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Sample records for activation molecule family

  1. Signaling Lymphocytic Activation Molecule Family Receptor Homologs in New World Monkey Cytomegaloviruses

    PubMed Central

    Pérez-Carmona, Natàlia; Farré, Domènec; Martínez-Vicente, Pablo; Terhorst, Cox; Engel, Pablo

    2015-01-01

    ABSTRACT Throughout evolution, large DNA viruses have been usurping genes from their hosts to equip themselves with proteins that restrain host immune defenses. Signaling lymphocytic activation molecule (SLAM) family (SLAMF) receptors are involved in the regulation of both innate and adaptive immunity, which occurs upon engagement with their ligands via homotypic or heterotypic interactions. Here we report a total of seven SLAMF genes encoded by the genomes of two cytomegalovirus (CMV) species, squirrel monkey CMV (SMCMV) and owl monkey CMV (OMCMV), that infect New World monkeys. Our results indicate that host genes were captured by retrotranscription at different stages of the CMV-host coevolution. The most recent acquisition led to S1 in SMCMV. S1 is a SLAMF6 homolog with an amino acid sequence identity of 97% to SLAMF6 in its ligand-binding N-terminal Ig domain. We demonstrate that S1 is a cell surface glycoprotein capable of binding to host SLAMF6. Furthermore, the OMCMV genome encodes A33, an LY9 (SLAMF3) homolog, and A43, a CD48 (SLAMF2) homolog, two soluble glycoproteins which recognize their respective cellular counterreceptors and thus are likely to be viral SLAMF decoy receptors. In addition, distinct copies of further divergent CD48 homologs were found to be encoded by both CMV genomes. Remarkably, all these molecules display a number of unique features, including cytoplasmic tails lacking characteristic SLAMF signaling motifs. Taken together, our findings indicate a novel immune evasion mechanism in which incorporation of host SLAMF receptors that retain their ligand-binding properties enables viruses to interfere with SLAMF functions and to supply themselves with convenient structural molds for expanding their immunomodulatory repertoires. IMPORTANCE The way in which viruses shape their genomes under the continual selective pressure exerted by the host immune system is central for their survival. Here, we report that New World monkey cytomegaloviruses

  2. Novel Small Molecule Activators of the Trk Family of Receptor Tyrosine Kinases

    PubMed Central

    Obianyo, Obiamaka; Ye, Keqiang

    2012-01-01

    The Tropomyosin-related kinase (Trk) receptors are a subset of the receptor tyrosine kinase family with an important functionality in the regulation of neurotrophic signaling in the peripheral and central nervous system. As the receptors are able to mediate neuronal survival by associating with their respective neurotrophin ligands, many studies have focused on the therapeutic potential of generating small-molecule mimetic compounds that elicit agonistic effects similar to those of the natural protein ligands. To this end, various structure-based studies have led to the generation of bivalent peptide-based agonists and antibodies that selectively initiate Trk receptor signaling; however, these compounds do not possess the ideal characteristics of a potential drug. Additionally, the reliance of structure-based data to generate the compound libraries, limits the potential identification of novel chemical structures with desirable activity. Therefore, subsequent investigations utilized a cell-based apoptotic screen to facilitate the analysis of large, diverse chemical libraries of small molecules and quickly identify compounds with Trk-dependent antiapoptotic activity. Herein, we describe the Trk agonists that have been identified by this screening methodology and summarize their in vitro and in vivo neurotrophic activity as well as their efficacy in various neurological disease models, implicating their future utility as therapeutic compounds. PMID:22982231

  3. NTB-A Receptor Crystal Structure: Insights into Homophilic Interactions in the Signaling Lymphocytic Activation Molecule Receptor Family

    SciTech Connect

    Cao,E.; Ramagopal, U.; Fedorov, A.; Fedorov, E.; Yan, Q.; Lary, J.; Cole, J.; Nathenson, S.; Almo, S.

