectroscopy using 44 residual dipolar couplings in addition to the conventional experimental restraints. Circular dichroism spectroscopy proved that the polyproline helix of Tip is already formed prior to SH3 binding and is conformationally stable. NMR titration experiments point out three major regions of the Tip-Lck interaction comprising the RT loop, the n-src loop, and a helical turn preceding the last strand of the beta-sheet. Further changes of the chemical shifts were observed for the N- and C-terminal beta-strands of the SH3 domain, indicating additional contacts outside the proline-rich segment or subtle structural rearrangements transmitted from the binding site of the proline helix. Fluorescence spectroscopy shows that Tip binds to the SH3 domains of several Src kinases (Lck, Hck, Lyn, Src, Fyn, Yes), exhibiting the highest affinities for Lyn, Hck, and Lck.

  16. Intramolecular interaction in the tail of Acanthamoeba myosin IC between the SH3 domain and a putative pleckstrin homology domain

    PubMed Central

    Hwang, Kae-Jung; Mahmoodian, Fatemeh; Ferretti, James A.; Korn, Edward D.; Gruschus, James M.

    2007-01-01

    The 466-aa tail of the heavy chain of Acanthamoeba myosin IC (AMIC) comprises an N-terminal 220-residue basic region (BR) followed by a 56-residue Gly/Pro/Ala-rich region (GPA1), a 55-residue Src homology 3 (SH3) domain, and a C-terminal 135-residue Gly/Pro/Ala-rich region (GPA2). Cryo-electron microscopy of AMIC had shown previously that the AMIC tail is folded back on itself, suggesting the possibility of interactions between its N- and C-terminal regions. We now show specific differences between the NMR spectrum of bacterially expressed full-length tail and the sum of the spectra of individually expressed BR and GPA1-SH3-GPA2 (GSG) regions. These results are indicative of interactions between the two subdomains in the full-length tail. From the NMR data, we could assign many of the residues in BR and GSG that are involved in these interactions. By combining homology modeling with the NMR data, we identify a putative pleckstrin homology (PH) domain within BR, and show that the PH domain interacts with the SH3 domain. PMID:17215368

  17. Recognition of lysine-rich peptide ligands by murine cortactin SH3 domain: CD, ITC, and NMR studies.

    PubMed

    Rubini, Chiara; Ruzza, Paolo; Spaller, Mark R; Siligardi, Giuliano; Hussain, Rohanah; Udugamasooriya, D Gomika; Bellanda, Massimo; Mammi, Stefano; Borgogno, Andrea; Calderan, Andrea; Cesaro, Luca; Brunati, Anna M; Donella-Deana, Arianna

    2010-01-01

    Cortactin is a ubiquitous actin-binding protein that regulates various aspects of cell dynamics and is implicated in the pathogenesis of human neoplasia. The sequence of cortactin contains a number of signaling motifs and an SH3 domain at the C-terminus, which mediates the interaction of the protein with several partners, including Shank2. A recombinant protein, comprising the murine cortactin SH3 domain fused to GST (GST-SH3(m-cort)), was prepared and used to assess the domain-binding affinity of potential peptide-ligands reproducing the proline-rich regions of human HPK1 and Shank2 proteins. The key residues involved in the SH3(m-cort) domain recognition were identified by three different approaches: non-immobilized ligand interaction assay by circular dichroism, isothermal titration calorimetry, and nuclear magnetic resonance. Our results show that the classical PxxPxK class II binding motif is not sufficient to mediate the interaction with GST-SH3(m-cort), an event that depends on the presence of additional basic residues located at either the N- or the C-terminus of the PxxPxK motif. Especially effective in promoting the peptide binding is a Lys residue at the -5 position, a determinant present in both P2 (HPK1 394-403) and S1 (Shank2 1168-1189) peptides. GST-SH3(m-cort) exhibits the highest affinity toward peptide S1, which contains additional Lys residues at the -3, -5, and -7 positions, indicating that the optimal consensus motif may be KPPxPxKxKxK. These results are supported by the in silico models of SH3(m-cort) complexed with P2 or S1, which highlight the domain residues that interact with the recognition determinants of the peptide-ligand and cooperate in binding stabilization.

  18. X-ray structure of the SH3 domain of the phosphoinositide 3-kinase p85β subunit

    PubMed Central

    Chen, Shuai; Xiao, Yibei; Ponnusamy, Rajesh; Tan, Jinzhi; Lei, Jian; Hilgenfeld, Rolf

    2011-01-01

    Src-homology 3 (SH3) domains are involved in extensive protein–protein interactions and constitute key elements of intracellular signal transduction. Three-dimensional structures have been reported for SH3 domains of various proteins, including the 85 kDa regulatory subunit (p85) of phosphoinositide 3-­kinase. However, all of the latter structures are of p85 isoform α and no crystal structure of the SH3 domain of the equally important isoform β has been reported to date. In this structural communication, the recombinant production, crystallization and X-ray structure determination at 2.0 Å resolution of the SH3 domain of human p85β is described. The structure reveals a compact β-barrel fold very similar to that of p85α. However, binding studies with two classes of proline-rich ligand peptides demonstrate that the ligand-binding specificity differs slightly between the SH3 domains of human p85β and p85α, despite their high structural similarity. PMID:22102226

  19. Autoinhibition of GEF activity in intersectin 1 is mediated by the short SH3-DH domain linker

    PubMed Central

    Kintscher, Carsten; Wuertenberger, Silvia; Eylenstein, Roy; Uhlendorf, Theresia; Groemping, Yvonne

    2010-01-01

    Intersectin 1L (ITSN1L) acts as a specific guanine nucleotide exchange factor (GEF) for the small guanine nucleotide binding protein Cdc42 via its C-terminal DH domain. Interestingly, constructs of ITSN1L that comprise additional domains, for instance the five SH3 domains amino-terminal of the DH domain, were shown to be inhibited in their exchange factor activity. Here, we investigate the inhibitory mechanism of ITSN1L in detail and identify a novel short amino acid motif which mediates autoinhibition. We found this motif to be located in the linker region between the SH3 domains and the DH domain, and we show that within this motif W1221 acts as key residue in establishing the inhibitory interaction. This assigns ITSN1L to a growing class of GEFs that are regulated by a short amino acid motif inhibiting GEF activity by an intramolecular interaction. Moreover, we quantify the interaction between the ITSN1L SH3 domains and the Cdc42 effector N-WASP using fluorescence anisotropy binding experiments. As the SH3 domains are not involved in autoinhibition, binding of N-WASP does not release inhibition of nucleotide exchange activity in kinetic experiments, in contrast to earlier observations. PMID:20842712

  20. 3BP-1, an SH3 domain binding protein, has GAP activity for Rac and inhibits growth factor-induced membrane ruffling in fibroblasts.

    PubMed Central

    Cicchetti, P; Ridley, A J; Zheng, Y; Cerione, R A; Baltimore, D

    1995-01-01

    The SH3 binding protein, 3BP-1, was originally cloned as a partial cDNA from an expression library using the Abl SH3 domain as a probe. In addition to an SH3 binding domain, 3BP-1 displayed homology to a class of GTPase activating proteins (GAPs) active against Rac and Rho proteins. We report here a full length cDNA of 3BP-1 which extends the homology to GAP proteins previously noted. 3BP-1 functions in vitro as a GAP with a specificity for Rac-related G proteins. Microinjection of the 3BP-1 protein into serum-starved fibroblasts produces an inhibition of platelet-derived growth factor (PDGF)-induced membrane ruffling mediated by Rac. Co-injection of 3BP-1 with an activated Rac mutant that is unresponsive to GAPs, counter-acts this inhibition. 3BP-1 does not show in vitro activity towards Rho and, in agreement with this finding, microinjection of 3BP-1 into fibroblasts has no effect on lysophosphatidic acid (LPA)-induced stress fiber assembly mediated by Rho. Thus 3BP-1 is a new and specific Rac GAP that can act in cells to counter Rac-mediated membrane ruffling. How its SH3 binding site interacts with its GAP activity remains to be understood. Images PMID:7621827

  1. 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.

  2. Muscarinic receptors transform NIH 3T3 cells through a Ras-dependent signalling pathway inhibited by the Ras-GTPase-activating protein SH3 domain.

    PubMed Central

    Mattingly, R R; Sorisky, A; Brann, M R; Macara, I G

    1994-01-01

    Expression of certain subtypes of human muscarinic receptors in NIH 3T3 cells provides an agonist-dependent model of cellular transformation by formation of foci in response to carbachol. Although focus formation correlates with the ability of the muscarinic receptors to activate phospholipase C, the actual mitogenic signal transduction pathway is unknown. Through cotransfection experiments and measurement of the activation state of native and epitope-tagged Ras proteins, the contributions of Ras and Ras GTPase-activating protein (Ras-GAP) to muscarinic receptor-dependent transformation were defined. Transforming muscarinic receptors were able to activate Ras, and such activation was required for transformation because focus formation was inhibited by coexpression of either Ras with a dominant-negative mutation or constructs of Ras-GAP that include the catalytic domain. Coexpression of the N-terminal region of GAP or of its isolated SH3 (Src homology 3) domain, but not its SH2 domain, was also sufficient to suppress muscarinic receptor-dependent focus formation. Point mutations at conserved residues in the Ras-GAP SH3 domain reversed its action, leading to an increase in carbachol-dependent transformation. The inhibitory effect of expression of the Ras-GAP SH3 domain occurs proximal to Ras activation and is selective for the mitogenic pathway activated by carbachol, as cellular transformation by either v-Ras or trkA/nerve growth factor is unaffected. Images PMID:7969134

  3. Design of peptoid analogue dimers and measure of their affinity for Grb2 SH3 domains.

    PubMed

    Vidal, M; Liu, W-Q; Lenoir, C; Salzmann, J; Gresh, N; Garbay, C

    2004-06-15

    This paper describes the design of the highest affinity ligands for Grb2 SH3 domains reported so far. These compounds were designed by combining N-alkyl amino acid incorporation in a proline-rich sequence with subsequent dimerization of the peptoid sequence based on structural data and molecular modeling. Optimization of the linker size is discussed, and the N-alkyl amino acid incorporation into both monomeric halves is reported. Because the affinity for Grb2 of the optimized compounds was too high to be measured using the fluorescent modifications that they induce on the Grb2 emission spectrum, a competition assay was developed. In this test, Grb2 is pulled down from a cellular extract by the initial VPPPVPPRRR peptide bound to Sepharose beads. In the presence of competitors, the test quantifies the amount of Grb2 displaced from the beads. It has enabled us to determine a K(i) value in the 10(-10) M range for the highest affinity Grb2 peptoid analogue dimer.

  4. 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.

  5. 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

  6. 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

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

    DOE PAGES

    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

  8. High-resolution structure of an α-spectrin SH3-domain mutant with a redesigned hydrophobic core

    PubMed Central

    Cámara-Artigas, Ana; Andújar-Sánchez, Monserrat; Ortiz-Salmerón, Emilia; Cuadri, Celia; Cobos, Eva S.; Martin-Garcia, Jose Manuel

    2010-01-01

    The α-spectrin SH3 domain (Spc-SH3) is a small modular domain which has been broadly used as a model protein in folding studies and these studies have sometimes been supported by structural information obtained from the coordinates of Spc-SH3 mutants. The structure of B5/D48G, a multiple mutant designed to improve the hydrophobic core and as a consequence the protein stability, has been solved at 1 Å resolution. The crystals belonged to the orthorhombic space group P212121, with unit-cell parameters a = 24.79, b = 37.23, c = 62.95 Å. This mutant also bears a D48G substitution in the distal loop and this mutation has also been reported to increase the stability of the protein by itself. The structure of the B5/D48G mutant shows a highly packed hydrophobic core and a more ordered distal loop compared with previous Spc-SH3 structures. PMID:20823517

  9. 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

  10. Mutational analysis of the Src SH3 domain: the same residues of the ligand binding surface are important for intra- and intermolecular interactions.

    PubMed Central

    Erpel, T; Superti-Furga, G; Courtneidge, S A

    1995-01-01

    The protein tyrosine kinase c-Src is negatively regulated by phosphorylation of Tyr527 in its C-terminal tail. The repressed state is achieved through intramolecular interactions involving the phosphorylated tail, the Src homology 2 (SH2) domain and the SH3 domain. Both the SH2 and SH3 domains have also been shown to mediate the intermolecular interaction of Src with several proteins. To test which amino acids of the Src SH3 domain are important for these interactions, and whether the intra- and intermolecular associations involve the same residues, we carried out a detailed mutational analysis of the presumptive interaction surface. All mutations of conserved hydrophobic residues had an effect on both inter- and intramolecular interactions of the Src SH3 domain, although not all amino acids were equally important. Chimeric molecules in which the Src SH3 domain was replaced with those of spectrin or Lck showed derepressed kinase activity, whereas a chimera containing the Fyn SH3 domain was fully regulated. Since spectrin and Lck SH3 domains share the conserved hydrophobic residues characteristic of SH3 domains, other amino acids must be important for specificity. Mutational analysis of non- or semi-conserved residues in the RT and n-Src loops showed that some of these were also involved in inter- and intramolecular interactions. Stable transfection of selected SH3 domain mutants into NIH-3T3 cells showed that despite elevated levels of phosphotyrosine, the cells were morphologically normal, indicating that the SH3 domain was required for efficient transformation of NIH-3T3 cells by Src. Images PMID:7534229

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

    PubMed

    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

    2013-12-01

    Nemaline myopathy (NM) is a congenital myopathy with an estimated incidence of 150,000 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

  12. Evidence for physical and functional interactions among two Saccharomyces cerevisiae SH3 domain proteins, an adenylyl cyclase-associated protein and the actin cytoskeleton.

    PubMed Central

    Lila, T; Drubin, D G

    1997-01-01

    In a variety of organisms, a number of proteins associated with the cortical actin cytoskeleton contain SH3 domains, suggesting that these domains may provide the physical basis for functional interactions among structural and regulatory proteins in the actin cytoskeleton. We present evidence that SH3 domains mediate at least two independent functions of the Saccharomyces cerevisiae actin-binding protein Abp1p in vivo. Abp1p contains a single SH3 domain that has recently been shown to bind in vitro to the adenylyl cyclase-associated protein Srv2p. Immunofluorescence analysis of Srv2p subcellular localization in strains carrying mutations in either ABP1 or SRV2 reveals that the Abp1p SH3 domain mediates the normal association of Srv2p with the cortical actin cytoskeleton. We also show that a site in Abp1p itself is specifically bound by the SH3 domain of the actin-associated protein Rvs167p. Genetic analysis provides evidence that Abp1p and Rvs167p have functions that are closely interrelated. Abp1 null mutations, like rvs167 mutations, result in defects in sporulation and reduced viability under certain suboptimal growth conditions. In addition, mutations in ABP1 and RVS167 yield similar profiles of genetic "synthetic lethal" interactions when combined with mutations in genes encoding other cytoskeletal components. Mutations which specifically disrupt the SH3 domain-mediated interaction between Abp1p and Srv2p, however, show none of the shared phenotypes of abp1 and rvs167 mutations. We conclude that the Abp1p SH3 domain mediates the association of Srv2p with the cortical actin cytoskeleton, and that Abp1p performs a distinct function that is likely to involve binding by the Rvs167p SH3 domain. Overall, work presented here illustrates how SH3 domains can integrate the activities of multiple actin cytoskeleton proteins in response to varying environmental conditions. Images PMID:9190214

  13. The auto-inhibitory state of Rho guanine nucleotide exchange factor ARHGEF5/TIM can be relieved by targeting its SH3 domain with rationally designed peptide aptamers.

    PubMed

    He, Ping; Tan, De-Li; Liu, Hong-Xiang; Lv, Feng-Lin; Wu, Wei

    2015-04-01

    The short isoform of Rho guanine nucleotide exchange factor ARHGEF5 is known as TIM, which plays diverse roles in, for example, tumorigenesis, neuronal development and Src-induced podosome formation through the activation of its substrates, the Rho family of GTPases. The activation is auto-inhibited by a putative helix N-terminal to the DH domain of TIM, which is stabilized by the intramolecular interaction of C-terminal SH3 domain with a poly-proline sequence between the putative helix and the DH domain. In this study, we systematically investigated the structural basis, energetic landscape and biological implication underlying TIM auto-inhibition by using atomistic molecular dynamics simulations and binding free energy analysis. The computational study revealed that the binding of SH3 domain to poly-proline sequence is the prerequisite for the stabilization of TIM auto-inhibition. Thus, it is suggested that targeting SH3 domain with competitors of the poly-proline sequence would be a promising strategy to relieve the auto-inhibitory state of TIM. In this consideration, we rationally designed a number of peptide aptamers for competitively inhibiting the SH3 domain based on modeled TIM structure and computationally generated data. Peptide binding test and guanine nucleotide exchange analysis solidified that these designed peptides can both bind to the SH3 domain potently and activate TIM-catalyzed RhoA exchange reaction effectively. Interestingly, a positive correlation between the peptide affinity and induced exchange activity was observed. In addition, separate mutation of three conserved residues Pro49, Pro52 and Lys54 - they are required for peptide recognition by SH3 domain -- in a designed peptide to Ala would completely abolish the capability of this peptide activating TIM. All these come together to suggest an intrinsic relationship between peptide binding to SH3 domain and the activation of TIM.

  14. The redundancy of NMR restraints can be used to accelerate the unfolding behavior of an SH3 domain during molecular dynamics simulations

    PubMed Central

    2011-01-01

    1 Abstract Background The simulation of protein unfolding usually requires recording long molecular dynamics trajectories. The present work aims to figure out whether NMR restraints data can be used to probe protein conformations in order to accelerate the unfolding simulation. The SH3 domain of nephrocystine (nph SH3) was shown by NMR to be destabilized by point mutations, and was thus chosen to illustrate the proposed method. Results The NMR restraints observed on the WT nph SH3 domain were sorted from the least redundant to the most redundant ones. Protein NMR conformations were then calculated with: (i) the set full including all NMR restraints measured on nph SH3, (ii) the set reduced where the least redundant restraints with respect to the set full were removed, (iii) the sets random where randomly picked-up restraints were removed. From each set of conformations, we recorded series of 5-ns MD trajectories. The β barrel architecture of nph SH3 in the trajectories starting from sets (i) and (iii) appears to be stable. On the contrary, on trajectories based on the set (ii), a displacement of the hydrophobic core residues and a variation of the β barrel inner cavity profile were observed. The overall nph SH3 destabilization agrees with previous experimental and simulation observations made on other SH3 domains. The destabilizing effect of mutations was also found to be enhanced by the removal of the least redundant restraints. Conclusions We conclude that the NMR restraint redundancy is connected to the instability of the SH3 nph domain. This restraint redundancy generalizes the contact order parameter, which is calculated from the contact map of a folded protein and was shown in the literature to be correlated to the protein folding rate. The relationship between the NMR restraint redundancy and the protein folding is also reminiscent of the previous use of the Gaussian Network Model to predict protein folding parameters. PMID:22115427

  15. Distinct and opposite roles for SH2 and SH3 domains of v-src in embryo survival and hemangiosarcoma formation.

    PubMed

    Morgan, John C; Majors, John E; Galileo, Deni S

    2005-01-01

    The cellular proto-oncogene c-src is thought to be involved in formation, progression, and metastasis of a variety of tumor cell types, although its exact role during tumor cell genesis is not well defined. v-src, the viral oncogene counterpart of c-src, causes metastatic sarcomas, hemorrhagic disease, and hemangiosarcomas in chicken embryos and, thus, can be used as a constitutively activated form of src for experimentally-induced tumorigenesis. Here, we used retroviral vectors to express wild-type v-src or SH2 or SH3 domain-deleted forms (DeltaSH2 or DeltaSH3) to determine if different pathogenic effects resulted. Vectors were injected into early chick embryo midbrain ventricles and embryos were sacrificed at various ages up to embryonic day (E) 18. Retroviral expression of all forms of v-src resulted in transformation of pial connective tissue cells into large, rounded abnormal-appearing cells. Surprisingly, all forms of v-src were lethal. The v-src retrovirus was lethal and killed most embryos by E15 with the development of hemangiosarcomas over the injection site between E10-E12. The DeltaSH3 retrovirus was the most deadly, killing most embryos by E12, however, it never resulted in hemangiosarcoma formation. The DeltaSH2 retrovirus injected embryos survived longer than v-src or DeltaSH3 embryos, and some of these embryos also developed large hemangiosarcomas over the injection site between E13 and E18. These results demonstrate that the src SH2 domain is required to be fully lethal, whereas the presence of the SH3 domain attenuated lethality. Furthermore, the formation of hemangiosarcomas absolutely required the presence of the src SH3 domain and to some extent required the SH2 domain. This implicates distinct and opposite roles for SH2 and SH3 domains of src and their cellular binding partners in tumorigenesis and hemorrhagic disease.

  16. Regions outside of conserved PxxPxR motifs drive the high affinity interaction of GRB2 with SH3 domain ligands.

    PubMed

    Bartelt, Rebekah R; Light, Jonathan; Vacaflores, Aldo; Butcher, Alayna; Pandian, Madhana; Nash, Piers; Houtman, Jon C D

    2015-10-01

    SH3 domains are evolutionarily conserved protein interaction domains that control nearly all cellular processes in eukaryotes. The current model is that most SH3 domains bind discreet PxxPxR motifs with weak affinity and relatively low selectivity. However, the interactions of full-length SH3 domain-containing proteins with ligands are highly specific and have much stronger affinity. This suggests that regions outside of PxxPxR motifs drive these interactions. In this study, we observed that PxxPxR motifs were required for the binding of the adaptor protein GRB2 to short peptides from its ligand SOS1. Surprisingly, PxxPxR motifs from the proline rich region of SOS1 or CBL were neither necessary nor sufficient for the in vitro or in vivo interaction with full-length GRB2. Together, our findings show that regions outside of the consensus PxxPxR sites drive the high affinity association of GRB2 with SH3 domain ligands, suggesting that the binding mechanism for this and other SH3 domain interactions may be more complex than originally thought.

  17. Intracellular cytoplasm-specific delivery of SH3 and SH2 domains of SLAP inhibits TcR-mediated signaling.

    PubMed

    Kim, Jung-Ho; Moon, Jae-Seung; Yu, JiSang; Lee, Sang-Kyou

    2015-05-01

    Signaling events triggered by T cell receptor (TcR) stimulation are important targets for the development of common therapeutics for various autoimmune diseases. SLAP is a negative regulator of TcR-mediated signaling cascade via targeting TcR zeta chain for degradation through recruiting the ubiquitin ligase c-Cbl. In this study, we generated a transducible form of SH3 and SH2 domains of SLAP (ctSLAPΔC) which can be specifically targeted to the cytoplasm of a cell. ctSLAPΔC inhibited tyrosine phosphorylation of signaling mediators such as ZAP-70 and LAT involved in T cell activation, and effectively suppressed transcriptional activity of NFAT and NFκB upon TcR stimulation. The transduced ctSLAPΔC in T cells blocked the secretion of T cell-specific cytokines such as IL-2, IFNγ, IL-17A, and IL-4 and induced the expression of CD69 and CD25 on effector T cells without influencing the cell viability. Inhibition of TcR-mediated signaling via SLAP blocked the differentiation of naïve T cells into Th1, Th2 or Treg cells with different sensitivity, suggesting that qualitative and quantitative intensity of TcR-mediated signaling in the context of polarizing cytokines environment may be a critical factor to determine the differentiation fate of naïve T cells. These results suggest that cytoplasm-specific transduction of the SH3 and SH2 domains of SLAP has a therapeutic potential of being an immunosuppressive reagent for the treatment of various autoimmune diseases. PMID:25800872

  18. Intracellular cytoplasm-specific delivery of SH3 and SH2 domains of SLAP inhibits TcR-mediated signaling.

    PubMed

    Kim, Jung-Ho; Moon, Jae-Seung; Yu, JiSang; Lee, Sang-Kyou

    2015-05-01

    Signaling events triggered by T cell receptor (TcR) stimulation are important targets for the development of common therapeutics for various autoimmune diseases. SLAP is a negative regulator of TcR-mediated signaling cascade via targeting TcR zeta chain for degradation through recruiting the ubiquitin ligase c-Cbl. In this study, we generated a transducible form of SH3 and SH2 domains of SLAP (ctSLAPΔC) which can be specifically targeted to the cytoplasm of a cell. ctSLAPΔC inhibited tyrosine phosphorylation of signaling mediators such as ZAP-70 and LAT involved in T cell activation, and effectively suppressed transcriptional activity of NFAT and NFκB upon TcR stimulation. The transduced ctSLAPΔC in T cells blocked the secretion of T cell-specific cytokines such as IL-2, IFNγ, IL-17A, and IL-4 and induced the expression of CD69 and CD25 on effector T cells without influencing the cell viability. Inhibition of TcR-mediated signaling via SLAP blocked the differentiation of naïve T cells into Th1, Th2 or Treg cells with different sensitivity, suggesting that qualitative and quantitative intensity of TcR-mediated signaling in the context of polarizing cytokines environment may be a critical factor to determine the differentiation fate of naïve T cells. These results suggest that cytoplasm-specific transduction of the SH3 and SH2 domains of SLAP has a therapeutic potential of being an immunosuppressive reagent for the treatment of various autoimmune diseases.

  19. Evidence for an Interaction between the SH3 Domain and the N-terminal Extension of the Essential Light Chain in Class II Myosins

    PubMed Central

    Lowey, Susan; Saraswat, Lakshmi D.; Liu, HongJun; Volkmann, Niels; Hanein, Dorit

    2009-01-01

    SUMMARY The function of the src-homology 3 (SH3) domain in class II myosins, a distinct β-barrel structure, remains unknown. Here we provide evidence, using electron cryomicroscopy, in conjunction with light scattering, fluorescence and kinetic analyses, that the SH3 domain facilitates the binding of the N-terminal extension of the essential light chain isoform (ELC-1) to actin. The 41-residue extension contains four conserved lysines followed by a repeating sequence of seven Pro/Ala residues. It is widely believed that the highly charged region interacts with actin, while the Pro/Ala-rich sequence forms a rigid tether that bridges the ~9 nm distance between the myosin lever arm and the thin filament. In order to localize the N-terminus of ELC in the actomyosin complex, an engineered Cys was reacted with undecagold-maleimide, and the labeled ELC was exchanged into myosin subfragment-1 (S1). Electron cryomicroscopy of S1-bound actin filaments, together with computer-based docking of the skeletal S1 crystal structure into 3D reconstructions, showed a well-defined peak for the gold cluster near the SH3 domain. Given that SH3 domains are known to bind proline-rich ligands, we suggest that the N-terminal extension of ELC interacts with actin and modulates myosin kinetics by binding to the SH3 domain during the ATPase cycle. PMID:17597155

  20. Structural Basis of the High Affinity Interaction between the Alphavirus Nonstructural Protein-3 (nsP3) and the SH3 Domain of Amphiphysin-2.

    PubMed

    Tossavainen, Helena; Aitio, Olli; Hellman, Maarit; Saksela, Kalle; Permi, Perttu

    2016-07-29

    We show that a peptide from Chikungunya virus nsP3 protein spanning residues 1728-1744 binds the amphiphysin-2 (BIN1) Src homology-3 (SH3) domain with an unusually high affinity (Kd 24 nm). Our NMR solution complex structure together with isothermal titration calorimetry data on several related viral and cellular peptide ligands reveal that this exceptional affinity originates from interactions between multiple basic residues in the target peptide and the extensive negatively charged binding surface of amphiphysin-2 SH3. Remarkably, these arginines show no fixed conformation in the complex structure, indicating that a transient or fluctuating polyelectrostatic interaction accounts for this affinity. Thus, via optimization of such dynamic electrostatic forces, viral peptides have evolved a superior binding affinity for amphiphysin-2 SH3 compared with typical cellular ligands, such as dynamin, thereby enabling hijacking of amphiphysin-2 SH3-regulated host cell processes by these viruses. Moreover, our data show that the previously described consensus sequence PXRPXR for amphiphysin SH3 ligands is inaccurate and instead define it as an extended Class II binding motif PXXPXRpXR, where additional positive charges between the two constant arginine residues can give rise to extraordinary high SH3 binding affinity.

  1. The neuronal proteins CIPP, Cypin and IRSp53 form a tripartite complex mediated by PDZ and SH3 domains.

    PubMed

    Barilari, Manuela; Dente, Luciana

    2010-10-01

    Here we report the dissection of a tripartite complex formed by CIPP (channel-interacting PDZ protein), IRSp53 (insulin receptor tyrosine kinase substrate protein) and Cypin (cytosolic PSD-95 interactor) in cultured cells. The three proteins are expressed in similar neuronal districts, where CIPP binds to different membrane channels and receptors, IRSp53 regulates the morphogenesis of actin-rich dendritic spines, and Cypin promotes dendrite branching and patterning by binding to tubulin heterodimers. We observed that the interaction among the three proteins is mediated by small binding domains: CIPP works as a bridge, linking the carboxy-termini of IRSp53 and Cypin with its PDZ domains; IRSp53 connects Cypin, through an unusual SH3-mediated association, which can be impaired by substituting two crucial positively charged residues of Cypin. The observation that the three engineered proteins co-localize in the cytoplasm, and at the tip of induced neurites in neuronal cells, raises the interesting possibility that they work together in the formation of neuronal protrusions.

  2. Artificial proteins as allosteric modulators of PDZ3 and SH3 in two-domain constructs: A computational characterization of novel chimeric proteins.

    PubMed

    Kirubakaran, Palani; Pfeiferová, Lucie; Boušová, Kristýna; Bednarova, Lucie; Obšilová, Veronika; Vondrášek, Jiří

    2016-10-01

    Artificial multidomain proteins with enhanced structural and functional properties can be utilized in a broad spectrum of applications. The design of chimeric fusion proteins utilizing protein domains or one-domain miniproteins as building blocks is an important advancement for the creation of new biomolecules for biotechnology and medical applications. However, computational studies to describe in detail the dynamics and geometry properties of two-domain constructs made from structurally and functionally different proteins are lacking. Here, we tested an in silico design strategy using all-atom explicit solvent molecular dynamics simulations. The well-characterized PDZ3 and SH3 domains of human zonula occludens (ZO-1) (3TSZ), along with 5 artificial domains and 2 types of molecular linkers, were selected to construct chimeric two-domain molecules. The influence of the artificial domains on the structure and dynamics of the PDZ3 and SH3 domains was determined using a range of analyses. We conclude that the artificial domains can function as allosteric modulators of the PDZ3 and SH3 domains. Proteins 2016; 84:1358-1374. © 2016 Wiley Periodicals, Inc.

  3. Resveratrol induces apoptosis by directly targeting Ras-GTPase-activating protein SH3 domain-binding protein 1.

    PubMed

    Oi, N; Yuan, J; Malakhova, M; Luo, K; Li, Y; Ryu, J; Zhang, L; Bode, A M; Xu, Z; Li, Y; Lou, Z; Dong, Z

    2015-05-14

    Resveratrol (trans-3,5,4'-truhydroxystilbene) 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-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 the 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.

  4. Palladin interacts with SH3 domains of SPIN90 and Src and is required for Src-induced cytoskeletal remodeling

    PubMed Central

    Rönty, Mikko; Taivainen, Anu; Heiska, Leena; Otey, Carol; Ehler, Elisabeth; Song, Woo Keun; Carpen, Olli

    2007-01-01

    Palladin and SPIN90 are widely expressed proteins, which participate in modulation of actin cytoskeleton by binding to a variety of scaffold and signaling molecules. Cytoskeletal reorganization can induced by activation of signaling pathways, including the PDGF receptor and Src tyrosine kinase pathways. In this study we have analyzed the interplay between palladin, SPIN90 and Src, and characterized the role of palladin and SPIN90 in PDGF and Src-induced cytoskeletal remodeling. We show that the SH3 domains of SPIN90 and Src directly bind palladin’s poly-proline sequence and the interaction controls intracellular targeting of SPIN90. In PDGF-treated cells, palladin and SPIN90 co-localize in actin rich membrane ruffles and lamellipodia. The effect of PDGF on the cytoskeleton is at least partly mediated by the Src kinase, since PP2, a selective Src kinase family inhibitor, blocked PDGF-induced changes. Furthermore, expression of active Src kinase resulted in coordinated translocation of both palladin and SPIN90 to membrane protrusions. Knock-down of endogenous SPIN90 did not inhibit Src-induced cytoskeletal rearrangement, whereas knock-down of palladin resulted in cytoskeletal disorganization and inhibition of remodeling. Further studies showed that palladin is tyrosine phosphorylated in cells expressing active Src indicating bidirectional interplay between palladin and Src. These results may have implications in understanding the invasive and metastatic phenotype of neoplastic cells induced by Src. PMID:17537434

  5. 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

  6. 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

  7. Structural study of hNck2 SH3 domain protein in solution by circular dichroism and X-ray solution scattering.

    PubMed

    Matsumura, Yoshitaka; Shinjo, Masaji; Matsui, Tsutomu; Ichimura, Kaoru; Song, Jianxing; Kihara, Hiroshi

    2013-01-01

    We have done conformational study of hNck2 SH3 domain by means of far-ultraviolet (far-UV) circular dichroism (CD) and X-ray solution scattering (XSS). The results indicated that the following: (1) hNck2 SH3 domain protein exhibited concentration dependent monomer-dimer transition at neutral pH, while the secondary structure of this protein was independent of the protein concentration. (2) The hNck2 SH3 domain also exhibited pH dependent monomer-dimer transition. This monomer-dimer transition was accompanied with helix-β transition of the secondary structural change. Moreover, the acid-induced conformation, which was previously studied by Liu and Song by CD and nuclear magnetic resonance (NMR), was found to be not compact, but the conformation of the protein at acidic pH was similar to the cold denatured state (C-state) reported by Yamada et al. for equine β-lactoglobulin. We calculated that a structure of the equilibrium helix-rich intermediate of the hNck2 SH3 domain by DAMMIF program.

  8. Structural study of hNck2 SH3 domain protein in solution by circular dichroism and X-ray solution scattering

    PubMed Central

    Matsumura, Yoshitaka; Shinjo, Masaji; Matsui, Tsutomu; Ichimura, Kaoru; Song, Jianxing; Kihara, Hiroshi

    2014-01-01

    We have done conformational study of hNck2 SH3 domain by means of far-ultraviolet (far-UV) circular dichroism (CD) and X-ray solution scattering (XSS). The results indicated that the following: (1) hNck2 SH3 domain protein exhibited concentration dependent monomer–dimer transition at neutral pH, while the secondary structure of this protein was independent of the protein concentration. (2) The hNck2 SH3 domain also exhibited pH dependent monomer–dimer transition. This monomer–dimer transition was accompanied with helix-β transition of the secondary structural change. Moreover, the acid-induced conformation, which was previously studied by Liu and Song by CD and nuclear magnetic resonance (NMR), was found to be not compact, but the conformation of the protein at acidic pH was similar to the cold denatured state (C-state) reported by Yamada et al. for equine β-lactoglobulin. We calculated that a structure of the equilibrium helix-rich intermediate of the hNck2 SH3 domain by DAMMIF program. PMID:23524290

  9. 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

  10. 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

  11. The protein product of the c-cbl protooncogene is phosphorylated after B cell receptor stimulation and binds the SH3 domain of Bruton's tyrosine kinase

    PubMed Central

    1995-01-01

    X-linked agammaglobulinemia, a B cell immunodeficiency, is caused by mutations in the Bruton's tyrosine kinase (Btk) gene. The absence of a functional Btk protein leads to a failure of B cell differentiation and antibody production. B cell receptor stimulation leads to the phosphorylation of the Btk protein and it is, therefore, likely that Btk is involved in B cell receptor signaling. As a nonreceptor tyrosine kinase, Btk is likely to interact with several proteins within the context of a signal transduction pathway. To understand such interactions, we have generated glutathione S-transferase fusion proteins corresponding to different domains of the human Btk protein. We have identified a 120-kD protein present in human B cells as being bound by the SH3 domain of Btk and which, after B cell receptor stimulation, is one of the major substrates of tyrosine phosphorylation. We have shown that this 120-kD protein is the protein product of c-cbl, a protooncogene, which is known to be phosphorylated in response to T cell receptor stimulation and to interact with several other tyrosine kinases. Association of the SH3 domain of Btk with p120cbl provides evidence for an analogous role for p120cbl in B cell signaling pathways. The p120cbl protein is the first identified ligand of the Btk SH3 domain. PMID:7629518

  12. Binding of the cSH3 domain of Grb2 adaptor to two distinct RXXK motifs within Gab1 docker employs differential mechanisms.

    PubMed

    McDonald, Caleb B; Seldeen, Kenneth L; Deegan, Brian J; Bhat, Vikas; Farooq, Amjad

    2011-01-01

    A ubiquitous component of cellular signaling machinery, Gab1 docker plays a pivotal role in routing extracellular information in the form of growth factors and cytokines to downstream targets such as transcription factors within the nucleus. Here, using isothermal titration calorimetry (ITC) in combination with macromolecular modeling (MM), we show that although Gab1 contains four distinct RXXK motifs, designated G1, G2, G3, and G4, only G1 and G2 motifs bind to the cSH3 domain of Grb2 adaptor and do so with distinct mechanisms. Thus, while the G1 motif strictly requires the PPRPPKP consensus sequence for high-affinity binding to the cSH3 domain, the G2 motif displays preference for the PXVXRXLKPXR consensus. Such sequential differences in the binding of G1 and G2 motifs arise from their ability to adopt distinct polyproline type II (PPII)- and 3(10) -helical conformations upon binding to the cSH3 domain, respectively. Collectively, our study provides detailed biophysical insights into a key protein-protein interaction involved in a diverse array of signaling cascades central to health and disease.

  13. 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

  14. Binding preference of carbon nanotube over proline-rich motif ligand on SH3-domain: a comparison with different force fields.

    PubMed

    Shi, Biyun; Zuo, Guanghong; Xiu, Peng; Zhou, Ruhong

    2013-04-01

    With the widespread applications of nanomaterials such as carbon nanotubes, there is a growing concern on the biosafety of these engineered nanoparticles, in particular their interactions with proteins. In molecular simulations of nanoparticle-protein interactions, the choice of empirical parameters (force fields) plays a decisive role, and thus is of great importance and should be examined carefully before wider applications. Here we compare three commonly used force fields, CHARMM, OPLSAA, and AMBER in study of the competitive binding of a single wall carbon nanotube (SWCNT) with a native proline-rich motif (PRM) ligand on its target protein SH3 domain, a ubiquitous protein-protein interaction mediator involved in signaling and regulatory pathways. We find that the SWCNT displays a general preference over the PRM in binding with SH3 domain in all the three force fields examined, although the degree of preference can be somewhat different, with the AMBER force field showing the highest preference. The SWCNT prevents the ligand from reaching its native binding pocket by (i) occupying the binding pocket directly, and (ii) binding with the ligand itself and then being trapped together onto some off-sites. The π-π stacking interactions between the SWCNT and aromatic residues are found to play a significant role in its binding to the SH3 domain in all the three force fields. Further analyses show that even the SWCNT-ligand binding can also be relatively more stable than the native ligand-protein binding, indicating a serious potential disruption to the protein SH3 function.

  15. Differential Recognition Preferences of the Three Src Homology 3 (SH3) Domains from the Adaptor CD2-associated Protein (CD2AP) and Direct Association with Ras and Rab Interactor 3 (RIN3).

    PubMed

    Rouka, Evgenia; Simister, Philip C; Janning, Melanie; Kumbrink, Joerg; Konstantinou, Tassos; Muniz, João R C; Joshi, Dhira; O'Reilly, Nicola; Volkmer, Rudolf; Ritter, Brigitte; Knapp, Stefan; von Delft, Frank; Kirsch, Kathrin H; Feller, Stephan M

    2015-10-16

    CD2AP is an adaptor protein involved in membrane trafficking, with essential roles in maintaining podocyte function within the kidney glomerulus. CD2AP contains three Src homology 3 (SH3) domains that mediate multiple protein-protein interactions. However, a detailed comparison of the molecular binding preferences of each SH3 remained unexplored, as well as the discovery of novel interactors. Thus, we studied the binding properties of each SH3 domain to the known interactor Casitas B-lineage lymphoma protein (c-CBL), conducted a peptide array screen based on the recognition motif PxPxPR and identified 40 known or novel candidate binding proteins, such as RIN3, a RAB5-activating guanine nucleotide exchange factor. CD2AP SH3 domains 1 and 2 generally bound with similar characteristics and specificities, whereas the SH3-3 domain bound more weakly to most peptide ligands tested yet recognized an unusually extended sequence in ALG-2-interacting protein X (ALIX). RIN3 peptide scanning arrays revealed two CD2AP binding sites, recognized by all three SH3 domains, but SH3-3 appeared non-functional in precipitation experiments. RIN3 recruited CD2AP to RAB5a-positive early endosomes via these interaction sites. Permutation arrays and isothermal titration calorimetry data showed that the preferred binding motif is Px(P/A)xPR. Two high-resolution crystal structures (1.65 and 1.11 Å) of CD2AP SH3-1 and SH3-2 solved in complex with RIN3 epitopes 1 and 2, respectively, indicated that another extended motif is relevant in epitope 2. In conclusion, we have discovered novel interaction candidates for CD2AP and characterized subtle yet significant differences in the recognition preferences of its three SH3 domains for c-CBL, ALIX, and RIN3. PMID:26296892

  16. Differential Recognition Preferences of the Three Src Homology 3 (SH3) Domains from the Adaptor CD2-associated Protein (CD2AP) and Direct Association with Ras and Rab Interactor 3 (RIN3)*

    PubMed Central

    Rouka, Evgenia; Simister, Philip C.; Janning, Melanie; Kumbrink, Joerg; Konstantinou, Tassos; Muniz, João R. C.; Joshi, Dhira; O'Reilly, Nicola; Volkmer, Rudolf; Ritter, Brigitte; Knapp, Stefan; von Delft, Frank; Kirsch, Kathrin H.; Feller, Stephan M.

    2015-01-01

    CD2AP is an adaptor protein involved in membrane trafficking, with essential roles in maintaining podocyte function within the kidney glomerulus. CD2AP contains three Src homology 3 (SH3) domains that mediate multiple protein-protein interactions. However, a detailed comparison of the molecular binding preferences of each SH3 remained unexplored, as well as the discovery of novel interactors. Thus, we studied the binding properties of each SH3 domain to the known interactor Casitas B-lineage lymphoma protein (c-CBL), conducted a peptide array screen based on the recognition motif PxPxPR and identified 40 known or novel candidate binding proteins, such as RIN3, a RAB5-activating guanine nucleotide exchange factor. CD2AP SH3 domains 1 and 2 generally bound with similar characteristics and specificities, whereas the SH3-3 domain bound more weakly to most peptide ligands tested yet recognized an unusually extended sequence in ALG-2-interacting protein X (ALIX). RIN3 peptide scanning arrays revealed two CD2AP binding sites, recognized by all three SH3 domains, but SH3-3 appeared non-functional in precipitation experiments. RIN3 recruited CD2AP to RAB5a-positive early endosomes via these interaction sites. Permutation arrays and isothermal titration calorimetry data showed that the preferred binding motif is Px(P/A)xPR. Two high-resolution crystal structures (1.65 and 1.11 Å) of CD2AP SH3-1 and SH3-2 solved in complex with RIN3 epitopes 1 and 2, respectively, indicated that another extended motif is relevant in epitope 2. In conclusion, we have discovered novel interaction candidates for CD2AP and characterized subtle yet significant differences in the recognition preferences of its three SH3 domains for c-CBL, ALIX, and RIN3. PMID:26296892

  17. 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

  18. A Sos-derived peptidimer blocks the Ras signaling pathway by binding both Grb2 SH3 domains and displays antiproliferative activity.

    PubMed

    Cussac, D; Vidal, M; Leprince, C; Liu, W Q; Cornille, F; Tiraboschi, G; Roques, B P; Garbay, C

    1999-01-01

    With the aim of interrupting the growth factor-stimulated Ras signaling pathway at the level of the Grb2-Sos interaction, a peptidimer, made of two identical proline-rich sequences from Sos linked by a lysine spacer, was designed using structural data from Grb2 and a proline-rich peptide complexed with its SH3 domains. The peptidimer affinity for Grb2 is 40 nM whereas that of the monomer is 16 microM, supporting the dual recognition of both Grb2 SH3 domains by the dimer. At 50 nM, the peptidimer blocks selectively Grb2-Sos complexation in ER 22 (CCL 39 fibroblasts overexpressing epidermal growth factor receptor) cellular extracts. The peptidimer specifically recognizes Grb2 and does not interact with PI3K or Nck, two SH3 domain-containing adaptors. The peptidimer was modified to enter cells by coupling to a fragment of Antennapedia homeodomain. At 10 microM, the conjugate inhibits the Grb2-Sos interaction (100%) and MAP kinase (ERK1 and ERK2) phosphorylation (60%) without modifying cellular growth of ER 22 cells. At the same concentration, the conjugate also inhibits both MAP kinase activation induced by nerve growth factor or epidermal growth factor in PC12 cells, and differentiation triggered by nerve growth factor. Finally, when tested for its antiproliferative activity, the conjugate was an efficient inhibitor of the colony formation of transformed NIH3T3/HER2 cells grown in soft agar, with an IC50 of around 1 microM. Thus, the designed peptidimers appear to be interesting leads to investigate signaling and intracellular processes and for designing selective inhibitors of tumorigenic Ras-dependent processes.

  19. Site-specific protein backbone and side-chain NMR chemical shift and relaxation analysis of human vinexin SH3 domain using a genetically encoded {sup 15}N/{sup 19}F-labeled unnatural amino acid

    SciTech Connect

    Shi, Pan; Xi, Zhaoyong; Wang, Hu; Shi, Chaowei; Xiong, Ying; Tian, Changlin

    2010-11-19

    Research highlights: {yields} Chemical synthesis of {sup 15}N/{sup 19}F-trifluomethyl phenylalanine. {yields} Site-specific incorporation of {sup 15}N/{sup 19}F-trifluomethyl phenylalanine to SH3. {yields} Site-specific backbone and side chain chemical shift and relaxation analysis. {yields} Different internal motions at different sites of SH3 domain upon ligand binding. -- Abstract: SH3 is a ubiquitous domain mediating protein-protein interactions. Recent solution NMR structural studies have shown that a proline-rich peptide is capable of binding to the human vinexin SH3 domain. Here, an orthogonal amber tRNA/tRNA synthetase pair for {sup 15}N/{sup 19}F-trifluoromethyl-phenylalanine ({sup 15}N/{sup 19}F-tfmF) has been applied to achieve site-specific labeling of SH3 at three different sites. One-dimensional solution NMR spectra of backbone amide ({sup 15}N){sup 1}H and side-chain {sup 19}F were obtained for SH3 with three different site-specific labels. Site-specific backbone amide ({sup 15}N){sup 1}H and side-chain {sup 19}F chemical shift and relaxation analysis of SH3 in the absence or presence of a peptide ligand demonstrated different internal motions upon ligand binding at the three different sites. This site-specific NMR analysis might be very useful for studying large-sized proteins or protein complexes.

  20. Suppression of c-Src activity by C-terminal Src kinase involves the c-Src SH2 and SH3 domains: analysis with Saccharomyces cerevisiae.

    PubMed Central

    Murphy, S M; Bergman, M; Morgan, D O

    1993-01-01

    The kinase activity of c-Src is normally repressed in vertebrate cells by extensive phosphorylation of Y-527. C-terminal Src kinase (CSK) is a candidate for the enzyme that catalyzes this phosphorylation. We have used budding yeast to study the regulation of c-Src activity by CSK in intact cells. Expression of c-Src in Saccharomyces cerevisiae, which lacks endogenous c-Src and Y-527 kinases, induces a kinase-dependent growth inhibition. Coexpression of CSK in these cells results in phosphorylation of c-Src on Y-527 and suppression of the c-Src phenotype. CSK does not fully suppress the activity of c-Src mutants lacking portions of the SH2 or SH3 domains, even though these mutant proteins are phosphorylated on Y-527 by CSK both in vivo and in vitro. These results suggest that both the SH2 and SH3 domains of c-Src are required for the suppression of c-Src activity by Y-527 phosphorylation. Images PMID:7689149

  1. The novel SH3 domain protein Dlish/CG10933 mediates fat signaling in Drosophila by binding and regulating Dachs

    PubMed Central

    Zhang, Yifei; Wang, Xing; Matakatsu, Hitoshi; Fehon, Richard; Blair, Seth S

    2016-01-01

    Much of the Hippo and planar cell polarity (PCP) signaling mediated by the Drosophila protocadherin Fat depends on its ability to change the subcellular localization, levels and activity of the unconventional myosin Dachs. To better understand this process, we have performed a structure-function analysis of Dachs, and used this to identify a novel and important mediator of Fat and Dachs activities, a Dachs-binding SH3 protein we have named Dlish. We found that Dlish is regulated by Fat and Dachs, that Dlish also binds Fat and the Dachs regulator Approximated, and that Dlish is required for Dachs localization, levels and activity in both wild type and fat mutant tissue. Our evidence supports dual roles for Dlish. Dlish tethers Dachs to the subapical cell cortex, an effect partly mediated by the palmitoyltransferase Approximated under the control of Fat. Conversely, Dlish promotes the Fat-mediated degradation of Dachs. DOI: http://dx.doi.org/10.7554/eLife.16624.001 PMID:27692068

  2. Role of the SH3-ligand domain of simian immunodeficiency virus Nef in interaction with Nef-associated kinase and simian AIDS in rhesus macaques.

    PubMed

    Khan, I H; Sawai, E T; Antonio, E; Weber, C J; Mandell, C P; Montbriand, P; Luciw, P A

    1998-07-01

    The nef gene of the human and simian immunodeficiency viruses (HIV and SIV) is dispensable for viral replication in T-cell lines; however, it is essential for high virus loads and progression to simian AIDS (SAIDS) in SIV-infected adult rhesus macaques. Nef proteins from HIV type 1 (HIV-1), HIV-2, and SIV contain a proline-Xaa-Xaa-proline (PxxP) motif. The region of Nef with this motif is similar to the Src homology region 3 (SH3) ligand domain found in many cell signaling proteins. In virus-infected lymphoid cells, Nef interacts with a cellular serine/threonine kinase, designated Nef-associated kinase (NAK). In this study, analysis of viral clones containing point mutations in the nef gene of the pathogenic clone SIVmac239 revealed that several strictly conserved residues in the PxxP region were essential for Nef-NAK interaction. The results of this analysis of Nef mutations in in vitro kinase assays indicated that the PxxP region in SIV Nef was strikingly similar to the consensus sequence for SH3 ligand domains possessing the minus orientation. To test the significance of the PxxP motif of Nef for viral pathogenesis, each proline was mutated to an alanine to produce the viral clone SIVmac239-P104A/P107A. This clone, expressing Nef that does not associate with NAK, was inoculated into seven juvenile rhesus macaques. In vitro kinase assays were performed on virus recovered from each animal; the ability of Nef to associate with NAK was restored in five of these animals as early as 8 weeks after infection. Analysis of nef genes from these viruses revealed patterns of genotypic reversion in the mutated PxxP motif. These revertant genotypes, which included a second-site suppressor mutation, restored the ability of Nef to interact with NAK. Additionally, the proportion of revertant viruses increased progressively during the course of infection in these animals, and two of these animals developed fatal SAIDS. Taken together, these results demonstrated that in vivo

  3. Exploring the activity space of peptides binding to diverse SH3 domains using principal property descriptors derived from amino acid rotamers.

    PubMed

    He, Ping; Wu, Wei; Yang, Kang; Jing, Tao; Liao, Ke-Long; Zhang, Wei; Wang, Hai-Dong; Hua, Xing

    2011-01-01

    Although there were intensive works addressed on multivariate extraction of the informative components from numerous physicochemical parameters of amino acids in isolated state, the various conformational behaviors of amino acids in complicated biological context have long been underappreciated in the field of quantitative structure-activity relationship (QSAR). In this work, the amino acid rotamers, which were derived from statistical survey of protein crystal structures, were used to reproduce the conformational variety of amino acid side-chains in real condition. In this procedure, these rotamers were superposed into a nx x ny x nz lattice and an artificial probe was employed to detect four kinds of nonbonding field potentials (i.e., electrostatic, steric, hydrophobic, and hydrogen bonds) at each lattice point using a Gaussian-type potential function; the generated massive data were then subjected to a principal component analysis (PCA) treatment to obtain a set of few, informative amino acid descriptors. We used this set of descriptors, that we named principal property descriptors derived from amino acid rotamers (PDAR), to characterize over 13,000 peptides with known binding affinities to 10 types of SH3 domains. Genetic algorithm/ partial least square regression (GA/PLS) modeling and Monte Carlo cross-validation (MCCV) demonstrated that the correlation between the PDAR descriptors and the binding affinities of peptides are comparable with or even better than previously published models. Furthermore, from the PDAR-based QSAR models we concluded that the core motif of peptides, particularly the electrostatic property, hydrophobicity, and hydrogen bond at residue positions P3, P2, and/or P0, contribute significantly to the hAmph SH3 domain-peptide binding, whereas two ends of the peptides, such as P6, P4, P-4, and P5, only play a secondary role in the binding.

  4. LytM Fusion with SH3b-Like Domain Expands Its Activity to Physiological Conditions

    PubMed Central

    Jagielska, Elzbieta; Chojnacka, Olga

    2016-01-01

    Staphylococcus aureus remains one of the most common and at the same time the most dangerous bacteria. The spreading antibiotic resistance calls for intensification of research on staphylococcal physiology and development of new strategies for combating this threatening pathogen. We have engineered new chimeric enzymes comprising the enzymatically active domain (EAD) of autolysin LytM from S. aureus and the cell wall binding domain (CBD) from bacteriocin lysostaphin. They display potent activity in extended environmental conditions. Our results exemplify the possibility of exploring autolytic enzymes in engineering lysins with desired features. Moreover, they suggest a possible mechanism of autolysin physiological activity regulation by local ionic environments in the cell wall. PMID:27351490

  5. Role of Interfacial Water Molecules in Proline-rich Ligand Recognition by the Src Homology 3 Domain of Abl*

    PubMed Central

    Palencia, Andres; Camara-Artigas, Ana; Pisabarro, M. Teresa; Martinez, Jose C.; Luque, Irene

    2010-01-01

    The interaction of Abl-Src homology 3 domain (SH3) with the high affinity peptide p41 is the most notable example of the inconsistency existing between the currently accepted description of SH3 complexes and their binding thermodynamic signature. We had previously hypothesized that the presence of interfacial water molecules is partially responsible for this thermodynamic behavior. We present here a thermodynamic, structural, and molecular dynamics simulation study of the interaction of p41 with Abl-SH3 and a set of mutants designed to alter the water-mediated interaction network. Our results provide a detailed description of the dynamic properties of the interfacial water molecules and a molecular interpretation of the thermodynamic effects elicited by the mutations in terms of the modulation of the water-mediated hydrogen bond network. In the light of these results, a new dual binding mechanism is proposed that provides a better description of proline-rich ligand recognition by Abl-SH3 and that has important implications for rational design. PMID:19906645

  6. 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

  7. The kinase, SH3, and SH2 domains of Lck play critical roles in T-cell activation after ZAP-70 membrane localization.

    PubMed Central

    Yamasaki, S; Takamatsu, M; Iwashima, M

    1996-01-01

    Antigenic stimulation of the T-cell antigen receptor initiates signal transduction through the immunoreceptor tyrosine-based activation motifs (ITAMs). When its two tyrosines are phosphorylated, ITAM forms a binding site for ZAP-70, one of the cytoplasmic protein tyrosine kinases essential for T-cell activation. The signaling process that follows ZAP-70 binding to ITAM has been analyzed by the construction of fusion proteins that localize ZAP-70 to the plasma membrane. We found that membrane-localized forms of ZAP-70 induce late signaling events such as activation of nuclear factor of activated T cells without any stimulation. This activity was observed only when Lck was expressed and functional. In addition, each mutation that affects the function of Lck in the kinase, Src homology 2 (SH2), and SH3 domains greatly impaired the signaling ability of the chimeric protein. Therefore, Lck functions in multiple manners in T-cell activation for the steps following ZAP-70 binding to ITAM. PMID:8943371

  8. The Best Disease-Linked Cl Channel hBest1 Regulates Cav1 (L-type) Ca2+ Channels Via SH3-binding Domains

    PubMed Central

    Yu, Kuai; Xiao, Qinghuan; Cui, Guiying; Lee, Amy; Hartzell, H. Criss

    2008-01-01

    Mutations in the bestrophin-1 (Best1) gene are linked to several kinds of macular degeneration in both humans and dogs. Although bestrophins have been shown clearly to be Cl− ion channels, it is controversial whether Cl− channel dysfunction can explain the diseases. It has been suggested that bestrophins are multi-functional proteins: they may regulate voltage-gated Ca2+ channels in addition to functioning as Cl− channels. Here we show that hBest1 differentially modulates Cav1.3 (L-type) voltage-gated Ca2+ channels through association with the Cavβ subunit. In transfected HEK-293 cells, hBest1 inhibited Cav1.3. Inhibition of Cav1.3 was not observed in the absence of the β subunit. Also, the hBest1 C-terminus binds to Cavβ subunits, suggesting that the effect of hBest1 was mediated by the Cavβ subunit. The region of hBest1 responsible for the effect was localized to a region (amino acids 330 − 370) in the cytoplasmic C-terminus that contains a predicted SH3-binding domain that is not present in other bestrophin subtypes. Mutation of Pro330 and Pro334 abolished the effects of hBest1 on Cav1.3. The effect was specific to hBest1: it was not observed with mBest1, -2, or -3. Wild type hBest1 and the disease-causing mutants R92S, G299R, and D312N inhibited Cav currents the same amount, whereas the A146K and G222E mutants were less effective. We propose that hBest1 regulates Cav channels by interacting with the Cavβ subunit and altering channel availability. Our findings reveal a novel function of bestrophin in regulation of Cav channels and suggest a possible mechanism for the role of hBest1 in macular degeneration. PMID:18509027

  9. 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

  10. Ligand-induced changes in dynamics in the RT loop of the C-terminal SH3 domain of Sem-5 indicate cooperative conformational coupling

    PubMed Central

    Ferreon, Josephine C.; Hilser, Vincent J.

    2003-01-01

    We report the effects of peptide binding on the 15N relaxation rates and chemical shifts of the C-SH3 of Sem-5. 15N spin-lattice relaxation time (T1), spin-spin relaxation time (T2), and {1H}-15N NOE were obtained from heteronuclear 2D NMR experiments. These parameters were then analyzed using the Lipari-Szabo model free formalism to obtain parameters that describe the internal motions of the protein. High-order parameters (S2 > 0.8) are found in elements of regular secondary structure, whereas some residues in the loop regions show relatively low-order parameters, notably the RT loop. Peptide binding is characterized by a significant decrease in the 15N relaxation in the RT loop. Concomitant with the change in dynamics is a cooperative change in chemical shifts. The agreement between the binding constants calculated from chemical shift differences and that obtained from ITC indicates that the binding of Sem-5 C-SH3 to its putative peptide ligand is coupled to a cooperative conformational change in which a portion of the binding site undergoes a significant reduction in conformational heterogeneity. PMID:12717021

  11. A thioredoxin fold protein Sh3bgr regulates Enah and is necessary for proper sarcomere formation

    PubMed Central

    Jang, Dong Gil; Sim, Hyo Jung; Song, Eun Kyung; Medina-Ruiz, Sofia; Seo, Jeong Kon; Park, Tae Joo

    2015-01-01

    The sh3bgr (SH3 domain binding glutamate-rich) gene encodes a small protein containing a thioredoxin-like fold, SH3 binding domain, and glutamate-rich domain. Originally, it was suggested that increased expression of Sh3bgr may cause the cardiac phenotypes in Down's syndrome. However, it was recently reported that the overexpression of Sh3bgr did not cause any disease phenotypes in mice. In this study, we have discovered that Sh3bgr is critical for sarcomere formation in striated muscle tissues and also for heart development. Sh3bgr is strongly expressed in the developing somites and heart in Xenopus. Morpholino mediated-knockdown of sh3bgr caused severe malformation of heart tissue and disrupted segmentation of the somites. Further analysis revealed that Sh3bgr specifically localized to the Z-line in mature sarcomeres and that knockdown of Sh3bgr completely disrupted sarcomere formation in the somites. Moreover, overexpression of Sh3bgr resulted in abnormally discontinues thick firmaments in the somitic sarcomeres. We suggest that Sh3bgr does its function at least partly by regulating localization of Enah for the sarcomere formation. In addition, we provide the data supporting Sh3bgr is also necessary for proper heart development in part by affecting the Enah protein level. PMID:26116879

  12. Measurement of multiple psi torsion angles in uniformly 13C,15N-labeled alpha-spectrin SH3 domain using 3D 15N-13C-13C-15N MAS dipolar-chemical shift correlation spectroscopy.

    PubMed

    Ladizhansky, Vladimir; Jaroniec, Christopher P; Diehl, Annette; Oschkinat, Hartmut; Griffin, Robert G

    2003-06-01

    We demonstrate the simultaneous measurement of several backbone torsion angles psi in the uniformly (13)C,(15)N-labeled alpha-Spectrin SH3 domain using two different 3D 15N-13C-13C-15N dipolar-chemical shift magic-angle spinning (MAS) NMR experiments. The first NCCN experiment utilizes double quantum (DQ) spectroscopy combined with the INADEQUATE type 13C-13C chemical shift correlation. The decay of the DQ coherences formed between 13C'(i) and 13C(alphai) spin pairs is determined by the "correlated" dipolar field due to 15N(i)-13C(alphai) and 13C'(i)-15N(i+1) dipolar couplings and is particularly sensitive to variations of the torsion angle in the regime |psi| > 140 degrees. However, the ability of this experiment to constrain multiple psi-torsion angles is limited by the resolution of the 13C(alpha)-(13)CO correlation spectrum. This problem is partially addressed in the second approach described here, which is an NCOCA NCCN experiment. In this case the resolution is enhanced by the superior spectral dispersion of the 15N resonances present in the 15N(i+1)-13C(alphai) part of the NCOCA chemical shift correlation spectrum. For the case of the 62-residue alpha-spectrin SH3 domain, we determined 13 psi angle constraints with the INADEQUATE NCCN experiment and 22 psi constraints were measured in the NCOCA NCCN experiment.

  13. Classic 18.5- and 21.5-kDa myelin basic protein isoforms associate with cytoskeletal and SH3-domain proteins in the immortalized N19-oligodendroglial cell line stimulated by phorbol ester and IGF-1.

    PubMed

    Smith, Graham S T; Homchaudhuri, Lopamudra; Boggs, Joan M; Harauz, George

    2012-06-01

    The 18.5-kDa classic myelin basic protein (MBP) is an intrinsically disordered protein arising from the Golli (Genes of Oligodendrocyte Lineage) gene complex and is responsible for compaction of the myelin sheath in the central nervous system. This MBP splice isoform also has a plethora of post-translational modifications including phosphorylation, deimination, methylation, and deamidation, that reduce its overall net charge and alter its protein and lipid associations within oligodendrocytes (OLGs). It was originally thought that MBP was simply a structural component of myelin; however, additional investigations have demonstrated that MBP is multi-functional, having numerous protein-protein interactions with Ca²⁺-calmodulin, actin, tubulin, and proteins with SH3-domains, and it can tether these proteins to a lipid membrane in vitro. Here, we have examined cytoskeletal interactions of classic 18.5-kDa MBP, in vivo, using early developmental N19-OLGs transfected with fluorescently-tagged MBP, actin, tubulin, and zonula occludens 1 (ZO-1). We show that MBP redistributes to distinct 'membrane-ruffled' regions of the plasma membrane where it co-localizes with actin and tubulin, and with the SH3-domain-containing proteins cortactin and ZO-1, when stimulated with PMA, a potent activator of the protein kinase C pathway. Moreover, using phospho-specific antibody staining, we show an increase in phosphorylated Thr98 MBP (human sequence numbering) in membrane-ruffled OLGs. Previously, Thr98 phosphorylation of MBP has been shown to affect its conformation, interactions with other proteins, and tethering of other proteins to the membrane in vitro. Here, MBP and actin were also co-localized in new focal adhesion contacts induced by IGF-1 stimulation in cells grown on laminin-2. This study supports a role for classic MBP isoforms in cytoskeletal and other protein-protein interactions during membrane and cytoskeletal remodeling in OLGs.

  14. 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

  15. 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

  16. 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.

  17. 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

  18. 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

  19. 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

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

    2014-12-08

    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.

  20. 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

  1. 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

  2. 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

  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

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

    2015-02-01

    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 (Sh3bp2(KI/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 Sh3bp2(KI/KI) mice. Bone marrow (BM) cells from wild-type (Sh3bp2(+/+)) mice were transplanted to 6-week-old Sh3bp2(KI/KI) mice with developing inflammation and to 10-week-old Sh3bp2(KI/KI) mice with established inflammation. Six-week-old Sh3bp2(KI/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 10weeks after BMT compared to Sh3bp2(KI/KI) mice transplanted with Sh3bp2(KI/KI) BM cells. Elevation of serum TNF-α levels was not detected after BMT. BMT was effective for up to 20weeks in 6-week-old Sh3bp2(KI/KI) mice transplanted with Sh3bp2(+/+) BM cells. BMT also ameliorated the inflammation and bone loss in 10-week-old Sh3bp2(KI/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.

  5. 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

  6. Abl Interactor 1 (Abi-1) Wave-Binding and SNARE Domains Regulate Its Nucleocytoplasmic Shuttling, Lamellipodium Localization, and Wave-1 Levels

    PubMed Central

    Echarri, Asier; Lai, Margaret J.; Robinson, Matthew R.; Pendergast, Ann Marie

    2004-01-01

    The Abl interactor 1 (Abi-1) protein has been implicated in the regulation of actin dynamics and localizes to the tips of lamellipodia and filopodia. Here, we show that Abi-1 binds the actin nucleator protein Wave-1 through an amino-terminal Wave-binding (WAB) domain and that disruption of the Abi-1-Wave-1 interaction prevents Abi-1 from reaching the tip of the lamellipodium. Abi-1 binds to the Wave homology domain of Wave-1, a region that is required for translocation of Wave-1 to the lamellipodium. Mouse embryo fibroblasts that lack one allele of Abi-1 and are homozygous null for the related Abi-2 protein exhibit decreased Wave-1 protein levels. This phenotype is rescued by Abi-1 proteins that retain Wave-1 binding but not by Abi-1 mutants that cannot bind to Wave-1. Moreover, we uncovered an overlapping SNARE domain in the amino terminus of Abi-1 that interacts with Syntaxin-1, a SNARE family member. Further, we demonstrated that Abi-1 shuttles in and out of the nucleus in a leptomycin B (LMB)-dependent manner and that complete nuclear translocation of Abi-1 in the absence of LMB requires the combined inactivation of the SNARE, WAB, and SH3 domains of Abi-1. Thus, Abi-1 undergoes nucleocytoplasmic shuttling and functions at the leading edge to regulate Wave-1 localization and protein levels. PMID:15143189

  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. A novel ∼34-kDa α-amylase from psychrotroph Exiguobacterium sp. SH3: production, purification, and characterization.

    PubMed

    Mojallali, Leila; Shahbani Zahiri, Hossein; Rajaei, Sarah; Akbari Noghabi, Kambiz; Haghbeen, Kamahldin

    2014-01-01

    An amylase-producing psychrotroph bacterium was isolated from soil and identified as belonging to the genus Exiguobacterium. A novel cold-adapted α-amylase, Amy SH3, was purified from culture medium of this bacterium using acetone precipitation and DEAE-Sepharose anion-exchange chromatography. The molecular mass of the enzyme was estimated about 34 kDa using SDS-PAGE. Biochemical characterization of Amy SH3 revealed that the optimum temperature for maximum activity of Amy SH3 was 37°C. However, Amy SH3 was also active at cold temperatures, showing 13% and 39% activity at 0 and 10°C, respectively. The optimum pH for maximum activity of Amy SH3 was pH 7, whereas the amylase was active over a pH range of 5 to 10. The activity of Amy SH3 was enhanced by Co²⁺ but decreased by Mg²⁺, Mn²⁺, Zn²⁺, Fe²⁺, and Ca²⁺. Amy SH3 was able to retain 76% of its activity in the presence of 0.5% SDS. The K(m) and V(max) of the enzyme were calculated to be 0.06 mg/mL and 4,010 U/mL, respectively. The cold-adapted Amy SH3 seems very promising for applications at ambient temperature.

  9. SH3 Domain–Based Phototrapping in Living Cells Reveals Rho Family GAP Signaling Complexes

    PubMed Central

    Okada, Hirokazu; Uezu, Akiyoshi; Mason, Frank M.; Soderblom, Erik J.; Moseley, M. Arthur; Soderling, Scott H.

    2012-01-01

    Rho family GAPs [guanosine triphosphatase (GTPase) activating proteins] negatively regulate Rho family GTPase activity and therefore modulate signaling events that control cytoskeletal dynamics. The spatial distribution of these GAPs and their specificity toward individual GTPases are controlled by their interactions with various proteins within signaling complexes. These interactions are likely mediated through the Src homology 3 (SH3) domain, which is abundant in the Rho family GAP proteome and exhibits a micromolar binding affinity, enabling the Rho family GAPs to participate in transient interactions with multiple binding partners. To capture these elusive GAP signaling complexes in situ, we developed a domain-based proteomics approach, starting with in vivo phototrapping of SH3 domain– binding proteins and the mass spectrometry identification of associated proteins for nine representative Rho family GAPs. After the selection of candidate binding proteins by cluster analysis, we performed peptide array–based high-throughput in vitro binding assays to confirm the direct interactions and map the SH3 domain–binding sequences. We thereby identified 54 SH3-mediated binding interactions (including 51 previously unidentified ones) for nine Rho family GAPs. We constructed Rho family GAP interactomes that provided insight into the functions of these GAPs. We further characterized one of the predicted functions for the Rac-specific GAP WRP and identified a role for WRP in mediating clustering of the postsynaptic scaffolding protein gephyrin and the GABAA (γ-aminobutyric acid type A) receptor at inhibitory synapses. PMID:22126966

  10. 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

  11. 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

  12. 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

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

    PubMed

    Badger, John; Grover, Prerna; Shi, Haibin; Panjarian, Shoghag B; Engen, John R; Smithgall, Thomas E; Makowski, Lee

    2016-06-14

    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

  14. 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

  15. 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

  16. 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.

  17. Identification of a Novel Coregulator, SH3YL1, That Interacts With the Androgen Receptor N-Terminus

    PubMed Central

    Blessing, Alicia M.; Ganesan, Sathya; Rajapakshe, Kimal; Ying Sung, Ying; Reddy Bollu, Lakshmi; Shi, Yan; Cheung, Edwin; Coarfa, Cristian; Chang, Jeffrey T.; McDonnell, Donald P.

    2015-01-01

    Nuclear receptor (NR)-mediated transcriptional activity is a dynamic process that is regulated by the binding of ligands that induce distinct conformational changes in the NR. These structural alterations lead to the differential recruitment of coregulators (coactivators or corepressors) that control the expression of NR-regulated genes. Here, we show that a stretch of proline residues located within the N-terminus of androgen receptor (AR) is a bona fide coregulator binding surface, the disruption of which reduces the androgen-dependent proliferation and migration of prostate cancer (PCa) cells. Using T7 phage display, we identified a novel AR-interacting protein, Src homology 3 (SH3)-domain containing, Ysc84-like 1 (SH3YL1), whose interaction with the receptor is dependent upon this polyproline domain. As with mutations within the AR polyproline domain, knockdown of SH3YL1 attenuated androgen-mediated cell growth and migration. RNA expression analysis revealed that SH3YL1 was required for the induction of a subset of AR-modulated genes. Notable was the observation that ubinuclein 1 (UBN1), a key member of a histone H3.3 chaperone complex, was a transcriptional target of the AR/SH3YL1 complex, correlated with aggressive PCa in patients, and was necessary for the maximal androgen-mediated proliferation and migration of PCa cells. Collectively, these data highlight the importance of an amino-terminal activation domain, its associated coregulator, and downstream transcriptional targets in regulating cellular processes of pathological importance in PCa. PMID:26305679

  18. 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.

  19. 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

  20. The impact of either 4-R-hydroxyproline or 4-R-fluoroproline on the conformation and SH3m-cort binding of HPK1 proline-rich peptide.

    PubMed

    Borgogno, Andrea; Ruzza, Paolo

    2013-02-01

    SH3 domains are probably the most abundant molecular-recognition modules of the proteome. A common feature of these domains is their interaction with ligand proteins containing Pro-rich sequences. Crystal and NMR structures of SH3 domains complexes with Pro-rich peptides show that the peptide ligands are bound over a range of up to seven residues in a PPII helix conformation. Short proline-rich peptides usually adopt little or no ordered secondary structure before binding interactions, and consequently their association with the SH3 domain is characterized by unfavorable binding entropy due to a loss of rotational freedom on forming the PPII helix. With the aim to stabilize the PPII helix conformation into the proline-rich decapeptide PPPLPPKPKF (P2), we replaced some proline residues either with the 4(R)-4-fluoro-L-proline (FPro) or the 4(R)-4-hydroxy-L-proline (Hyp). The interactions of P2 analogues with the SH3 domain of cortactin (SH3(m-cort)) were analyzed by circular dichroism spectroscopy, while CD thermal transition experiments have been used to determine their propensity to adopt a PPII helix conformation. Results show that the introduction of three residues of Hyp efficiently stabilizes the PPII helix conformation, while it does not improve the affinity towards the SH3 domain, suggesting that additional forces, e.g., electrostatic interactions, are involved in the SH3(m-cort) substrate recognition.

  1. Identification and characterization of Csh3 as an SH3 protein that interacts with fission yeast Cap1.

    PubMed

    Yamamoto, Takaharu; Kobayashi-Ooka, Yasuyo; Zhou, Guo-Lei; Kawamukai, Makoto

    2015-12-01

    Schizosaccharomyces pombe Cap1 has been identified as the (adenylyl) cyclase-associated protein. Cap1 was able to bind Cap1 itself and actin. Cap1 localized at the growing tip, and this localization was dependent on the Cap1 P2 region. In a two-hybrid screening using cap1 as bait, we isolated csh3, which encodes a protein of 296 amino acids with an SH3 domain and a proline/glutamine-rich region. The binding of Csh3 and Cap1 was confirmed by in vivo pull down assays. Cooperative functions of Csh3 and Cap1 were observed. Deletion of both csh3 and cap1 resulted in heightened sensitivity to CaCl2, while disruption of either gene alone did not have any effect in this regard. In addition, over-expression of csh3 or cap1 alone did not affect cell growth, while over-expression of both genes resulted in growth retardation. Finally, while Csh3-GFP localized to the cytoplasm in wild-type cells, its localization was altered in cap1Δ cells, suggesting that the interaction between Csh3 and Cap1 controls the cellular localization of Csh3. These results demonstrate that Cap1 in Schizo. pombe is a multifunctional protein that functions through interaction with Cap1 itself and other proteins including adenylyl cyclase, actin and Csh3.

  2. Structure of Crumbs tail in complex with the PALS1 PDZ-SH3-GK tandem reveals a highly specific assembly mechanism for the apical Crumbs complex.

    PubMed

    Li, Youjun; Wei, Zhiyi; Yan, Yan; Wan, Qingwen; Du, Quansheng; Zhang, Mingjie

    2014-12-01

    The Crumbs (Crb) complex, formed by Crb, PALS1, and PATJ, is evolutionarily conserved in metazoans and acts as a master cell-growth and -polarity regulator at the apical membranes in polarized epithelia. Crb intracellular functions, including its direct binding to PALS1, are mediated by Crb's highly conserved 37-residue cytoplasmic tail. However, the mechanistic basis governing the highly specific Crb-PALS1 complex formation is unclear, as reported interaction between the Crb tail (Crb-CT) and PALS1 PSD-95/DLG/ZO-1 (PDZ) domain is weak and promiscuous. Here we have discovered that the PDZ-Src homolgy 3 (SH3)-Guanylate kinase (GK) tandem of PALS1 binds to Crb-CT with a dissociation constant of 70 nM, which is ∼ 100-fold stronger than the PALS1 PDZ-Crb-CT interaction. The crystal structure of the PALS1 PDZ-SH3-GK-Crb-CT complex reveals that PDZ-SH3-GK forms a structural supramodule with all three domains contributing to the tight binding to Crb. Mutations disrupting the tertiary interactions of the PDZ-SH3-GK supramodule weaken the PALS1-Crb interaction and compromise PALS1-mediated polarity establishment in Madin-Darby canine kidney (MDCK) cysts. We further show that specific target binding of other members of membrane-associated guanylate kinases (MAGUKs) (e.g., CASK binding to neurexin) also requires the presence of their PDZ-SH3-GK tandems.

  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. The dominant folding route minimizes backbone distortion in SH3.

    PubMed

    Lammert, Heiko; Noel, Jeffrey K; Onuchic, José N

    2012-01-01

    Energetic frustration in protein folding is minimized by evolution to create a smooth and robust energy landscape. As a result the geometry of the native structure provides key constraints that shape protein folding mechanisms. Chain connectivity in particular has been identified as an essential component for realistic behavior of protein folding models. We study the quantitative balance of energetic and geometrical influences on the folding of SH3 in a structure-based model with minimal energetic frustration. A decomposition of the two-dimensional free energy landscape for the folding reaction into relevant energy and entropy contributions reveals that the entropy of the chain is not responsible for the folding mechanism. Instead the preferred folding route through the transition state arises from a cooperative energetic effect. Off-pathway structures are penalized by excess distortion in local backbone configurations and contact pair distances. This energy cost is a new ingredient in the malleable balance of interactions that controls the choice of routes during protein folding.

  5. 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

  6. The SH2 domain regulates c-Abl kinase activation by a cyclin-like mechanism and remodulation of the hinge motion.

    PubMed

    Dölker, Nicole; Górna, Maria W; Sutto, Ludovico; Torralba, Antonio S; Superti-Furga, Giulio; Gervasio, Francesco L

    2014-10-01

    Regulation of the c-Abl (ABL1) tyrosine kinase is important because of its role in cellular signaling, and its relevance in the leukemiogenic counterpart (BCR-ABL). Both auto-inhibition and full activation of c-Abl are regulated by the interaction of the catalytic domain with the Src Homology 2 (SH2) domain. The mechanism by which this interaction enhances catalysis is not known. We combined computational simulations with mutagenesis and functional analysis to find that the SH2 domain conveys both local and global effects on the dynamics of the catalytic domain. Locally, it regulates the flexibility of the αC helix in a fashion reminiscent of cyclins in cyclin-dependent kinases, reorienting catalytically important motifs. At a more global level, SH2 binding redirects the hinge motion of the N and C lobes and changes the conformational equilibrium of the activation loop. The complex network of subtle structural shifts that link the SH2 domain with the activation loop and the active site may be partially conserved with other SH2-domain containing kinases and therefore offer additional parameters for the design of conformation-specific inhibitors.

  7. Abl promotes cadherin-dependent adhesion and signaling in myoblasts.

    PubMed

    Lu, Min; Krauss, Robert S

    2010-07-15

    Cell-cell contact promotes myogenic differentiation but the mechanisms that regulate this phenomenon are not well understood. Cdo (also known as Cdon), an Ig superfamily member, functions as a component of cell surface complexes to promote myogenic differentiation through activation of p38alpha/beta MAP kinase. We recently showed that N-cadherin ligation activated p38alpha/beta in a Cdo-dependent manner, whereas N-cadherin ligation-dependent activation of ERK MAP kinase was not affected by loss of Cdo. The non-receptor tyrosine kinase Abl associates with Cdo during myoblast differentiation and is necessary for full activition of p38alpha/beta during this process. The Abl SH3 domain binds to a PxxP motif in the Cdo intracellular domain, and both these motifs are required for their promyogenic activity. Here we show that Abl is necessary for p38alpha/beta activation initiated by N-cadherin ligation, but in contrast to Cdo, Abl is also required for N-cadherin-dependent ERK activation. Moreover, Abl is required for efficient cadherin-mediated myoblast aggregation via modulation of RhoA-ROCK signaling. Therefore, Abl regulates N-cadherin-mediated p38alpha/beta activation by multiple mechanisms, more generally through regulation of cell-cell adhesion and specifically as a component of Cdo-containing complexes. The role of Cdo as a multifunctional coreceptor with roles in several pathways is also discussed.

  8. A C-terminal protein-binding domain in the retinoblastoma protein regulates nuclear c-Abl tyrosine kinase in the cell cycle.

    PubMed

    Welch, P J; Wang, J Y

    1993-11-19

    The ubiquitously expressed c-Abl tyrosine kinase is localized to the nucleus and binds to DNA. The DNA binding activity is regulated by cdc2-mediated phosphorylation, suggesting a cell cycle function for c-Abl. Here we show that the tyrosine kinase activity of nuclear c-Abl is regulated in the cell cycle through a specific interaction with the retinoblastoma protein (RB). A domain in the C-terminus of RB, outside of the A/B pocket, binds to the ATP-binding lobe of the c-Abl tyrosine kinase, resulting in kinase inhibition. The RB-c-Abl interaction is not affected by the viral oncoproteins that bind to RB. Hyperphosphorylation of RB correlates with release of c-Abl and activation of the tyrosine kinase in S phase cells. The nuclear c-Abl tyrosine kinase can enhance transcription, and this activity is inhibited by RB. Nuclear c-Abl is an S phase-activated tyrosine kinase that may participate directly in the regulation of transcription. PMID:8242749

  9. Identical Mutation in SH3BP2 Gene Causes Clinical Phenotypes with Different Severity in Mother and Daughter – Case Report

    PubMed Central

    Preda, L.; Dinca, O.; Bucur, A.; Dragomir, C.; Severin, E.