    2006-01-01

    The signaling lymphocytic activation molecule (SLAM) family includes homophilic and heterophilic receptors that regulate both innate and adaptive immunity. The ectodomains of most SLAM family members are composed of an N-terminal IgV domain and a C-terminal IgC2 domain. NK-T-B-antigen (NTB-A) is a homophilic receptor that stimulates cytotoxicity in natural killer (NK) cells, regulates bactericidal activities in neutrophils, and potentiates T helper 2 (Th2) responses. The 3.0 {angstrom} crystal structure of the complete NTB-A ectodomain revealed a rod-like monomer that self-associates to form a highly kinked dimer spanning an end-to-end distance of {approx}100 {angstrom}. The NTB-A homophilic and CD2-CD58 heterophilic dimers show overall structural similarities but differ in detailed organization and physicochemical properties of their respective interfaces. The NTB-A structure suggests a mechanism responsible for binding specificity within the SLAM family and imposes physical constraints relevant to the colocalization of SLAM-family proteins with other signaling molecules in the immunological synapse.

  4. Fragment-based discovery of a new family of non-peptidic small-molecule cyclophilin inhibitors with potent antiviral activities

    PubMed Central

    Ahmed-Belkacem, Abdelhakim; Colliandre, Lionel; Ahnou, Nazim; Nevers, Quentin; Gelin, Muriel; Bessin, Yannick; Brillet, Rozenn; Cala, Olivier; Douguet, Dominique; Bourguet, William; Krimm, Isabelle; Pawlotsky, Jean-Michel; Guichou, Jean- François

    2016-01-01

    Cyclophilins are peptidyl-prolyl cis/trans isomerases (PPIase) that catalyse the interconversion of the peptide bond at proline residues. Several cyclophilins play a pivotal role in the life cycle of a number of viruses. The existing cyclophilin inhibitors, all derived from cyclosporine A or sanglifehrin A, have disadvantages, including their size, potential for side effects unrelated to cyclophilin inhibition and drug–drug interactions, unclear antiviral spectrum and manufacturing issues. Here we use a fragment-based drug discovery approach using nucleic magnetic resonance, X-ray crystallography and structure-based compound optimization to generate a new family of non-peptidic, small-molecule cyclophilin inhibitors with potent in vitro PPIase inhibitory activity and antiviral activity against hepatitis C virus, human immunodeficiency virus and coronaviruses. This family of compounds has the potential for broad-spectrum, high-barrier-to-resistance treatment of viral infections. PMID:27652979

  5. Family Activities for Fitness

    ERIC Educational Resources Information Center

    Grosse, Susan J.

    2009-01-01

    This article discusses how families can increase family togetherness and improve physical fitness. The author provides easy ways to implement family friendly activities for improving and maintaining physical health. These activities include: walking, backyard games, and fitness challenges.

  6. Structure of Natural Killer Receptor 2B4 Bound to CD48 Reveals Basis for Heterophilic Recognition in Signaling Lymphocyte Activation Molecule Family

    SciTech Connect

    Velikovsky,C.; Deng, L.; Chlewicki, L.; Fernandez, M.; Kumar, V.; Mariuzza, R.

    2007-01-01

    Natural killer (NK) cells eliminate virally infected and tumor cells. Among the receptors regulating NK cell function is 2B4 (CD244), a member of the signaling lymphocyte-activation molecule (SLAM) family that binds CD48. 2B4 is the only heterophilic receptor of the SLAM family, whose other members, e.g., NK-T-B-antigen (NTB-A), are self-ligands. We determined the structure of the complex between the N-terminal domains of mouse 2B4 and CD48, as well as the structures of unbound 2B4 and CD48. The complex displayed an association mode related to, yet distinct from, that of the NTB-A dimer. Binding was accompanied by the rigidification of flexible 2B4 regions containing most of the polymorphic residues across different species and receptor isoforms. We propose a model for 2B4-CD48 interactions that permits the intermixing of SLAM receptors with major histocompatibility complex-specific receptors in the NK cell immune synapse. This analysis revealed the basis for heterophilic recognition within the SLAM family.

  7. Valuing Families. Activity Guide.

    ERIC Educational Resources Information Center

    Glashagel, Jerry; Glashagel, Char

    Developed as a resource for family life education, this activity guide can be used to lead experiential learning situations for intergenerational groups by a counselor, in a course, in a family organization like the YMCA, or in the home. The goals of this guide are to increase the self-esteem of each person and to strengthen the family as a human…

  8. New families of adhesion molecules play a vital role in platelet functions.

    PubMed

    Parmentier, S; Kaplan, C; Catimel, B; McGregor, J L

    1990-07-01

    Adhesion molecules play a crucial part in cell-matrix and in cell-cell interactions. These interactions, which are essential to the body's defense processes, involve adhesion molecules belonging to different families: integrins, immunoglobulins and selectins. Integrins are expressed by a large number of tissues, whereas other adhesion molecule families are restricted to a small number of cell types. A recent symposium dealt with the recruitment of circulating platelets at specific sites, their adhesion to extracellular matrix components and their activation by agonists leading to aggregation or attachment to other cells. These events, supporting hemostasis and thrombosis, involve integrins, selectins and other adhesion molecules. This report focuses on newly reported integrins (GPIa, GPIc, GPIIa), selectins (GMP-140) and GPIIIb, previously known as 'minor' surface oriented platelet glycoproteins. Major membrane glycoproteins such as GPIIb-IIIa (an integrin) and GPIb, which also play a vital role in platelet functions, have been extensively reviewed elsewhere.

  9. Single-molecule studies of kinesin family motor proteins

    NASA Astrophysics Data System (ADS)

    Fordyce, Polly

    Kinesin family motor proteins drive many essential cellular processes, including cargo transport and mitotic spindle assembly and regulation. They accomplish these tasks by converting the chemical energy released from the hydrolysis of adenosine triphosphate (ATP) directly into mechanical motion along microtubules in cells. Optical traps allow us to track and apply force to individual motor proteins, and have already revealed many details of the movement of conventional kinesin, although the precise mechanism by which chemical energy is converted into mechanical motion is unclear. Other kinesin family members remain largely uncharacterized. This dissertation details the use of a novel optical-trapping assay to study Eg5, a Kinesin-5 family member involved in both spindle assembly and pole separation during mitosis. We demonstrate that individual Eg5 dimers are relatively slow and force-insensitive motors that take about 8 steps, on average, before detaching from the microtubule. Key differences in processivity and force-response between Eg5 and conventional kinesin suggest ways in which the two motors might have evolved to perform very different tasks in cells. This dissertation also details efforts to unravel how chemical energy is converted into mechanical motion by simultaneously measuring mechanical transitions (with an optical trap) and nucleotide binding and release (with single-molecule fluorescence) for individual conventional kinesin motors. We constructed a combined instrument, demonstrated its capabilities by unzipping fluorescently-labeled DNA duplexes, and used this instrument to record the motion of individual conventional kinesin motors powered by the hydrolysis of fluorescent nucleotides. Preliminary data reveal the challenges inherent in such measurements and guide proposals for future experimental approaches. Finally, this dissertation includes several chapters intended to serve as practical guides to understanding, constructing, and maintaining

  10. Control of autoimmune diseases by the B7-CD28 family molecules.

    PubMed

    Anand, Sudarshan; Chen, Lieping

    2004-01-01

    The roles of B7-CD28 family molecules in the regulation of immune responses have been intensively studied over the past decade. The findings resulting from these studies not only broaden our understanding in the control of immune responses at the molecular level, but also lead to identification of molecular targets for future manipulation and potential treatment of human diseases. There is convincing evidence that the B7-CD28 family molecules play critical roles in the control of initiation, progression and pathogenesis of autoimmune diseases, which is the focus of this review. Currently, five molecular pathways within this family have been identified and each of them appears to overlap but have distinct functions in the control of priming, activation, maturation and amplification of cellular and humoral immune responses. Rationale-based design of intervention, targeting on multiple pathways should lead to new methods and approaches for management of autoimmune diseases. PMID:14754392