    2010-01-01

    Cherubism is a particular form of fibrous dysplasia of the jaws. Familial occurrence was reported in most cases. The condition is a rare hereditary disorder with autosomal dominant inheritance, with complete penetrance in males and incomplete penetrance in females and variable expressivity. It is known to be caused by mutations in the gene encoding SH3-domain binding protein 2, SH3BP2 gene. Major diagnostic criteria are cherubic facial appearance, painless hard enlargement of the jaws, and frequently associated dental abnormalities. The aim of the study was to analyze clinical and genetic features of cherubism in a family with 3 daughters in which the youngest one was affected. Clinical and radiographic examinations, hematological and biochemical evaluations and biopsy were performed. Molecular genetic analysis consisted of PCR amplification and direct sequencing of selected exons of the SH3BP2 gene. Cherubism was suspected based on clinical and radiographic examinations of the 9-year-old daughter. She presented asymmetrical enlargement of the mandible, speech and swallowing problems and dental abnormalities on the lower jaw. There was no history of similar clinical findings in any of the daughters or the parents of the affected girl. Abnormal results were obtained by genetic analysis. A c.1244G>A mutation was identified in exon 9 of the SH3BP2 gene in the asymptomatic mother and her affected daughter. The identified mutation in the SH3BP2 gene is probably disease-causing. The asymptomatic mother transmitted the gene mutation to her affected daughter. Our results confirm the reduced penetrance and variable expression of the gene mutation. PMID:21045962

  10. Evolution of BCR/ABL Gene Mutation in CML Is Time Dependent and Dependent on the Pressure Exerted by Tyrosine Kinase Inhibitor

    PubMed Central

    Vaidya, Shantashri; Vundinti, Babu Rao; Shanmukhaiah, Chandrakala; Chakrabarti, Prantar; Ghosh, Kanjaksha

    2015-01-01

    Background Mutations in the ABL kinase domain and SH3-SH2 domain of the BCR/ABL gene and amplification of the Philadelphia chromosome are the two important BCR/ABL dependent mechanisms of imatinib resistance. Here, we intended to study the role played by TKI, imatinib, in selection of gene mutations and development of chromosomal abnormalities in Indian CML patients. Methods Direct sequencing methodology was employed to detect mutations and conventional cytogenetics was done to identify Philadelphia duplication. Results Among the different mechanisms of imatinib resistance, kinase domain mutations (39%) of the BCR/ABL gene were seen to be more prevalent, followed by mutations in the SH3-SH2 domain (4%) and then BCR/ABL amplification with the least frequency (1%). The median duration of occurrence of mutation was significantly shorter for patients with front line imatinib than those pre-treated with hydroxyurea. Patients with high Sokal score (p = 0.003) showed significantly higher incidence of mutations, as compared to patients with low/intermediate score. Impact of mutations on the clinical outcome in AP and BC was observed to be insignificant. Of the 94 imatinib resistant patients, only 1 patient exhibited duplication of Philadelphia chromosome, suggesting a less frequent occurrence of this abnormality in Indian CML patients. Conclusion Close monitoring at regular intervals and proper analysis of the disease resistance would facilitate early detection of resistance and thus aid in the selection of the most appropriate therapy. PMID:25629972

  11. 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.

  12. Structural and Spectroscopic Analysis of the Kinase Inhibitor Bosutinib and an Isomer of Bosutinib Binding to the Abl Tyrosine Kinase Domain

    PubMed Central

    Levinson, Nicholas M.; Boxer, Steven G.

    2012-01-01

    Chronic myeloid leukemia (CML) is caused by the kinase activity of the BCR-Abl fusion protein. The Abl inhibitors imatinib, nilotinib and dasatinib are currently used to treat CML, but resistance to these inhibitors is a significant clinical problem. The kinase inhibitor bosutinib has shown efficacy in clinical trials for imatinib-resistant CML, but its binding mode is unknown. We present the 2.4 Å structure of bosutinib bound to the kinase domain of Abl, which explains the inhibitor's activity against several imatinib-resistant mutants, and reveals that similar inhibitors that lack a nitrile moiety could be effective against the common T315I mutant. We also report that two distinct chemical compounds are currently being sold under the name “bosutinib”, and report spectroscopic and structural characterizations of both. We show that the fluorescence properties of these compounds allow inhibitor binding to be measured quantitatively, and that the infrared absorption of the nitrile group reveals a different electrostatic environment in the conserved ATP-binding sites of Abl and Src kinases. Exploiting such differences could lead to inhibitors with improved selectivity. PMID:22493660

  13. Quantitative comparison of CrkL-SH3 binding proteins from embryonic murine brain and liver: Implications for developmental signaling and the quantification of protein species variants in bottom-up proteomics.

    PubMed

    Cheerathodi, Mujeeburahim; Vincent, James J; Ballif, Bryan A

    2015-07-01

    A major aim of proteomics is to comprehensively identify and quantify all protein species variants from a given biological source. However, in spite of its tremendous utility, bottom-up proteomic strategies can do little to provide true quantification of distinct whole protein species variants given its reliance on proteolysis. This is particularly true when molecular size information is lost as in gel-free proteomics. Crk and CrkL comprise a family of adaptor proteins that couple upstream phosphotyrosine signals to downstream effectors by virtue of their SH2 and SH3 domains respectively. Here we compare the identification and quantification of CrkL-SH3 binding partners between embryonic murine brain and liver. We also uncover and quantify tissue-specific variants in CrkL-SH3 binding proteins.

  14. dRAGging Amino Acid-mTORC1 Signaling by SH3BP4

    PubMed Central

    Kim, Young-Mi; Kim, Do-Hyung

    2013-01-01

    Mammalian target of rapamycin complex 1 (mTORC1) is a master regulator of cell growth and autophagy. Its activity is regulated by the availability of amino acids and growth factors. The activation of mTORC1 by growth factors, such as insulin and insulin-like growth factor-1 (IGF-1), is mediated by tuberous sclerosis complex (TSC) 1 and 2 and Rheb GTPase. Relative to the growth factor-regulated mTORC1 pathway, the evolutionarily ancient amino acid-mTORC1 pathway remains not yet clearly defined. The amino acid-mTORC1 pathway is mediated by Rag GTPase heterodimers. Several binding proteins of Rag GTPases were discovered in recent studies. Here, we discuss the functions and mechanisms of the newly-identified binders of Rag GTPases. In particular, this review focuses on SH3 binding protein 4 (SH3BP4), the protein recently identifed as a negative regulator of Rag GTPases. PMID:23274731

  15. 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.

  16. Interaction with the Src Homology (SH3-SH2) Region of the Src-family Kinase Hck Structures the HIV-1 Nef Dimer for Kinase Activation and Effector Recruitment*

    PubMed Central

    Alvarado, John Jeff; Tarafdar, Sreya; Yeh, Joanne I.; Smithgall, Thomas E.

    2014-01-01

    HIV-1 Nef supports high titer viral replication in vivo and is essential for AIDS progression. Nef function depends on interactions with multiple host cell effectors, including Hck and other Src-family kinases. Here we describe the x-ray crystal structure of Nef in complex with the Hck SH3-SH2 regulatory region to a resolution of 1.86 Å. The complex crystallized as a dimer of complexes, with the conserved Nef PXXPXR motif engaging the Hck SH3 domain. A new intercomplex contact was found between SH3 Glu-93, and Nef Arg-105. Mutagenesis of Hck SH3 Glu-93 interfered with Nef·Hck complex formation and kinase activation in cells. The Hck SH2 domains impinge on the N-terminal region of Nef to stabilize a dimer conformation that exposes Asp-123, a residue critical for Nef function. Our results suggest that in addition to serving as a kinase effector for Nef, Hck binding may reorganize the Nef dimer for functional interaction with other signaling partners. PMID:25122770

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

    PubMed

    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

    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.

  18. 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

  19. Efficacy of the dual PI3K and mTOR inhibitor NVP-BEZ235 in combination with nilotinib against BCR-ABL-positive leukemia cells involves the ABL kinase domain mutation

    PubMed Central

    Okabe, Seiichi; Tauchi, Tetsuzo; Tanaka, Yuko; Kitahara, Toshihiko; Kimura, Shinya; Maekawa, Taira; Ohyashiki, Kazuma

    2014-01-01

    Imatinib, an ABL tyrosine kinase inhibitor (TKI), has shown clinical efficacy against chronic myeloid leukemia (CML). However, a substantial number of patients develop resistance to imatinib treatment due to the emergence of clones carrying mutations in the protein BCR-ABL. The phosphoinositide 3 kinase (PI3K)/Akt/mammalian target of rapamycin (mTOR) pathway regulates various processes, including cell proliferation, cell survival, and antiapoptosis activity. In this study, we investigated the efficacy of NVP-BEZ235, a dual PI3K and mTOR inhibitor, using BCR-ABL-positive cell lines. Treatment with NVP-BEZ235 for 48 h inhibited cell growth and induced apoptosis. The phosphorylation of the AKT kinase, eukaryotic initiation factor 4-binding protein 1 (4E-BP1), and p70 S6 kinase were decreased after NVP-BEZ235 treatment. The combination of NVP-BEZ235 with a BCR-ABL kinase inhibitor, imatinib, or nilotinib, induced a more pronounced colony growth inhibition, whereas the combination of NVP-BEZ235 and nilotinib was more effective in inducing apoptosis and reducing the phosphorylation of AKT, 4E-BP1, and S6 kinase. NVP-BEZ235 in combination with nilotinib also inhibited tumor growth in a xenograft model and inhibited the growth of primary T315I mutant cells and ponatinib-resistant cells. Taken together, these results suggest that administration of the dual PI3K and mTOR inhibitor NVP-BEZ235 may be an effective strategy against BCR-ABL mutant cells and may enhance the cytotoxic effects of nilotinib in ABL TKI-resistant BCR-ABL mutant cells. PMID:24100660

  20. 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

  1. The murine Nck SH2/SH3 adaptors are important for the development of mesoderm-derived embryonic structures and for regulating the cellular actin network.

    PubMed

    Bladt, Friedhelm; Aippersbach, Elke; Gelkop, Sigal; Strasser, Geraldine A; Nash, Piers; Tafuri, Anna; Gertler, Frank B; Pawson, Tony

    2003-07-01

    Mammalian Nck1 and Nck2 are closely related adaptor proteins that possess three SH3 domains, followed by an SH2 domain, and are implicated in coupling phosphotyrosine signals to polypeptides that regulate the actin cytoskeleton. However, the in vivo functions of Nck1 and Nck2 have not been defined. We have mutated the murine Nck1 and Nck2 genes and incorporated beta-galactosidase reporters into the mutant loci. In mouse embryos, the two Nck genes have broad and overlapping expression patterns. They are functionally redundant in the sense that mice deficient for either Nck1 or Nck2 are viable, whereas inactivation of both Nck1 and Nck2 results in profound defects in mesoderm-derived notochord and embryonic lethality at embryonic day 9.5. Fibroblast cell lines derived from Nck1(-/-) Nck2(-/-) embryos have defects in cell motility and in the organization of the lamellipodial actin network. These data suggest that the Nck SH2/SH3 adaptors have important functions in the development of mesodermal structures during embryogenesis, potentially linked to a role in cell movement and cytoskeletal organization.

  2. Structure of Crumbs tail in complex with the PALS1 PDZ–SH3–GK tandem reveals a highly specific assembly mechanism for the apical Crumbs complex

    PubMed Central

    Li, Youjun; Wei, Zhiyi; Yan, Yan; Wan, Qingwen; Du, Quansheng; Zhang, Mingjie

    2014-01-01

    The Crumbs (Crb) complex, formed by Crb, PALS1, and PATJ, is evolutionarily conserved in metazoans and acts as a master cell-growth and -polarity regulator at the apical membranes in polarized epithelia. Crb intracellular functions, including its direct binding to PALS1, are mediated by Crb’s highly conserved 37-residue cytoplasmic tail. However, the mechanistic basis governing the highly specific Crb–PALS1 complex formation is unclear, as reported interaction between the Crb tail (Crb-CT) and PALS1 PSD-95/DLG/ZO-1 (PDZ) domain is weak and promiscuous. Here we have discovered that the PDZ–Src homolgy 3 (SH3)–Guanylate kinase (GK) tandem of PALS1 binds to Crb-CT with a dissociation constant of 70 nM, which is ∼100-fold stronger than the PALS1 PDZ–Crb-CT interaction. The crystal structure of the PALS1 PDZ–SH3–GK–Crb-CT complex reveals that PDZ–SH3–GK forms a structural supramodule with all three domains contributing to the tight binding to Crb. Mutations disrupting the tertiary interactions of the PDZ–SH3–GK supramodule weaken the PALS1–Crb interaction and compromise PALS1-mediated polarity establishment in Madin–Darby canine kidney (MDCK) cysts. We further show that specific target binding of other members of membrane-associated guanylate kinases (MAGUKs) (e.g., CASK binding to neurexin) also requires the presence of their PDZ–SH3–GK tandems. PMID:25385611

  3. 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

  4. 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.

  5. Pex13p is an SH3 protein of the peroxisome membrane and a docking factor for the predominantly cytoplasmic PTs1 receptor.

    PubMed

    Gould, S J; Kalish, J E; Morrell, J C; Bjorkman, J; Urquhart, A J; Crane, D I

    1996-10-01

    Import of newly synthesized PTS1 proteins into the peroxisome requires the PTS1 receptor (Pex5p), a predominantly cytoplasmic protein that cycles between the cytoplasm and peroxisome. We have identified Pex13p, a novel integral peroxisomal membrane from both yeast and humans that binds the PTS1 receptor via a cytoplasmically oriented SH3 domain. Although only a small amount of Pex5p is bound to peroxisomes at steady state (< 5%), loss of Pex13p further reduces the amount of peroxisome-associated Pex5p by approximately 40-fold. Furthermore, loss of Pex13p eliminates import of peroxisomal matrix proteins that contain either the type-1 or type-2 peroxisomal targeting signal but does not affect targeting and insertion of integral peroxisomal membrane proteins. We conclude that Pex13p functions as a docking factor for the predominantly cytoplasmic PTS1 receptor.

  6. Functional interaction between nonreceptor tyrosine kinase c-Abl and SR-Rich protein RBM39.

    PubMed

    Mai, Sanyue; Qu, Xiuhua; Li, Ping; Ma, Qingjun; Liu, Xuan; Cao, Cheng

    2016-04-22

    RBM39, also known as splicing factor HCC1.4, acts as a transcriptional coactivator for the steroid nuclear receptors JUN/AP-1, ESR1/ER-α and ESR2/ER-β. RBM39 is involved in the regulation of the transcriptional responses of these steroid nuclear receptors and promotes transcriptional initiation. In this paper, we report that RBM39 interacts with the nonreceptor tyrosine kinase c-Abl. Both the Src homology (SH) 2 and SH3 domains of c-Abl interact with RBM39. The major tyrosine phosphorylation sites on RBM39 that are phosphorylated by c-Abl are Y95 and Y99, as demonstrated by liquid chromatography coupled with tandem mass spectrometry (LC/MS/MS) and mutational analysis. c-Abl was shown boost the transcriptional coactivation activity of RBM39 for ERα and PRβ in a tyrosine kinase-dependent manner. The results suggest that mammalian c-Abl plays an important role in steroid hormone receptor-mediated transcription by regulating RBM39.

  7. LASP1 is a novel BCR-ABL substrate and a phosphorylation-dependent binding partner of CRKL in chronic myeloid leukemia

    PubMed Central

    Grunewald, Thomas G. P.; Schweigel, Hardy; Nollau, Peter; Ziermann, Janine; Clement, Joachim H.; La Rosée, Paul; Hochhaus, Andreas; Butt, Elke

    2014-01-01

    Chronic myeloid leukemia (CML) is characterized by a genomic translocation generating a permanently active BCR-ABL oncogene with a complex pattern of atypically tyrosine-phosphorylated proteins that drive the malignant phenotype of CML. Recently, the LIM and SH3 domain protein 1 (LASP1) was identified as a component of a six gene signature that is strongly predictive for disease progression and relapse in CML patients. However, the underlying mechanisms why LASP1 expression correlates with dismal outcome remained unresolved. Here, we identified LASP1 as a novel and overexpressed direct substrate of BCR-ABL in CML. We demonstrate that LASP1 is specifically phosphorylated by BCR-ABL at tyrosine-171 in CML patients, which is abolished by tyrosine kinase inhibitor therapy. Further studies revealed that LASP1 phosphorylation results in an association with CRKL – another specific BCR-ABL substrate and bona fide biomarker for BCR-ABL activity. pLASP1-Y171 binds to non-phosphorylated CRKL at its SH2 domain. Accordingly, the BCR-ABL-mediated pathophysiological hyper-phosphorylation of LASP1 in CML disrupts normal regulation of CRKL and LASP1, which likely has implications on downstream BCR-ABL signaling. Collectively, our results suggest that LASP1 phosphorylation might serve as an additional candidate biomarker for assessment of BCR-ABL activity and provide a first step toward a molecular understanding of LASP1 function in CML. PMID:24913448

  8. Up-regulated expression and aberrant DNA methylation of LEP and SH3PXD2A in pre-eclampsia.

    PubMed

    Xiang, Yuqian; Cheng, Yan; Li, Xiaotian; Li, Qiaoli; Xu, Jiawei; Zhang, Junyu; Liu, Yun; Xing, Qinghe; Wang, Lei; He, Lin; Zhao, Xinzhi

    2013-01-01

    The primary mechanism underlying pre-eclampsia (PE) remains one of the most burning problems in the obstetrics and gynecology. In this study, we performed an expression profiling screen and detected 1312 genes that were differentially expressed (p<0.05 and fold change >1.5) in PE placentas, including LEP and SH3PXD2A. After validating the microarray results, we conducted the quantitative methylation analysis of LEP and SH3PXD2A in preeclamptic (n = 16) versus normal placentas (n = 16). Our results showed that many CpG sites close to the transcriptional start site (TSS) of LEP gene were hypomethylated in placentas from pregnancies with PE compared with those of in controls, including the TSS position (p = 0.001), the binding sites of Sp1 (p = 1.57×10(-4)), LP1 (p = 0.023) and CEBPα (p = 0.031). Luciferase reporter analysis confirmed the aberrant methylation of LEP promoter and CEBPα co-transfection had a role in the regulation of gene expression. Our results indicated the aberrant LEP promoter methylation was involved in the development of PE. We did not find a significant methylation differences between groups in the promoter region of SH3PXD2A, however, a CGI region in the gene body (CGI34) presented a higher methylation in preeclamptic placentas (p = 1.57×10(-4)), which might promote the efficiency of gene transcription. We speculated that SH3PXD2A may take part in the pathogenesis of PE through its role in the regulation of trophoblast cell invasion in the period of placenta formation.

  9. C3G forms complexes with Bcr-Abl and p38α MAPK at the focal adhesions in chronic myeloid leukemia cells: implication in the regulation of leukemic cell adhesion

    PubMed Central

    2013-01-01

    Background Previous studies by our group and others have shown that C3G interacts with Bcr-Abl through its SH3-b domain. Results In this work we show that C3G and Bcr-Abl form complexes with the focal adhesion (FA) proteins CrkL, p130Cas, Cbl and Abi1 through SH3/SH3-b interactions. The association between C3G and Bcr-Abl decreased upon Abi1 or p130Cas knock-down in K562 cells, which suggests that Abi1 and p130Cas are essential partners in this interaction. On the other hand, C3G, Abi1 or Cbl knock-down impaired adhesion to fibronectin, while p130Cas silencing enhanced it. C3G, Cbl and p130Cas-SH3-b domains interact directly with common proteins involved in the regulation of cell adhesion and migration. Immunoprecipitation and immunofluorescence studies revealed that C3G form complexes with the FA proteins paxillin and FAK and their phosphorylated forms. Additionally, C3G, Abi1, Cbl and p130Cas regulate the expression and phosphorylation of paxillin and FAK. p38α MAPK also participates in the regulation of adhesion in chronic myeloid leukemia cells. It interacts with C3G, CrkL, FAK and paxillin and regulates the expression of paxillin, CrkL and α5 integrin, as well as paxillin phosphorylation. Moreover, double knock-down of C3G/p38α decreased adhesion to fibronectin, similarly to the single silencing of one of these genes, either C3G or p38α. These suggest that C3G and p38α MAPK are acting through a common pathway to regulate cell adhesion in K562 cells, as previously described for the regulation of apoptosis. Conclusions Our results indicate that C3G-p38αMAPK pathway regulates K562 cell adhesion through the interaction with FA proteins and Bcr-Abl, modulating the formation of different protein complexes at FA. PMID:23343344

  10. 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

  11. Experimental and Analytic Evaluation of the Effects of Visual and Motion Simulation in SH-3 Helicopter Training. Technical Report 85-002.

    ERIC Educational Resources Information Center

    Pfeiffer, Mark G.; Scott, Paul G.

    A fly-only group (N=16) of Navy replacement pilots undergoing fleet readiness training in the SH-3 helicopter was compared with groups pre-trained on Device 2F64C with: (1) visual only (N=13); (2) no visual/no motion (N=14); and (3) one visual plus motion group (N=19). Groups were compared for their SH-3 helicopter performance in the transition…

  12. Overexpression of EGFR in Head and Neck Squamous Cell Carcinoma Is Associated with Inactivation of SH3GL2 and CDC25A Genes

    PubMed Central

    Maiti, Guru Prasad; Mondal, Pinaki; Mukherjee, Nupur; Ghosh, Amlan; Ghosh, Susmita; Dey, Sanjib; Chakrabarty, Jayanta; Roy, Anup; Biswas, Jaydip; Roychoudhury, Susanta; Panda, Chinmay Kumar

    2013-01-01

    The aim of this study is to understand the mechanism of EGFR overexpression in head and neck squamous cell carcinoma (HNSCC). For this reason, expression/mutation of EGFR were analyzed in 30 dysplastic head and neck lesions and 148 HNSCC samples of Indian patients along with 3 HNSCC cell lines. In addition, deletion/methylation/mutation/expression of SH3GL2 (associated with EGFR degradation) and CDC25A (associated with dephosphorylation of EGFR) were analyzed in the same set of samples. Our study revealed high frequency of EGFR overexpression (66–84%), low frequency of gene amplification (10–32.5%) and absence of functional mutation in the dysplastic lesions and HNSCC samples. No correlation was found between protein overexpression and mRNA expression/gene amplification status of EGFR. On the other hand, frequent alterations (deletion/methylation) of SH3GL2 (63–77%) and CDC25A (37–64%) were seen in the dysplastic and HNSCC samples. Two novel single nucleotide polymorphism (SNPs) were found in the promoter region of SH3GL2. Reduced expression of these genes showed concordance with their alterations. Overexpression of EGFR and p-EGFR were significantly associated with reduced expression and alterations of SH3GL2 and CDC25A respectively. In-vitro demethylation experiment by 5-aza-2′-deoxycytidine (5-aza-dC) showed upregulation of SH3GL2 and CDC25A and downregulation of EGFR expression in Hep2 cell line. Poor patient outcome was predicted in the cases with alterations of SH3GL2 and CDC25A in presence of human papilloma virus (HPV) infection. Also, low SH3GL2 and high EGFR expression was a predictor of poor patient survival. Thus, our data suggests that overexpression of EGFR due to its reduced degradation and dephosphorylation is needed for development of HNSCC. PMID:23675485

  13. Clinical, in silico, and experimental evidence for pathogenicity of two novel splice site mutations in the SH3TC2 gene.

    PubMed

    Laššuthová, Petra; Gregor, Martin; Sarnová, Lenka; Machalová, Eliška; Sedláček, Radek; Seeman, Pavel

    2012-09-01

    Charcot-Marie-Tooth (CMT) neuropathy is the most common inherited neuromuscular disorder. CMT is genetically very heterogeneous. Mutations in the SH3TC2 gene cause Charcot-Marie-Tooth neuropathy type 4C (CMT4C), a demyelinating form with autosomal recessive inheritance. In this study, two novel splice site mutations in the SH3TC2 gene have been studied (c.279G → A, c.3676-8G → A). Mutation c.279G → A was detected on one allele in two unrelated families with CMT4C in combination with a known pathogenic mutation (c.2860 C →T in one family, c.505T → C in the other) on the second allele of SH3TC2 gene. Variant c.3676-8G → A was detected in two patients from unrelated families on one allele of the SH3TC2 gene in combination with c.2860C →T mutation on the other allele. Several in silico tests were performed and exon trap experiments were undertaken in order to prove the effect of both mutations on proper splicing of SH3TC2. Fragments of SH3TC2 were subcloned into pET01 exon trap vector (Mobitec) and transfected into COS-7 cells. Aberrant splicing was predicted in silico for both mutations, which was confirmed by exon trap analysis. For c.279G → A mutation, 19 bases from intron 3 are retained in cDNA. The mutation c.3676-8G→ A produces a novel splice acceptor site for exon 17 and complex changes in splicing were observed. We present evidence that mutations c.279G → A and c.3676-8G →A in the SH3TC2 gene cause aberrant splicing and are therefore pathogenic and causal for CMT4C.

  14. BCR-ABL kinase domain mutation analysis in chronic myeloid leukemia patients treated with tyrosine kinase inhibitors: recommendations from an expert panel on behalf of European LeukemiaNet.

    PubMed

    Soverini, Simona; Hochhaus, Andreas; Nicolini, Franck E; Gruber, Franz; Lange, Thoralf; Saglio, Giuseppe; Pane, Fabrizio; Müller, Martin C; Ernst, Thomas; Rosti, Gianantonio; Porkka, Kimmo; Baccarani, Michele; Cross, Nicholas C P; Martinelli, Giovanni

    2011-08-01

    Mutations in the Bcr-Abl kinase domain may cause, or contribute to, resistance to tyrosine kinase inhibitors (TKIs) in chronic myeloid leukemia patients. Recommendations aimed to rationalize the use of BCR-ABL mutation testing in chronic myeloid leukemia have been compiled by a panel of experts appointed by the European LeukemiaNet (ELN) and European Treatment and Outcome Study and are here reported. Based on a critical review of the literature and, whenever necessary, on panelists' experience, key issues were identified and discussed concerning: (1) when to perform mutation analysis, (2) how to perform it, and (3) how to translate results into clinical practice. In chronic phase patients receiving imatinib first-line, mutation analysis is recommended only in case of failure or suboptimal response according to the ELN criteria. In imatinib-resistant patients receiving an alternative TKI, mutation analysis is recommended in case of hematologic or cytogenetic failure as provisionally defined by the ELN. The recommended methodology is direct sequencing, although it may be preceded by screening with other techniques, such as denaturing-high performance liquid chromatography. In all the cases outlined within this abstract, a positive result is an indication for therapeutic change. Some specific mutations weigh on TKI selection. PMID:21562040

  15. 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.

  16. Screening for SH3TC2 gene mutations in a series of demyelinating recessive Charcot-Marie-Tooth disease (CMT4).

    PubMed

    Piscosquito, Giuseppe; Saveri, Paola; Magri, Stefania; Ciano, Claudia; Gandioli, Claudia; Morbin, Michela; Bella, Daniela D; Moroni, Isabella; Taroni, Franco; Pareyson, Davide

    2016-09-01

    Charcot-Marie-Tooth disease type 4C (CMT4C) is an autosomal recessive (AR) demyelinating neuropathy associated to SH3TC2 mutations, characterized by early onset, spine deformities, and cranial nerve involvement. We screened 43 CMT4 patients (36 index cases) with AR inheritance, demyelinating nerve conductions, and negative testing for PMP22 duplication, GJB1 and MPZ mutations, for SH3TC2 mutations. Twelve patients (11 index cases) had CMT4C as they carried homozygous or compound heterozygous mutations in SH3TC2. We found six mutations: three nonsense (p.R1109*, p.R954*, p.Q892*), one splice site (c.805+2T>C), one synonymous variant (p.K93K) predicting altered splicing, and one frameshift (p.F491Lfs*32) mutation. The splice site and the frameshift mutations are novel. Mean onset age was 7 years (range: 1-14). Neuropathy was moderate-to-severe. Scoliosis was present in 11 patients (severe in 4), and cranial nerve deficits in 9 (hearing loss in 7). Scoliosis and cranial nerve involvement are frequent features of this CMT4 subtype, and their presence should prompt the clinician to look for SH3TC2 gene mutations. In our series of undiagnosed CMT4 patients, SH3TC2 mutation frequency is 30%, confirming that CMT4C may be the most common AR-CMT type.

  17. 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

  18. 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.

  19. 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...

  20. Affinity and specificity requirements for the first Src homology 3 domain of the Crk proteins.

    PubMed Central

    Knudsen, B S; Zheng, J; Feller, S M; Mayer, J P; Burrell, S K; Cowburn, D; Hanafusa, H

    1995-01-01

    The specificity of SH3 domain complex formation plays an important role in determining signal transduction events. We have previously identified a highly specific interaction between the first CrkSH3 domain [CrkSH3(1)] and proline-rich sequences in the guanine nucleotide exchange factor C3G. A 10 amino acid peptide derived from the first proline-rich sequence (P3P4P5A6L7P8P9K10K11R12) bound with a Kd of 1.89 +/- 0.06 microM and fully retained the high affinity and unique selectivity for the CrkSH3(1) domain. Mutational analysis showed that P5, P8, L7 and K10 are critical for high affinity binding. A conservative mutation, K10R, significantly decreased the affinity for the CrkSH3(1) domain while increasing the affinity for Grb2. Comparative binding studies with the K10R and K10A mutant peptides to c-Crk and v-Crk further suggested that K10 binds via a charge-dependent and a charge-independent interaction to the RT loop of the CrkSH3(1) domain. Besides determining important structural features necessary for high affinity and specificity binding to the CrkSH3(1) domain, our results also demonstrate that a conservative mutation in a single amino acid can significantly alter the specificity of an SH3 binding peptide. Images PMID:7774577

  1. 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.

  2. Sorbin and SH3 Domain‐Containing Protein 2 Is Released From Infarcted Heart in the Very Early Phase: Proteomic Analysis of Cardiac Tissues From Patients

    PubMed Central

    Kakimoto, Yu; Ito, Shinji; Abiru, Hitoshi; Kotani, Hirokazu; Ozeki, Munetaka; Tamaki, Keiji; Tsuruyama, Tatsuaki

    2013-01-01

    Background Few proteomic studies have examined human cardiac tissue following acute lethal infarction. Here, we applied a novel proteomic approach to formalin‐fixed, paraffin‐embedded human tissue and aimed to reveal the molecular changes in the very early phase of acute myocardial infarction. Methods and Results Heart tissue samples were collected from 5 patients who died within 7 hours of myocardial infarction and from 5 age‐ and sex‐matched control cases. Infarcted and control myocardia were histopathologically diagnosed and captured using laser microdissection. Proteins were extracted using an originally established method and analyzed using liquid chromatography–tandem mass spectrometry. The label‐free quantification demonstrated that the levels of 21 proteins differed significantly between patients and controls. In addition to known biomarkers, the sarcoplasmic protein sorbin and SH3 domain‐containing protein 2 (SORBS2) was greatly reduced in infarcted myocardia. Immunohistochemical analysis of cardiac tissues confirmed the decrease, and Western blot analysis showed a significant increase in serum sorbin and SH3 domain‐containing protein 2 in acute myocardial infarction patients (n=10) compared with control cases (n=11). Conclusions Our advanced comprehensive analysis using patient tissues and serums indicated that sarcoplasmic sorbin and SH3 domain‐containing protein 2 is released from damaged cardiac tissue into the bloodstream upon lethal acute myocardial infarction. The proteomic strategy presented here is based on precise microscopic findings and is quite useful for candidate biomarker discovery using human tissue samples stored in depositories. PMID:24342996

  3. 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

  4. Ras-GAP SH3 domain binding protein (G3BP) is a modulator of USP10, a novel human ubiquitin specific protease.

    PubMed

    Soncini, C; Berdo, I; Draetta, G

    2001-06-28

    Degradation of cellular proteins through ubiquitination is a fundamental strategy for regulating biological pathways. De-ubiquitination, i.e. the removal of ubiquitin from proteins and peptides to which ubiquitin is attached, is catalyzed by processing proteases known as de-ubiquitinating enzymes. We are studying the biology of a family of de-ubiquitinating enzymes, the mammalian ubiquitin-specific proteases (USPs), some of which appear to play a role in growth control. Given the fact that the modes of regulation of USPs and of their substrate specificity are poorly understood, we decided to attempt the identification of USP interacting proteins. Using the yeast two-hybrid system (2HS), we have isolated a cDNA clone whose product specifically interacts with USP10 but not with other USP baits tested. The isolated clone encodes a protein known to interact with the Ras-GTPase activating protein (G3BP). This interaction was further confirmed by performing a 2HS with G3BP, which led to the isolation of USP10 encoding cDNAs. We validated the interaction between the two proteins by performing in vitro binding assays and immunoprecipitations in human cells. G3BP does not appear to be a substrate of USP10; it rather inhibits the ability of USP10 to disassemble ubiquitin chains. The USP10/G3BP complex appears to co-immunoprecipitate with ubiquitinated species that could be substrates of USP10.

  5. 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 ...

  6. Src-homology 3 domain of protein kinase p59fyn mediates binding to phosphatidylinositol 3-kinase in T cells.

    PubMed Central

    Prasad, K V; Janssen, O; Kapeller, R; Raab, M; Cantley, L C; Rudd, C E

    1993-01-01

    The Src-related tyrosine kinase p59fyn(T) plays an important role in the generation of intracellular signals from the T-cell antigen receptor TCR zeta/CD3 complex. A key question concerns the nature and the binding sites of downstream components that interact with this Src-related kinase. p59fyn(T) contains Src-homology 2 and 3 domains (SH2 and SH3) with a capacity to bind to intracellular proteins. One potential downstream target is phosphatidylinositol 3-kinase (PI 3-kinase). In this study, we demonstrate that anti-CD3 and anti-Fyn immunoprecipitates possess PI 3-kinase activity as assessed by TLC and HPLC. Both free and receptor-bound p59fyn(T) were found to bind to the lipid kinase. Further, our results indicate that Src-related kinases have developed a novel mechanism to interact with PI 3-kinase. Precipitation using GST fusion proteins containing Fyn SH2, SH3, and SH2/SH3 domains revealed that PI 3-kinase bound principally to the SH3 domain of Fyn. Fyn SH3 bound directly to the p85 subunit of PI 3-kinase as expressed in a baculoviral system. Anti-CD3 crosslinking induced an increase in the detection of Fyn SH3-associated PI 3-kinase activity. Thus PI 3-kinase is a target of SH3 domains and is likely to play a major role in the signals derived from the TCR zeta/CD3-p59fyn complex. Images Fig. 1 Fig. 2 Fig. 3 Fig. 4 PMID:8394019

  7. 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

  8. Progesterone receptor (PR) polyproline domain (PPD) mediates inhibition of epidermal growth factor receptor (EGFR) signaling in non-small cell lung cancer cells.

    PubMed

    Kawprasertsri, Sornsawan; Pietras, Richard J; Marquez-Garban, Diana C; Boonyaratanakornkit, Viroj

    2016-05-01

    Recent evidence has suggested a possible role for progesterone receptor (PR) in the progression of non-small cell lung cancer (NSCLC). However, little is known concerning roles of PR in NSCLC. PR contains a polyproline domain (PPD), which directly binds to the SH3 domain of signaling molecules. Because PPD-SH3 interactions are essential for EGFR signaling, we hypothesized that the presence of PR-PPD interfered with EGFR-mediated signaling and cell proliferation. We examined the role of PR-PPD in cell proliferation and signaling by stably expressing PR-B, or PR-B with disrupting mutations in the PPD (PR-BΔSH3), from a tetracycline-regulated promoter in A549 NSCLC cells. PR-B dose-dependently inhibited cell growth in the absence of ligand, and progestin (R5020) treatment further suppressed the growth. Treatment with RU486 abolished PR-B- and R5020-mediated inhibition of cell proliferation. Expression of PR-BΔSH3 and treatment with R5020 or RU486 had no effect on cell proliferation. Furthermore, PR-B expression but not PR-BΔSH3 expression reduced EGF-induced A549 proliferation and activation of ERK1/2, in the absence of ligand. Taken together, our data demonstrated the significance of PR extranuclear signaling through PPD interactions in EGFR-mediated proliferation and signaling in NSCLC.

  9. Multifunctional Abl kinases in health and disease.

    PubMed

    Khatri, Aaditya; Wang, Jun; Pendergast, Ann Marie

    2016-01-01

    The Abelson tyrosine kinases were initially identified as drivers of leukemia in mice and humans. The Abl family kinases Abl1 and Abl2 regulate diverse cellular processes during development and normal homeostasis, and their functions are subverted during inflammation, cancer and other pathologies. Abl kinases can be activated by multiple stimuli leading to cytoskeletal reorganization required for cell morphogenesis, motility, adhesion and polarity. Depending on the cellular context, Abl kinases regulate cell survival and proliferation. Emerging data support important roles for Abl kinases in pathologies linked to inflammation. Among these are neurodegenerative diseases and inflammatory pathologies. Unexpectedly, Abl kinases have also been identified as important players in mammalian host cells during microbial pathogenesis. Thus, the use of Abl kinase inhibitors might prove to be effective in the treatment of pathologies beyond leukemia and solid tumors. In this Cell Science at a Glance article and in the accompanying poster, we highlight the emerging roles of Abl kinases in the regulation of cellular processes in normal cells and diverse pathologies ranging from cancer to microbial pathogenesis.

  10. Teaching English to Less-Able Learners.

    ERIC Educational Resources Information Center

    Gefen, Raphael

    1981-01-01

    Discusses the "less-able" student via a description of the strategies used by the good language learner. Such strategies include a positive, active, and tolerant approach to learning the language, with a willingness to practice the language. The less-able needs a constant sense of achievement to continue. (PJM)

  11. 46 CFR 15.840 - Able seamen.

    Code of Federal Regulations, 2010 CFR

    2010-10-01

    ... 46 Shipping 1 2010-10-01 2010-10-01 false Able seamen. 15.840 Section 15.840 Shipping COAST GUARD, DEPARTMENT OF HOMELAND SECURITY MERCHANT MARINE OFFICERS AND SEAMEN MANNING REQUIREMENTS Computations § 15.840 Able seamen. (a) With certain exceptions, 46 U.S.C. 8702 applies to all vessels of at least...

  12. 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.

  13. c-Abl antagonizes the YAP oncogenic function

    PubMed Central

    Keshet, R; Adler, J; Ricardo Lax, I; Shanzer, M; Porat, Z; Reuven, N; Shaul, Y

    2015-01-01

    YES-associated protein (YAP) is a central transcription coactivator that functions as an oncogene in a number of experimental systems. However, under DNA damage, YAP activates pro-apoptotic genes in conjunction with p73. This program switching is mediated by c-Abl (Abelson murine leukemia viral oncogene) via phosphorylation of YAP at the Y357 residue (pY357). YAP as an oncogene coactivates the TEAD (transcriptional enhancer activator domain) family transcription factors. Here we asked whether c-Abl regulates the YAP–TEAD functional module. We found that DNA damage, through c-Abl activation, specifically depressed YAP–TEAD-induced transcription. Remarkably, c-Abl counteracts YAP-induced transformation by interfering with the YAP–TEAD transcriptional program. c-Abl induced TEAD1 phosphorylation, but the YAP–TEAD complex remained unaffected. In contrast, TEAD coactivation was compromised by phosphomimetic YAP Y357E mutation but not Y357F, as demonstrated at the level of reporter genes and endogenous TEAD target genes. Furthermore, YAP Y357E also severely compromised the role of YAP in cell transformation, migration, anchorage-independent growth, and epithelial-to-mesenchymal transition (EMT) in human mammary MCF10A cells. These results suggest that YAP pY357 lost TEAD transcription activation function. Our results demonstrate that YAP pY357 inactivates YAP oncogenic function and establish a role for YAP Y357 phosphorylation in cell-fate decision. PMID:25361080

  14. Project ABLE: Descriptive Summaries of Project ABLE Programs. 1968-1969.

    ERIC Educational Resources Information Center

    New York State Education Dept., Albany. Div. of Pupil Personnel Services.

    Project ABLE is a series of demonstration educational programs initiated in 1961 for the purpose of identifying and encouraging potential abilities among pupils from culturally deprived groups and low socioeconomic backgrounds. The word ABLE was chosen to indicate a belief that, given adequate resources, schools are ABLE to devise educational…

  15. Able-Disabled Battling against Barriers.

    ERIC Educational Resources Information Center

    Neill, Shirley Boes

    1978-01-01

    Disabled persons manage a program in San Diego, California, called "Able-Disabled Advocacy," which offers, with federal aid, work experience, counseling, and placement services to the area's disabled through experience in publishing a monthly magazine promoting opportunities for the disabled and through an on-the-job training project. (MF)

  16. On not being able to dream.

    PubMed

    Ogden, Thomas H

    2003-02-01

    In this paper, the author explores the phenomenon of not being able to dream (as opposed to not being able to remember one's dreams) from three different vantage points. First, from the point of view of psychoanalytic theory, he discusses Bion's idea that the work of dreaming creates the conscious and unconscious mind (and not the other way around). A person who cannot dream is unable to generate differentiable conscious and unconscious experience and, consequently, lives in a psychic state in which he is unable to differentiate waking from sleeping, dreaming from perceiving. The author then approaches the problem of the inability to dream from the perspective achieved by a literary work. He discusses a Borges fiction that creates, in a singularly artful way, the experience of not being able to dream. Finally, the author utilises the vantage point of a detailed account of a clinical experience to explore what it means not to be able to dream. He describes an initial state characterised by the patient's proliferation of unutilisable 'psychic noise' which, over a period of years, led to the analyst's experiencing 'reverie-deprivation' and brief periods of countertransference psychosis. Two analytic sessions are presented and discussed in which psychological work was done that contributed to an enhanced capacity on the part of both patient and analyst for genuine dreaming - both in sleep and in analytic reverie states.

  17. ABL and BAM Friction Analysis Comparison

    DOE PAGES

    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.

  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. Energetics of Src homology domain interactions in receptor tyrosine kinase-mediated signaling.

    PubMed

    Ladbury, John E; Arold, Stefan T

    2011-01-01

    Intracellular signaling from receptor tyrosine kinases (RTK) on extracellular stimulation is fundamental to all cellular processes. The protein-protein interactions which form the basis of this signaling are mediated through a limited number of polypeptide domains. For signal transduction without corruption, based on a model where signaling pathways are considered as linear bimolecular relays, these interactions have to be highly specific. This is particularly the case when one considers that any cell may have copies of similar binding domains found in numerous proteins. In this work, an overview of the thermodynamics of binding of two of the most common of these domains (SH2 and SH3 domains) is given. This, coupled with insight from high-resolution structural detail, provides a comprehensive survey of how recognition of cognate binding sites for these domains occurs. Based on the data presented, we conclude that specificity offered by these interactions of SH2 and SH3 domains is limited and not sufficient to enforce mutual exclusivity in RTK-mediated signaling. This may explain the current lack of success in pharmaceutical intervention to inhibit the interactions of these domains when they are responsible for aberrant signaling and the resulting disease states such as cancer.