  11. Raman Optical Activity Spectra for Large Molecules through Molecules-in-Molecules Fragment-Based Approach.

    PubMed

    Jovan Jose, K V; Raghavachari, Krishnan

    2016-02-01

    We present an efficient method for the calculation of the Raman optical activity (ROA) spectra for large molecules through the molecules-in-molecules (MIM) fragment-based method. The relevant higher energy derivatives from smaller fragments are used to build the property tensors of the parent molecule to enable the extension of the MIM method for evaluating ROA spectra (MIM-ROA). Two factors were found to be particularly important in yielding accurate results. First, the link-atom tensor components are projected back onto the corresponding host and supporting atoms through the Jacobian projection method, yielding a mathematically rigorous method. Second, the long-range interactions between fragments are taken into account by using a less computationally expensive lower level of theory. The performance of the MIM-ROA model is calibrated on the enantiomeric pairs of 10 carbohydrate benchmark molecules, with strong intramolecular interactions. The vibrational frequencies and ROA intensities are accurately reproduced relative to the full, unfragmented, results for these systems. In addition, the MIM-ROA method is employed to predict the ROA spectra of d-maltose, α-D-cyclodextrin, and cryptophane-A, yielding spectra in excellent agreement with experiment. The accuracy and performance of the benchmark systems validate the MIM-ROA model for exploring ROA spectra of large molecules.

  12. Uranium-mediated activation of small molecules.

    PubMed

    Arnold, Polly L

    2011-08-28

    Molecular complexes of uranium are capable of activating a range of industrially and economically important small molecules such as CO, CO(2), and N(2); new and often unexpected reactions provide insight into an element that needs to be well-understood if future clean-energy solutions are to involve nuclear power.

  13. A novel approach to predict active sites of enzyme molecules.

    PubMed

    Chou, Kuo-Chen; Cai, Yu-dong

    2004-04-01

    Enzymes are critical in many cellular signaling cascades. With many enzyme structures being solved, there is an increasing need to develop an automated method for identifying their active sites. However, given the atomic coordinates of an enzyme molecule, how can we predict its active site? This is a vitally important problem because the core of an enzyme molecule is its active site from the viewpoints of both pure scientific research and industrial application. In this article, a topological entity was introduced to characterize the enzymatic active site. Based on such a concept, the covariant discriminant algorithm was formulated for identifying the active site. As a paradigm, the serine hydrolase family was demonstrated. The overall success rate by jackknife test for a data set of 88 enzyme molecules was 99.92%, and that for a data set of 50 independent enzyme molecules was 99.91%. Meanwhile, it was shown through an example that the prediction algorithm can also be used to find any typographic error of a PDB file in annotating the constituent amino acids of catalytic triad and to suggest a possible correction. The very high success rates are due to the introduction of a covariance matrix in the prediction algorithm that makes allowance for taking into account the coupling effects among the key constituent atoms of active site. It is anticipated that the novel approach is quite promising and may become a useful high throughput tool in enzymology, proteomics, and structural bioinformatics. PMID:14997541

  14. Wildfire: A Family Activity Book.

    ERIC Educational Resources Information Center

    WGBH-TV, Boston, MA.

    This family activity book provides information for discovering and demonstrating the science of fire--how firefighters decide which fires to let burn and which to put out, how fires start and spread, and what to do when they flare up. Chapters include: (1) "A Game about Wildfire"; (2) "Create a Fire Safety Commercial"; (3) "Make a Fire Escape…

  15. Small molecules inhibitors of plasminogen activator inhibitor-1 - an overview.

    PubMed

    Rouch, Anne; Vanucci-Bacqué, Corinne; Bedos-Belval, Florence; Baltas, Michel

    2015-03-01

    PAI-1, a glycoprotein from the serpin family and the main inhibitor of tPA and uPA, plays an essential role in the regulation of intra and extravascular fibrinolysis by inhibiting the formation of plasmin from plasminogen. PAI-1 is also involved in pathological processes such as thromboembolic diseases, atherosclerosis, fibrosis and cancer. The inhibition of PAI-1 activity by small organic molecules has been observed in vitro and with some in vivo models. Based on these findings, PAI-1 appears as a potential therapeutic target for several pathological conditions. Over the past decades, many efforts have therefore been devoted to developing PAI-1 inhibitors. This article provides an overview of the publishing activity on small organic molecules used as PAI-1 inhibitors. The chemical synthesis of the most potent inhibitors as well as their biological and biochemical evaluations is also presented.

  16. Vibrational Raman optical activity of biological molecules

    NASA Astrophysics Data System (ADS)

    Barron, L. D.; Gargaro, A. R.; Hecht, Lutz; Wen, Z. Q.; Hug, W.

    1991-05-01

    Advances in Raman optical activity (ROA) instrumentation based on the employment of a backscattering geometry together with a cooled CCD detector have now enhanced the sensitivity to the level necessary to provide vibrational ROA spectra of biological molecules in aqueous solution. Preliminary results on peptides and proteins show features originating in coupled Ca-H and N-H deformations of the peptide backbone which appear to be sensitive to the secondary conformation. Also carbohydrates show many features that appear to be characteristic of the central aspects of carbohydrate architecture with effects from the glycosidic link in di- and oligosaccharides particularly prominent. 1.

  17. Attachment of second harmonic-active moiety to molecules for detection of molecules at interfaces

    DOEpatents

    Salafsky, Joshua S.; Eisenthal, Kenneth B.

    2005-10-11

    This invention provides methods of detecting molecules at an interface, which comprise labeling the molecules with a second harmonic-active moiety and detecting the labeled molecules at the interface using a surface selective technique. The invention also provides methods for detecting a molecule in a medium and for determining the orientation of a molecular species within a planar surface using a second harmonic-active moiety and a surface selective technique.

  18. Connecting Family Learning and Active Citizenship

    ERIC Educational Resources Information Center

    Flanagan, Mary

    2009-01-01

    In Ireland family learning and active citizenship has not been linked together until 2006. It was while the Clare Family Learning Project was involved in a family learning EU learning network project, that a suggestion to create a new partnership project linking both areas was made and FACE IT! was born (Families and Active Citizenship…

  19. Small Molecule Inhibitors of BAF; A Promising Family of Compounds in HIV-1 Latency Reversal

    PubMed Central

    Stoszko, Mateusz; De Crignis, Elisa; Rokx, Casper; Khalid, Mir Mubashir; Lungu, Cynthia; Palstra, Robert-Jan; Kan, Tsung Wai; Boucher, Charles; Verbon, Annelies; Dykhuizen, Emily C.; Mahmoudi, Tokameh

    2015-01-01

    Persistence of latently infected cells in presence of Anti-Retroviral Therapy presents the main obstacle to HIV-1 eradication. Much effort is thus placed on identification of compounds capable of HIV-1 latency reversal in order to render infected cells susceptible to viral cytopathic effects and immune clearance. We identified the BAF chromatin remodeling complex as a key player required for maintenance of HIV-1 latency, highlighting its potential as a molecular target for inhibition in latency reversal. Here, we screened a recently identified panel of small molecule inhibitors of BAF (BAFi's) for potential to activate latent HIV-1. Latency reversal was strongly induced by BAFi's Caffeic Acid Phenethyl Ester and Pyrimethamine, two molecules previously characterized for clinical application. BAFi's reversed HIV-1 latency in cell line based latency models, in two ex vivo infected primary cell models of latency, as well as in HIV-1 infected patient's CD4 + T cells, without inducing T cell proliferation or activation. BAFi-induced HIV-1 latency reversal was synergistically enhanced upon PKC pathway activation and HDAC-inhibition. Therefore BAFi's constitute a promising family of molecules for inclusion in therapeutic combinatorial HIV-1 latency reversal. PMID:26870822