  20. Bacteriophage-derived CHAP domain protein, P128, kills Staphylococcus cells by cleaving interpeptide cross-bridge of peptidoglycan.

    PubMed

    Sundarrajan, Sudarson; Raghupatil, Junjappa; Vipra, Aradhana; Narasimhaswamy, Nagalakshmi; Saravanan, Sanjeev; Appaiah, Chemira; Poonacha, Nethravathi; Desai, Srividya; Nair, Sandhya; Bhatt, Rajagopala Narayana; Roy, Panchali; Chikkamadaiah, Ravisha; Durgaiah, Murali; Sriram, Bharathi; Padmanabhan, Sriram; Sharma, Umender

    2014-10-01

    P128 is an anti-staphylococcal protein consisting of the Staphylococcus aureus phage-K-derived tail-associated muralytic enzyme (TAME) catalytic domain (Lys16) fused with the cell-wall-binding SH3b domain of lysostaphin. In order to understand the mechanism of action and emergence of resistance to P128, we isolated mutants of Staphylococcus spp., including meticillin-resistant Staphylococcus aureus (MRSA), resistant to P128. In addition to P128, the mutants also showed resistance to Lys16, the catalytic domain of P128. The mutants showed loss of fitness as shown by reduced rate of growth in vitro. One of the mutants tested was found to show reduced virulence in animal models of S. aureus septicaemia suggesting loss of fitness in vivo as well. Analysis of the antibiotic sensitivity pattern showed that the mutants derived from MRSA strains had become sensitive to meticillin and other β-lactams. Interestingly, the mutant cells were resistant to the lytic action of phage K, although the phage was able to adsorb to these cells. Sequencing of the femA gene of three P128-resistant mutants showed either a truncation or deletion in femA, suggesting that improper cross-bridge formation in S. aureus could be causing resistance to P128. Using glutathione S-transferase (GST) fusion peptides as substrates it was found that both P128 and Lys16 were capable of cleaving a pentaglycine sequence, suggesting that P128 might be killing S. aureus by cleaving the pentaglycine cross-bridge of peptidoglycan. Moreover, peptides corresponding to the reported cross-bridge of Staphylococcus haemolyticus (GGSGG, AGSGG), which were not cleaved by lysostaphin, were cleaved efficiently by P128. This was also reflected in high sensitivity of S. haemolyticus to P128. This showed that in spite of sharing a common mechanism of action with lysostaphin, P128 has unique properties, which allow it to act on certain lysostaphin-resistant Staphylococcus strains.

  1. Structure of the second PDZ domain from human zonula occludens 2

    PubMed Central

    Chen, Hui; Tong, Shuilong; Li, Xu; Wu, Jiawen; Zhu, Zhiqiang; Niu, Liwen; Teng, Maikun

    2009-01-01

    Human zonula occludens 2 (ZO-2) protein is a multi-domain protein that consists of an SH3 domain, a GK domain and three copies of a PDZ domain with slight divergence. The three PDZ domains act as protein-recognition modules that may mediate protein assembly and subunit localization. The crystal structure of the second PDZ domain of ZO-2 (ZO-2 PDZ2) was determined by molecular replacement at 1.75 Å resolution, revealing a dimer in the asymmetric unit. The dimer is stabilized by extensive symmetrical domain-swapping of the β1 and β2 strands. Structural comparison shows that the ZO-2 PDZ2 homodimer may have a similar ligand-binding pattern to the ZO-1 PDZ2–connexin 43 complex. PMID:19342771

  2. 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

  3. A Coiled-Coil Mimetic Intercepts BCR-ABL1 Dimerization in Native and Kinase-Mutant Chronic Myeloid Leukemia

    PubMed Central

    Woessner, David W.; Eiring, Anna M.; Bruno, Benjamin J.; Zabriskie, Matthew S.; Reynolds, Kimberly R.; Miller, Geoffrey D.; O’Hare, Thomas; Deininger, Michael W.; Lim, Carol S.

    2015-01-01

    Targeted therapy of chronic myeloid leukemia is currently based on small-molecule inhibitors that directly bind the tyrosine kinase domain of BCR-ABL1. This strategy has generally been successful, but is subject to drug resistance due to point mutations in the kinase domain. Kinase activity requires transactivation of BCR-ABL1 following an oligomerization event, which is mediated by the coiled-coil (CC) domain at the N-terminus of the protein. Here, we describe a rationally engineered mutant version of the CC domain, called CCmut3, which interferes with BCR-ABL1 oligomerization and promotes apoptosis in BCR-ABL1-expressing cells, regardless of kinase domain mutation status. CCmut3 exhibits strong pro-apoptotic and anti-proliferative activity in cell lines expressing native BCR-ABL1, single kinase domain mutant BCR-ABL1 (E255V and T315I) or compound mutant BCR-ABL1 (E255V/T315I). Moreover, CCmut3 inhibits colony formation by primary CML CD34+ cells ex vivo, including a sample expressing the T315I mutant. These data suggest that targeting BCR-ABL1 with CC mutants may provide a novel alternative strategy for treating patients with resistance to current targeted therapies. PMID:25721898

  4. Molecular dynamics reveal BCR-ABL1 polymutants as a unique mechanism of resistance to PAN-BCR-ABL1 kinase inhibitor therapy

    PubMed Central

    Gibbons, Don L.; Pricl, Sabrina; Posocco, Paola; Laurini, Erik; Fermeglia, Maurizio; Sun, Hanshi; Talpaz, Moshe; Donato, Nicholas; Quintás-Cardama, Alfonso

    2014-01-01

    The acquisition of mutations within the BCR-ABL1 kinase domain is frequently associated with tyrosine kinase inhibitor (TKI) failure in chronic myeloid leukemia. Sensitive sequencing techniques have revealed a high prevalence of compound BCR-ABL1 mutations (polymutants) in patients failing TKI therapy. To investigate the molecular consequences of such complex mutant proteins with regards to TKI resistance, we determined by cloning techniques the presence of polymutants in a cohort of chronic-phase patients receiving imatinib followed by dasatinib therapy. The analysis revealed a high frequency of polymutant BCR-ABL1 alleles even after failure of frontline imatinib, and also the progressive exhaustion of the pool of unmutated BCR-ABL1 alleles over the course of sequential TKI therapy. Molecular dynamics analyses of the most frequent polymutants in complex with TKIs revealed the basis of TKI resistance. Modeling of BCR-ABL1 in complex with the potent pan-BCR-ABL1 TKI ponatinib highlighted potentially effective therapeutic strategies for patients carrying these recalcitrant and complex BCR-ABL1 mutant proteins while unveiling unique mechanisms of escape to ponatinib therapy. PMID:24550512

  5. 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

  6. 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

  7. 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

  8. 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.

  9. 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

  10. c-Abl modulates AICD dependent cellular responses: transcriptional induction and apoptosis.

    PubMed

    Vázquez, Mary C; Vargas, Lina M; Inestrosa, Nibaldo C; Alvarez, Alejandra R

    2009-07-01

    APP intracellular domain (AICD) has been proposed as a transcriptional inductor that moves to the nucleus with the adaptor protein Fe65 and regulates transcription. The two proteins, APP and Fe65, can be phosphorylated by c-Abl kinase. Neprilysin has been proposed as a target gene for AICD. We found that AICD expression is decreased by treatment with STI-571, a c-Abl inhibitor, suggesting a modulation of AICD transcription by c-Abl kinase. We observed interaction between c-Abl kinase, the AICD fragment and the Fe65 adaptor protein. In addition, STI-571 reduces apoptosis in APPSw, and the apoptotic response induced by Fe65 over-expression was inhibited by with the expression of a kinase dead (KD) c-Abl and enhanced by over-expression of WT-c-Abl. However, in the APPSw cells, the ability of the KD-c-Abl to protect against Fe65 was reduced. Finally, in APPSw clone, we detected higher trans-activation of the pro-apoptotic p73 isoform, TAp73 promoter. Our results show that c-Abl modulates AICD dependent cellular responses, transcriptional induction as well as the apoptotic response, which could participate in the onset and progression of the neurodegenerative pathology, observed in Alzheimer's disease (AD).

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

    PubMed

    Vognsen, Tina; Kristensen, Ole

    2012-03-30

    The crystal structure of the NTF2-like domain of the Drosophila homolog of Ras GTPase SH3 Binding Protein (G3BP), Rasputin, was determined at 2.7Å resolution. The overall structure is highly similar to nuclear transport factor 2: It is a homodimer comprised of a β-sheet and three α-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.

  12. 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.

  13. 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

  14. The Capable ABL: What Is Its Biological Function?

    PubMed Central

    2014-01-01

    The mammalian ABL1 gene encodes the ubiquitously expressed nonreceptor tyrosine kinase ABL. In response to growth factors, cytokines, cell adhesion, DNA damage, oxidative stress, and other signals, ABL is activated to stimulate cell proliferation or differentiation, survival or death, retraction, or migration. ABL also regulates specialized functions such as antigen receptor signaling in lymphocytes, synapse formation in neurons, and bacterial adhesion to intestinal epithelial cells. Although discovered as the proto-oncogene from which the Abelson leukemia virus derived its Gag–v-Abl oncogene, recent results have linked ABL kinase activation to neuronal degeneration. This body of knowledge on ABL seems confusing because it does not fit the one-gene-one-function paradigm. Without question, ABL capabilities are encoded by its gene sequence and that molecular blueprint designs this kinase to be regulated by subcellular location-dependent interactions with inhibitors and substrate activators. Furthermore, ABL shuttles between the nucleus and the cytoplasm where it binds DNA and actin—two biopolymers with fundamental roles in almost all biological processes. Taken together, the cumulated results from analyses of ABL structure-function, ABL mutant mouse phenotypes, and ABL substrates suggest that this tyrosine kinase does not have its own agenda but that, instead, it has evolved to serve a variety of tissue-specific and context-dependent biological functions. PMID:24421390

  15. Cloning and expression analysis of two novel paraflagellar rod domain genes found in Trypanosoma cruzi.

    PubMed

    Clark, April K; Kovtunovych, Gennadiy; Kandlikar, Sachin; Lal, Shailesh; Stryker, Gabrielle A

    2005-07-01

    The eukaryotic flagellum is one of the most complex macromolecular structures found in cells, containing more than 250 proteins. One unique structure in the flagella of trypanomastids is the paraflagellar rod (PFR). The PFR constitutes a lattice of cytoskeletal filaments that lies alongside the axoneme in the flagella. This unique and complex structure is critical for cell motility, though little is known about its molecular assembly or its role in the lifecycle of trypanosomatids. These proteins are of particular importance in Trypanosoma cruzi, as purified or recombinant PFR proteins have been demonstrated to be immunogenic, protecting mice from a lethal challenge with the parasite. We have searched the T. cruzi databases and discovered two novel genes containing PFR domains. Both these genes are transcribed in vivo and are significantly larger than the previously described PFR genes identified in T. cruzi (>2 Kb). Real-time PCR was used to examine the relative expression levels of six PFR genes, including the two we describe here, in all three stages of T. cruzi's lifecycle. Database searches have further provided EST and genomic sequence support for the presence of these genes in two other pathogenic trypanosomatids, Trypanosoma brucei and Leishmania spp. One of these genes, designated PFR5 contains a carboxy terminal SH3 domain not previously seen in PFR family genes. We propose that this proline-binding SH3 domain may play an important role in the assembly of the PFR. PMID:15918067

  16. 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.…

  17. Abl suppresses cell extrusion and intercalation during epithelium folding.

    PubMed

    Jodoin, Jeanne N; Martin, Adam C

    2016-09-15

    Tissue morphogenesis requires control over cell shape changes and rearrangements. In the Drosophila mesoderm, linked epithelial cells apically constrict, without cell extrusion or intercalation, to fold the epithelium into a tube that will then undergo epithelial-to-mesenchymal transition (EMT). Apical constriction drives tissue folding or cell extrusion in different contexts, but the mechanisms that dictate the specific outcomes are poorly understood. Using live imaging, we found that Abelson (Abl) tyrosine kinase depletion causes apically constricting cells to undergo aberrant basal cell extrusion and cell intercalation. abl depletion disrupted apical-basal polarity and adherens junction organization in mesoderm cells, suggesting that extruding cells undergo premature EMT. The polarity loss was associated with abnormal basolateral contractile actomyosin and Enabled (Ena) accumulation. Depletion of the Abl effector Enabled (Ena) in abl-depleted embryos suppressed the abl phenotype, consistent with cell extrusion resulting from misregulated ena Our work provides new insight into how Abl loss and Ena misregulation promote cell extrusion and EMT.

  18. 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

  19. 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

  20. Characterization of leukemias with ETV6-ABL1 fusion

    PubMed Central

    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

    2016-01-01

    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

  1. 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.

  2. Comparison of mutated JAK2 and ABL1 as oncogenes and drug targets in myeloproliferative disorders

    PubMed Central

    Walz, Christoph; Cross, Nicholas C. P.; Van Etten, Richard A.; Reiter, Andreas

    2012-01-01

    Constitutively activated mutants of the non-receptor tyrosine kinases (TK) ABL1 and JAK2 play a central role in the pathogenesis of clinically and morphologically distinct chronic myeloproliferative disorders but are also found in some cases of de novo acute leukemia and lymphoma. Ligand-independent activation occurs as a consequence of point mutations or insertions/deletions within functionally relevant regulatory domains (JAK2), or the creation of TK fusion proteins by balanced reciprocal translocations, insertions or episomal amplification (ABL1 and JAK2). Specific abnormalities are correlated with clinical phenotype, although some are broad and encompass several WHO-defined entities. TKs are excellent drug targets as exemplified by the activity of imatinib in BCR-ABL1-positive disease, particularly chronic myeloid leukemia. Resistance to imatinib is seen in a minority of cases and is often associated with the appearance of secondary point mutations within the TK domain of BCR-ABL1. These mutations are highly variable in their sensitivity to increased doses of imatinib or alternative TK-inhibitors such as nilotinib or dasatinib. Selective and non-selective inhibitors of JAK2 are currently being developed and encouraging data from pre-clinical experiments and initial phase-I-studies regarding efficacy and potential toxicity of these compounds have already been reported. PMID:18528425

  3. 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.

  4. 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…

  5. Modular organization of the PDZ domains in the human discs-large protein suggests a mechanism for coupling PDZ domain-binding proteins to ATP and the membrane cytoskeleton

    PubMed Central

    1996-01-01

    The human homologue (hDIg) of the Drosophila discs-large tumor suppressor (DIg) is a multidomain protein consisting of a carboxyl- terminal guanylate kinase-like domain, an SH3 domain, and three slightly divergent copies of the PDZ (DHR/GLGF) domain. Here have examined the structural organization of the three PDZ domains of hDIg using a combination of protease digestion and in vitro binding measurements. Our results show that the PDZ domains are organized into two conformationally stable modules one (PDZ, consisting of PDZ domains 1 and 2, and the other (PDZ) corresponding to the third PDZ domain. Using amino acid sequencing and mass spectrometry, we determined the boundaries of the PDZ domains after digestion with endoproteinase Asp- N, trypsin, and alpha-chymotrypsin. The purified PDZ1+2, but not the PDZ3 domain, contains a high affinity binding site for the cytoplasmic domain of Shaker-type K+ channels. Similarly, we demonstrate that the PDZ1+2 domain can also specifically bind to ATP. Furthermore, we provide evidence for an in vivo interaction between hDIg and protein 4.1 and show that the hDIg protein contains a single high affinity protein 4.1-binding site that is not located within the PDZ domains. The results suggest a mechanism by which PDZ domain-binding proteins may be coupled to ATP and the membrane cytoskeleton via hDlg. PMID:8909548

  6. Heat shock protein-70 neutralizes apoptosis inducing factor in Bcr/Abl expressing cells.

    PubMed

    Wang, Fang; Dai, An-Ya; Tao, Kun; Xiao, Qing; Huang, Zheng-Lan; Gao, Miao; Li, Hui; Wang, Xin; Cao, Wei-Xi; Feng, Wen-Li

    2015-10-01

    Bcr/Abl fusion protein is a hallmark of human chronic myeloid leukemia (CML). The protein can activate various signaling pathways to make normal cells transform malignantly and thus to facilitate tumorigenesis. It has been reported that heat shock protein-70 (HSP-70) can be served as an anti-apoptotic protein that suppresses Bax and Apo-2L/TRAIL. But it is unclear whether HSP-70 affects AIF-initiated apoptosis in Bcr/Abl expressing cells considering that HSP-70 is coincidentally over-regulated in these cells. Our findings supported that abundant HSP-70 in Bcr/Abl cells neutralizes AIF by segregating it from nucleus via direct interaction, leading to the failure of AIF initiating cell death and the silence of caspase-independent apoptotic pathway upon apoptotic induction. Moderate inhibition of HSP-70 expression by siRNA leads to Vp-16 triggered re-distribution of AIF in nucleus. In addition, AIF bears a HSP-70 binding domain allowing association with HSP-70. Therefore, disruption of the association using an AIF mutant lacking this domain can restore the potential of AIF importing into nucleus, and finally triggers cell death in a time dependent manner.

  7. 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.

  8. 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.

  9. 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.

  10. 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.

  11. 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

  12. 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)…

  13. The "Project ABLE" General Woodworking Curriculum. (Interim Product).

    ERIC Educational Resources Information Center

    American Institutes for Research in the Behavioral Sciences, Pittsburgh, PA.

    A sample of student tasks and topic objectives which were developed for woodworking courses in Project ABLE are given. A larger collection of these were identified for grades 10, 11, and 12. Prior to this activity an analysis of selected occupations was made, and a frequency count of skills and knowledges was utilized to determine the necessary…

  14. 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

  15. Melissotarsus ants are likely able to digest plant polysaccharides.

    PubMed

    Mony, Ruth; Dejean, Alain; Bilong, Charles Félix Bilong; Kenne, Martin; Rouland-Lefèvre, Corinne

    2013-10-01

    Melissotarsus ants have an extremely specialized set of behaviours. Both workers and gynes tunnel galleries in their host tree bark. Workers walk with their mesothoracic legs pointing upwards and tend Diaspididae hemiptera for their flesh. The ants use their forelegs to plug the galleries with silk that they secrete themselves. We hypothesised that the ants' energetic needs for nearly constant gallery digging could be satisfied through the absorption of host tree tissues; so, using basic techniques, we examined the digestive capacities of workers from two species. We show that workers are able to degrade oligosaccharides and heterosides as well as, to a lesser degree, polysaccharides. This is one of the rare reports on ants able to digest plant polysaccharides other than starch.

  16. 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

  17. Enhanced and selective killing of chronic myelogenous leukemia cells with an engineered BCR-ABL binding protein and imatinib.

    PubMed

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

    2012-11-01

    The oncoprotein Bcr-Abl stimulates prosurvival 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 relocalize 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 10 μM imatinib at 48 h 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

  18. Communication barriers between 'the able-bodied' and 'the handicapped'.

    PubMed

    Zola, I K

    1981-08-01

    The specific details and hardships of having a handicap or chronic disease vary from person to person. But not the core problem. The story is inevitably difficult both to hear and to tell. The hearing brings up many basic fears of personal and social failure to the listener. The telling confronts the individual who has a handicap with the depressing realities to be faced every day. Moreover, the problems that both "the able-bodied" and "the handicapped" have in communicating with one another are rooted deep in Western culture. PMID:6455101

  19. Isolation of oxalotrophic bacteria able to disperse on fungal mycelium.

    PubMed

    Bravo, Daniel; Cailleau, Guillaume; Bindschedler, Saskia; Simon, Anaele; Job, Daniel; Verrecchia, Eric; Junier, Pilar

    2013-11-01

    A technique based on an inverted Petri dish system was developed for the growth and isolation of soil oxalotrophic bacteria able to disperse on fungal mycelia. The method is related to the 'fungal highways' dispersion theory in which mycelial fungal networks allow active movement of bacteria in soil. Quantification of this phenomenon showed that bacterial dispersal occurs preferentially in upper soil horizons. Eight bacteria and one fungal strain were isolated by this method. The oxalotrophic activity of the isolated bacteria was confirmed through calcium oxalate dissolution in solid selective medium. After separation of the bacteria-fungus couple, partial sequencing of the 16S and the ITS1 and ITS2 sequences of the ribosomal RNA genes were used for the identification of bacteria and the associated fungus. The isolated oxalotrophic bacteria included strains related to Stenotrophomonas, Achromobacter, Lysobacter, Pseudomonas, Agrobacterium, Cohnella, and Variovorax. The recovered fungus corresponded to Trichoderma sp. A test carried out to verify bacterial transport in an unsaturated medium showed that all the isolated bacteria were able to migrate on Trichoderma hyphae or glass fibers to re-colonize an oxalate-rich medium. The results highlight the importance of fungus-driven bacterial dispersal to understand the functional role of oxalotrophic bacteria and fungi in soils. PMID:24106816

  20. 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

  1. 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.

  2. BCR-ABL tyrosine kinase inhibitors in the treatment of Philadelphia chromosome positive chronic myeloid leukemia: a review.

    PubMed

    An, Xin; Tiwari, Amit K; Sun, Yibo; Ding, Pei-Rong; Ashby, Charles R; Chen, Zhe-Sheng

    2010-10-01

    Chronic Myeloid Leukemia (CML) is a clonal disease characterized by the presence of the Philadelphia (Ph+) chromosome and its oncogenic product, BCR-ABL, a constitutively active tyrosine kinase, that is present in >90% of the patients. Epidemiologic data indicates that almost 5000 new cases are reported every year and 10% of these patients eventually succumb to the disease. The treatment of CML was revolutionized by the introduction of imatinib mesylate (IM, Gleevec), a BCR-ABL tyrosine kinase inhibitor (TKI). The clinical use of specific BCR-ABL inhibitors has resulted in a significantly improved prognosis, response rate, overall survival, and patient outcome in CML patients compared to previous therapeutic regimens. However, the complete eradication of CML in patients receiving imatinib was limited by the emergence of resistance mostly due to mutations in the ABL kinase domain and to a lesser extent by molecular residual disease after treatment. The second-generation BCR-ABL TKIs nilotinib (Tasigna) and dasatinib (Sprycel), showed significant activity in clinical trials in patients intolerant or resistant to imatinib therapy, except in those patients with the T315I BCR-ABL mutation. Identifying key components involved in the CML pathogenesis may lead to the exploration of new approaches that might eventually overcome resistance mediated to the BCR-ABL TKIs. Here, we present an overview about the current treatment of Ph+ CML patients with the TKIs and the obstacles to successful treatment with these drugs.

  3. 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

  4. 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

  5. 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.

  6. TAT-CC fusion protein depresses the oncogenicity of BCR-ABL in vitro and in vivo through interrupting its oligomerization.

    PubMed

    Huang, Zheng-Lan; Gao, Miao; Ji, Mao-Sheng; Tao, Kun; Xiao, Qing; Zhong, Liang; Zeng, Jian-Ming; Feng, Wen-Li

    2013-02-01

    Chronic myeloid leukemia (CML) is a clonal hematologic malignancy characterized by the BCR-ABL protein. BCR-ABL is a constitutively active tyrosine kinase and plays a critical role in the pathogenesis of CML. Imatinib mesylate, a selective tyrosine kinase inhibitor, is effective in CML, but drug resistance and relapse occur. The coiled-coil (CC) domain located in BCR(1-72) mediates BCR-ABL tetramerization, which is essential for the activation of tyrosine kinase and transformation potential of BCR-ABL. CC domain is supposed to be a therapeutic target for CML. We purified a TAT-CC protein competively binding with the endogenous CC domain to reduce BCR-ABL kinase activity. We found that TAT-CC co-located and interacted with BCR-ABL in Ba/F3-p210 and K562 cells. It induced apoptosis and inhibited proliferation in these cells. It increased the sensitivity of these cells to imatinib and reduced the phosphorylation of BCR-ABL, CRKL and STAT5. We confirmed that TAT-CC could attenuate the oncogenicity of Ba/F3-p210 cells and diminish the volume of K562 solid tumor in mice. We conclude targeting the CC may provide a complementary therapy to inhibit BCR-ABL oncogenicity.

  7. 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

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

    PubMed

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

    2015-09-21

    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.

  9. 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

  10. Incidence and clinical importance of BCR-ABL1 mutations in Iranian patients with chronic myeloid leukemia on imatinib.

    PubMed

    Rostami, Golale; Hamid, Mohammad; Yaran, Majid; Khani, Mohsen; Karimipoor, Morteza

    2015-05-01

    Mutations of the BCR-ABL1 kinase domain seem to be the most common cause of imatinib mesylate resistance in chronic myeloid leukemia (CML). We screened BCR-ABL1 kinase domain mutations using nested reverse transcriptase polymerase chain reaction and direct sequencing in 30 CML patients including 22 resistant patients and 8 patients with optimal response to imatinib. Three mutations of two different types were identified in 3 of 22 (13.6%) resistant patients. Two patients had p.E355G mutation in the catalytic domain, and the third patient had p.G398R in the activation loop that is reported here for the first time. No mutation was found in patients with optimal response to imatinib. The frequency of mutations was similar in patients with primary resistance compared with patients with secondary resistance (25 vs 11%; P=1). Mutation status had no impact on the overall survival and progression-free survival. p.E355G mutation was correlated with shorter survival (P=0.047) in resistant patients. We conclude that BCR- ABL1 mutations are associated with the clinical resistance, but may not be considered the only cause of resistance to imatinib. Mutational analysis may identify resistant patients at risk of disease progression. PMID:25740611

  11. 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

  12. Practical management of patients with chronic myeloid leukemia who develop tyrosine kinase inhibitor-resistant BCR-ABL1 mutations

    PubMed Central

    Ai, Jing

    2014-01-01

    Five BCR-ABL1 tyrosine kinase inhibitors (TKIs), imatinib, nilotinib, dasatinib, bosutinib, and ponatinib, are currently approved for the treatment of chronic myeloid leukemia (CML). Standard treatment of CML with TKIs is highly effective in reducing disease burden, delaying disease progression, and prolonging overall survival of patients; however, resistance to TKI treatment has become an increasingly important cause of treatment failure. The emergence of mutations in the BCR-ABL1 kinase domain is a common mechanism of TKI resistance, and laboratory testing to detect these mutations is currently available for clinical use. Patients who do not respond or have lost their response to TKI therapy should be considered for mutational testing. Despite clinical practice guidelines that recommend testing for BCR-ABL1 mutations in patients with clinical signs of TKI resistance, many oncologists and hematologists managing patients with CML do not perform such testing. This review addresses outstanding questions related to when testing should be conducted, what type of testing should be done, and how testing results should be applied to subsequent therapeutic decisions. It describes how BCR-ABL1 kinase domain mutations confer resistance, outlines the prevalence of mutations in patients with resistance to TKIs, summarizes the common and investigational methods used in mutational testing, and presents an algorithm reflecting a clinical perspective on how and when to conduct mutational testing, and what to do with test results. PMID:25360237

  13. 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.

  14. 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.

  15. A novel pyridopyrimidine inhibitor of abl kinase is a picomolar inhibitor of Bcr-abl-driven K562 cells and is effective against STI571-resistant Bcr-abl mutants.

    PubMed

    Huron, David R; Gorre, Mercedes E; Kraker, Alan J; Sawyers, Charles L; Rosen, Neal; Moasser, Mark M

    2003-04-01

    Inhibition of the constitutively active Bcr-abl tyrosine kinase(TK) by STI571 has proven to be a highly effective treatment for chronic myelogenous leukemia (CML). However, STI571 is only transiently effective in blast crisis, and drug resistance emerges by amplification of or development of mutational changes in Bcr-abl. We have screened a family of TK inhibitors of the pyrido [2,3-d]pyrimidine class, unrelated to STI571, and describe here a compound with substantial activity against STI-resistant mutant Bcr-abl proteins. This compound, PD166326, is a dual specificity TK inhibitor and inhibits src and abl in vitro with IC(50)s of 6 and 8 nM respectively. PD166326 inhibits the growth of K562 cells with IC(50) of 300 pM, leading to apoptotic G(1) arrest, whereas non-Bcr-abl cell types require >1000 times higher concentrations. We tested the effects of PD166326 on two of the clinically observed STI571-resistant Bcr-abl mutants. PD166326 potently inhibits the E255K mutant Bcr-abl protein and the growth of Bcr-ablE255K-driven cells. The T315I mutant Bcr-abl protein, which is mutated within the ATP-binding pocket, is resistant to PD166326; however, the growth of Bcr-ablT315I-driven cells is partially sensitive to this compound, likely through the inhibition of Bcr-abl effector pathways. These findings show that TK drug resistance is a structure-specific phenomenon and can be overcome by TK inhibitors of other structural classes, suggesting new approaches for future anticancer drug development. PD166326 is a prototype of a new generation of anti-Bcr-abl compounds with picomolar potency and substantial activity against STI571-resistant mutants.

  16. 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.

  17. 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

  18. Protein domain architectures.

    PubMed

    Mulder, Nicola J

    2010-01-01

    Proteins are composed of functional units, or domains, that can be found alone or in combination with other domains. Analysis of protein domain architectures and the movement of protein domains within and across different genomes provide clues about the evolution of protein function. The classification of proteins into families and domains is provided through publicly available tools and databases that use known protein domains to predict other members in new proteins sequences. Currently at least 80% of the main protein sequence databases can be classified using these tools, thus providing a large data set to work from for analyzing protein domain architectures. Each of the protein domain databases provide intuitive web interfaces for viewing and analyzing their domain classifications and provide their data freely for downloading. Some of the main protein family and domain databases are described here, along with their Web-based tools for analyzing domain architectures.

  19. A thirty-five nucleotides BCR-ABL1 insertion mutation of controversial significance confers resistance to imatinib in a patient with chronic myeloid leukemia (CML).

    PubMed

    Marcé, Silvia; Cortés, Montserrat; Zamora, Lurdes; Cabezón, Marta; Grau, Javier; Millá, Fuensanta; Feliu, Evarist

    2015-08-01

    Tyrosine kinase inhibitors (TKI) have improved the management of patients with chronic myeloid leukemia (CML). However, a significant proportion of patients does not achieve the optimal response or are resistant to TKI. ABL1 kinase domain mutations have been extensively implicated in the pathogenesis of TKI resistance. Although deletion or insertion of nucleotides in BCR-ABL1 has rarely been described, we identified a CML patient with an already described 35 nucleotides insertion (BCR-ABL1(35INS)) of controversial significance, that confers resistance to imatinib but sensitivity to dasatinib. PMID:25913326

  20. Understanding the Public Domain.

    ERIC Educational Resources Information Center

    Russell, Carrie

    2003-01-01

    This overview of the public domain covers: defining the public domain; figuring out if a work is protected by copyright; being sure a work is in the public domain; asserting the copyright protection and term; the Creative Commons initiative; building the Information Commons; when permission is needed for using a public domain work; and special…

  1. Domain Differences in Early Social Interactions

    ERIC Educational Resources Information Center

    Dahl, Audun; Campos, Joseph J.

    2013-01-01

    Different social experiences help children develop distinctions between domains of norms. This study investigated whether mothers respond differently to moral, prudential, and pragmatic norms during the 2nd year, a period that precedes the time when children are able to make explicit distinctions between these norms. Sixty mothers of infants…

  2. Subcellular localization of Bcr, Abl, and Bcr-Abl proteins in normal and leukemic cells and correlation of expression with myeloid differentiation.

    PubMed Central

    Wetzler, M; Talpaz, M; Van Etten, R A; Hirsh-Ginsberg, C; Beran, M; Kurzrock, R

    1993-01-01

    We used specific antisera and immunohistochemical methods to investigate the subcellular localization and expression of Bcr, Abl, and Bcr-Abl proteins in leukemic cell lines and in fresh human leukemic and normal samples at various stages of myeloid differentiation. Earlier studies of the subcellular localization of transfected murine type IV c-Abl protein in fibroblasts have shown that this molecule resides largely in the nucleus, whereas transforming deletion variants are localized exclusively in the cytoplasm. Here, we demonstrate that the murine type IV c-Abl protein is also found in the nucleus when overexpressed in a mouse hematopoietic cell line. However, in both normal and leukemic human hematopoietic cells, c-Abl is discerned predominantly in the cytoplasm, with nuclear staining present, albeit at a lower level. In contrast, normal endogenous Bcr protein, as well as the aberrant p210BCR-ABL and p190BCR-ABL proteins consistently localize to the cytoplasm in both cell lines and fresh cells. The results with p210BCR-ABL were confirmed in a unique Ph1-positive chronic myelogenous leukemia (CML) cell line, KBM5, which lacks the normal chromosome 9 and hence the normal c-Abl product. Because the p210BCR-ABL protein appears cytoplasmic in both chronic phase and blast crisis CML cells, as does the p190BCR-ABL in Ph1-positive acute leukemia, a change in subcellular location of Bcr-Abl proteins between cytoplasm and nucleus cannot explain the different spectrum of leukemias associated with p210 and p190, nor the transition from the chronic to the acute leukemia phenotype seen in CML. Further analysis of fresh CML and normal hematopoietic bone marrow cells reveals that p210BCR-ABL, as well as the normal Bcr and Abl proteins, are expressed primarily in the early stages of myeloid maturation, and that levels of expression are reduced significantly as the cells mature to polymorphonuclear leukocytes. Similarly, a decrease in Bcr and Abl levels occurs in HL-60 cells

  3. 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

  4. 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

  5. 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.

  6. Major Molecular Response Achievement in CML Patients Can Be Predicted by BCR-ABL1/ABL1 or BCR-ABL1/GUS Ratio at an Earlier Time Point of Follow-Up than Currently Recommended

    PubMed Central

    Huet, Sarah; Cony-Makhoul, Pascale; Heiblig, Maël; Tigaud, Isabelle; Gazzo, Sophie; Belhabri, Amine; Souche, Denis; Michallet, Mauricette; Magaud, Jean-Pierre; Hayette, Sandrine; Nicolini, Franck

    2014-01-01

    Recent studies demonstrate that early molecular response to tyrosine-kinase inhibitors is strongly predictive of outcome in chronic myeloid leukemia patients and that early response landmarks may identify patients at higher risk for transformation who would benefit from an early switch to second-line therapy. In this study, we evaluated the ability of the control gene GUS to identify relevant thresholds for known therapeutic decision levels (BCR-ABL1/ABL1IS  = 10% and 0.1%). We then defined the most relevant cut-offs for early molecular response markers (transcript level at 3 months, halving time and log reduction between diagnosis and 3 months of treatment) using GUS or ABL1. We demonstrated that, although both control genes could be used (in an equivalent way) to accurately assess early molecular response, the BCR-ABL1/GUS level at diagnosis is impacted by the higher GUS copy number over-expressed in CML cells, thus negatively impacting its ability to completely replace ABL1 at diagnosis. Furthermore, we pointed out, for the first time, that it would be helpful to monitor BCR-ABL1 levels at an earlier time point than that currently performed, in order to assess response to first-line tyrosine-kinase inhibitors and consider a potential switch of therapy as early as possible. We evaluated this optimal time point as being 19 days after the start of treatment in our cohort. PMID:25203717

  7. The intracellular domain of teneurin-1 interacts with MBD1 and CAP/ponsin resulting in subcellular codistribution and translocation to the nuclear matrix

    SciTech Connect

    Nunes, Samantha M.; Ferralli, Jacqueline; Choi, Karen; Brown-Luedi, Marianne; Minet, Ariane D.; Chiquet-Ehrismann, Ruth . E-mail: chiquet@fmi.ch

    2005-04-15

    Teneurin-1 is a type II transmembrane protein expressed in neurons of the developing and adult central nervous system. To investigate the intracellular signaling of teneurin-1, we searched for proteins interacting with its intracellular domain. One of the proteins identified is the c-Cbl-associated protein CAP/ponsin, an adaptor protein containing SH3 domains. This interaction results on one hand in the recruitment of the soluble intracellular domain of teneurin-1 to the cell membrane enriched in CAP/ponsin. On the other hand, it leads to the translocation of CAP/ponsin to the nucleus, the major site of accumulation of the intracellular domain of teneurin-1. The second interacting protein identified is the methyl-CpG binding protein MBD1. In the nucleus, the intracellular domain of teneurin-1 colocalizes with this transcriptional repressor in foci associated with the nuclear matrix. We propose that these interactions are part of a specific signaling pathway. Evidence for cleavage and nuclear translocation of the intracellular domain has been obtained by the detection of endogenous teneurin-1 immunoreactivity in nuclear speckles in chick embryo fibroblasts. Furthermore, in the nuclear matrix fraction of these cells as well as in cells expressing a hormone-inducible full-length teneurin-1 protein, a teneurin-1 fragment of identical size could be detected as in cells transfected with the intracellular domain alone.

  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 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.

  10. Complex network inference from P300 signals: Decoding brain state under visual stimulus for able-bodied and disabled subjects

    NASA Astrophysics Data System (ADS)

    Gao, Zhong-Ke; Cai, Qing; Dong, Na; Zhang, Shan-Shan; Bo, Yun; Zhang, Jie

    2016-10-01

    Distinguishing brain cognitive behavior underlying disabled and able-bodied subjects constitutes a challenging problem of significant importance. Complex network has established itself as a powerful tool for exploring functional brain networks, which sheds light on the inner workings of the human brain. Most existing works in constructing brain network focus on phase-synchronization measures between regional neural activities. In contrast, we propose a novel approach for inferring functional networks from P300 event-related potentials by integrating time and frequency domain information extracted from each channel signal, which we show to be efficient in subsequent pattern recognition. In particular, we construct brain network by regarding each channel signal as a node and determining the edges in terms of correlation of the extracted feature vectors. A six-choice P300 paradigm with six different images is used in testing our new approach, involving one able-bodied subject and three disabled subjects suffering from multiple sclerosis, cerebral palsy, traumatic brain and spinal-cord injury, respectively. We then exploit global efficiency, local efficiency and small-world indices from the derived brain networks to assess the network topological structure associated with different target images. The findings suggest that our method allows identifying brain cognitive behaviors related to visual stimulus between able-bodied and disabled subjects.

  11. 20 CFR 604.3 - Able and available requirement-general principles.

    Code of Federal Regulations, 2014 CFR

    2014-04-01

    ...—general principles. (a) A State may pay UC only to an individual who is able to work and available for work for the week for which UC is claimed. (b) Whether an individual is able to work and available for... for an individual under its UC law. (c) The requirement that an individual be able to work...

  12. 20 CFR 604.3 - Able and available requirement-general principles.

    Code of Federal Regulations, 2013 CFR

    2013-04-01

    ...—general principles. (a) A State may pay UC only to an individual who is able to work and available for work for the week for which UC is claimed. (b) Whether an individual is able to work and available for... for an individual under its UC law. (c) The requirement that an individual be able to work...

  13. 20 CFR 604.3 - Able and available requirement-general principles.

    Code of Federal Regulations, 2011 CFR

    2011-04-01

    ...—general principles. (a) A State may pay UC only to an individual who is able to work and available for work for the week for which UC is claimed. (b) Whether an individual is able to work and available for... for an individual under its UC law. (c) The requirement that an individual be able to work...

  14. 20 CFR 604.3 - Able and available requirement-general principles.

    Code of Federal Regulations, 2010 CFR

    2010-04-01

    ...—general principles. (a) A State may pay UC only to an individual who is able to work and available for work for the week for which UC is claimed. (b) Whether an individual is able to work and available for... for an individual under its UC law. (c) The requirement that an individual be able to work...

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

    Code of Federal Regulations, 2012 CFR

    2012-04-01

    ...—general principles. (a) A State may pay UC only to an individual who is able to work and available for work for the week for which UC is claimed. (b) Whether an individual is able to work and available for... for an individual under its UC law. (c) The requirement that an individual be able to work...

  16. Positive Outcomes of Group Learning in the ABLE Classroom. Research to Practice.

    ERIC Educational Resources Information Center

    Crites, Beverly J.; McKenna, Gail Kaylor

    In the fall of 1993, a study was begun on how adult basic and literacy education (ABLE) students reacted to working in groups. The research was conducted through a joint vocational school's ABLE program using three target groups at two of its ABLE centers. The groups met two times per week and were facilitated by three different teachers. More…

  17. Crystal Structures of the Human G3BP1 NTF2-Like Domain Visualize FxFG Nup Repeat Specificity

    PubMed Central

    Vognsen, Tina; Møller, Ingvar Runár; Kristensen, Ole

    2013-01-01

    Ras GTPase Activating Protein SH3 Domain Binding Protein (G3BP) is a potential anti-cancer drug target implicated in several cellular functions. We have used protein crystallography to solve crystal structures of the human G3BP1 NTF2-like domain both alone and in complex with an FxFG Nup repeat peptide. Despite high structural similarity, the FxFG binding site is located between two alpha helices in the G3BP1 NTF2-like domain and not at the dimer interface as observed for nuclear transport factor 2. ITC studies showed specificity towards the FxFG motif but not FG and GLFG motifs. The unliganded form of the G3BP1 NTF2-like domain was solved in two crystal forms to resolutions of 1.6 and 3.3 Å in space groups P212121 and P6322 based on two different constructs, residues 1–139 and 11–139, respectively. Crystal packing of the N-terminal residues against a symmetry related molecule in the P212121 crystal form might indicate a novel ligand binding site that, however, remains to be validated. The crystal structures give insight into the nuclear transportation mechanisms of G3BP and provide a basis for future structure based drug design. PMID:24324649

  18. 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

  19. Genomic organization and chromosomal localization of the murine 2 P domain potassium channel gene Kcnk8: conservation of gene structure in 2 P domain potassium channels.

    PubMed

    Bockenhauer, D; Nimmakayalu, M A; Ward, D C; Goldstein, S A; Gallagher, P G

    2000-12-31

    A 2 P domain potassium channel expressed in eye, lung, and stomach, Kcnk8, has recently been identified. To initiate further biochemical and genetic studies of this channel, we assembled the murine Kcnk8 cDNA sequence, characterized the genomic structure of the Kcnk8 gene, determined its chromosomal localization, and analyzed its activity in a Xenopus laevis oocyte expression system. The composite cDNA has an open reading frame of 1029 bp and encodes a protein of 343 amino acids with a predicted molecular mass of 36 kDa. Structure analyses predict 2 P domains and four potential transmembrane helices with a potential single EF-hand motif and four potential SH3-binding motifs in the COOH-terminus. Cloning of the Kcnk8 chromosomal gene revealed that it is composed of three exons distributed over 4 kb of genomic DNA. Genome database searching revealed that one of the intron/exon boundaries identified in Kcnk8 is present in other mammalian 2 P domain potassium channels genes and many C. elegans 2P domain potassium channel genes, revealing evolutionary conservation of gene structure. Using fluorescence in situ hybridization, the murine Kcnk8 gene was mapped to chromosome 19, 2B, the locus of the murine dancer phenotype, and syntenic to 11q11-11q13, the location of the human homologue. No significant currents were generated in a Xenopus laevis oocyte expression system using the composite Kcnk8 cDNA sequence, suggesting, like many potassium channels, additional channel subunits, modulator substances, or cellular chaperones are required for channel function.

  20. Discovery of allosteric BCR-ABL inhibitors from phenotypic screen to clinical candidate.

    PubMed

    Gray, Nathanael S; Fabbro, Doriano

    2014-01-01

    The development of imatinib, an ATP-competitive inhibitor of the BCR-ABL oncoprotein, has revolutionized the treatment of chronic myelogenous leukemia (CML). Unfortunately, the leukemia eventually becomes resistant imatinib as a result of emergence of cells expressing drug insensitive BCR-ABL mutant proteins. This has motivated the development of several next-generation ATP-competitive drugs. This chapter describes the discovery and development of a complementary strategy involving inhibiting BCR-ABL by targeting an allosteric binding site. Compounds that bind to the myristate-binding pocket of BCR-ABL are able to induce formation of an "inactive" state and are able to overcome resistance mutations located in the ATP-binding pocket including the recalcitrant T315I "gatekeeper" mutation. Myristate-pocket inhibitors are also able to function synergistically with ATP-competitive inhibitors in cellular and murine models of CML and this dual inhibitory strategy is currently being investigated in the clinic.

  1. Characterization of ABL exon 7 deletion by molecular genetic and bioinformatic methods reveals no association with imatinib resistance in chronic myeloid leukemia.

    PubMed

    Meggyesi, Nóra; Kalmár, Lajos; Fekete, Sándor; Masszi, Tamás; Tordai, Attila; Andrikovics, Hajnalka

    2012-09-01

    In chronic myeloid leukemia (CML), the best characterized imatinib resistance mechanisms are BCR-ABL tyrosine kinase domain mutations and clonal evolution, but recently alternative splicing of BCR-ABL was also proposed as a mechanism for imatinib resistance. Among recently reported BCR-ABL splice variants, exon 7 deletion (Δexon7) was characterized in this study. The frequency of Δexon7 was investigated in 30 healthy controls and in 76 CML patients at different time points of the disease course by four different molecular genetic methods (direct sequencing, fragment analysis, allele-specific and quantitative PCR). The functionality and viability of the variant protein was tested by bioinformatic prediction. The Δexon7 was abundantly detected with similar frequency in healthy controls, in imatinib naive and resistant CML patients on BCR-ABL and also on the nontranslocated ABL. The detection rate of Δexon7 (varying between 17 and 100%) was highly dependent on the expression levels of BCR-ABL or ABL and the sensitivity of detection method. According to secondary structure prediction by bioinformatic methods, the exon 7 deleted mRNA is a target for nonsense-mediated decay, and the translated protein is likely to be nonfunctional and unstable. Taken together all the above observations, we concluded that Δexon7 is a common splice variant not associating with imatinib resistance.

  2. Abl family kinases regulate FcγR-mediated phagocytosis in murine macrophages.

    PubMed

    Greuber, Emileigh K; Pendergast, Ann Marie

    2012-12-01

    Phagocytosis of Ab-coated pathogens is mediated through FcγRs, which activate intracellular signaling pathways to drive actin cytoskeletal rearrangements. Abl and Arg define a family of nonreceptor tyrosine kinases that regulate actin-dependent processes in a variety of cell types, including those important in the adaptive immune response. Using pharmacological inhibition as well as dominant negative and knockout approaches, we demonstrate a role for the Abl family kinases in phagocytosis by macrophages and define a mechanism whereby Abl kinases regulate this process. Bone marrow-derived macrophages from mice lacking Abl and Arg kinases exhibit inefficient phagocytosis of sheep erythrocytes and zymosan particles. Treatment with the Abl kinase inhibitors imatinib and GNF-2 or overexpression of kinase-inactive forms of the Abl family kinases also impairs particle internalization in murine macrophages, indicating Abl kinase activity is required for efficient phagocytosis. Further, Arg kinase is present at the phagocytic cup, and Abl family kinases are activated by FcγR engagement. The regulation of phagocytosis by Abl family kinases is mediated in part by the spleen tyrosine kinase (Syk). Loss of Abl and Arg expression or treatment with Abl inhibitors reduced Syk phosphorylation in response to FcγR ligation. The link between Abl family kinases and Syk may be direct, as purified Arg kinase phosphorylates Syk in vitro. Further, overexpression of membrane-targeted Syk in cells treated with Abl kinase inhibitors partially rescues the impairment in phagocytosis. Together, these findings reveal that Abl family kinases control the efficiency of phagocytosis in part through the regulation of Syk function.

  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. 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.

  5. 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.

  6. 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

  7. T cell survival and function requires the c-Abl tyrosine kinase.

    PubMed

    Silberman, Isabelle; Sionov, Ronit Vogt; Zuckerman, Valentina; Haupt, Sue; Goldberg, Zehavit; Strasser, Andreas; Ben-Sasson, Zami S; Baniyash, Michal; Koleske, Anthony J; Haupt, Ygal

    2008-12-15

    C-Abl (Abl) regulates multiple cellular processes, including proliferation, survival, shape determination and motility, and participates in cellular responses to genotoxic and oxidative stress stimuli. Mice lacking Abl exhibit retarded growth, osteoporosis and defects in the immune system resulting in lymphopoenia and susceptibility to infections, leading to early death. To define the role of Abl in the regulation of adult T cells we ablated Abl exclusively in T cells by generating mice with floxed abl alleles and expressing an Lck-Cre transgene (Abl-T(-/-)). These mice exhibited thymic atrophy and abnormally reduced T cell numbers in the periphery. The thymic atrophy was caused by increased susceptibility of thymocytes to cell death. Importantly, Abl deficient T cells displayed abnormally reduced response to mitogenic stimulation in vitro. Consequently, Abl-T(-/-) mice exhibited impaired ability to reject syngeneic tumor, to induce T-mediated tumor cell killing, and to generate anti-tumor antibodies. These results demonstrate a cell-autonomous role for Abl in T cell function and survival.

  8. Novel redox-dependent regulation of NOX5 by the tyrosine kinase c-Abl.

    PubMed

    El Jamali, Amina; Valente, Anthony J; Lechleiter, James D; Gamez, Maria J; Pearson, Doran W; Nauseef, William M; Clark, Robert A

    2008-03-01

    We investigated the mechanism of H(2)O(2) activation of the Ca(2+)-regulated NADPH oxidase NOX5. H(2)O(2) induced a transient, dose-dependent increase in superoxide production in K562 cells expressing NOX5. Confocal studies demonstrated that the initial calcium influx generated by H(2)O(2) is amplified by a feedback mechanism involving NOX5-dependent superoxide production and H(2)O(2). H(2)O(2) NOX5 activation was inhibited by extracellular Ca(2+) chelators, a pharmacological inhibitor of c-Abl, and overexpression of kinase-dead c-Abl. Transfected kinase-active GFP-c-Abl colocalized with vesicular sites of superoxide production in a Ca(2+)-dependent manner. In contrast to H(2)O(2), the Ca(2+) ionophore ionomycin induced NOX5 activity independent of c-Abl. Immunoprecipitation of cell lysates revealed that active GFP-c-Abl formed oligomers with endogenous c-Abl and that phosphorylation of both proteins was increased by H(2)O(2) treatment. Furthermore, H(2)O(2)-induced NOX5 activity correlated with increased localization of c-Abl to the membrane fraction, and NOX5 proteins could be coimmunoprecipitated with GFP-Abl proteins. Our data demonstrate for the first time that NOX5 is activated by c-Abl through a Ca(2+)-mediated, redox-dependent signaling pathway and suggest a functional association between NOX5 NADPH oxidase and c-Abl.

  9. 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

  10. 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 Central

    Panuzzo, Cristina; Volpe, Gisella; Rocchietti, Elisa Cibrario; 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

  11. 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

  12. 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).

  13. Visualizing domain wall and reverse domain superconductivity.

    PubMed

    Iavarone, M; Moore, S A; Fedor, J; Ciocys, S T; Karapetrov, G; Pearson, J; Novosad, V; Bader, S D

    2014-08-28

    In magnetically coupled, planar ferromagnet-superconductor (F/S) hybrid structures, magnetic domain walls can be used to spatially confine the superconductivity. In contrast to a superconductor in a uniform applied magnetic field, the nucleation of the superconducting order parameter in F/S structures is governed by the inhomogeneous magnetic field distribution. The interplay between the superconductivity localized at the domain walls and far from the walls leads to effects such as re-entrant superconductivity and reverse domain superconductivity with the critical temperature depending upon the location. Here we use scanning tunnelling spectroscopy to directly image the nucleation of superconductivity at the domain wall in F/S structures realized with Co-Pd multilayers and Pb thin films. Our results demonstrate that such F/S structures are attractive model systems that offer the possibility to control the strength and the location of the superconducting nucleus by applying an external magnetic field, potentially useful to guide vortices for computing application.

  14. Visualizing domain wall and reverse domain superconductivity

    PubMed Central

    Iavarone, M.; Moore, S. A.; Fedor, J.; Ciocys, S. T.; Karapetrov, G.; Pearson, J.; Novosad, V.; Bader, S. D.

    2014-01-01

    In magnetically coupled, planar ferromagnet-superconductor (F/S) hybrid structures, magnetic domain walls can be used to spatially confine the superconductivity. In contrast to a superconductor in a uniform applied magnetic field, the nucleation of the superconducting order parameter in F/S structures is governed by the inhomogeneous magnetic field distribution. The interplay between the superconductivity localized at the domain walls and far from the walls leads to effects such as re-entrant superconductivity and reverse domain superconductivity with the critical temperature depending upon the location. Here we use scanning tunnelling spectroscopy to directly image the nucleation of superconductivity at the domain wall in F/S structures realized with Co-Pd multilayers and Pb thin films. Our results demonstrate that such F/S structures are attractive model systems that offer the possibility to control the strength and the location of the superconducting nucleus by applying an external magnetic field, potentially useful to guide vortices for computing application. PMID:25164004

  15. Direct interaction of v-Src with the focal adhesion kinase mediated by the Src SH2 domain.

    PubMed Central

    Xing, Z; Chen, H C; Nowlen, J K; Taylor, S J; Shalloway, D; Guan, J L

    1994-01-01

    The recently described focal adhesion kinase (FAK) has been implicated in signal transduction pathways initiated by cell adhesion receptor integrins and by neuropeptide growth factors. To examine the mechanisms by which FAK relays signals from the membrane to the cell interior, we carried out a series of experiments to detect potential FAK interactions with proteins containing Src homology 2 (SH2) domains that are important intracellular signaling molecules. Using v-Src-transformed NIH3T3 cells, we showed that FAK was present in the immune-complex precipitated by anti-Src antibody, suggesting potential interaction of FAK with v-Src in vivo. We also showed potentially direct interaction of FAK with v-Src in vivo using the yeast two-hybrid system. Using recombinant FAK expressed in insect cells and bacterial fusion proteins containing Src SH2 domains, we showed direct binding of FAK to the Src SH2 domain but not to the SH3 domain in vitro. A kinase-defective mutant of FAK, which is not autophosphorylated, did not interact with the Src SH2 domain under the same conditions, suggesting the involvement of the FAK autophosphorylation sites. Treatment of FAK with a protein-tyrosine phosphatase decreased its binding to the Src SH2 domain, whereas autophosphorylation in vitro increased its binding. These results confirm the importance of FAK autophosphorylation sites in its interaction with SH2 domain-containing proteins. Taken together, these results suggest that FAK may mediate signal transduction events initiated on the cell surface by kinase activation and autophosphorylation that result in its binding to other key intracellular signaling molecules. Images PMID:8054685

  16. COOH-terminal association of human smooth muscle calcium channel Ca(v)1.2b with Src kinase protein binding domains: effect of nitrotyrosylation.

    PubMed

    Kang, Minho; Ross, Gracious R; Akbarali, Hamid I

    2007-12-01

    The carboxyl terminus of the calcium channel plays an important role in the regulation of calcium entry, signal transduction, and gene expression. Potential protein-protein interaction sites within the COOH terminus of the L-type calcium channel include those for the SH3 and SH2 binding domains of c-Src kinase that regulates calcium currents in smooth muscle. In this study, we examined the binding sites involved in Src kinase-mediated phosphorylation of the human voltage-gated calcium channel (Ca(v)) 1.2b (hCav1.2b) and the effect of nitrotyrosylation. Cotransfection of human embryonic kidney (HEK)-293 cells with hCa(v)1.2b and c-Src resulted in tyrosine phosphorylation of the calcium channel, which was prevented by nitration of tyrosine residues by peroxynitrite. Whole cell calcium currents were reduced by 58 + 5% by the Src kinase inhibitor PP2 and 64 + 6% by peroxynitrite. Nitrotyrosylation prevented Src-mediated regulation of the currents. Glutathione S-transferase fusion protein of the distal COOH terminus of hCa(v)1.2b (1809-2138) bound to SH2 domain of Src following tyrosine phosphorylation, while binding to SH3 required the presence of the proline-rich motif. Site-directed mutation of Y(2134) prevented SH2 binding and resulted in reduced phosphorylation of hCa(v)1.2b. Within the distal COOH terminus, single, double, or triple mutations of Y(1837), Y(1861), and Y(2134) were constructed and expressed in HEK-293 cells. The inhibitory effects of PP2 and peroxynitrite on calcium currents were significantly reduced in the double mutant Y(1837-2134F). These data demonstrate that the COOH terminus of hCa(v)1.2b contains sites for the SH2 and SH3 binding of Src kinase. Nitrotyrosylation of these sites prevents Src kinase regulation and may be importantly involved in calcium influx regulation during inflammation.

  17. Functional domains of the poliovirus receptor

    SciTech Connect

    Koike, Satoshi; Ise, Iku; Nomoto, Akio )

    1991-05-15

    A number of mutant cDNAs of the human poliovirus receptor were constructed to identify essential regions of the molecule as the receptor. All mutant cDNAs carrying the sequence coding for the entire N-terminal immunoglobulin-like domain (domain I) confer permissiveness for poliovirus to mouse L cells, but a mutant cDNA lacking the sequence for domain I does not. The transformants permissive for poliovirus were able to bind the virus and were also recognized by monoclonal antibody D171, which competes with poliovirus for the cellular receptor. These results strongly suggest that the poliovirus binding site resides in domain I of the receptor. Mutant cDNAs for the sequence encoding the intracellular peptide were also constructed and expressed in mouse L cells. Susceptibility of these cells to poliovirus revealed that the entire putative cytoplasmic domain is not essential for virus infection. Thus, the cytoplasmic domain of the molecule appears not to play a role in the penetration of poliovirus.

  18. 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…

  19. 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.

  20. 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…

  1. Semi-supervised domain adaptation on manifolds.

    PubMed

    Cheng, Li; Pan, Sinno Jialin

    2014-12-01

    In real-life problems, the following semi-supervised domain adaptation scenario is often encountered: we have full access to some source data, which is usually very large; the target data distribution is under certain unknown transformation of the source data distribution; meanwhile, only a small fraction of the target instances come with labels. The goal is to learn a prediction model by incorporating information from the source domain that is able to generalize well on the target test instances. We consider an explicit form of transformation functions and especially linear transformations that maps examples from the source to the target domain, and we argue that by proper preprocessing of the data from both source and target domains, the feasible transformation functions can be characterized by a set of rotation matrices. This naturally leads to an optimization formulation under the special orthogonal group constraints. We present an iterative coordinate descent solver that is able to jointly learn the transformation as well as the model parameters, while the geodesic update ensures the manifold constraints are always satisfied. Our framework is sufficiently general to work with a variety of loss functions and prediction problems. Empirical evaluations on synthetic and real-world experiments demonstrate the competitive performance of our method with respect to the state-of-the-art. PMID:25314712

  2. One Health Core Competency Domains.

    PubMed

    Frankson, Rebekah; Hueston, William; Christian, Kira; Olson, Debra; Lee, Mary; Valeri, Linda; Hyatt, Raymond; Annelli, Joseph; Rubin, Carol

    2016-01-01

    The emergence of complex global challenges at the convergence of human, animal, and environmental health has catalyzed a movement supporting "One Health" approaches. Despite recognition of the importance of One Health approaches to address these complex challenges, little effort has been directed at identifying the seminal knowledge, skills, and attitudes necessary for individuals to successfully contribute to One Health efforts. Between 2008 and 2011, three groups independently embarked on separate initiatives to identify core competencies for professionals involved with One Health approaches. Core competencies were considered critically important for guiding curriculum development and continuing professional education, as they describe the knowledge, skills, and attitudes required to be effective. A workshop was convened in 2012 to synthesize the various strands of work on One Health competencies. Despite having different mandates, participants, and approaches, all of these initiatives identified similar core competency domains: management; communication and informatics; values and ethics; leadership; teams and collaboration; roles and responsibilities; and systems thinking. These core competency domains have been used to develop new continuing professional education programs for One Health professionals and help university curricula prepare new graduates to be able to contribute more effectively to One Health approaches. PMID:27679794

  3. One Health Core Competency Domains

    PubMed Central

    Frankson, Rebekah; Hueston, William; Christian, Kira; Olson, Debra; Lee, Mary; Valeri, Linda; Hyatt, Raymond; Annelli, Joseph; Rubin, Carol

    2016-01-01

    The emergence of complex global challenges at the convergence of human, animal, and environmental health has catalyzed a movement supporting “One Health” approaches. Despite recognition of the importance of One Health approaches to address these complex challenges, little effort has been directed at identifying the seminal knowledge, skills, and attitudes necessary for individuals to successfully contribute to One Health efforts. Between 2008 and 2011, three groups independently embarked on separate initiatives to identify core competencies for professionals involved with One Health approaches. Core competencies were considered critically important for guiding curriculum development and continuing professional education, as they describe the knowledge, skills, and attitudes required to be effective. A workshop was convened in 2012 to synthesize the various strands of work on One Health competencies. Despite having different mandates, participants, and approaches, all of these initiatives identified similar core competency domains: management; communication and informatics; values and ethics; leadership; teams and collaboration; roles and responsibilities; and systems thinking. These core competency domains have been used to develop new continuing professional education programs for One Health professionals and help university curricula prepare new graduates to be able to contribute more effectively to One Health approaches. PMID:27679794

  4. One Health Core Competency Domains

    PubMed Central

    Frankson, Rebekah; Hueston, William; Christian, Kira; Olson, Debra; Lee, Mary; Valeri, Linda; Hyatt, Raymond; Annelli, Joseph; Rubin, Carol

    2016-01-01

    The emergence of complex global challenges at the convergence of human, animal, and environmental health has catalyzed a movement supporting “One Health” approaches. Despite recognition of the importance of One Health approaches to address these complex challenges, little effort has been directed at identifying the seminal knowledge, skills, and attitudes necessary for individuals to successfully contribute to One Health efforts. Between 2008 and 2011, three groups independently embarked on separate initiatives to identify core competencies for professionals involved with One Health approaches. Core competencies were considered critically important for guiding curriculum development and continuing professional education, as they describe the knowledge, skills, and attitudes required to be effective. A workshop was convened in 2012 to synthesize the various strands of work on One Health competencies. Despite having different mandates, participants, and approaches, all of these initiatives identified similar core competency domains: management; communication and informatics; values and ethics; leadership; teams and collaboration; roles and responsibilities; and systems thinking. These core competency domains have been used to develop new continuing professional education programs for One Health professionals and help university curricula prepare new graduates to be able to contribute more effectively to One Health approaches.

  5. One Health Core Competency Domains.

    PubMed

    Frankson, Rebekah; Hueston, William; Christian, Kira; Olson, Debra; Lee, Mary; Valeri, Linda; Hyatt, Raymond; Annelli, Joseph; Rubin, Carol

    2016-01-01

    The emergence of complex global challenges at the convergence of human, animal, and environmental health has catalyzed a movement supporting "One Health" approaches. Despite recognition of the importance of One Health approaches to address these complex challenges, little effort has been directed at identifying the seminal knowledge, skills, and attitudes necessary for individuals to successfully contribute to One Health efforts. Between 2008 and 2011, three groups independently embarked on separate initiatives to identify core competencies for professionals involved with One Health approaches. Core competencies were considered critically important for guiding curriculum development and continuing professional education, as they describe the knowledge, skills, and attitudes required to be effective. A workshop was convened in 2012 to synthesize the various strands of work on One Health competencies. Despite having different mandates, participants, and approaches, all of these initiatives identified similar core competency domains: management; communication and informatics; values and ethics; leadership; teams and collaboration; roles and responsibilities; and systems thinking. These core competency domains have been used to develop new continuing professional education programs for One Health professionals and help university curricula prepare new graduates to be able to contribute more effectively to One Health approaches.

  6. 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)…

  7. 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…

  8. BCR/ABL stimulates WRN to promote survival and genomic instability

    PubMed Central

    Slupianek, Artur; Poplawski, Tomasz; Jozwiakowski, Stanislaw K.; Cramer, Kimberly; Pytel, Dariusz; Stoczynska, Ewelina; Nowicki, Michal O.; Blasiak, Janusz; Skorski, Tomasz

    2010-01-01

    BCR/ABL-transformed chronic myeloid leukemia (CML) cells accumulate numerous DNA double-strand breaks (DSBs) induced by reactive oxygen species (ROS) and genotoxic agents. To repair these lesions BCR/ABL stimulate unfaithful DSB repair pathways, homologous recombination repair (HRR), non-homologous end-joining (NHEJ) and single-strand annealing (SSA). Here we show that BCR/ABL enhances the expression and increase nuclear localization of WRN (mutated in Werner syndrome), which is required for processing DSB ends during the repair. Other fusion tyrosine kinases (FTKs) such as TEL/ABL, TEL/JAK2, TEL/PDGFβR, and NPM/ALK also elevate WRN. BCR/ABL induces WRN mRNA and protein expression in part by c-MYC -mediated activation of transcription and Bcl-xL –dependent inhibition of caspase-dependent cleavage, respectively. WRN is in complex with BCR/ABL resulting in WRN tyrosine phosphorylation and stimulation of its helicase and exonuclease activities. Activated WRN protects BCR/ABL-positive cells from the lethal effect of oxidative and genotoxic stresses, which causes DSBs. In addition, WRN promotes unfaithful recombination-dependent repair mechanisms HRR and SSA, and enhances the loss of DNA bases during NHEJ in leukemia cells. In summary, we postulate that BCR/ABL-mediated stimulation of WRN modulates the efficiency and fidelity of major DSB repair mechanisms to protect leukemia cells from apoptosis and to facilitate genomic instability. PMID:21123451

  9. BCR/ABL stimulates WRN to promote survival and genomic instability.

    PubMed

    Slupianek, Artur; Poplawski, Tomasz; Jozwiakowski, Stanislaw K; Cramer, Kimberly; Pytel, Dariusz; Stoczynska, Ewelina; Nowicki, Michal O; Blasiak, Janusz; Skorski, Tomasz

    2011-02-01

    BCR/ABL-transformed chronic myeloid leukemia (CML) cells accumulate numerous DNA double-strand breaks (DSB) induced by reactive oxygen species (ROS) and genotoxic agents. To repair these lesions BCR/ABL stimulate unfaithful DSB repair pathways, homologous recombination repair (HRR), nonhomologous end-joining (NHEJ), and single-strand annealing (SSA). Here, we show that BCR/ABL enhances the expression and increase nuclear localization of WRN (mutated in Werner syndrome), which is required for processing DSB ends during the repair. Other fusion tyrosine kinases (FTK), such as TEL/ABL, TEL/JAK2, TEL/PDGFβR, and NPM/ALK also elevate WRN. BCR/ABL induces WRN mRNA and protein expression in part by c-MYC-mediated activation of transcription and Bcl-xL-dependent inhibition of caspase-dependent cleavage, respectively. WRN is in complex with BCR/ABL resulting in WRN tyrosine phosphorylation and stimulation of its helicase and exonuclease activities. Activated WRN protects BCR/ABL-positive cells from the lethal effect of oxidative and genotoxic stresses, which causes DSBs. In addition, WRN promotes unfaithful recombination-dependent repair mechanisms HRR and SSA, and enhances the loss of DNA bases during NHEJ in leukemia cells. In summary, we postulate that BCR/ABL-mediated stimulation of WRN modulates the efficiency and fidelity of major DSB repair mechanisms to protect leukemia cells from apoptosis and to facilitate genomic instability.

  10. 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

  11. Identifying and Selecting Able Students for the NAGTY Summer School: Emerging Issues and Future Considerations

    ERIC Educational Resources Information Center

    Hartas, Dimitra; Lindsay, Geoff; Muijs, Daniel

    2008-01-01

    In recent years, there has been an increasing recognition that the educational needs of able students were not being adequately met in British schools resulting in a series of governmental educational initiatives aiming at improving the education of able students. The establishment of the National Academy for Gifted and Talented Youth (NAGTY) at…

  12. 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

  13. Novel pyrazolo[3,4-d]pyrimidines as dual Src-Abl inhibitors active against mutant form of Abl and the leukemia K-562 cell line.

    PubMed

    El-Moghazy, Samir M; George, Riham F; Osman, Essam Eldin A; Elbatrawy, Ahmed A; Kissova, Miroslava; Colombo, Ambra; Crespan, Emmanuele; Maga, Giovanni

    2016-11-10

    Some novel 6-substituted pyrazolo[3,4-d]pyrimidines 4, 5, 6a-d, 7a-c, 8 and pyrazolo[4,3-e][1,2,4]triazolo[4,3-a]pyrimidines 9a-c, 10a-c, 11, 12a,b, 13a-c and 14 were synthesized and characterized by spectral and elemental analyses. They were screened for their biological activity in vitro against Abl and Src kinases. Compounds 7a and 7b revealed the highest activity against both wild and mutant Abl kinases as well as the Src kinase and the leukemia K-562 cell line. They can be considered as new hits for further structural optimization to obtain better activity.

  14. Domain fusion analysis by applying relational algebra to protein sequence and domain databases

    PubMed Central

    Truong, Kevin; Ikura, Mitsuhiko

    2003-01-01

    Background Domain fusion analysis is a useful method to predict functionally linked proteins that may be involved in direct protein-protein interactions or in the same metabolic or signaling pathway. As separate domain databases like BLOCKS, PROSITE, Pfam, SMART, PRINTS-S, ProDom, TIGRFAMs, and amalgamated domain databases like InterPro continue to grow in size and quality, a computational method to perform domain fusion analysis that leverages on these efforts will become increasingly powerful. Results This paper proposes a computational method employing relational algebra to find domain fusions in protein sequence databases. The feasibility of this method was illustrated on the SWISS-PROT+TrEMBL sequence database using domain predictions from the Pfam HMM (hidden Markov model) database. We identified 235 and 189 putative functionally linked protein partners in H. sapiens and S. cerevisiae, respectively. From scientific literature, we were able to confirm many of these functional linkages, while the remainder offer testable experimental hypothesis. Results can be viewed at . Conclusion As the analysis can be computed quickly on any relational database that supports standard SQL (structured query language), it can be dynamically updated along with the sequence and domain databases, thereby improving the quality of predictions over time. PMID:12734020

  15. Purification of TAT-CC-HA protein under native condition, and its transduction analysis and biological effects on BCR-ABL positive cells.

    PubMed

    Huang, Zhenglan; Ji, Maosheng; Peng, Zhi; Huang, Shifeng; Xiao, Qing; Li, Chunli; Zeng, Jianming; Gao, Miao; Feng, Wenli

    2011-06-01

    BCR-ABL oncoprotein is the cause of chronic myeloid leukemia. The homologous oligomerization of BCR-ABL protein mediated by BCR coiled-coil (CC) domain plays an important role in ABL kinase activation. The HIV-1 TAT peptide has been used extensively for the introduction of proteins into cells. We recombinated a TAT-CC-HA protein to interrupt the homologous oligomerization of BCR-ABL. The expression conditions for TAT-CC-HA were optimized. The TAT-CC-HA fusion protein was purified with Ni+-NTA resin. TAT-CC-HA fusion protein was added into the cultures of Ba/F3-p210, 32D-p210, K562, KU812, Ba/F3, 32D, and HL-60 cells. It was found that TAT-CC-HA could transduce into these cells. It was confirmed that TAT-CC-HA fusion protein was internalized by Ba/F3-p210, K562, and Ba/F3 cells and located in the cytoplasm observed by confocal laser scanning fluorescence microscope. The transduction of TAT-CC-HA fusion protein into K562 cells was in a dose-dependent and time-dependent manner. The result of coimmunoprecipitation assay indicated that TAT-CC-HA could interact with BCR-ABL in K562 cells. The effects of TAT-CC-HA fusion protein on cell growth and apoptosis were detected by MTT test and flow cytometry. Our findings suggested that TAT-CC-HA fusion protein could specifically inhibit the growth of BCR-ABL positive cells, and specifically induce apoptosis of BCR-ABL positive cells, while not affect the growth and apoptosis of BCR-ABL negative cells.

  16. Productive replication of Ebola virus is regulated by the c-Abl1 tyrosine kinase.

    PubMed

    García, Mayra; Cooper, Arik; Shi, Wei; Bornmann, William; Carrion, Ricardo; Kalman, Daniel; Nabel, Gary J

    2012-02-29

    Ebola virus causes a fulminant infection in humans resulting in diffuse bleeding, vascular instability, hypotensive shock, and often death. Because of its high mortality and ease of transmission from human to human, Ebola virus remains a biological threat for which effective preventive and therapeutic interventions are needed. An understanding of the mechanisms of Ebola virus pathogenesis is critical for developing antiviral therapeutics. Here, we report that productive replication of Ebola virus is modulated by the c-Abl1 tyrosine kinase. Release of Ebola virus-like particles (VLPs) in a cell culture cotransfection system was inhibited by c-Abl1-specific small interfering RNA (siRNA) or by Abl-specific kinase inhibitors and required tyrosine phosphorylation of the Ebola matrix protein VP40. Expression of c-Abl1 stimulated an increase in phosphorylation of tyrosine 13 (Y(13)) of VP40, and mutation of Y(13) to alanine decreased the release of Ebola VLPs. Productive replication of the highly pathogenic Ebola virus Zaire strain was inhibited by c-Abl1-specific siRNAs or by the Abl-family inhibitor nilotinib by up to four orders of magnitude. These data indicate that c-Abl1 regulates budding or release of filoviruses through a mechanism involving phosphorylation of VP40. This step of the virus life cycle therefore may represent a target for antiviral therapy.

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

    PubMed Central

    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

    2016-01-01

    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

  18. 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

  19. 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;…

  20. 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

  1. A rapid RT-PCR based method for the detection of BCR-ABL translocation.

    PubMed Central

    Sidorova JYu; Saltykova, L B; Lyschov, A A; Zaritskey AYu; Abdulkadyrov, K M; Blinov, M N

    1997-01-01

    AIMS: To optimise a one step reverse transcriptase polymerase chain reaction (RT-PCR) protocol for BCR-ABL chimaera detection. METHODS: Compared with published RT-PCR procedures, this novel approach has at least two advantages. First, the same enzyme is used for both reverse transcription and PCR. Second, amplification of the target (BCR-ABL chimaera) and control gene (ABL) is performed simultaneously in the same tube. RESULTS: On testing 40 chronic myelogenous leukaemia patients and 10 healthy donors there was a specificity for the newly developed technique. In addition, dilution experiments demonstrated that the protocol was highly sensitive. CONCLUSIONS: The suggested one step PCR strategy is a simple and reliable way to reveal BCR-ABL chimaeras. Images PMID:9497918

  2. BCR-ABL fusion peptides and cytotoxic T cells in chronic myeloid leukaemia.

    PubMed

    Clark, R E; Christmas, S E

    2001-01-01

    The BCR-ABL gene that arises in chronic myeloid leukaemia (CML) is a neoantigen. Peptides derived from the BCR-ABL fusion junction may therefore be immunogenic, if appropriately presented to the immune system. This article reviews data demonstrating that certain junctional peptides will bind to HLA molecules, and that these peptides will elicit specific T-lymphocyte responses in vitro, in both normal subjects and in CML patients. The clinical relevance of these observations is discussed.

  3. Allosteric Interactions between the Myristate- and ATP-Site of the Abl Kinase

    PubMed Central

    Iacob, Roxana E.; Zhang, Jianming; Gray, Nathanael S.; Engen, John R.

    2011-01-01

    Abl kinase inhibitors targeting the ATP binding pocket are currently employed as potent anti-leukemogenic agents but drug resistance has become a significant clinical limitation. Recently, a compound that binds to the myristate pocket of Abl (GNF-5) was shown to act cooperatively with nilotinib, an ATP-competitive inhibitor to target the recalcitrant “T315I” gatekeeper mutant of Bcr-Abl. To uncover an explanation for how drug binding at a distance from the kinase active site could lead to inhibition and how inhibitors could combine their effects, hydrogen exchange mass spectrometry (HX MS) was employed to monitor conformational effects in the presence of both dasatinib, a clinically approved ATP-site inhibitor, and GNF-5. While dasatinib binding to wild type Abl clearly influenced Abl conformation, no binding was detected between dasatinib and T315I. GNF-5, however, elicited the same conformational changes in both wild type and T315I, including changes to dynamics within the ATP site located approximately 25 Å from the site of GNF-5 interaction. Simultaneous binding of dasatinib and GNF-5 to T315I caused conformational and/or dynamics changes in Abl such that effects of dasatinib on T315I were the same as when it bound to wild type Abl. These results provide strong biophysical evidence that allosteric interactions play a role in Abl kinase downregulation and that targeting sites outside the ATP binding site can provide an important pharmacological tool to overcome mutations that cause resistance to ATP-competitive inhibitors. PMID:21264348

  4. Bcr-Abl stabilizes β-catenin in chronic myeloid leukemia through its tyrosine phosphorylation

    PubMed Central

    Coluccia, Addolorata Maria Luce; Vacca, Angelo; Duñach, Mireia; Mologni, Luca; Redaelli, Sara; Bustos, Victor H; Benati, Daniela; Pinna, Lorenzo A; Gambacorti-Passerini, Carlo

    2007-01-01

    Self-renewal of Bcr-Abl+ chronic myeloid leukemia (CML) cells is sustained by a nuclear activated serine/threonine-(S/T) unphosphorylated β-catenin. Although β-catenin can be tyrosine (Y)-phosphorylated, the occurrence and biological relevance of this covalent modification in Bcr-Abl-associated leukemogenesis is unknown. Here we show that Bcr-Abl levels control the degree of β-catenin protein stabilization by affecting its Y/S/T-phospho content in CML cells. Bcr-Abl physically interacts with β-catenin, and its oncogenic tyrosine kinase activity is required to phosphorylate β-catenin at Y86 and Y654 residues. This Y-phospho β-catenin binds to the TCF4 transcription factor, thus representing a transcriptionally active pool. Imatinib, a Bcr-Abl antagonist, impairs the β-catenin/TCF-related transcription causing a rapid cytosolic retention of Y-unphosphorylated β-catenin, which presents an increased binding affinity for the Axin/GSK3β complex. Although Bcr-Abl does not affect GSK3β autophosphorylation, it prevents, through its effect on β-catenin Y phosphorylation, Axin/GSK3β binding to β-catenin and its subsequent S/T phosphorylation. Silencing of β-catenin by small interfering RNA inhibited proliferation and clonogenicity of Bcr-Abl+ CML cells, in synergism with Imatinib. These findings indicate the Bcr-Abl triggered Y phosphorylation of β-catenin as a new mechanism responsible for its protein stabilization and nuclear signalling activation in CML. PMID:17318191

  5. Novel Regulation of Parkin Function Through c-Abl-Mediated Tyrosine Phosphorylation: Implications for Parkinson's Disease

    PubMed Central

    Imam, Syed Z.; Zhou, Qing; Yamamoto, Ayako; Valente, Anthony J.; Ali, Syed F.; Bains, Mona; Roberts, James L.; Kahle, Philipp J.; Clark, Robert A.; Li, Senlin

    2011-01-01

    Mutations in parkin, an E3 ubiquitin ligase, are most common cause of autosomal-recessive Parkinson's disease (PD). Here, we show that the stress-signaling non-receptor tyrosine-kinase c-Abl links parkin to sporadic forms of PD via tyrosine phosphorylation. Under oxidative and dopaminergic stress, c-Abl was activated in cultured neuronal cells and in striatum of adult C57 mice. Activated c-Abl was found in the striatum of PD patients. Concomitantly, parkin was tyrosine-phosphorylated, causing loss ofit's ubiquitin ligase and cytoprotective activities, and the accumulation of parkin substrates, AIMP2 (p38/JTV-1) and FBP-1. STI-571, a selective c-Abl inhibitor, prevented tyrosine phosphorylation of parkin and restored its E3 ligase activity and cytoprotective function both in vitro and in vivo. Our results suggest that tyrosine phosphorylation of parkin by c-Abl is a major post-translational modification that leads to loss of parkin function and disease progression in sporadic PD. Moreover, inhibition of c-Abl offers new therapeutic opportunities for blocking PD progression. PMID:21209200

  6. A solid-phase Bcr-Abl kinase assay in 96-well hydrogel plates.

    PubMed

    Wu, Ding; Mand, Michael R; Veach, Darren R; Parker, Laurie L; Clarkson, Bayard; Kron, Stephen J

    2008-04-01

    Regulated phosphorylation by protein tyrosine kinases (PTKs), such as c-Abl, is critical to cellular homeostasis. In turn, once deregulated as in the chronic myeloid leukemia (CML) fusion protein Bcr-Abl, PTKs can promote cancer onset and progression. The dramatic success of the Bcr-Abl inhibitor imatinib as therapy for CML has inspired interest in other PTKs as targets for cancer drug discovery. Here we report a novel PTK activity and inhibition screening method using hydrogel-immobilized peptide substrates. Using acrylate crosslinkers, we tether peptides via terminal cysteines to thiol-presenting hydrogels in 96-well plates. These surfaces display low background and high reproducibility, allowing semiquantitative detection of peptide phosphorylation by recombinant c-Abl or by Bcr-Abl activity in cell extracts using traditional anti-phosphotyrosine immunodetection and chemifluorescence. The capabilities of this assay are demonstrated by performing model screens for inhibition with several commercially available PTK inhibitors and a collection of pyridopyrimidine Src/Abl dual inhibitors. This assay provides a practical method to measure the activity of a single kinase present in a whole cell lysate with high sensitivity and specificity as a valuable means for efficient small molecule screening.

  7. The Abl/Enabled signaling pathway regulates Golgi architecture in Drosophila photoreceptor neurons

    PubMed Central

    Kannan, Ramakrishnan; Kuzina, Irina; Wincovitch, Stephen; Nowotarski, Stephanie H.; Giniger, Edward

    2014-01-01

    The Golgi apparatus is optimized separately in different tissues for efficient protein trafficking, but we know little of how cell signaling shapes this organelle. We now find that the Abl tyrosine kinase signaling pathway controls the architecture of the Golgi complex in Drosophila photoreceptor (PR) neurons. The Abl effector, Enabled (Ena), selectively labels the cis-Golgi in developing PRs. Overexpression or loss of function of Ena increases the number of cis- and trans-Golgi cisternae per cell, and Ena overexpression also redistributes Golgi to the most basal portion of the cell soma. Loss of Abl or its upstream regulator, the adaptor protein Disabled, lead to the same alterations of Golgi as does overexpression of Ena. The increase in Golgi number in Abl mutants arises in part from increased frequency of Golgi fission events and a decrease in fusions, as revealed by live imaging. Finally, we demonstrate that the effects of Abl signaling on Golgi are mediated via regulation of the actin cytoskeleton. Together, these data reveal a direct link between cell signaling and Golgi architecture. Moreover, they raise the possibility that some of the effects of Abl signaling may arise, in part, from alterations of protein trafficking and secretion. PMID:25103244

  8. 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

  9. 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.

  10. 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

  11. 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

  12. 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

  13. The N-terminal end of the catalytic domain of SRC kinase Hck is a conformational switch implicated in long-range allosteric regulation.

    PubMed

    Banavali, Nilesh K; Roux, Benoît

    2005-11-01

    Signal transduction in cell growth and proliferation involves regulation of kinases through long-range allostery between remote protein regions. Molecular dynamics free energy calculations are used to clarify the coupling between the catalytic domain of Src kinase Hck and its N-terminal end connecting to the regulatory SH2 and SH3 modules. The N-terminal end is stable in the orientation required for the regulatory modules to remain properly bound only in the inactive catalytic domain. In the active catalytic domain, the N-terminal end prefers a different conformation consistent with dissociation of the regulatory modules. The free energy surface shows that the N-terminal end acts as a reversible two-state conformational switch coupling the catalytic domain to the regulatory modules. Structural analogy with insulin receptor kinase and c-Src suggests that such reversible conformational switching in a critical hinge region could be a common mechanism in long-range allosteric regulation of protein kinase activity.

  14. 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

  15. The nebulette repeat domain is necessary for proper maintenance of tropomyosin with the cardiac sarcomere.

    PubMed

    Bonzo, Jeremy R; Norris, Andrea A; Esham, Michael; Moncman, Carole L

    2008-11-15

    Nebulette is a cardiac-specific isoform of the giant actin-binding protein nebulin. Nebulette, having a mass of approximately 100 kDa, is only predicted to extend 150 nm from the edge of the Z-lines. Overexpression of the nebulette C-terminal linker and/or SH3 domains in chicken cardiomyocytes results in a loss of endogenous nebulette with a concomitant loss of tropomyosin (TPM) and troponin, as well as a shortening of the thin filaments. These data suggest that nebulette's position in the sarcomere is important for the maintenance of TPM, troponin and thin filament length. To evaluate this hypothesis, N-terminal nested truncations tagged with GFP were expressed in chicken cardiomyocytes and the cells were analyzed for the distribution of myofilament proteins. Minimal effects on the myofilaments were observed with N-terminal deletions of up to 10 modules; however, deletion of 15 modules replicated the phenotype observed with expression of the C-terminal fragments. Expression of internal deletions of nebulette verifies that a site between module 10 and 15 is important for TPM maintenance within the sarcomeric lattice. We have additionally isolated TPM cDNAs from a yeast two hybrid (Y2H) analysis. These data indicate the importance of the nebulette-TPM interactions in the maintenance and stability of the thin filaments.

  16. Two Amino Acid Residues Confer Different Binding Affinities of Abelson Family Kinase Src Homology 2 Domains for Phosphorylated Cortactin*

    PubMed Central

    Gifford, Stacey M.; Liu, Weizhi; Mader, Christopher C.; Halo, Tiffany L.; Machida, Kazuya; Boggon, Titus J.; Koleske, Anthony J.

    2014-01-01

    The closely related Abl family kinases, Arg and Abl, play important non-redundant roles in the regulation of cell morphogenesis and motility. Despite similar N-terminal sequences, Arg and Abl interact with different substrates and binding partners with varying affinities. This selectivity may be due to slight differences in amino acid sequence leading to differential interactions with target proteins. We report that the Arg Src homology (SH) 2 domain binds two specific phosphotyrosines on cortactin, a known Abl/Arg substrate, with over 10-fold higher affinity than the Abl SH2 domain. We show that this significant affinity difference is due to the substitution of arginine 161 and serine 187 in Abl to leucine 207 and threonine 233 in Arg, respectively. We constructed Abl SH2 domains with R161L and S187T mutations alone and in combination and find that these substitutions are sufficient to convert the low affinity Abl SH2 domain to a higher affinity “Arg-like” SH2 domain in binding to a phospho-cortactin peptide. We crystallized the Arg SH2 domain for structural comparison to existing crystal structures of the Abl SH2 domain. We show that these two residues are important determinants of Arg and Abl SH2 domain binding specificity. Finally, we expressed Arg containing an “Abl-like” low affinity mutant Arg SH2 domain (L207R/T233S) and find that this mutant, although properly localized to the cell periphery, does not support wild type levels of cell edge protrusion. Together, these observations indicate that these two amino acid positions confer different binding affinities and cellular functions on the distinct Abl family kinases. PMID:24891505

  17. Just how versatile are domains?

    PubMed Central

    2008-01-01

    Background Creating new protein domain arrangements is a frequent mechanism of evolutionary innovation. While some domains always form the same combinations, others form many different arrangements. This ability, which is often referred to as versatility or promiscuity of domains, its a random evolutionary model in which a domain's promiscuity is based on its relative frequency of domains. Results We show that there is a clear relationship across genomes between the promiscuity of a given domain and its frequency. However, the strength of this relationship differs for different domains. We thus redefine domain promiscuity by defining a new index, DV I ("domain versatility index"), which eliminates the effect of domain frequency. We explore links between a domain's versatility, when unlinked from abundance, and its biological properties. Conclusion Our results indicate that domains occurring as single domain proteins and domains appearing frequently at protein termini have a higher DV I. This is consistent with previous observations that the evolution of domain re-arrangements is primarily driven by fusion of pre-existing arrangements and single domains as well as loss of domains at protein termini. Furthermore, we studied the link between domain age, defined as the first appearance of a domain in the species tree, and the DV I. Contrary to previous studies based on domain promiscuity, it seems as if the DV I is age independent. Finally, we find that contrary to previously reported findings, versatility is lower in Eukaryotes. In summary, our measure of domain versatility indicates that a random attachment process is sufficient to explain the observed distribution of domain arrangements and that several views on domain promiscuity need to be revised. PMID:18854028

  18. 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

  19. 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.

  20. S6K1 determines the metabolic requirements for BCR-ABL survival.

    PubMed

    Barger, J F; Gallo, C A; Tandon, P; Liu, H; Sullivan, A; Grimes, H L; Plas, D R

    2013-01-24

    In chronic myelogenous leukemia, the constitutive activation of the BCR-ABL kinase transforms cells to an addicted state that requires glucose metabolism for survival. We investigated S6K1, a protein kinase that drives glycolysis in leukemia cells, as a target for counteracting glucose-dependent survival induced by BCR-ABL. BCR-ABL potently activated S6K1-dependent signaling and glycolysis. Although S6K1 knockdown or rapamycin treatment suppressed glycolysis in BCR-ABL-transformed cells, these treatments did not induce cell death. Instead, loss of S6K1 triggered compensatory activation of fatty-acid oxidation, a metabolic program that can support glucose-independent cell survival. Fatty-acid oxidation in response to S6K1 inactivation required the expression of the fatty-acid transporter carnitine palmitoyl transferase 1c, which was recently linked to rapamycin resistance in cancer. Finally, addition of an inhibitor of fatty-acid oxidation significantly enhanced cytotoxicity in response to S6K1 inactivation. These data indicate that S6K1 dictates the metabolic requirements mediating BCR-ABL survival and provide a rationale for combining targeted inhibitors of signal transduction, with strategies to interrupt oncogene-induced metabolism. PMID:22391570

  1. Spatiotemporal Variables of Able-bodied and Amputee Sprinters in Men's 100-m Sprint.

    PubMed

    Hobara, H; Kobayashi, Y; Mochimaru, M

    2015-06-01

    The difference in world records set by able-bodied sprinters and amputee sprinters in the men's 100-m sprint is still approximately 1 s (as of 28 March 2014). Theoretically, forward velocity in a 100-m sprint is the product of step frequency and step length. The goal of this study was to examine the hypothesis that differences in the sprint performance of able-bodied and amputee sprinters would be due to a shorter step length rather than lower step frequency. Men's elite-level 100-m races with a total of 36 able-bodied, 25 unilateral and 17 bilateral amputee sprinters were analyzed from the publicly available internet broadcasts of 11 races. For each run of each sprinter, the average forward velocity, step frequency and step length over the whole 100-m distance were analyzed. The average forward velocity of able-bodied sprinters was faster than that of the other 2 groups, but there was no significant difference in average step frequency among the 3 groups. However, the average step length of able-bodied sprinters was significantly longer than that of the other 2 groups. These results suggest that the differences in sprint performance between 2 groups would be due to a shorter step length rather than lower step frequency.

  2. Abl tyrosine kinases modulate cadherin-dependent adhesion upstream and downstream of Rho family GTPases.

    PubMed

    Zandy, Nicole L; Pendergast, Ann Marie

    2008-02-15

    Formation and dissolution of intercellular adhesions are processes of paramount importance during tissue morphogenesis and for pathological conditions such as tumor metastasis. Cadherin-mediated intercellular adhesion requires dynamic regulation of the actin cytoskeleton. The pathways that link cadherin signaling to cytoskeletal regulation remain poorly defined. We have recently uncovered a novel role for the Abl family of tyrosine kinases linking cadherin-mediated adhesion to actin dynamics via the regulation of Rho family GTPases. Abl kinases are activated by cadherin engagement, localize to cell-cell junctions and are required for the formation of adherens junctions. Notably, we showed that Abl kinases are required for Rac activation during formation of adherens junctions, and also regulate a Rho-ROCK-myosin signaling pathway that is required for the maintenance of intercellular adhesion. Here we show that Abl kinases regulate the formation and strengthening of adherens junctions downstream of active Rac, and that Abl tyrosine kinases are components of a positive feed-back loop that employs the Crk/CrkL adaptor proteins to promote the formation and maturation of adherens junctions.

  3. 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.

  4. 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.

  5. 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

  6. BCR-ABL Translocation in Pediatric Acute Lymphoblastic Leukemia in Southern India.

    PubMed

    Sugapriya, D; Preethi, S; Shanthi, P; Chandra, N; Jeyaraman, G; Sachdanandam, P; Thilagavathy, S; Venkatadesilalu, S

    2012-03-01

    Cytogenetics and polymerase chain reaction (PCR) based assays provide important information regarding biologically defined and prognostically relevant subgroups in acute leukemias. We utilized karyotyping and molecular analysis by reverse transcriptase PCR for the BCR-ABL translocation, in addition to morphological study, cytochemistry and immunophenotyping, to study 24 cases of pediatric acute lymphoblastic leukemia (ALL). Our objective was to determine the frequency of the BCRABL translocation in childhood ALL from southern India. Karyotyping showed one case of hyperdiploidy, one case of t (12; 21) translocation and one case of 46, XY-21+mar. The BCR-ABL translocation was found in 8.3% of these cases. One of these was a cryptic translocation, the karyotype being normal. BCR-ABL positivity in ALL is associated with aggressive disease and has been shown to be a poor prognostic factor, especially in children. PMID:23449388

  7. The Interface between BCR-ABL-Dependent and -Independent Resistance Signaling Pathways in Chronic Myeloid Leukemia

    PubMed Central

    Nestal de Moraes, Gabriela; Souza, Paloma Silva; Costas, Fernanda Casal de Faria; Vasconcelos, Flavia Cunha; Reis, Flaviana Ruade Souza; Maia, Raquel Ciuvalschi

    2012-01-01

    Chronic myeloid leukemia (CML) is a clonal hematopoietic disorder characterized by the presence of the Philadelphia chromosome which resulted from the reciprocal translocation between chromosomes 9 and 22. The pathogenesis of CML involves the constitutive activation of the BCR-ABL tyrosine kinase, which governs malignant disease by activating multiple signal transduction pathways. The BCR-ABL kinase inhibitor, imatinib, is the front-line treatment for CML, but the emergence of imatinib resistance and other tyrosine kinase inhibitors (TKIs) has called attention for additional resistance mechanisms and has led to the search for alternative drug treatments. In this paper, we discuss our current understanding of mechanisms, related or unrelated to BCR-ABL, which have been shown to account for chemoresistance and treatment failure. We focus on the potential role of the influx and efflux transporters, the inhibitor of apoptosis proteins, and transcription factor-mediated signals as feasible molecular targets to overcome the development of TKIs resistance in CML. PMID:23259070

  8. 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

  9. 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.

  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. 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.

  12. 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.

  13. 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.

  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. Whole Language in ABLE--Just Do It! Research to Practice.

    ERIC Educational Resources Information Center

    Baker, Karen

    The whole language approach was used with a "well" or successful adult basic and literacy education (ABLE) group. Literature was chosen as a curriculum for which group work could be devised that was geared to all levels of students. The book used was "Choices" by George Ella Lyon, a book of short stories written by characters who live in a small…

  16. 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…

  17. 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…

  18. 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)…

  19. 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…

  20. We Are ABLE: Success Stories from Northwest Ohio Adult Basic and Literacy Education Programs.

    ERIC Educational Resources Information Center

    Northwest ABLE Resource Center, Toledo, OH.

    This publication provides photographs and the success stories of seven students in Adult Basic and Literacy Education (ABLE) in Northwest Ohio. The stories detail the sacrifices, dedication, and hard work that contributed to the students' success, as well as the work of the dedicated, hard-working instructors who facilitated their learning.…

  1. Frequency of BCR-ABL Transcript Types in Syrian CML Patients.

    PubMed

    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

  2. Crystal structure of the G3BP2 NTF2-like domain in complex with a canonical FGDF motif peptide.

    PubMed

    Kristensen, Ole

    2015-11-01

    The crystal structure of the NTF2-like domain of the human Ras GTPase SH3 Binding Protein (G3BP), isoform 2, was determined at a resolution of 2.75 Å in complex with a peptide containing a FGDF sequence motif. The overall structure of the protein is highly similar to the homodimeric N-terminal domains of the G3BP1 and Rasputin proteins. Recently, a subset of G3BP interacting proteins was recognized to share a common sequence motif, FGDF. The most studied binding partners, USP10 and viral nsP3, interfere with essential G3BP functions related to assembly of cellular stress granules. Reported molecular modeling suggested that FGDF-motif containing peptides bind in an extended conformation into a hydrophobic groove on the surface of the G3BP NTF2-like domain in a manner similar to the known binding of FxFG nucleoporin repeats. The results in this paper provide evidence for a different binding mode. The FGDF peptide binds and changes conformation of the protruding N-terminal residues by providing hydrophobic interactions to a symmetry related molecule that facilitated crystallization of the G3BP2 isoform.

  3. c-Abl is an effector of Src for growth factor-induced c-myc expression and DNA synthesis

    PubMed Central

    Furstoss, Olivia; Dorey, Karel; Simon, Valérie; Barilà, Daniela; Superti-Furga, Giulio; Roche, Serge

    2002-01-01

    The mechanism by which the ubiquitously expressed Src family kinases regulate mitogenesis is not well understood. Here we report that cytoplasmic tyrosine kinase c-Abl is an important effector of c-Src for PDGF- and serum-induced DNA synthesis. Inactivation of cytoplasmic c-Abl by the kinase- inactive Abl-PP-K– (AblP242E/P249E/K290M) or by microinjection of Abl neutralizing antibodies inhibited mitogenesis. The kinase-inactive SrcK295M induced a G1 block that was overcome by the constitutively active Abl-PP (AblP242E/P249E). Conversely, the inhibitory effect of Abl-PP-K– was not compensated by Src. c-Src-induced c-Abl activation involves phosphorylation of Y245 and Y412, two residues required for c-Abl mitogenic function. Finally, we found that p53 inactivation and c-myc expression, two cell cycle events regulated by Src during mitogenesis, also implied c-Abl: c-Abl function was dispensable in cells deficient in active p53 and inhibition of c-Abl reduced mitogen-induced c-myc expression. These data identify a novel function of cytoplasmic c-Abl in the signalling pathways regulating growth factor-induced c-myc expression and we propose the existence of a tyro sine kinase signalling cascade (PDGFR/c-Src/c-Abl) important for mitogenesis. PMID:11847100

  4. 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.

  5. TIE2-mediated tyrosine phosphorylation of H4 regulates DNA damage response by recruiting ABL1

    PubMed Central

    Hossain, Mohammad B.; Shifat, Rehnuma; Johnson, David G.; Bedford, Mark T.; Gabrusiewicz, Konrad R.; Cortes-Santiago, Nahir; Luo, Xuemei; Lu, Zhimin; Ezhilarasan, Ravesanker; Sulman, Erik P.; Jiang, Hong; Li, Shawn S. C.; Lang, Frederick F.; Tyler, Jessica; Hung, Mien-Chie; Fueyo, Juan; Gomez-Manzano, Candelaria

    2016-01-01

    DNA repair pathways enable cancer cells to survive DNA damage induced after genotoxic therapies. Tyrosine kinase receptors (TKRs) have been reported as regulators of the DNA repair machinery. TIE2 is a TKR overexpressed in human gliomas at levels that correlate with the degree of increasing malignancy. Following ionizing radiation, TIE2 translocates to the nucleus, conferring cells with an enhanced nonhomologous end-joining mechanism of DNA repair that results in a radioresistant phenotype. Nuclear TIE2 binds to key components of DNA repair and phosphorylates H4 at tyrosine 51, which, in turn, is recognized by the proto-oncogene ABL1, indicating a role for nuclear TIE2 as a sensor for genotoxic stress by action as a histone modifier. H4Y51 constitutes the first tyrosine phosphorylation of core histones recognized by ABL1, defining this histone modification as a direct signal to couple genotoxic stress with the DNA repair machinery.

  6. 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.

  7. 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.

  8. Detection of BCR-ABL using one step reverse transcriptase- polymerase chain reaction and microchip electrophoresis.

    PubMed

    Lin, Xuexia; Wu, Jing; Liu, Wu; Li, Haifang; Wang, Zhihua; Lin, Jin-Ming

    2013-12-15

    One-step reverse transcriptase polymerase chain reaction (RT-PCR) coupled with microchip electrophoresis (MCE) was established to analyze BCR-ABL fusion gene. The use of one-step RT-PCR could simplify the RT-PCR procedure and thus reduced the risk of contamination and sample consumption. This method also enhanced the sensitivity for amplified target DNA and dramatically shorted the analysis time. Moreover, this assay can simultaneously identify b2a2 and b3a2. Orthogonal array design, which can investigated mutual effects of PCR parameters, was used to optimize the reaction system. This approach was highly effective, reproducible and sensitive, and would be suitable for the determination of BCR-ABL in clinic diagnosis.

  9. Real-time quantification assay to monitor BCR-ABL1 transcripts in chronic myeloid leukemia.

    PubMed

    Foskett, Pierre; Gerrard, Gareth; Foroni, Letizia

    2014-01-01

    The BCR-ABL1 fusion gene, the causative lesion of chronic myeloid leukemia (CML) in >95 % of newly presenting patients, offers both a therapeutic and diagnostic target. Reverse-transcription quantitative polymerase chain reaction technology (RT-qPCR), utilizing primer-probe combinations directed to exons flanking the breakpoint junctional region, offers very high levels of both specificity and sensitivity, in a scalable, robust, and cost-effective assay.

  10. BCR-ABL1-Associated Reduction of Beta Catenin Antagonist Chibby1 in Chronic Myeloid Leukemia

    PubMed Central

    Aluigi, Michela; Luatti, Simona; Castagnetti, Fausto; Testoni, Nicoletta; Soverini, Simona; Santucci, Maria Alessandra; Martinelli, Giovanni

    2013-01-01

    Beta Catenin signaling is critical for the self-renewal of leukemic stem cells in chronic myeloid leukemia. It is driven by multiple events, enhancing beta catenin stability and promoting its transcriptional co-activating function. We investigated the impact of BCR-ABL1 on Chibby1, a beta catenin antagonist involved in cell differentiation and transformation. Relative proximity of the Chibby1 encoding gene (C22orf2) on chromosome 22q12 to the BCR breakpoint (22q11) lets assume its involvement in beta catenin activation in chronic myeloid leukemia as a consequence of deletions of distal BCR sequences encompassing one C22orf2 allele. Forty patients with chronic myeloid leukemia in chronic phase were analyzed for C22orf2 relocation and Chibby1 expression. Fluorescent in situ hybridization analyses established that the entire C22orf2 follows BCR regardless of chromosomes involved in the translocation. In differentiated hematopoietic progenitors (bone marrow mononuclear cell fractions) of 30/40 patients, the expression of Chibby1 protein was reduced below 50% of the reference value (peripheral blood mononuclear cell fractions of healthy persons). In such cell context, Chibby1 protein reduction is not dependent on C22orf2 transcriptional downmodulation; however, it is strictly dependent upon BCR-ABL1 expression because it was not observed at the moment of major molecular response under tyrosine kinase inhibitor therapy. Moreover, it was not correlated with the disease prognosis or response to therapy. Most importantly, a remarkable Chibby1 reduction was apparent in a putative BCR-ABL1+ leukemic stem cell compartment identified by a CD34+ phenotype compared to more differentiated hematopoietic progenitors. In CD34+ cells, Chibby1 reduction arises from transcriptional events and is driven by C22orf2 promoter hypermethylation. These results advance low Chibby1 expression associated with BCR-ABL1 as a component of beta catenin signaling in leukemic stem cells. PMID:24339928

  11. Molecular Imaging of Bcr-Abl Phosphokinase in a Xenograft Model*

    PubMed Central

    Wu, Ji Yuan; Yang, David J.; Angelo, Laura S.; Kohanim, Saady; Kurzrock, Razelle

    2009-01-01

    The purpose of this study was to determine whether the Bcr-Abl tyrosine kinase can be assessed by gamma imaging using an 111Indium-labeled anti-phosphotyrosine antibody, and if the response to treatment with imatinib could be detected using this imaging technique. Anti-phosphotyrosine antibody (APT) was labeled with indium (111In) using ethylenedicysteine (EC) as a chelator. To determine if 111In-EC-APT could assess a non-receptor tyrosine kinase, xenografts of the human chronic myelogenous leukemia (CML) cell line K562 were used. Gamma scintigraphy of the tumor-bearing mice, before and after imatinib treatment, was obtained 1, 24, and 48 hours after they were given 111In-EC-APT (100 uCi/mouse, i.v.). 111In-EC-APT is preferentially taken up by Bcr-Abl-bearing tumor cells when compared to 111In-EC-BSA or 111In-EC-IgG1 controls, and comparable to the level of uptake of 111In-EC-Bcr-Abl. Imatinib treatment resulted in decreased expression of phosphorylated Bcr-Abl by Western blot analysis, which correlated with early (four days after starting imatinib) kinase down-regulation as assessed by imaging using 111In-EC-APT. The optimal time to imaging was 24 and 48 hours after injection of 111In-EC-APT. Although tumor regression was insignificant on day 4 after starting imatinib treatment, it was marked by day 14. 111In-EC-APT can assess intracellular phosphokinase activity, and down-regulation of phosphokinase activity predates tumor regression. This technique may therefore be useful in the clinic to detect the presence of phosphokinase activity, and for early prediction of response. PMID:19258427

  12. Comparison of sport achievement orientation between wheelchair and able-bodied basketball athletes.

    PubMed

    Skordilis, E K; Koutsouki, D; Asonitou, K; Evans, E; Jensen, B

    2002-02-01

    Differences in sport achievement orientations between 31 recreational wheelchair and 76 able-bodied basketball athletes were tested. Athletes from the New England region completed the three subscales of the Sport Orientation Questionnaire (competitiveness, win orientation, and goal orientation). Wheelchair athletes responded higher on the Competitiveness and Goal Orientation subscales. In discriminative function analysis competitiveness scores were the only significant discriminator between the two groups.

  13. RNAi Screen Reveals an Abl Kinase-Dependent Host Cell Pathway Involved in Pseudomonas aeruginosa Internalization

    PubMed Central

    Pielage, Julia F.; Powell, Kimberly R.; Kalman, Daniel; Engel, Joanne N.

    2008-01-01

    Internalization of the pathogenic bacterium Pseudomonas aeruginosa by non-phagocytic cells is promoted by rearrangements of the actin cytoskeleton, but the host pathways usurped by this bacterium are not clearly understood. We used RNAi-mediated gene inactivation of ∼80 genes known to regulate the actin cytoskeleton in Drosophila S2 cells to identify host molecules essential for entry of P. aeruginosa. This work revealed Abl tyrosine kinase, the adaptor protein Crk, the small GTPases Rac1 and Cdc42, and p21-activated kinase as components of a host signaling pathway that leads to internalization of P. aeruginosa. Using a variety of complementary approaches, we validated the role of this pathway in mammalian cells. Remarkably, ExoS and ExoT, type III secreted toxins of P. aeruginosa, target this pathway by interfering with GTPase function and, in the case of ExoT, by abrogating P. aeruginosa–induced Abl-dependent Crk phosphorylation. Altogether, this work reveals that P. aeruginosa utilizes the Abl pathway for entering host cells and reveals unexpected complexity by which the P. aeruginosa type III secretion system modulates this internalization pathway. Our results furthermore demonstrate the applicability of using RNAi screens to identify host signaling cascades usurped by microbial pathogens that may be potential targets for novel therapies directed against treatment of antibiotic-resistant infections. PMID:18369477

  14. 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

  15. Ras complements the carboxyl terminus of v-Abl protein in lymphoid transformation.

    PubMed Central

    Parmar, K; Rosenberg, N

    1996-01-01

    Abelson murine leukemia virus (Ab-MLV) mutants expressing v-Abl proteins lacking the carboxyl terminus are compromised in the ability to transform lymphoid but not NIH 3T3 cells. This feature correlates with the presence of low levels of phosphotyrosine in lymphoid cells infected with carboxyl-terminal truncation mutants. In contrast, high levels of phosphotyrosine are observed in NIH 3T3 cells infected with wild-type and mutant Ab-MLV. Two downstream targets affected in lymphoid transformants are the GTPase-activating protein and GTPase-activating protein-associated protein p62, molecules which are heavily tyrosine phosphorylated in lymphoid cells transformed by wild-type Ab-MLV but not carboxyl-terminal truncation mutants of Ab-MLV. This difference suggested that signaling mediated via the Ras pathway may be compromised in lymphoid cells expressing the carboxyl-terminal truncation mutants. Consistent with this idea, expression of v-Ha-ras complemented these mutants in primary bone marrow transformation assays and increased transformation frequencies obtained with the Ab-MLV mutants 8- to 20-fold. These data suggest that a biologically important link exists between the carboxyl terminus of v-Abl protein and the Ras pathway. Signals transmitted via this connection may enhance those mediated via other regions of the v-Abl protein and facilitate transformation of primary, nonimmortalized cells such as pre-B lymphocytes. PMID:8551558

  16. 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.

  17. 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.

  18. Stat5 is indispensable for the maintenance of bcr/abl-positive leukaemia

    PubMed Central

    Hoelbl, Andrea; Schuster, Christian; Kovacic, Boris; Zhu, Bingmei; Wickre, Mark; Hoelzl, Maria A; Fajmann, Sabine; Grebien, Florian; Warsch, Wolfgang; Stengl, Gabriele; Hennighausen, Lothar; Poli, Valeria; Beug, Hartmut; Moriggl, Richard; Sexl, Veronika

    2010-01-01

    Tumourigenesis caused by the Bcr/Abl oncoprotein is a multi-step process proceeding from initial to tumour-maintaining events and finally results in a complex tumour-supporting network. A key to successful cancer therapy is the identification of critical functional nodes in an oncogenic network required for disease maintenance. So far, the transcription factors Stat3 and Stat5a/b have been implicated in bcr/abl-induced initial transformation. However, to qualify as a potential drug target, a signalling pathway must be required for the maintenance of the leukaemic state. Data on the roles of Stat3 or Stat5a/b in leukaemia maintenance are elusive. Here, we show that both, Stat3 and Stat5 are necessary for initial transformation. However, Stat5- but not Stat3-deletion induces G0/G1 cell cycle arrest and apoptosis of imatinib-sensitive and imatinib-resistant stable leukaemic cells in vitro. Accordingly, Stat5-abrogation led to effective elimination of myeloid and lymphoid leukaemia maintenance in vivo. Hence, we identified Stat5 as a vulnerable point in the oncogenic network downstream of Bcr/Abl representing a case of non-oncogene addiction (NOA). PMID:20201032

  19. 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

  20. Metabolic efficiency of volitional and electrically stimulated cycling in able-bodied subjects.

    PubMed

    Hunt, K J; Hosmann, D; Grob, M; Saengsuwan, J

    2013-07-01

    This study compared the metabolic efficiency of volitional cycling and functional-electrical-stimulation (FES) cycling within a subject group of able-bodied individuals, with a view to further elucidating the mechanisms underlying the low efficiency of FES cycling. Previous studies estimated the metabolic efficiency of volitional cycling and anaesthetised FES cycling in able-bodied subjects, and of FES cycling in subjects paralysed by spinal cord injury. The rationale for the experimental model chosen here, i.e. non-anaesthetised able-bodied subjects, was that this lies between normal cycling and paralysed cycling: while using FES, this group has artificial muscle activation and timing like the paralysed group; but it does not have disrupted sensory feedback and vasomotor control; this measurement therefore allows delineation of the magnitude of reduction in metabolic efficiency resulting from: (i) the FES itself and (ii) paralysis (where there is disrupted sensory feedback and vasomotor control). Furthermore, we used the same methods employed previously for estimation of metabolic efficiency in subjects with motor- and sensory-complete paraplegia. The mean metabolic efficiency of volitional cycling was found to be 29.8% and that of FES cycling was 16.4% (n=11). The low efficiency of FES cycling can be explained in large part by the crude timing of muscle activation and by non-physiological muscle fibre recruitment. In FES cycling with paralysed subjects, disrupted sensory feedback and vasomotor control may play a further, albeit smaller, role in the reduced efficiency. PMID:23253953

  1. 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.

  2. 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

  3. Comparison of the domain and frequency domain state feedbacks

    SciTech Connect

    Zhang, S.Y.

    1986-01-01

    In this paper, we present explicitly the equivalence of the time domain and frequency domain state feedbacks, as well as the dynamic state feedback and a modified frequency domain state feedback, from the closed-loop transfer function point of view. The difference of the two approaches is also shown.

  4. 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…

  5. 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

  6. Acute Effects of Caffeine on Heart Rate Variability, Blood Pressure and Tidal Volume in Paraplegic and Tetraplegic Compared to Able-Bodied Individuals: A Randomized, Blinded Trial

    PubMed Central

    Flueck, Joelle Leonie; Schaufelberger, Fabienne; Lienert, Martina; Schäfer Olstad, Daniela; Wilhelm, Matthias; Perret, Claudio

    2016-01-01

    Caffeine increases sympathetic nerve activity in healthy individuals. Such modulation of nervous system activity can be tracked by assessing the heart rate variability. This study aimed to investigate the influence of caffeine on time- and frequency-domain heart rate variability parameters, blood pressure and tidal volume in paraplegic and tetraplegic compared to able-bodied participants. Heart rate variability was measured in supine and sitting position pre and post ingestion of either placebo or 6 mg caffeine in 12 able-bodied, 9 paraplegic and 7 tetraplegic participants in a placebo-controlled, randomized and double-blind study design. Metronomic breathing was applied (0.25 Hz) and tidal volume was recorded during heart rate variability assessment. Blood pressure, plasma caffeine and epinephrine concentrations were analyzed pre and post ingestion. Most parameters of heart rate variability did not significantly change post caffeine ingestion compared to placebo. Tidal volume significantly increased post caffeine ingestion in able-bodied (p = 0.021) and paraplegic (p = 0.036) but not in tetraplegic participants (p = 0.34). Systolic and diastolic blood pressure increased significantly post caffeine in able-bodied (systolic: p = 0.003; diastolic: p = 0.021) and tetraplegic (systolic: p = 0.043; diastolic: p = 0.042) but not in paraplegic participants (systolic: p = 0.09; diastolic: p = 0.33). Plasma caffeine concentrations were significantly increased post caffeine ingestion in all three groups of participants (p<0.05). Plasma epinephrine concentrations increased significantly in able-bodied (p = 0.002) and paraplegic (p = 0.032) but not in tetraplegic participants (p = 0.63). The influence of caffeine on the autonomic nervous system seems to depend on the level of lesion and the extent of the impairment. Therefore, tetraplegic participants may be less influenced by caffeine ingestion. Trial Registration ClinicalTrials.gov NCT02083328 PMID:27776149

  7. 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

  8. 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.

  9. Multiple domains of Stardust differentially mediate localisation of the Crumbs-Stardust complex during photoreceptor development in Drosophila.

    PubMed

    Bulgakova, Natalia A; Kempkens, Ozlem; Knust, Elisabeth

    2008-06-15

    Drosophila Stardust (Sdt), a member of the MAGUK family of scaffolding proteins, is a constituent of the evolutionarily conserved Crumbs-Stardust (Crb-Sdt) complex that controls epithelial cell polarity in the embryo and morphogenesis of photoreceptor cells. Although apical localisation is a hallmark of the complex in all cell types and in all organisms analysed, only little is known about how individual components are targeted to the apical membrane. We have performed a structure-function analysis of Sdt by constructing transgenic flies that express altered forms of Sdt to determine the roles of individual domains for localisation and function in photoreceptor cells. The results corroborate the observation that the organisation of the Crb-Sdt complex is differentially regulated in pupal and adult photoreceptors. In pupal photoreceptors, only the PDZ domain of Sdt - the binding site of Crb - is required for apical targeting. In adult photoreceptors, by contrast, targeting of Sdt to the stalk membrane, a distinct compartment of the apical membrane between the rhabdomere and the zonula adherens, depends on several domains, and seems to be a two-step process. The N-terminus, including the two ECR domains and a divergent N-terminal L27 domain that binds the multi-PDZ domain protein PATJ in vitro, is necessary for targeting the protein to the apical pole of the cell. The PDZ-, the SH3- and the GUK-domains are required to restrict the protein to the stalk membrane. Drosophila PATJ or Drosophila Lin-7 are stabilised whenever a Sdt variant that contains the respective binding site is present, independently of where the variant is localised. By contrast, only full-length Sdt, confined to the stalk membrane, stabilises and localises Crb, although only in reduced amounts. The amount of Crumbs recruited to the stalk membrane correlates with its length. Our results highlight the importance of the different Sdt domains and point to a more intricate regulation of the Crb

  10. 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.

  11. 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.

  12. 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.

  13. 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

  14. 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

  15. 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.

  16. 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

  17. 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

  18. 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.

  19. 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

  20. 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

  1. 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

  2. Screening of wild type Streptomyces isolates able to overproduce clavulanic acid

    PubMed Central

    Viana Marques, Daniela A.; Santos-Ebinuma, Valéria de Carvalho; de Oliveira, Patrícia Maria Sobral; Lima, Gláucia Manoella de Souza; Araújo, Janete M.; Lima-Filho, José L.; Converti, Attilio; Pessoa-Júnior, Adalberto; Porto, Ana L.F.

    2014-01-01

    The selection of new microorganisms able to produce antimicrobial compounds is hoped for to reduce their production costs and the side effects caused by synthetic drugs. Clavulanic acid is a β-lactam antibiotic produced by submerged culture, which is widely used in medicine as a powerful inhibitor of β-lactamases, enzymes produced by bacteria resistant to antibiotics such penicillin and cephalosporin. The purpose of this work was to select the best clavulanic acid producer among strains of Streptomyces belonging to the Microorganism Collection of the Department of Antibiotics of the Federal University of Pernambuco (DAUFPE). Initially, the strains were studied for their capacity to inhibit the action of β-lactamases produced by Klebsiella aerogenes ATCC 15380. From these results, five strains were selected to investigate the batch kinetics of growth and clavulanic acid production in submerged culture carried out in flasks. The results were compared with the ones obtained by Streptomyces clavuligerus ATCC 27064 selected as a control strain. The best clavulanic acid producer was Streptomyces DAUFPE 3060, molecularly identified as Streptomyces variabilis, which increased the clavulanic acid production by 28% compared to the control strain. This work contributes to the enlargement of knowledge on new Streptomyces wild strains able to produce clavulanic acid by submerged culture. PMID:25477926

  3. Establishment of Immortalized Human Erythroid Progenitor Cell Lines Able to Produce Enucleated Red Blood Cells

    PubMed Central

    Kurita, Ryo; Suda, Noriko; Sudo, Kazuhiro; Miharada, Kenichi; Hiroyama, Takashi; Miyoshi, Hiroyuki; Tani, Kenzaburo; Nakamura, Yukio

    2013-01-01

    Transfusion of red blood cells (RBCs) is a standard and indispensable therapy in current clinical practice. In vitro production of RBCs offers a potential means to overcome a shortage of transfusable RBCs in some clinical situations and also to provide a source of cells free from possible infection or contamination by microorganisms. Thus, in vitro production of RBCs may become a standard procedure in the future. We previously reported the successful establishment of immortalized mouse erythroid progenitor cell lines that were able to produce mature RBCs very efficiently. Here, we have developed a reliable protocol for establishing immortalized human erythroid progenitor cell lines that are able to produce enucleated RBCs. These immortalized cell lines produce functional hemoglobin and express erythroid-specific markers, and these markers are upregulated following induction of differentiation in vitro. Most importantly, these immortalized cell lines all produce enucleated RBCs after induction of differentiation in vitro, although the efficiency of producing enucleated RBCs remains to be improved further. To the best of our knowledge, this is the first demonstration of the feasibility of using immortalized human erythroid progenitor cell lines as an ex vivo source for production of enucleated RBCs. PMID:23533656

  4. 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

  5. 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

  6. Screening of wild type Streptomyces isolates able to overproduce clavulanic acid.

    PubMed

    Viana Marques, Daniela A; Santos-Ebinuma, Valéria de Carvalho; de Oliveira, Patrícia Maria Sobral; Lima, Gláucia Manoella de Souza; Araújo, Janete M; Lima-Filho, José L; Converti, Attilio; Pessoa-Júnior, Adalberto; Porto, Ana L F

    2014-01-01

    The selection of new microorganisms able to produce antimicrobial compounds is hoped for to reduce their production costs and the side effects caused by synthetic drugs. Clavulanic acid is a β-lactam antibiotic produced by submerged culture, which is widely used in medicine as a powerful inhibitor of β-lactamases, enzymes produced by bacteria resistant to antibiotics such penicillin and cephalosporin. The purpose of this work was to select the best clavulanic acid producer among strains of Streptomyces belonging to the Microorganism Collection of the Department of Antibiotics of the Federal University of Pernambuco (DAUFPE). Initially, the strains were studied for their capacity to inhibit the action of β-lactamases produced by Klebsiella aerogenes ATCC 15380. From these results, five strains were selected to investigate the batch kinetics of growth and clavulanic acid production in submerged culture carried out in flasks. The results were compared with the ones obtained by Streptomyces clavuligerus ATCC 27064 selected as a control strain. The best clavulanic acid producer was Streptomyces DAUFPE 3060, molecularly identified as Streptomyces variabilis, which increased the clavulanic acid production by 28% compared to the control strain. This work contributes to the enlargement of knowledge on new Streptomyces wild strains able to produce clavulanic acid by submerged culture.

  7. 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.

  8. 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.

  9. 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

  10. A Cell-Based Assay for Measuring Endogenous BcrAbl Kinase Activity and Inhibitor Resistance

    PubMed Central

    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

  11. New Betaproteobacterial Rhizobium Strains Able To Efficiently Nodulate Parapiptadenia rigida (Benth.) Brenan

    PubMed Central

    Taulé, Cecilia; Zabaleta, María; Mareque, Cintia; Platero, Raúl; Sanjurjo, Lucía; Sicardi, Margarita; Frioni, Lillian; Battistoni, Federico

    2012-01-01

    Among the leguminous trees native to Uruguay, Parapiptadenia rigida (Angico), a Mimosoideae legume, is one of the most promising species for agroforestry. Like many other legumes, it is able to establish symbiotic associations with rhizobia and belongs to the group known as nitrogen-fixing trees, which are major components of agroforestry systems. Information about rhizobial symbionts for this genus is scarce, and thus, the aim of this work was to identify and characterize rhizobia associated with P. rigida. A collection of Angico-nodulating isolates was obtained, and 47 isolates were selected for genetic studies. According to enterobacterial repetitive intergenic consensus PCR patterns and restriction fragment length polymorphism analysis of their nifH and 16S rRNA genes, the isolates could be grouped into seven genotypes, including the genera Burkholderia, Cupriavidus, and Rhizobium, among which the Burkholderia genotypes were the predominant group. Phylogenetic studies of nifH, nodA, and nodC sequences from the Burkholderia and the Cupriavidus isolates indicated a close relationship of these genes with those from betaproteobacterial rhizobia (beta-rhizobia) rather than from alphaproteobacterial rhizobia (alpha-rhizobia). In addition, nodulation assays with representative isolates showed that while the Cupriavidus isolates were able to effectively nodulate Mimosa pudica, the Burkholderia isolates produced white and ineffective nodules on this host. PMID:22226956

  12. 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

  13. The EH-domain-containing protein Pan1 is required for normal organization of the actin cytoskeleton in Saccharomyces cerevisiae.

    PubMed Central

    Tang, H Y; Cai, M

    1996-01-01

    Normal cell growth and division in the yeast Saccharomyces cerevisiae involve dramatic and frequent changes in the organization of the actin cytoskeleton. Previous studies have suggested that the reorganization of the actin cytoskeleton in accordance with cell cycle progression is controlled, directly or indirectly, by the cyclin-dependent kinase Cdc28. Here we report that by isolating rapid-death mutants in the background of the Start-deficient cdc28-4 mutation, the essential yeast gene PAN1, previously thought to encode the yeast poly(A) nuclease, is identified as a new factor required for normal organization of the actin cytoskeleton. We show that at restrictive temperature, the pan1 mutant exhibited abnormal bud growth, failed to maintain a proper distribution of the actin cytoskeleton, was unable to reorganize actin the cytoskeleton during cell cycle, and was defective in cytokinesis. The mutant also displayed a random pattern of budding even at permissive temperature. Ectopic expression of PAN1 by the GAL promoter caused abnormal distribution of the actin cytoskeleton when a single-copy vector was used. Immunofluorescence staining revealed that the Pan1 protein colocalized with the cortical actin patches, suggesting that it may be a filamentous actin-binding protein. The Pan1 protein contains an EF-hand calcium-binding domain, a putative Src homology 3 (SH3)-binding domain, a region similar to the actin cytoskeleton assembly control protein Sla1, and two repeats of a newly identified protein motif known as the EH domain. These findings suggest that Pan1, recently recognized as not responsible for the poly(A) nuclease activity (A. B. Sachs and J. A. Deardorff, erratum, Cell 83:1059, 1995; R. Boeck, S. Tarun, Jr., M. Rieger, J. A. Deardorff, S. Muller-Auer, and A. B. Sachs, J. Biol. Chem. 271:432-438, 1996), plays an important role in the organization of the actin cytoskeleton in S. cerevisiae. PMID:8756649

  14. 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.

  15. Crystal structure of the lytic CHAPK domain of the endolysin LysK from Staphylococcus aureus bacteriophage K

    PubMed Central

    2014-01-01

    Background Bacteriophages encode endolysins to lyse their host cell and allow escape of their progeny. Endolysins are also active against Gram-positive bacteria when applied from the outside and are thus attractive anti-bacterial agents. LysK, an endolysin from staphylococcal phage K, contains an N-terminal cysteine-histidine dependent amido-hydrolase/peptidase domain (CHAPK), a central amidase domain and a C-terminal SH3b cell wall-binding domain. CHAPK cleaves bacterial peptidoglycan between the tetra-peptide stem and the penta-glycine bridge. Methods The CHAPK domain of LysK was crystallized and high-resolution diffraction data was collected both from a native protein crystal and a methylmercury chloride derivatized crystal. The anomalous signal contained in the derivative data allowed the location of heavy atom sites and phase determination. The resulting structures were completed, refined and analyzed. The presence of calcium and zinc ions in the structure was confirmed by X-ray fluorescence emission spectroscopy. Zymogram analysis was performed on the enzyme and selected site-directed mutants. Results The structure of CHAPK revealed a papain-like topology with a hydrophobic cleft, where the catalytic triad is located. Ordered buffer molecules present in this groove may mimic the peptidoglycan substrate. When compared to previously solved CHAP domains, CHAPK contains an additional lobe in its N-terminal domain, with a structural calcium ion, coordinated by residues Asp45, Asp47, Tyr49, His51 and Asp56. The presence of a zinc ion in the active site was also apparent, coordinated by the catalytic residue Cys54 and a possible substrate analogue. Site-directed mutagenesis was used to demonstrate that residues involved in calcium binding and of the proposed active site were important for enzyme activity. Conclusions The high-resolution structure of the CHAPK domain of LysK was determined, suggesting the location of the active site, the substrate-binding groove and

  16. Spectral Domain Phase Microscopy

    NASA Astrophysics Data System (ADS)

    Hendargo, Hansford C.; Ellerbee, Audrey K.; Izatt, Joseph A.

    Spectral domain phase microscopy (SDPM) is a functional extension of optical coherence tomography (OCT) using common-path interferometry to produce phase-referenced images of dynamic samples. Like OCT, axial resolution in SDPM is determined by the source coherence length, while lateral resolution is limited by diffraction in the microscope optics. However, the quantitative phase information SDPM generates is sensitive to nanometer-scale displacements of scattering structures. The use of a common-path optical geometry yields an imaging system with high phase stability. Due to coherence gating, SDPM can achieve full depth discrimination, allowing for independent motion resolution of subcellular structures throughout the sample volume. Here we review the basic theory of OCT and SDPM along with applications of SDPM in cellular imaging to measure topology, Doppler flow in single-celled organisms, time-resolved motions, rheological information of the cytoskeleton, and optical signaling of neural activation. Phase imaging limitations, artifacts, and sensitivity considerations are discussed.

  17. 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

  18. Adult acute lymphoblastic leukemia with a rare b3a3 type BCR/ABL1 fusion transcript.

    PubMed

    Kurita, Daisuke; Hatta, Yoshihiro; Hojo, Atsuko; Kura, Yoshimasa; Sawada, Umihiko; Kanda, Yoshinobu; Takei, Masami

    2016-04-01

    The Philadelphia chromosome (Ph) is the most frequent chromosomal abnormality detected in adult acute lymphoblastic leukemia (ALL). This chromosome forms the BCR/ABL1 fusion gene; thus, ABL1 exon a2 is generally used as a primer-binding region for the detection of the fusion transcript via reverse transcription polymerase chain reaction (RT-PCR). We observed a rare case of adult Ph-positive (Ph(+)) ALL, in which the BCR/ABL1 fusion transcript was not detected using the ABL1 exon a2 region primer. However, we were able to isolate a PCR product by RT-PCR with the BCR exon 13 (b2) and ABL1 exon a3 primers. Analysis of the sequence of the RT-PCR product revealed that the fusion point was between BCR exon 14 (b3) and ABL1 exon a3, and that the transcript lacked ABL1 exon a2. The patient achieved cytogenetic remission through combination chemotherapies, but relapse occurred before hematopoietic stem cell transplantation and the patient died 11 months after the initialization of chemotherapies. If the BCR/ABL1 fusion transcript is undetected with the ABL1 exon a2 region primer in Ph(+) ALL cases, an RT-PCR analysis that can detect the b3a3 type BCR/ABL1 fusion transcript should be considered to improve diagnosis. PMID:26854094

  19. 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

  20. Imaging coexisting fluid domains in biomembrane models coupling curvature and line tension.

    PubMed

    Baumgart, Tobias; Hess, Samuel T; Webb, Watt W

    2003-10-23

    Lipid bilayer membranes--ubiquitous in biological systems and closely associated with cell function--exhibit rich shape-transition behaviour, including bud formation and vesicle fission. Membranes formed from multiple lipid components can laterally separate into coexisting liquid phases, or domains, with distinct compositions. This process, which may resemble raft formation in cell membranes, has been directly observed in giant unilamellar vesicles. Detailed theoretical frameworks link the elasticity of domains and their boundary properties to the shape adopted by membranes and the formation of particular domain patterns, but it has been difficult to experimentally probe and validate these theories. Here we show that high-resolution fluorescence imaging using two dyes preferentially labelling different fluid phases directly provides a correlation between domain composition and local membrane curvature. Using freely suspended membranes of giant unilamellar vesicles, we are able to optically resolve curvature and line tension interactions of circular, stripe and ring domains. We observe long-range domain ordering in the form of locally parallel stripes and hexagonal arrays of circular domains, curvature-dependent domain sorting, and membrane fission into separate vesicles at domain boundaries. By analysing our observations using available membrane theory, we are able to provide experimental estimates of boundary tension between fluid bilayer domains. PMID:14574408

  1. 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

  2. Modeling Interdependencies between power and economic sectors using the N-ABLE agent-based model.

    SciTech Connect

    Ehlen, Mark Andrew; Scholand, Andrew Joseph

    2005-01-01

    The nation's electric power sector is highly interdependent with the economic sectors it serves; electric power needs are driven by economic activity while the economy itself depends on reliable and sustainable electric power. To advance higher level understandings of the vulnerabilities that result from these interdependencies and to identify the loss prevention and loss mitigation policies that best serve the nation, the National Infrastructure Simulation and Analysis Center is developing and using N-ABLE{trademark}, an agent-based microeconomic framework and simulation tool that models these interdependencies at the level of collections of individual economic firms. Current projects that capture components of these electric power and economic sector interdependencies illustrate some of the public policy issues that should be addressed for combined power sector reliability and national economic security.

  3. 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

  4. Gait phase detection in able-bodied subjects and dementia patients.

    PubMed

    Meng, Xiaoli; Yu, Haoyong; Tham, Ming Po

    2013-01-01

    Accurate detection of gait phases allows identification of specific functional deficits at each phase of the gait cycle for motor function assessment. This paper proposes a robust gait phase detection method to identify the seven gait phases in overground walking for normal and pathologic gaits. Four inertial sensors are used to obtain knee angles, tibia angles and feet angular rate patterns in the sagittal plane. The key events segmenting the gait cycles are searched using an adaptive threshold in adaptive searching intervals to make sure it works well for different subjects with high variation in cadence and step length during walking. The subjects involved in this study are categorized into three groups: five healthy adult subjects, two healthy elderly subjects and two severe dementia patients. The experimental results have shown our method can reliably detect all gait phases for able-bodied subjects and dementia patients without subject-specific calibration.

  5. 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.

  6. 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.

  7. 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.

  8. In vitro screening of antifungal compounds able to counteract biofilm development.

    PubMed

    Girardot, Marion; Imbert, Christine

    2014-01-01

    Fungi are able to grow as a single-species or a more complex biofilm attached to inert surfaces (catheters…) or tissues (lung…). This last form is a microbial niche which must be considered as a major risk factor of developing a human fungal infection. Nowadays, only a few therapeutic agents have been shown to be active against fungal biofilms in vitro and/or in vivo. So there is a real need to find new anti-biofilm molecules. Here we describe in detail some rapid, 96-well microtiter plate-based methods, for the screening of compounds with anti-biofilm activity against Candida spp. yeasts. Two approaches will be considered: prophylactic or curative effects of the tested compounds by producing biofilms on two supports - polystyrene well surfaces and catheter sections. PMID:24664834

  9. Polysaccharides immobilized in polypyrrole matrices are able to induce osteogenic differentiation in mouse mesenchymal stem cells.

    PubMed

    Moreno, Judith Serra; Sabbieti, Maria Giovanna; Agas, Dimitrios; Marchetti, Luigi; Panero, Stefania

    2014-12-01

    Bone marrow mesenchymal stem cells (MSCs) have attracted considerable interest due to their ability to differentiate and contribute to the regeneration of mesenchymal tissues. The present study illustrates that the proper immobilization of heparin (Hep) and hyaluronic acid (HA) into a polypyrrole (PPy) matrix by electropolymerization results in an optimal interface for MSC differentiation towards osteoblast lineage. The obtained thin films showed good thermal stability, hydrophilicity and slow controlled polysaccharide release. The in vitro tests showed the main role of the interface chemical composition. Indeed, PPyHep and PPyHA thin films were able to induce osteogenic differentiation as determined by levels of specific early osteogenic markers (Runx2 and osterix) even in the absence of differentiating medium. Increased levels of ALP and Alizarin red staining, both indicating mineralization processes, confirmed the presence of mature osteoblasts.

  10. 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.

  11. 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

  12. 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.

  13. 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

  14. Colorimetric assessment of BCR-ABL1 transcripts in clinical samples via gold nanoprobes.

    PubMed

    Vinhas, Raquel; Correia, Cláudia; Ribeiro, Patricia; Lourenço, Alexandra; Botelho de Sousa, Aida; Fernandes, Alexandra R; Baptista, Pedro V

    2016-07-01

    Gold nanoparticles functionalized with thiolated oligonucleotides (Au-nanoprobes) have been used in a range of applications for the detection of bioanalytes of interest, from ions to proteins and DNA targets. These detection strategies are based on the unique optical properties of gold nanoparticles, in particular, the intense color that is subject to modulation by modification of the medium dieletric. Au-nanoprobes have been applied for the detection and characterization of specific DNA sequences of interest, namely pathogens and disease biomarkers. Nevertheless, despite its relevance, only a few reports exist on the detection of RNA targets. Among these strategies, the colorimetric detection of DNA has been proven to work for several different targets in controlled samples but demonstration in real clinical bioanalysis has been elusive. Here, we used a colorimetric method based on Au-nanoprobes for the direct detection of the e14a2 BCR-ABL fusion transcript in myeloid leukemia patient samples without the need for retro-transcription. Au-nanoprobes directly assessed total RNA from 38 clinical samples, and results were validated against reverse transcription-nested polymerase chain reaction (RT-nested PCR) and reverse transcription-quantitative polymerase chain reaction (RT-qPCR). The colorimetric Au-nanoprobe assay is a simple yet reliable strategy to scrutinize myeloid leukemia patients at diagnosis and evaluate progression, with obvious advantages in terms of time and cost, particularly in low- to medium-income countries where molecular screening is not routinely feasible. Graphical abstract Gold nanoprobe for colorimetric detection of BCR-ABL1 fusion transcripts originating from the Philadelphia chromosome. PMID:27225178

  15. The Arctic Boundary Layer Expedition (ABLE-3B): July-August 1990

    NASA Astrophysics Data System (ADS)

    Harriss, R. C.; Wofsy, S. C.; Hoell, J. M.; 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.

  16. 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.

  17. 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

  18. 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

  19. 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.

  20. 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.

  1. 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].

  2. How many people are able to control a P300-based brain-computer interface (BCI)?

    PubMed

    Guger, Christoph; Daban, Shahab; Sellers, Eric; Holzner, Clemens; Krausz, Gunther; Carabalona, Roberta; Gramatica, Furio; Edlinger, Guenter

    2009-10-01

    An EEG-based brain-computer system can be used to control external devices such as computers, wheelchairs or Virtual Environments. One of the most important applications is a spelling device to aid severely disabled individuals with communication, for example people disabled by amyotrophic lateral sclerosis (ALS). P300-based BCI systems are optimal for spelling characters with high speed and accuracy, as compared to other BCI paradigms such as motor imagery. In this study, 100 subjects tested a P300-based BCI system to spell a 5-character word with only 5 min of training. EEG data were acquired while the subject looked at a 36-character matrix to spell the word WATER. Two different versions of the P300 speller were used: (i) the row/column speller (RC) that flashes an entire column or row of characters and (ii) a single character speller (SC) that flashes each character individually. The subjects were free to decide which version to test. Nineteen subjects opted to test both versions. The BCI system classifier was trained on the data collected for the word WATER. During the real-time phase of the experiment, the subject spelled the word LUCAS, and was provided with the classifier selection accuracy after each of the five letters. Additionally, subjects filled out a questionnaire about age, sex, education, sleep duration, working duration, cigarette consumption, coffee consumption, and level of disturbance that the flashing characters produced. 72.8% (N=81) of the subjects were able to spell with 100% accuracy in the RC paradigm and 55.3% (N=38) of the subjects spelled with 100% accuracy in the SC paradigm. Less than 3% of the subjects did not spell any character correctly. People who slept less than 8h performed significantly better than other subjects. Sex, education, working duration, and cigarette and coffee consumption were not statistically related to differences in accuracy. The disturbance of the flashing characters was rated with a median score of 1 on a

  3. Acoustic remote sensing of the ABL wind structure in Moscow city

    NASA Astrophysics Data System (ADS)

    Akhiyarova, Ksenia; Lokoshchenko, Mikhail

    2014-05-01

    The dynamics of wind velocity V in the atmospheric boundary layer (ABL) above Moscow city have been analyzed by long-term data of sodar measurements. The Doppler 'MODOS' sodar of METEK firm (Germany) production operates at Moscow University since 2004. Besides, data of two cup anemometers on 7 and 15 m heights inside 'dead zone' of the sodar have been added to analysis. The methodical questions of comparison between in situ and sodar data about V are discussed in details. The profile of wind velocity V in the air layer from 7 to 500 m has been received and analyzed in average of eight last years from 2004 to 2012. In average it is close to logarithmical law up to 60 m so that this value seems to be equal to the surface air layer height. It should be noted that sodar due to its high spatial resolution (20 m) allows studying the ABL fine-structure. Among others, the daily course of V has been investigated in details at different heights. It demonstrates clear daily maximum and nocturnal minimum at any height below 80 m and, vice versa, nocturnal maximum and daily minimum above 140 m everywhere. The air layer from 80 to 140 m represents intermediate zone of smoothed daily course of V. In general this zone corresponds to cross-over height (ideal level where the daily course of wind velocity is absent) but it is noted by important additional feature - minimum in the morning which is statistically significant. Besides, with using of the sodar data it's possible to study mostly interesting weather phenomena such as thunderstorm. Total sampling of this weather event was equal to 137 cases in Moscow from 2004 to 2012. Averaged values both of V, and of its vertical component W have been analyzed during these thunderstorms. As it was shown both V, and W values are increased at the moment of this phenomenon starting. The wind velocity at this moment is in average nearly on 1 m/s higher than three hours before thunderstorm and this increase is statistically significant with 0

  4. Summertime Tropospheric Nonmethane Hydrocarbon and Halocarbon Concentrations Over Central and Eastern Canada during ABLE-3B.

    NASA Astrophysics Data System (ADS)

    Smith, Tyrrel Wayne, Jr.

    Global tropospheric change is now a reality and a focus of international scientific and policy concern. An attempt to understand this tropospheric change was initiated at UCI with the development of a trace gas apparatus to analyze simultaneously nonmethane hydrocarbon (NMHC) and chlorocarbon trace gas species with atmospheric lifetimes ranging from minutes to tens and even hundreds of years. The experimental design of the equipment used to collect, transfer, and analyze ambient air samples is described in detail, and the data obtained show the usefulness of such a system. Approximately 1400 whole air samples were collected on the NASA Electra aircraft and at ground locations and were assayed for selected C_2-C _{10} hydrocarbons and seven halocarbons during the five-week Arctic Boundary Layer Experiment (ABLE-3B) conducted in the eastern Canadian Wetlands. Urban plumes, enhanced in many NMHCs, were identified by their high correlation with elevated levels of perchloroethene (C_2Cl_4). Meteorological isentropic back-trajectories were used in conjunction with NMHC and halocarbon chemical tracers to confirm that the identity of a clean air mass transported to the Canadian Wetlands was attributed to tropical marine air. In more than half of the 46 vertical profiles flown, enhanced NMHC concentrations attributable to plumes from Canadian forest fires were observed. Emission factors relative to ethane were determined for 22 hydrocarbons released from circumpolar biomass burning. Using these data for rm C_2H_6, C_2H _2, C_3H_8, and n- rm C_4H_{10} and CO enhancement estimates from the literature, emissions from biomass burning were calculated to be significant on a global scale. Because of its very short atmospheric lifetime and its below detection background mixing ratio, 1,3-butadiene is an excellent indicator of recent combustion. No statistically significant emissions of N_2O, isoprene (rm C_5H_8), CCl_2F _2 (CFC-12) or any other halocarbon were observed in the

  5. 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

  6. 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

  7. [Disallowed conformations of polypeptide chain exemplified by the β-bend of the β-hairpin in the α-spectrin CH3-domain].

    PubMed

    Uroshlev, L A; Torshin, I Iu; Batianovskiĭ, A V; Esipova, N G; Tumanian, V G

    2015-01-01

    The work presents the results of an exhaustive conformational analysis of β-turns involving amino acid residues with disallowed backbone conformation of the polypeptide chain. It is known that the first residue of the β-turn (Asn47) of the distal β-hairpin in the α-spectrin SH3-domain is characterized by sterically disallowed main chain conformation (values of the dihedral angles (φ and ψ are in the right bottom quadrant of the Ramachandran plot). All α-spectrin structures with the anomalous elements deposited in the PDB were analysed. We hypothesized that the formation of disallowed conformation may occur through the fixation (due to the SH3 domain structure) of the adjacent to the β-turn amino acid residues with the β-structure. These residues are disposed in such a manner that β-turn conformation of the residues contributes just to the disallowed local conformation of this residue whereas any other β-turn conformations (with allowed local conformation) are impossible. To test this hypothesis an exhaustive conformational analysis of the β-bend has been performed by altering internal coordinates (two pairs of φ and ψ angles and two Ω angles). The conformations were selected as a result of grid search procedure with. 1 degrees step that corresponded to stereochemically allowed local deformations of the polypeptide chain segment forming the β-turn. In all conformations obtained the local conformation of Asn47 rests in the disallowed region. The conformations found include conformations coinciding with experimentally determined structures from the PDB as well as an additional variant that differs from X-ray structure in values of a pair of φ and ψ angles of the second residue belonging to the β-bend. Values of these angles fall in the region of the Ramachandran plot near the line φ = 0 (and negative values of ψ) i.e. in strongly disallowed region without experimental points. Therefore the additional variants of the β-turn local deformation are

  8. 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.

  9. 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

  10. Are Plant Species Able to Keep Pace with the Rapidly Changing Climate?

    PubMed Central

    Cunze, Sarah; Heydel, Felix; Tackenberg, Oliver

    2013-01-01

    Future climate change is predicted to advance faster than the postglacial warming. Migration may therefore become a key driver for future development of biodiversity and ecosystem functioning. For 140 European plant species we computed past range shifts since the last glacial maximum and future range shifts for a variety of Intergovernmental Panel on Climate Change (IPCC) scenarios and global circulation models (GCMs). Range shift rates were estimated by means of species distribution modelling (SDM). With process-based seed dispersal models we estimated species-specific migration rates for 27 dispersal modes addressing dispersal by wind (anemochory) for different wind conditions, as well as dispersal by mammals (dispersal on animal's coat – epizoochory and dispersal by animals after feeding and digestion – endozoochory) considering different animal species. Our process-based modelled migration rates generally exceeded the postglacial range shift rates indicating that the process-based models we used are capable of predicting migration rates that are in accordance with realized past migration. For most of the considered species, the modelled migration rates were considerably lower than the expected future climate change induced range shift rates. This implies that most plant species will not entirely be able to follow future climate-change-induced range shifts due to dispersal limitation. Animals with large day- and home-ranges are highly important for achieving high migration rates for many plant species, whereas anemochory is relevant for only few species. PMID:23894290

  11. Cattle egrets are less able to cope with light refraction than are other herons.

    PubMed

    Katzir; Strod; Schechtman; Hareli; Arad

    1999-03-01

    The majority of heron species (Aves, Ardeidae) forage on aquatic prey in shallow water. Prey detection, aiming and the beginning of the capture strikes are performed while the heron's eyes are above water. For most angles, as a result of air/water light refraction, the apparent image available to a heron is vertically displaced from the prey's real position. Herons must therefore correct for refraction. We tested the hypothesis that species that forage in aquatic habitats should be more able to correct for image disparity than those of terrestrial habitats. The ability of hand-reared herons of four species to capture stationary prey (fish) underwater (submerged) or in air (aerial) was tested. Three species (little egret Egretta garzetta, squacco heron Ardeola ralloides, and night heron Nycticorax nycticorax) normally forage in aquatic habitats while the fourth (cattle egret Bubulcus ibis) forages in terrestrial habitats. No individuals missed aerial prey. Success rates of little egrets and of squacco herons with submerged prey were high, while night herons became less successful with increased prey depth and/or distance. In cattle egrets, success rate was low and negatively correlated with prey depth. The observed interspecific differences may thus be related to (1) differential ability to correct for air/water light refraction and (2) the species' foraging behaviour. We suggest that cattle egrets are in the process of losing their ability to cope with submerged prey. Copyright 1999 The Association for the Study of Animal Behaviour.

  12. Are biomarkers able to determine the holistic integrity of ecosystem health?

    SciTech Connect

    Weeks, J.M.

    1995-12-31

    The term ecosystem health has been adopted with much fervor by ecotoxicologists in recent years. Possibly because it offers an all encompassing statement about ones apparent environment. However, the term may best be described as a misalliance, only in so much as that in order for it to exist one must also have ecosystem unhealth. In recent years the use of biomarkers (perhaps another term fit for a semantic discussion) to describe the effects of toxicants and chemicals at different levels of biological organization ranging from biochemical reactions, sub-cellular components, cells, tissues, organs, physiological levels, individuals and groups of individuals has been attempted. However, very few biomarkers could possibly hope to encompass the temporal and spatial variability and scale or the combination of biotic and abiotic influences constituting a healthy ecosystem. So what values or attributes do biomarkers have that lend them to the task of recognizing unhealthy systems. If the authors consider the holistic approach to ecosystem health then biomarkers fall at the first hurdle, having never been truly tested at a level greater than the individual. So do biomarkers play a role at the complex system level or should one restrict their use purely to simple screening and early warning at the lower levels of biological and ecological organization such that one is able to infer effects at the higher levels? The following presents arguments both for and against the routine use of biomarkers for assessing ecosystem health.

  13. 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

  14. 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

  15. Research note: exceptional absolute pitch perception for spoken words in an able adult with autism.

    PubMed

    Heaton, Pamela; Davis, Robert E; Happé, Francesca G E

    2008-01-01

    Autism is a neurodevelopmental disorder, characterised by deficits in socialisation and communication, with repetitive and stereotyped behaviours [American Psychiatric Association (1994). Diagnostic and statistical manual for mental disorders (4th ed.). Washington, DC: APA]. Whilst intellectual and language impairment is observed in a significant proportion of diagnosed individuals [Gillberg, C., & Coleman, M. (2000). The biology of the autistic syndromes (3rd ed.). London: Mac Keith Press; Klinger, L., Dawson, G., & Renner, P. (2002). Autistic disorder. In E. Masn, & R. Barkley (Eds.), Child pyschopathology (2nd ed., pp. 409-454). New York: Guildford Press], the disorder is also strongly associated with the presence of highly developed, idiosyncratic, or savant skills [Heaton, P., & Wallace, G. (2004) Annotation: The savant syndrome. Journal of Child Psychology and Psychiatry, 45 (5), 899-911]. We tested identification of fundamental pitch frequencies in complex tones, sine tones and words in AC, an intellectually able man with autism and absolute pitch (AP) and a group of healthy controls with self-reported AP. The analysis showed that AC's naming of speech pitch was highly superior in comparison to controls. The results suggest that explicit access to perceptual information in speech is retained to a significantly higher degree in autism.

  16. 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.

  17. 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

  18. Genetic characterization of fast-growing rhizobia able to nodulate Prosopis alba in North Spain.

    PubMed

    Iglesias, Olga; Rivas, Raúl; García-Fraile, Paula; Abril, Adriana; Mateos, Pedro F; Martinez-Molina, Eustoquio; Velázquez, Encarna

    2007-12-01

    Prosopis is a Mimosaceae legume tree indigenous to South America and not naturalized in Europe. In this work 18 rhizobial strains nodulating Prosopis alba roots were isolated from a soil in North Spain that belong to eight different randomly amplified polymorphic DNA groups phylogenetically related to Sinorhizobium medicae, Sinorhizobium meliloti and Rhizobium giardinii according to their intergenic spacer and 16S rRNA gene sequences. The nodC genes of isolates close to S. medicae and S. meliloti were identical to those of S. medicae USDA 1,037(T) and S. meliloti LMG 6,133(T) and accordingly all these strains were able to nodulate both alfalfa and Prosopis. These nodC genes were phylogenetically divergent from those of the isolates close to R. giardinii that were identical to that of R. giardinii H152(T) and therefore all these strains formed nodules in common beans and Prosopis. The nodC genes of the strains isolated in Spain were phylogenetically divergent from that carried by Mesorhizobium chacoense Pr-5(T) and Sinorhizobium arboris LMG 1,4919(T) nodulating Prosopis in America and Africa, respectively. Therefore, Prosopis is a promiscuous host which can establish symbiosis with strains carrying very divergent nodC genes and this promiscuity may be an important advantage for this legume tree to be used in reforestation.

  19. Overcoming drug resistance with alginate oligosaccharides able to potentiate the action of selected antibiotics.

    PubMed

    Khan, Saira; Tøndervik, Anne; Sletta, Håvard; Klinkenberg, Geir; Emanuel, Charlotte; Onsøyen, Edvar; Myrvold, Rolf; Howe, Robin A; Walsh, Timothy R; Hill, Katja E; Thomas, David W

    2012-10-01

    The uncontrolled, often inappropriate use of antibiotics has resulted in the increasing prevalence of antibiotic-resistant pathogens, with major cost implications for both United States and European health care systems. We describe the utilization of a low-molecular-weight oligosaccharide nanomedicine (OligoG), based on the biopolymer alginate, which is able to perturb multidrug-resistant (MDR) bacteria by modulating biofilm formation and persistence and reducing resistance to antibiotic treatment, as evident using conventional and robotic MIC screening and microscopic analyses of biofilm structure. OligoG increased (up to 512-fold) the efficacy of conventional antibiotics against important MDR pathogens, including Pseudomonas, Acinetobacter, and Burkholderia spp., appearing to be effective with several classes of antibiotic (i.e., macrolides, β-lactams, and tetracyclines). Using confocal laser scanning microscopy (CLSM) and scanning electron microscopy (SEM), increasing concentrations (2%, 6%, and 10%) of alginate oligomer were shown to have a direct effect on the quality of the biofilms produced and on the health of the cells within that biofilm. Biofilm growth was visibly weakened in the presence of 10% OligoG, as seen by decreased biomass and increased intercellular spaces, with the bacterial cells themselves becoming distorted and uneven due to apparently damaged cell membranes. This report demonstrates the feasibility of reducing the tolerance of wound biofilms to antibiotics with the use of specific alginate preparations.

  20. 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

  1. 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

  2. Hydrophobic Compounds Reshape Membrane Domains

    PubMed Central

    Barnoud, Jonathan; Rossi, Giulia; Marrink, Siewert J.; Monticelli, Luca

    2014-01-01

    Cell membranes have a complex lateral organization featuring domains with distinct composition, also known as rafts, which play an essential role in cellular processes such as signal transduction and protein trafficking. In vivo, perturbations of membrane domains (e.g., by drugs or lipophilic compounds) have major effects on the activity of raft-associated proteins and on signaling pathways, but they are difficult to characterize because of the small size of the domains, typically below optical resolution. Model membranes, instead, can show macroscopic phase separation between liquid-ordered and liquid-disordered domains, and they are often used to investigate the driving forces of membrane lateral organization. Studies in model membranes have shown that some lipophilic compounds perturb membrane domains, but it is not clear which chemical and physical properties determine domain perturbation. The mechanisms of domain stabilization and destabilization are also unknown. Here we describe the effect of six simple hydrophobic compounds on the lateral organization of phase-separated model membranes consisting of saturated and unsaturated phospholipids and cholesterol. Using molecular simulations, we identify two groups of molecules with distinct behavior: aliphatic compounds promote lipid mixing by distributing at the interface between liquid-ordered and liquid-disordered domains; aromatic compounds, instead, stabilize phase separation by partitioning into liquid-disordered domains and excluding cholesterol from the disordered domains. We predict that relatively small concentrations of hydrophobic species can have a broad impact on domain stability in model systems, which suggests possible mechanisms of action for hydrophobic compounds in vivo. PMID:25299598

  3. 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.

  4. 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

  5. On Probability Domains

    NASA Astrophysics Data System (ADS)

    Frič, Roman; Papčo, Martin

    2010-12-01

    Motivated by IF-probability theory (intuitionistic fuzzy), we study n-component probability domains in which each event represents a body of competing components and the range of a state represents a simplex S n of n-tuples of possible rewards-the sum of the rewards is a number from [0,1]. For n=1 we get fuzzy events, for example a bold algebra, and the corresponding fuzzy probability theory can be developed within the category ID of D-posets (equivalently effect algebras) of fuzzy sets and sequentially continuous D-homomorphisms. For n=2 we get IF-events, i.e., pairs ( μ, ν) of fuzzy sets μ, ν∈[0,1] X such that μ( x)+ ν( x)≤1 for all x∈ X, but we order our pairs (events) coordinatewise. Hence the structure of IF-events (where ( μ 1, ν 1)≤( μ 2, ν 2) whenever μ 1≤ μ 2 and ν 2≤ ν 1) is different and, consequently, the resulting IF-probability theory models a different principle. The category ID is cogenerated by I=[0,1] (objects of ID are subobjects of powers I X ), has nice properties and basic probabilistic notions and constructions are categorical. For example, states are morphisms. We introduce the category S n D cogenerated by Sn=\\{(x1,x2,ldots ,xn)in In;sum_{i=1}nxi≤ 1\\} carrying the coordinatewise partial order, difference, and sequential convergence and we show how basic probability notions can be defined within S n D.

  6. Fractional diffusion on bounded domains

    SciTech Connect

    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.

  7. Nucleophosmin contains amyloidogenic regions that are able to form toxic aggregates under physiological conditions.

    PubMed

    Di Natale, Concetta; Scognamiglio, Pasqualina Liana; Cascella, Roberta; Cecchi, Cristina; Russo, Anna; Leone, Marilisa; Penco, Amanda; Relini, Annalisa; Federici, Luca; Di Matteo, Adele; Chiti, Fabrizio; Vitagliano, Luigi; Marasco, Daniela

    2015-09-01

    Nucleophosmin (NPM)-1 is a multifunctional protein involved in a variety of biologic processes and has been implicated in the pathogenesis of several human malignancies. To gain insight into the role of isolated fragments in NPM1 activities, we dissected the C-terminal domain (CTD) into its helical fragments. In this study, we observed the unexpected structural behavior of the peptide fragment corresponding to helix (H)2 (residues 264-277). This peptide has a strong tendency to form amyloidlike assemblies endowed with fibrillar morphology and β-sheet structure, under physiologic conditions, as shown by circular dichroism, thioflavin T, and Congo red binding assays; dynamic light scattering; and atomic force microscopy. The aggregates are also toxic to neuroblastoma cells, as determined using 3-(4;5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide reduction and Ca(2+) influx assays. We also found that the extension of the H2 sequence beyond its N terminus, comprising the connecting loop with H1, delayed aggregation and its associated cytotoxicity, suggesting that contiguous regions of H2 have a protective role in preventing aggregation. Our findings and those in the literature suggest that the helical structures present in the CTD are important in preventing harmful aggregation. These findings could elucidate the pathogenesis of acute myeloid leukemia (AML) caused by NPM1 mutants. Because the CTD is not properly folded in these mutants, we hypothesize that the aggregation propensity of this NPM1 region is involved in the pathogenesis of AML. Preliminary assays on NPM1-Cter-MutA, the most frequent AML-CTD mutation, revealed its significant propensity for aggregation. Thus, the aggregation phenomena should be seriously considered in studies aimed at unveiling the molecular mechanisms of this pathology.

  8. 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…

  9. Characterization of Amyloid Cores in Prion Domains

    PubMed Central

    Sant’Anna, Ricardo; Fernández, Maria Rosario; Batlle, Cristina; Navarro, Susanna; de Groot, Natalia S.; Serpell, Louise; Ventura, Salvador

    2016-01-01

    Amyloids consist of repetitions of a specific polypeptide chain in a regular cross-β-sheet conformation. Amyloid propensity is largely determined by the protein sequence, the aggregation process being nucleated by specific and short segments. Prions are special amyloids that become self-perpetuating after aggregation. Prions are responsible for neuropathology in mammals, but they can also be functional, as in yeast prions. The conversion of these last proteins to the prion state is driven by prion forming domains (PFDs), which are generally large, intrinsically disordered, enriched in glutamines/asparagines and depleted in hydrophobic residues. The self-assembly of PFDs has been thought to rely mostly on their particular amino acid composition, rather than on their sequence. Instead, we have recently proposed that specific amyloid-prone sequences within PFDs might be key to their prion behaviour. Here, we demonstrate experimentally the existence of these amyloid stretches inside the PFDs of the canonical Sup35, Swi1, Mot3 and Ure2 prions. These sequences self-assemble efficiently into highly ordered amyloid fibrils, that are functionally competent, being able to promote the PFD amyloid conversion in vitro and in vivo. Computational analyses indicate that these kind of amyloid stretches may act as typical nucleating signals in a number of different prion domains. PMID:27686217

  10. 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

  11. Synthesis and characterization of image-able polyvinyl alcohol microspheres for image-guided chemoembolization.

    PubMed

    Negussie, Ayele H; Dreher, Matthew R; Johnson, Carmen Gacchina; Tang, Yiqing; Lewis, Andrew L; Storm, Gert; Sharma, Karun V; Wood, Bradford J

    2015-06-01

    Therapeutic embolization of blood vessels is a minimally invasive, catheter-based procedure performed with solid or liquid emboli to treat bleeding, vascular malformations, and vascular tumors. Hepatocellular carcinoma (HCC) affects about half a million people per year. When unresectable, HCC is treated with embolization and local drug therapy by transarterial chemoembolization (TACE). For TACE, drug eluting beads (DC Bead(®)) may be used to occlude or reduce arterial blood supply and deliver chemotherapeutics locally to the tumor. Although this treatment has been shown to be safe and to improve patient survival, the procedure lacks imaging feedback regarding the location of embolic agent and drug coverage. To address this shortcoming, herein we report the synthesis and characterization of image-able drug eluting beads (iBeads) from the commercial DC Bead(®) product. Two different radiopaque beads were synthesized. In one approach, embolic beads were conjugated with 2,3,5-triiodobenzyl alcohol in the presence of 1,1'-carbonyldiimidazol to give iBead I. iBead II was synthesized with a similar approach but instead using a trimethylenediamine spacer and 2,3,5-triiodobenzoic acid. Doxorubicin was loaded into the iBeads II using a previously reported method. Size and shape of iBeads were evaluated using an upright microscope and their conspicuity assessed using a clinical CT and micro-CT. Bland and Dox-loaded iBeads II visualized with both clinical CT and microCT. Under microCT, individual bland and Dox loaded beads had a mean attenuation of 7904 ± 804 and 11,873.96 ± 706.12 HU, respectively. These iBeads have the potential to enhance image-guided TACE procedures by providing localization of embolic-particle and drug. PMID:26105830

  12. Postural stability and history of falls in cognitively able older adults: the Canton Ticino study.

    PubMed

    Merlo, Andrea; Zemp, Damiano; Zanda, Enrica; Rocchi, Sabrina; Meroni, Fabiano; Tettamanti, Mauro; Recchia, Angela; Lucca, Ugo; Quadri, Pierluigi

    2012-09-01

    Falls are common events in the elderly and represent the main risk factor for fractures and other injuries. Strategies for fall prevention rely on the multifactorial assessment of the risk of falling. The contribution of instrumented balance assessment to the prediction of falls remains unclear in the literature. In this study, we analyzed the association between the fall-history of a wide sample of older people without dementia and the values of a set of posturographic parameters acquired in different visual, proprioceptive and mental conditions. A consecutive sample of 130 cognitively able elderly subjects, age≥70 years, was analyzed. Based on their fall-history in the last year, subjects were categorized into non-fallers (NF), fallers (F) and recurrent fallers (RF>2 falls). Each subject was assessed by measurements of cognition and functional ability. Static posturography tests were performed in five conditions: with eyes open/close (EO/EC) on a firm/compliant (FS/CS) surface and while performing a cognitive task. The center of pressure (COP) mean position referred to the mid-point of the heels, area of the 95% confidence ellipse, sway mean velocities and RMS displacements in the antero-posterior (AP) and medio-lateral (ML) directions were computed and their association with the fall-history was assessed. The mean position of the COP in the AP direction and the confidence ellipse area were associated with the fall-history in the EOFS, ECFS and EOCS conditions (P<0.05). RMS displacements were also associated with the fall-history in the EOCS condition (P<0.05). Significant group differences (P<0.05) were found in the EOCS conditions, which greatly enhanced the differences among NF, F and RF. The ability to control balance while standing with eyes open on a compliant surface showed a high degree of association with the fall-history of older people with no or mild cognitive impairment.

  13. 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

  14. 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.

  15. 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

  16. 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

  17. A meteorological interpretation of the Arctic Boundary Layer Expedition (ABLE) 3B flight series

    NASA Technical Reports Server (NTRS)

    Shipham, Mark C.; Bachmeier, A. Scott; Cahoon, Donald R., Jr.; Gregory, Gerald L.; Anderson, Bruce E.; Browell, Edward V.

    1994-01-01

    The Arctic Boundary Layer Expedition (ABLE) 3B was conducted to determine the summertime tropospheric distribution, sources, and sinks of important trace gas and aerosol species over the wetlands and boreal forests of central and eastern Canada. Isentropic trajectories and analyzed midtropospheric circulation patterns were used to group flights according to the transport histories of polar, midlatitude, or tropical air masses which were sampled. These data were then divided into bands of potential temperature levels representing the low, middle, and maximum aircraft altitudes to assess the effects of both local and long distance transport and natural and man-made pollutants to the measured chemical species. Detailed case studies are provided to depict the complex three-dimensional airflow regimes that transported air with differing chemical signatures to the study area. Mission 6 details the large-scale movement of smoke in the generally prevailing west to northwesterly airflow that was observed on the majority of flights. Mission 1 analyzes the horizontal and vertical motions of maritime Pacific air in the upper troposphere that was routinely mixed downward to the aircraft altitude. Finally, mission 14 tracks the far northward excursion of tropical air that had been associated with a Pacific typhoon. The following three factors all had important influences on the collected chemical data sets: (1) local and distant stratospheric in puts into the upper and middle troposphere; (2) biomass-burning plumes from active fires in Alaska and Canada; (3) a band of 'low ozone' upper tropospheric air that was observed by airborne differential absorption lidar (DIAL) above the aircraft maximum altitude. Other modification factors observed on some flights included urban pollution from U.S. and Canadian cities, tropical air that had been associated with a Pacific typhoon, and precipitation scavenging by clouds and rain. Many flights were affected by several of the above factors

  18. Diversity in protein domain superfamilies

    PubMed Central

    Das, Sayoni; Dawson, Natalie L; Orengo, Christine A

    2015-01-01

    Whilst ∼93% of domain superfamilies appear to be relatively structurally and functionally conserved based on the available data from the CATH-Gene3D domain classification resource, the remainder are much more diverse. In this review, we consider how domains in some of the most ubiquitous and promiscuous superfamilies have evolved, in particular the plasticity in their functional sites and surfaces which expands the repertoire of molecules they interact with and actions performed on them. To what extent can we identify a core function for these superfamilies which would allow us to develop a ‘domain grammar of function’ whereby a protein's biological role can be proposed from its constituent domains? Clearly the first step is to understand the extent to which these components vary and how changes in their molecular make-up modifies function. PMID:26451979

  19. SMART, a simple modular architecture research tool: identification of signaling domains.

    PubMed

    Schultz, J; Milpetz, F; Bork, P; Ponting, C P

    1998-05-26

    Accurate multiple alignments of 86 domains that occur in signaling proteins have been constructed and used to provide a Web-based tool (SMART: simple modular architecture research tool) that allows rapid identification and annotation of signaling domain sequences. The majority of signaling proteins are multidomain in character with a considerable variety of domain combinations known. Comparison with established databases showed that 25% of our domain set could not be deduced from SwissProt and 41% could not be annotated by Pfam. SMART is able to determine the modular architectures of single sequences or genomes; application to the entire yeast genome revealed that at least 6.7% of its genes contain one or more signaling domains, approximately 350 greater than previously annotated. The process of constructing SMART predicted (i) novel domain homologues in unexpected locations such as band 4.1-homologous domains in focal adhesion kinases; (ii) previously unknown domain families, including a citron-homology domain; (iii) putative functions of domain families after identification of additional family members, for example, a ubiquitin-binding role for ubiquitin-associated domains (UBA); (iv) cellular roles for proteins, such predicted DEATH domains in netrin receptors further implicating these molecules in axonal guidance; (v) signaling domains in known disease genes such as SPRY domains in both marenostrin/pyrin and Midline 1; (vi) domains in unexpected phylogenetic contexts such as diacylglycerol kinase homologues in yeast and bacteria; and (vii) likely protein misclassifications exemplified by a predicted pleckstrin homology domain in a Candida albicans protein, previously described as an integrin.

  20. 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

  1. 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.

  2. 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

  3. Separated matter and antimatter domains with vanishing domain walls

    SciTech Connect

    Dolgov, A.D.; Godunov, S.I.; Rudenko, A.S.; Tkachev, I.I. E-mail: sgodunov@itep.ru E-mail: tkachev@ms2.inr.ac.ru

    2015-10-01

    We present a model of spontaneous (or dynamical) C and CP violation where it is possible to generate domains of matter and antimatter separated by cosmologically large distances. Such C(CP) violation existed only in the early universe and later it disappeared with the only trace of generated baryonic and/or antibaryonic domains. So the problem of domain walls in this model does not exist. These features are achieved through a postulated form of interaction between inflaton and a new scalar field, realizing short time C(CP) violation.

  4. The Tail Domain Is Essential but the Head Domain Dispensable for C. elegans Intermediate Filament IFA-2 Function

    PubMed Central

    Williams, Kyle; Williams, Kristen; Baucher, Hallie M.; Plenefisch, John

    2015-01-01

    The intermediate filament protein IFA-2 is essential for the structural integrity of the Caenorhabditis elegans epidermis. It is one of the major components of the fibrous organelle, an epidermal structure comprised of apical and basal hemidesmosomes linked by cytoplasmic intermediate filaments that serve to transmit force from the muscle to the cuticle. Mutations of IFA-2 result in epidermal fragility and separation of the apical and basal epidermal surfaces during postembryonic growth. An IFA-2 lacking the head domain fully rescues the IFA-2 null mutant, whereas an IFA-2 lacking the tail domain cannot. Conversely, an isolated IFA-2 head was able to localize to fibrous organelles whereas the tail was not. Taken together these results suggest that the head domain contains redundant signals for IF localization, whereas non-redundant essential functions map to the IFA-2, tail, although the tail is unlikely to be directly involved in fibrous organelle localization. PMID:25742641

  5. Comparative quantitative analysis of BCR-ABL transcripts with the T315I mutant clone by polymerase chain reaction (PCR)-Invader method.

    PubMed

    Tadokoro, Kenichi; Ishikawa, Maho; Suzuki, Makoto; Saito, Tomoyoshi; Suzuki, Yoshie; Yamaguchi, Toshikazu; Yagasaki, Fumiharu

    2011-09-01

    Drug resistance is a serious complication in the treatment of chronic myeloid leukemia (CML). The most common and best-characterized mechanism of secondary imatinib resistance in CML is the development of kinase domain mutations in the BCR-ABL gene. Second-generation tyrosine kinase inhibitors, such as dasatinib or nilotinib, overcome most of these mutations, but they are not effective against the T315I mutant. To determine whether these mutations contribute to clinical resistance, it is necessary to monitor the ratio of the mutant and wild-type forms. Here, we developed a polymerase chain reaction (PCR)-Invader assay for comparative quantitative analysis (qPI assay) of BCR-ABL transcripts with the T315I mutant clone. T315I ratios were calculated for the wild-type and mutant fold-over-zero (FOZ) values. In examination with 2 kinds of plasmids containing wild-type or T315I mutant PCR amplicons, mutant FOZ values were detected down to 1% of the total. The results of 12 serial samples from 2 patients (case A: Philadelphia-positive acute lymphoblastic leukemia and case B: CML) with the T315I mutant clone were compared with those of direct sequencing or 2 kinds of allele-specific oligonucleotide (ASO)-PCR. All samples showed the T315I mutation by qPI assay and ASO-PCR, and 10 samples showed it by direct sequencing. Significant correlation (correlation coefficient; r2 = 0.951) was noted between the qPI assay and quantitative ASO-PCR to analyze T315I mutant ratios. Thus, the qPI assay is a useful method for evaluating the T315I mutant clone in BCR-ABL transcripts.

  6. Comparative quantitative analysis of BCR-ABL transcripts with the T315I mutant clone by polymerase chain reaction (PCR)-Invader method.

    PubMed

    Tadokoro, Kenichi; Ishikawa, Maho; Suzuki, Makoto; Saito, Tomoyoshi; Suzuki, Yoshie; Yamaguchi, Toshikazu; Yagasaki, Fumiharu

    2011-09-01

    Drug resistance is a serious complication in the treatment of chronic myeloid leukemia (CML). The most common and best-characterized mechanism of secondary imatinib resistance in CML is the development of kinase domain mutations in the BCR-ABL gene. Second-generation tyrosine kinase inhibitors, such as dasatinib or nilotinib, overcome most of these mutations, but they are not effective against the T315I mutant. To determine whether these mutations contribute to clinical resistance, it is necessary to monitor the ratio of the mutant and wild-type forms. Here, we developed a polymerase chain reaction (PCR)-Invader assay for comparative quantitative analysis (qPI assay) of BCR-ABL transcripts with the T315I mutant clone. T315I ratios were calculated for the wild-type and mutant fold-over-zero (FOZ) values. In examination with 2 kinds of plasmids containing wild-type or T315I mutant PCR amplicons, mutant FOZ values were detected down to 1% of the total. The results of 12 serial samples from 2 patients (case A: Philadelphia-positive acute lymphoblastic leukemia and case B: CML) with the T315I mutant clone were compared with those of direct sequencing or 2 kinds of allele-specific oligonucleotide (ASO)-PCR. All samples showed the T315I mutation by qPI assay and ASO-PCR, and 10 samples showed it by direct sequencing. Significant correlation (correlation coefficient; r2 = 0.951) was noted between the qPI assay and quantitative ASO-PCR to analyze T315I mutant ratios. Thus, the qPI assay is a useful method for evaluating the T315I mutant clone in BCR-ABL transcripts. PMID:21867983

  7. 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

  8. 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 (

  9. Weighted mutual information analysis substantially improves domain-based functional network models

    PubMed Central

    Shim, Jung Eun; Lee, Insuk

    2016-01-01

    Motivation: Functional protein–protein interaction (PPI) networks elucidate molecular pathways underlying complex phenotypes, including those of human diseases. Extrapolation of domain–domain interactions (DDIs) from known PPIs is a major domain-based method for inferring functional PPI networks. However, the protein domain is a functional unit of the protein. Therefore, we should be able to effectively infer functional interactions between proteins based on the co-occurrence of domains. Results: Here, we present a method for inferring accurate functional PPIs based on the similarity of domain composition between proteins by weighted mutual information (MI) that assigned different weights to the domains based on their genome-wide frequencies. Weighted MI outperforms other domain-based network inference methods and is highly predictive for pathways as well as phenotypes. A genome-scale human functional network determined by our method reveals numerous communities that are significantly associated with known pathways and diseases. Domain-based functional networks may, therefore, have potential applications in mapping domain-to-pathway or domain-to-phenotype associations. Availability and Implementation: Source code for calculating weighted mutual information based on the domain profile matrix is available from www.netbiolab.org/w/WMI. Contact: Insuklee@yonsei.ac.kr Supplementary information: Supplementary data are available at Bioinformatics online. PMID:27207946

  10. Modeling software systems by domains

    NASA Technical Reports Server (NTRS)

    Dippolito, Richard; Lee, Kenneth

    1992-01-01

    The Software Architectures Engineering (SAE) Project at the Software Engineering Institute (SEI) has developed engineering modeling techniques that both reduce the complexity of software for domain-specific computer systems and result in systems that are easier to build and maintain. These techniques allow maximum freedom for system developers to apply their domain expertise to software. We have applied these techniques to several types of applications, including training simulators operating in real time, engineering simulators operating in non-real time, and real-time embedded computer systems. Our modeling techniques result in software that mirrors both the complexity of the application and the domain knowledge requirements. We submit that the proper measure of software complexity reflects neither the number of software component units nor the code count, but the locus of and amount of domain knowledge. As a result of using these techniques, domain knowledge is isolated by fields of engineering expertise and removed from the concern of the software engineer. In this paper, we will describe kinds of domain expertise, describe engineering by domains, and provide relevant examples of software developed for simulator applications using the techniques.

  11. The protein kinase C-responsive inhibitory domain of CARD11 functions in NF-kappaB activation to regulate the association of multiple signaling cofactors that differentially depend on Bcl10 and MALT1 for association.

    PubMed

    McCully, Ryan R; Pomerantz, Joel L

    2008-09-01

    The activation of NF-kappaB by T-cell receptor (TCR) signaling is critical for T-cell activation during the adaptive immune response. CARD11 is a multidomain adapter that is required for TCR signaling to the IkappaB kinase (IKK) complex. During TCR signaling, the region in CARD11 between the coiled-coil and PDZ domains is phosphorylated by protein kinase Ctheta (PKCtheta) in a required step in NF-kappaB activation. In this report, we demonstrate that this region functions as an inhibitory domain (ID) that controls the association of CARD11 with multiple signaling cofactors, including Bcl10, TRAF6, TAK1, IKKgamma, and caspase-8, through an interaction that requires both the caspase recruitment domain (CARD) and the coiled-coil domain. Consistent with the ID-mediated control of their association, we demonstrate that TRAF6 and caspase-8 associate with CARD11 in T cells in a signal-inducible manner. Using an RNA interference rescue assay, we demonstrate that the CARD, linker 1, coiled-coil, linker 3, SH3, linker 4, and GUK domains are each required for TCR signaling to NF-kappaB downstream of ID neutralization. Requirements for the CARD, linker 1, and coiled-coil domains in signaling are consistent with their roles in the association of CARD11 with Bcl10, TRAF6, TAK1, caspase-8, and IKKgamma. Using Bcl10- and MALT1-deficient cells, we show that CARD11 can recruit signaling cofactors independently of one another in a signal-inducible manner.

  12. What Researchers Should Know and be Able to do When Contemplating Involvement in Education and Outreach

    NASA Astrophysics Data System (ADS)

    Ridky, R. W.

    2004-12-01

    At some point in their careers, many researchers are motivated to share what they have learned with a wider audience. As their studies mature, and national awareness for more effective integration of research and education intensifies, researchers are increasingly directing efforts toward informal and pre-college educational sectors. Each initiative comes with good intentions, but many fall short of intended benefit. Quality education and outreach programs develop from the same precepts that shape research programs of high professional standing. A researcher is most likely to make useful contributions when they are willing and able to implement familiar research principles to broader educational endeavors. As with research endeavors, principles of significance, literacy, design, feasibility, analysis and dissemination need to be regarded as essential indicators of education program quality. It is helpful to provide researchers who are contemplating more active educational involvement with more than casual understanding of the purposes underlying their pending contributions. Such understanding is premised on the tenet that education and research are always in the public service and therefore inextricably bound at all levels. Both research and education have, as their ultimate goal, enhanced scientific literacy of the citizenry. By example, it can be shown that the best-supported programs, within government and academia, recognize that the way they translate knowledge and make it available to scientific organizations and the public is critical to their intrinsic societal value and level of support. As education conjures up a host of operational meanings arising from one's own values and experiences, the knowledge researchers bring to pre-college and informal educational settings is often based on personal experience rather than on education research, practice and policy. Researchers may believe that because they spent 13 years in school, an additional 4 years at a

  13. Archaeal flagellin combines a bacterial type IV pilin domain with an Ig-like domain.

    PubMed

    Braun, Tatjana; Vos, Matthijn R; Kalisman, Nir; Sherman, Nicholas E; Rachel, Reinhard; Wirth, Reinhard; Schröder, Gunnar F; Egelman, Edward H

    2016-09-13

    The bacterial flagellar apparatus, which involves ∼40 different proteins, has been a model system for understanding motility and chemotaxis. The bacterial flagellar filament, largely composed of a single protein, flagellin, has been a model for understanding protein assembly. This system has no homology to the eukaryotic flagellum, in which the filament alone, composed of a microtubule-based axoneme, contains more than 400 different proteins. The archaeal flagellar system is simpler still, in some cases having ∼13 different proteins with a single flagellar filament protein. The archaeal flagellar system has no homology to the bacterial one and must have arisen by convergent evolution. However, it has been understood that the N-terminal domain of the archaeal flagellin is a homolog of the N-terminal domain of bacterial type IV pilin, showing once again how proteins can be repurposed in evolution for different functions. Using cryo-EM, we have been able to generate a nearly complete atomic model for a flagellar-like filament of the archaeon Ignicoccus hospitalis from a reconstruction at ∼4-Å resolution. We can now show that the archaeal flagellar filament contains a β-sandwich, previously seen in the FlaF protein that forms the anchor for the archaeal flagellar filament. In contrast to the bacterial flagellar filament, where the outer globular domains make no contact with each other and are not necessary for either assembly or motility, the archaeal flagellin outer domains make extensive contacts with each other that largely determine the interesting mechanical properties of these filaments, allowing these filaments to flex. PMID:27578865

  14. MicroRNA-320a acts as a tumor suppressor by targeting BCR/ABL oncogene in chronic myeloid leukemia.

    PubMed

    Xishan, Zhu; Ziying, Lin; Jing, Du; Gang, Liu

    2015-07-31

    Accumulating evidences demonstrated that the induction of epithelial-mesenchymal transition (EMT) and aberrant expression of microRNAs (miRNAs) are associated with tumorigenesis, tumor progression, metastasis and relapse in cancers, including chronic myeloid leukemia (CML). We found that miR-320a expression was reduced in K562 and in CML cancer stem cells. Moreover, we found that miR-320a inhibited K562 cell migration, invasion, proliferation and promoted apoptosis by targeting BCR/ABL oncogene. As an upstream regulator of BCR/ABL, miR-320a directly targets BCR/ABL. The enhanced expression of miR-320a inhibited the phosphorylation of PI3K, AKT and NF-κB; however, the expression of phosphorylated PI3K, AKT and NF-κB were restored by the overexpression of BCR/ABL. In K562, infected with miR-320a or transfected with SiBCR/ABL, the protein levels of fibronectin, vimentin, and N-cadherin were decreased, but the expression of E-cadherin was increased. The expression of mesenchymal markers in miR-320a-expressing cells was restored to normal levels by the restoration of BCR/ABL expression. Generally speaking, miR-320a acts as a novel tumor suppressor gene in CML and miR-320a can decrease migratory, invasive, proliferative and apoptotic behaviors, as well as CML EMT, by attenuating the expression of BCR/ABL oncogene.

  15. ABL kinases promote breast cancer osteolytic metastasis by modulating tumor-bone interactions through TAZ and STAT5 signaling

    PubMed Central

    Wang, Jun; Rouse, Clay; Jasper, Jeff S.; Pendergast, Ann Marie

    2016-01-01

    Bone metastases occur in up to 70% of advanced breast cancer. For most patients with breast cancer, bone metastases are predominantly osteolytic. Interactions between tumor cells and stromal cells in the bone microenvironment drive osteolytic bone metastasis, a process that requires the activation of osteoclasts, cells that break down bone. Here, we report that ABL kinases promoted metastasis of breast cancer cells to bone by regulating the crosstalk between tumor and the bone microenvironment. ABL kinases protected tumor cells from apoptosis induced by TRAIL (TNF-related apoptosis-inducing ligand), activated the transcription factor STAT5, and promoted osteolysis through the STAT5-dependent expression of genes encoding the osteoclast activating factors interleukin 6 (IL6) and matrix metalloproteinase-1 (MMP1). Furthermore, ABL kinases increased the abundance of the Hippo pathway mediator TAZ and the expression of TAZ-dependent target genes that promote bone metastasis. Knockdown of ABL kinases or treatment with ABL-specific allosteric inhibitor impaired osteolytic metastasis of breast cancer cells in mice. These findings revealed a role for ABL kinases in regulating tumor-bone interactions and provide a rationale for targeting both tumor and the bone microenvironment with ABL-specific inhibitors. PMID:26838548

  16. ABL kinases promote breast cancer osteolytic metastasis by modulating tumor-bone interactions through TAZ and STAT5 signaling.

    PubMed

    Wang, Jun; Rouse, Clay; Jasper, Jeff S; Pendergast, Ann Marie

    2016-02-01

    Bone metastases occur in up to 70% of advanced breast cancer. For most patients with breast cancer, bone metastases are predominantly osteolytic. Interactions between tumor cells and stromal cells in the bone microenvironment drive osteolytic bone metastasis, a process that requires the activation of osteoclasts, cells that break down bone. We report that ABL kinases promoted metastasis of breast cancer cells to bone by regulating the crosstalk between tumor cells and the bone microenvironment. ABL kinases protected tumor cells from apoptosis induced by TRAIL (TNF-related apoptosis-inducing ligand), activated the transcription factor STAT5, and promoted osteolysis through the STAT5-dependent expression of genes encoding the osteoclast-activating factors interleukin-6 (IL-6) and matrix metalloproteinase 1 (MMP1). Furthermore, in breast cancer cells, ABL kinases increased the abundance of the Hippo pathway mediator TAZ and the expression of TAZ-dependent target genes that promote bone metastasis. Knockdown of ABL kinases or treatment with ABL-specific allosteric inhibitor impaired osteolytic metastasis of breast cancer cells in mice. These findings revealed a role for ABL kinases in regulating tumor-bone interactions and provide a rationale for using ABL-specific inhibitors to limit breast cancer metastasis to bone. PMID:26838548

  17. The presence of the Rb c-box peptide in the cytoplasm inhibits p210bcr-abl transforming function.

    PubMed

    Guo, X Y; Balague, C; Wang, T; Randhawa, G; Yuan, Z; Bachier, C; Greenberger, J; Arlinghaus, R; Kufe, D; Deisseroth, A B

    1999-02-25

    In order to test if the carboxyl terminal polypeptide of the Retinoblastoma (Rb) tumor suppressor protein, could be used to suppress the growth factor-independent growth phenotype of p210bcr-abl positive myeloid cells, we introduced a truncated form of the 3' end of the Rb cDNA encoding its last 173 amino acid residues (Rb C-box) which localize into the cytoplasm where the p210bcr-abl transforming protein is found, into myeloid cells (32D) which depends on the p210bcr-abl protein for IL3 growth factor-independent growth (32D-p210). The expression of the plasmid vectors carrying the Rb C-box cDNAs was shown to inhibit the abl tyrosine specific protein kinase activity of the p210(bcr-abl) oncoprotein and to suppress the IL3-independent growth phenotype of the 32D-p210 cells. The Rb C-box polypeptides did not suppress the growth of the untransfected 32D parental cell line in methylcellulose in the presence of IL3-conditioned medium. These results suggest that the cytoplasmic localization of the p210(bcr-abl) allows it to escape the effect of intranuclear proteins such as Rb which negatively regulate the p145(c-abl) kinase. PMID:10102629

  18. Accurate prediction of interfacial residues in two-domain proteins using evolutionary information: implications for three-dimensional modeling.

    PubMed

    Bhaskara, Ramachandra M; Padhi, Amrita; Srinivasan, Narayanaswamy

    2014-07-01

    With the preponderance of multidomain proteins in eukaryotic genomes, it is essential to recognize the constituent domains and their functions. Often function involves communications across the domain interfaces, and the knowledge of the interacting sites is essential to our understanding of the structure-function relationship. Using evolutionary information extracted from homologous domains in at least two diverse domain architectures (single and multidomain), we predict the interface residues corresponding to domains from the two-domain proteins. We also use information from the three-dimensional structures of individual domains of two-domain proteins to train naïve Bayes classifier model to predict the interfacial residues. Our predictions are highly accurate (∼85%) and specific (∼95%) to the domain-domain interfaces. This method is specific to multidomain proteins which contain domains in at least more than one protein architectural context. Using predicted residues to constrain domain-domain interaction, rigid-body docking was able to provide us with accurate full-length protein structures with correct orientation of domains. We believe that these results can be of considerable interest toward rational protein and interaction design, apart from providing us with valuable information on the nature of interactions.

  19. MAPK15 mediates BCR-ABL1-induced autophagy and regulates oncogene-dependent cell proliferation and tumor formation

    PubMed Central

    Colecchia, David; Rossi, Matteo; Sasdelli, Federica; Sanzone, Sveva; Strambi, Angela; Chiariello, Mario

    2015-01-01

    A reciprocal translocation of the ABL1 gene to the BCR gene results in the expression of the oncogenic BCR-ABL1 fusion protein, which characterizes human chronic myeloid leukemia (CML), a myeloproliferative disorder considered invariably fatal until the introduction of the imatinib family of tyrosine kinase inhibitors (TKI). Nonetheless, insensitivity of CML stem cells to TKI treatment and intrinsic or acquired resistance are still frequent causes for disease persistence and blastic phase progression experienced in patients after initial successful therapies. Here, we investigated a possible role for the MAPK15/ERK8 kinase in BCR-ABL1-dependent autophagy, a key process for oncogene-induced leukemogenesis. In this context, we showed the ability of MAPK15 to physically recruit the oncogene to autophagic vesicles, confirming our hypothesis of a biologically relevant role for this MAP kinase in signal transduction by this oncogene. Indeed, by modeling BCR-ABL1 signaling in HeLa cells and taking advantage of a physiologically relevant model for human CML, i.e. K562 cells, we demonstrated that BCR-ABL1-induced autophagy is mediated by MAPK15 through its ability to interact with LC3-family proteins, in a LIR-dependent manner. Interestingly, we were also able to interfere with BCR-ABL1-induced autophagy by a pharmacological approach aimed at inhibiting MAPK15, opening the possibility of acting on this kinase to affect autophagy and diseases depending on this cellular function. Indeed, to support the feasibility of this approach, we demonstrated that depletion of endogenous MAPK15 expression inhibited BCR-ABL1-dependent cell proliferation, in vitro, and tumor formation, in vivo, therefore providing a novel “druggable” link between BCR-ABL1 and human CML. PMID:26291129

  20. Concept Convergence in Empirical Domains

    NASA Astrophysics Data System (ADS)

    Ontañón, Santiago; Plaza, Enric

    How to achieve shared meaning is a significant issue when more than one intelligent agent is involved in the same domain. We define the task of concept convergence, by which intelligent agents can achieve a shared, agreed-upon meaning of a concept (restricted to empirical domains). For this purpose we present a framework that, integrating computational argumentation and inductive concept learning, allows a pair of agents to (1) learn a concept in an empirical domain, (2) argue about the concept's meaning, and (3) reach a shared agreed-upon concept definition. We apply this framework to marine sponges, a biological domain where the actual definitions of concepts such as orders, families and species are currently open to discussion. An experimental evaluation on marine sponges shows that concept convergence is achieved, within a reasonable number of interchanged arguments, and reaching short and accurate definitions (with respect to precision and recall).

  1. Inhibition of the Raf-1 kinase by cyclic AMP agonists causes apoptosis of v-abl-transformed cells.

    PubMed Central

    Weissinger, E M; Eissner, G; Grammer, C; Fackler, S; Haefner, B; Yoon, L S; Lu, K S; Bazarov, A; Sedivy, J M; Mischak, H; Kolch, W

    1997-01-01

    Here we investigate the role of the Raf-1 kinase in transformation by the v-abl oncogene. Raf-1 can activate a transforming signalling cascade comprising the consecutive activation of Mek and extracellular-signal-regulated kinases (Erks). In v-abl-transformed cells the endogenous Raf-1 protein was phosphorylated on tyrosine and displayed high constitutive kinase activity. The activities of the Erks were constitutively elevated in both v-raf- and v-abl-transformed cells. In both cell types the activities of Raf-1 and v-raf were almost completely suppressed after activation of the cyclic AMP-dependent kinase (protein kinase A [PKA]), whereas the v-abl kinase was not affected. Raf inhibition substantially diminished the activities of Erks in v-raf-transformed cells but not in v-abl-transformed cells, indicating that v-abl can activate Erks by a Raf-1-independent pathway. PKA activation induced apoptosis in v-abl-transformed cells while reverting v-raf transformation without severe cytopathic effects. Overexpression of Raf-1 in v-abl-transformed cells partially protected the cells from apoptosis induced by PKA activation. In contrast to PKA activators, a Mek inhibitor did not induce apoptosis. The diverse biological responses correlated with the status of c-myc gene expression. v-abl-transformed cells featured high constitutive levels of expression of c-myc, which were not reduced following PKA activation. Myc activation has been previously shown to be essential for transformation by oncogenic Abl proteins. Using estrogen-regulated c-myc and temperature-sensitive Raf-1 mutants, we found that Raf-1 activation could protect cells from c-myc-induced apoptosis. In conclusion, these results suggest (i) that Raf-1 participates in v-abl transformation via an Erk-independent pathway by providing a survival signal which complements c-myc in transformation, and (ii) that cAMP agonists might become useful for the treatment of malignancies where abl oncogenes are involved, such as

  2. Are general practitioners able to accurately diagnose dementia and identify Alzheimer's disease? A comparison with an outpatient memory clinic.

    PubMed Central

    van Hout, H; Vernooij-Dassen, M; Poels, P; Hoefnagels, W; Grol, R

    2000-01-01

    Since the introduction of agents for the treatment of Alzheimer's disease, and in order to increase understanding of a patient's changed behaviour, it has become particularly important that dementia is both diagnosed at an early stage and differentiated into its subtypes. This study aims to ascertain whether GPs were able to diagnose dementia and identify the type of dementia accurately and confidently. GPs were well able to assess the firmness of their own dementia diagnoses, which supposes that they are able to make appropriate selection for referral. Diagnostic support from a specialised team can particularly contribute to identifying the type of dementia. PMID:10897518

  3. Establishment and characterization of A novel Philadelphia-chromosome positive chronic myeloid leukemia cell line, TCC-S, expressing P210 and P190 BCR/ABL transcripts but missing normal ABL gene.

    PubMed

    Van, Phan Nguyen Thanh; Xinh, Phan Thi; Kano, Yasuhiko; Tokunaga, Katsushi; Sato, Yuko

    2005-03-01

    A novel Philadelphia-chromosome positive (Ph+) cell line, TCC-S, has been established from a patient with Ph+ chronic myeloid leukemia (CML) in the blastic crisis. TCC-S cells were shown to express both P210 and P190 BCR/ABL transcripts by reverse transcriptase-polymerase chain reaction (PCR), although quantitative-PCR revealed that TCC-S cells mainly expressed P210 BCR/ABL transcript. Karyotype analysis revealed several triploid clones which constantly harbored two der(9)del(9) (p12)t(9;22) (q34;qll)s and two del(9) (q21)s. The der(9)del(9) (p12)t(9;22) (q34;q11) is rarely found in other CML cell lines. Moreover, to the best of our knowledge, del(9) (q21) resulting in missing of a restrict region including normal ABL gene has not been found among CML cell lines previously described. Thus, TCC-S cells with only BCR/ABL gene and no normal ABL gene may be a useful tool for functional study of ABL in Ph+ CML.

  4. MicroRNA-203 negatively regulates c-Abl, ERK1/2 phosphorylation, and proliferation in smooth muscle cells.

    PubMed

    Liao, Guoning; Panettieri, Reynold A; Tang, Dale D

    2015-09-01

    The nonreceptor tyrosine kinase c-Abl has a role in regulating smooth muscle cell proliferation, which contributes to the development of airway remodeling in chronic asthma. MicroRNAs (miRs) are small noncoding RNA molecules that regulate gene expression by binding to complementary sequences in the 3' untranslated regions (3' UTR) of target mRNAs. Previous analysis suggests that miR-203 is able to bind to the 3' UTR of human c-Abl mRNA. In this report, treatment with miR-203 attenuated the expression of c-Abl mRNA and protein in human airway smooth muscle (HASM) cells. Furthermore, transfection with an miR-203 inhibitor enhanced the expression of c-Abl at mRNA and protein levels in HASM cells. Treatment with platelet-derived growth factor (PDGF) induced the proliferation and ERK1/2 phosphorylation in HASM cells. Exposure to miR-203 attenuated the PDGF-stimulated proliferation and ERK1/2 phosphorylation in HASM cells. The expression of c-Abl at protein and mRNA levels was higher in asthmatic HASM cells, whereas the level of miR-203 was reduced in asthmatic HASM cells as compared to control HASM cells. Taken together, our present results suggest that miR-203 is a negative regulator of c-Abl expression in smooth muscle cells. miR-203 regulates smooth muscle cell proliferation by controlling c-Abl expression, which in turn modulates the activation of ERK1/2.

  5. Detection of BCR-ABL Fusion mRNA Using Reverse Transcriptase Loop-mediated Isothermal Amplification

    SciTech Connect

    Dugan, L C; Hall, S; Kohlgruber, A; Urbin, S; Torres, C; Wilson, P

    2011-12-08

    RT-PCR is commonly used for the detection of Bcr-Abl fusion transcripts in patients diagnosed with chronic myelogenous leukemia, CML. Two fusion transcripts predominate in CML, Br-Abl e13a2 and e14a2. They have developed reverse transcriptase isothermal loop-mediated amplification (RT-LAMP) assays to detect these two fusion transcripts along with the normal Bcr transcript.

  6. Hypoxia-Like Signatures Induced by BCR-ABL Potentially Alter the Glutamine Uptake for Maintaining Oxidative Phosphorylation.

    PubMed

    Sontakke, Pallavi; Koczula, Katarzyna M; Jaques, Jennifer; Wierenga, Albertus T J; Brouwers-Vos, Annet Z; Pruis, Maurien; Günther, Ulrich L; Vellenga, Edo; Schuringa, Jan Jacob

    2016-01-01

    The Warburg effect is probably the most prominent metabolic feature of cancer cells, although little is known about the underlying mechanisms and consequences. Here, we set out to study these features in detail in a number of leukemia backgrounds. The transcriptomes of human CB CD34+ cells transduced with various oncogenes, including BCR-ABL, MLL-AF9, FLT3-ITD, NUP98-HOXA9, STAT5A and KRASG12V were analyzed in detail. Our data indicate that in particular BCR-ABL, KRASG12V and STAT5 could impose hypoxic signaling under normoxic conditions. This coincided with an upregulation of glucose importers SLC2A1/3, hexokinases and HIF1 and 2. NMR-based metabolic profiling was performed in CB CD34+ cells transduced with BCR-ABL versus controls, both cultured under normoxia and hypoxia. Lactate and pyruvate levels were increased in BCR-ABL-expressing cells even under normoxia, coinciding with enhanced glutaminolysis which occurred in an HIF1/2-dependent manner. Expression of the glutamine importer SLC1A5 was increased in BCR-ABL+ cells, coinciding with an increased susceptibility to the glutaminase inhibitor BPTES. Oxygen consumption rates also decreased upon BPTES treatment, indicating a glutamine dependency for oxidative phosphorylation. The current study suggests that BCR-ABL-positive cancer cells make use of enhanced glutamine metabolism to maintain TCA cell cycle activity in glycolytic cells. PMID:27055152

  7. Hypoxia-Like Signatures Induced by BCR-ABL Potentially Alter the Glutamine Uptake for Maintaining Oxidative Phosphorylation

    PubMed Central

    Sontakke, Pallavi; Koczula, Katarzyna M.; Jaques, Jennifer; Wierenga, Albertus T. J.; Brouwers-Vos, Annet Z.; Pruis, Maurien; Günther, Ulrich L.; Vellenga, Edo; Schuringa, Jan Jacob

    2016-01-01

    The Warburg effect is probably the most prominent metabolic feature of cancer cells, although little is known about the underlying mechanisms and consequences. Here, we set out to study these features in detail in a number of leukemia backgrounds. The transcriptomes of human CB CD34+ cells transduced with various oncogenes, including BCR-ABL, MLL-AF9, FLT3-ITD, NUP98-HOXA9, STAT5A and KRASG12V were analyzed in detail. Our data indicate that in particular BCR-ABL, KRASG12V and STAT5 could impose hypoxic signaling under normoxic conditions. This coincided with an upregulation of glucose importers SLC2A1/3, hexokinases and HIF1 and 2. NMR-based metabolic profiling was performed in CB CD34+ cells transduced with BCR-ABL versus controls, both cultured under normoxia and hypoxia. Lactate and pyruvate levels were increased in BCR-ABL-expressing cells even under normoxia, coinciding with enhanced glutaminolysis which occurred in an HIF1/2-dependent manner. Expression of the glutamine importer SLC1A5 was increased in BCR-ABL+ cells, coinciding with an increased susceptibility to the glutaminase inhibitor BPTES. Oxygen consumption rates also decreased upon BPTES treatment, indicating a glutamine dependency for oxidative phosphorylation. The current study suggests that BCR-ABL-positive cancer cells make use of enhanced glutamine metabolism to maintain TCA cell cycle activity in glycolytic cells. PMID:27055152

  8. Domain and Specification Models for Software Engineering

    NASA Technical Reports Server (NTRS)

    Iscoe, Neil; Liu, Zheng-Yang; Feng, Guohui

    1992-01-01

    This paper discusses our approach to representing application domain knowledge for specific software engineering tasks. Application domain knowledge is embodied in a domain model. Domain models are used to assist in the creation of specification models. Although many different specification models can be created from any particular domain model, each specification model is consistent and correct with respect to the domain model. One aspect of the system-hierarchical organization is described in detail.

  9. The Phage Lytic Proteins from the Staphylococcus aureus Bacteriophage vB_SauS-phiIPLA88 Display Multiple Active Catalytic Domains and Do Not Trigger Staphylococcal Resistance

    PubMed Central

    Rodríguez-Rubio, Lorena; Martínez, Beatriz; Rodríguez, Ana; Donovan, David M.; Götz, Friedrich; García, Pilar

    2013-01-01

    The increase in antibiotic resistance world-wide revitalized the interest in the use of phage lysins to combat pathogenic bacteria. In this work, we analyzed the specific cleavage sites on the staphylococcal peptidoglycan produced by three phage lytic proteins. The investigated cell wall lytic enzymes were the endolysin LysH5 derived from the S. aureus bacteriophage vB_SauS-phi-IPLA88 (phi-IPLA88) and two fusion proteins between lysostaphin and the virion-associated peptidoglycan hydrolase HydH5 (HydH5SH3b and HydH5Lyso). We determined that all catalytic domains present in these proteins were active. Additionally, we tested for the emergence of resistant Staphylococcus aureus to any of the three phage lytic proteins constructs. Resistant S. aureus could not be identified after 10 cycles of bacterial exposure to phage lytic proteins either in liquid or plate cultures. However, a quick increase in lysostaphin resistance (up to 1000-fold in liquid culture) was observed. The lack of resistant development supports the use of phage lytic proteins as future therapeutics to treat staphylococcal infections. PMID:23724076

  10. Localization of resistive domains in inhomogeneous superconductors

    SciTech Connect

    Gurevich, A.V.; Mints, R.G.

    1981-01-01

    The properties of resistive domains due to the Joule heating in inhomogeneous superconductors with transport currents are studied. The equilibrium of a domain at an inhomogeneity of arbitrary type and with dimensions much smaller than the dimensions of the domain is investigated. It is shown that resistive domains can become localized at inhomogeneities. The temperature distribution in a domain and the current--voltage characteristic of the domain are determined. The stability of localized domains is discussed. It is shown that such domains give rise to a hysteresis in the destruction (recovery) of the superconductivity by the transport current.

  11. Predicting cognitive change within domains

    PubMed Central

    Duff, Kevin; Beglinger, Leigh J.; Moser, David J.; Paulsen, Jane S.

    2010-01-01

    Standardized regression based (SRB) formulas, a method for predicting cognitive change across time, traditionally use baseline performance on a neuropsychological measure to predict future performance on that same measure. However, there are instances in which the same tests may not be given at follow-up assessments (e.g., lack of continuity of provider, avoiding practice effects). The current study sought to expand this methodology by developing SRBs to predict performance on different tests within the same cognitive domain. Using a sample of 127 non-demented community-dwelling older adults assessed at baseline and after one year, two sets of SRBs were developed: 1. those predicting performance on the same test, and 2. those predicting performance on a different test within the same cognitive domain. The domains examined were learning and memory, processing speed, and language. Across both sets of SRBs, one year scores were significantly predicted by baseline scores, especially for the learning and memory and processing speed measures. Although SRBs developed for the same test were comparable to those developed for different tests within the same domain, less variance was accounted for as tests became less similar. The current results lend preliminary support for additional development of SRBs, both for same- and different-tests, as well as beginning to examine domain-based SRBs. PMID:20358479

  12. Functional domain walls in multiferroics

    NASA Astrophysics Data System (ADS)

    Meier, Dennis

    2015-11-01

    During the last decade a wide variety of novel and fascinating correlation phenomena has been discovered at domain walls in multiferroic bulk systems, ranging from unusual electronic conductance to inseparably entangled spin and charge degrees of freedom. The domain walls represent quasi-2D functional objects that can be induced, positioned, and erased on demand, bearing considerable technological potential for future nanoelectronics. Most of the challenges that remain to be solved before turning related device paradigms into reality, however, still fall in the field of fundamental condensed matter physics and materials science. In this topical review seminal experimental findings gained on electric and magnetic domain walls in multiferroic bulk materials are addressed. A special focus is put on the physical properties that emerge at so-called charged domain walls and the added functionality that arises from coexisting magnetic order. The research presented in this review highlights that we are just entering a whole new world of intriguing nanoscale physics that is yet to be explored in all its details. The goal is to draw attention to the persistent challenges and identify future key directions for the research on functional domain walls in multiferroics.

  13. G-protein coupled receptor 34 activates Erk and phosphatidylinositol 3-kinase/Akt pathways and functions as alternative pathway to mediate p185Bcr-Abl-induced transformation and leukemogenesis.

    PubMed

    Zuo, Bo; Li, Mei; Liu, Yulan; Li, Kun; Ma, Shuyun; Cui, Meihua; Qin, Yazhen; Zhu, Honghu; Pan, Xiuying; Guo, Jingzhu; Dai, Zonghan; Yu, Weidong

    2015-07-01

    Tyrosine 177 and the Src homology 2 (SH2) domain play important roles in linking p185Bcr-Abl to downstream pathways critical for cell growth and survival. However, a mutant p185(Y177FR552L) (p185(YR)), in which tyrosine 177 and arginine 552 in the SH2 domain are mutated, is still capable of transforming hematopoietic cells in vitro. Transplant of these cells into syngeneic mice also leads to leukemogenesis, albeit with a phenotype distinct from that produced by wild-type p185Bcr-Abl (p185(wt))-transformed cells. Here we show that G-protein coupled receptor 34 (Gpr34) expression is markedly up-regulated in p185(YR)-transformed cells compared to those transformed by p185(wt). Knockdown of Gpr34 in p185(YR) cells is sufficient to suppress growth factor-independent proliferation and survival in vitro and attenuate leukemogenesis in vivo. The Erk and phosphatidylinositol 3-kinase/Akt pathways are activated in p185(YR) cells and the activation is dependent on Gpr34 expression. These studies identify Gpr34 as an alternative pathway that may mediate p185Bcr-Abl-induced transformation and leukemogenesis. PMID:25363403

  14. miR-29b suppresses CML cell proliferation and induces apoptosis via regulation of BCR/ABL1 protein

    SciTech Connect

    Li, Yajuan; Wang, Haixia; Tao, Kun; Xiao, Qing; Huang, Zhenglan; Zhong, Liang; Cao, Weixi; Wen, Jianping; Feng, Wenli

    2013-05-01

    MicroRNAs (miRNAs) are small RNAs that regulate gene expression posttranscriptionally and are critical for many cellular pathways. Recent evidence has shown that aberrant miRNA expression profiles and unique miRNA signaling pathways are present in many cancers. Here, we demonstrate that miR-29b is markedly lower expressed in CML patient samples. Bioinformatics analysis reveals a conserved target site for miR-29b in the 3′-untranslated region (UTR) of ABL1. miR-29b significantly suppresses the activity of a luciferase reporter containing ABL1-3′UTR and this activity is not observed in cells transfected with mutated ABL1-3′UTR. Enforced expression of miR-29b in K562 cells inhibits cell growth and colony formation ability thereby inducing apoptosis through cleavage of procaspase 3 and PARP. Furthermore, K562 cells transfected with a siRNA targeting ABL1 show similar growth and apoptosis phenotypes as cells overexpression of miR-29b. Collectively, our results suggest that miR-29b may function as a tumor suppressor by targeting ABL1 and BCR/ABL1. - Highlights: ► miR-29b expression was downregulated in CML patients. ► ABL1 was identified as a direct target gene of miR-29b. ► Enforced expression of miR-29b inhibits cell proliferation and induces apoptosis. ► miR-29b might be a therapeutic target to CML.

  15. MicroRNA-320a acts as a tumor suppressor by targeting BCR/ABL oncogene in chronic myeloid leukemia

    PubMed Central

    Xishan, Zhu; Ziying, Lin; Jing, Du; Gang, Liu

    2015-01-01

    Accumulating evidences demonstrated that the induction of epithelial-mesenchymal transition (EMT) and aberrant expression of microRNAs (miRNAs) are associated with tumor