  20. Small Molecule Inhibitors of BAF; A Promising Family of Compounds in HIV-1 Latency Reversal.

    PubMed

    Stoszko, Mateusz; De Crignis, Elisa; Rokx, Casper; Khalid, Mir Mubashir; Lungu, Cynthia; Palstra, Robert-Jan; Kan, Tsung Wai; Boucher, Charles; Verbon, Annelies; Dykhuizen, Emily C; Mahmoudi, Tokameh

    2016-01-01

    Persistence of latently infected cells in presence of Anti-Retroviral Therapy presents the main obstacle to HIV-1 eradication. Much effort is thus placed on identification of compounds capable of HIV-1 latency reversal in order to render infected cells susceptible to viral cytopathic effects and immune clearance. We identified the BAF chromatin remodeling complex as a key player required for maintenance of HIV-1 latency, highlighting its potential as a molecular target for inhibition in latency reversal. Here, we screened a recently identified panel of small molecule inhibitors of BAF (BAFi's) for potential to activate latent HIV-1. Latency reversal was strongly induced by BAFi's Caffeic Acid Phenethyl Ester and Pyrimethamine, two molecules previously characterized for clinical application. BAFi's reversed HIV-1 latency in cell line based latency models, in two ex vivo infected primary cell models of latency, as well as in HIV-1 infected patient's CD4 + T cells, without inducing T cell proliferation or activation. BAFi-induced HIV-1 latency reversal was synergistically enhanced upon PKC pathway activation and HDAC-inhibition. Therefore BAFi's constitute a promising family of molecules for inclusion in therapeutic combinatorial HIV-1 latency reversal. PMID:26870822

  1. Vibrational Raman optical activity of biological molecules

    NASA Astrophysics Data System (ADS)

    Barron, L. D.; Hecht, Lutz; Wen, Z. Q.; Ford, Steven J.; Bell, A. F.

    1993-06-01

    Advances in Raman optical activity (ROA) instrumentation based on the employment of a backscattering geometry together with a cooled backthinned CCD detector, a holographic notch filter, and a high-efficiency single-grating spectrograph have now enhanced the sensitivity to the level necessary to provide vibrational ROA spectra of most biological molecules in aqueous solution. Results on peptides and proteins show features originating in coupled C(alpha )-H and N-H deformations of the peptide backbone which appear to be sensitive to the secondary conformation including loop and turn structures. Also carbohydrates show many features characteristic of the central aspects of carbohydrate architecture, with effects from the glycosidic link in oligosaccharides particularly prominent. Preliminary ROA spectra of pyrimidine nucleosides appear to reflect the mutual orientation of the sugar and base rings and the dominant furanose conformations.

  2. Activation of small molecules by phosphorus biradicaloids.

    PubMed

    Hinz, Alexander; Kuzora, Rene; Rosenthal, Uwe; Schulz, Axel; Villinger, Alexander

    2014-11-01

    The reactivity of biradicaloid [P(μ-NTer)]2 was employed to activate small molecules bearing single, double, and triple bonds. Addition of chalcogens (O2 , S8 , Sex and Tex ) led to the formation of dichalcogen-bridged P2 N2 heterocycles, except from the reaction with molecular oxygen, which gave a P2 N2 ring featuring a dicoordinated P(III) and a four-coordinated P(V) center. In formal [2πe+2πe] addition reactions, small unsaturated compounds such as ethylene, acetylene, acetone, acetonitrile, tolane, diphenylcarbodiimide, and bis(trimethylsilyl)sulfurdiimide are readily added to the P2 N2 heterocycle of the biradicaloid [P(μ-NTer)]2 , yielding novel heteroatom cage compounds. The synthesis, reactivity, and bonding of the biradicaloid [P(μ-NTer)]2 were studied in detail as well as the synthesis, properties, and structural features of all addition products. PMID:25266101

  3. The role of B7 family molecules in hematologic malignancy

    PubMed Central

    Greaves, Paul

    2013-01-01

    The B7 family consists of structurally related, cell-surface proteins that regulate immune responses by delivering costimulatory or coinhibitory signals through their ligands. Eight family members have been identified to date including CD80 (B7-1), CD86 (B7-2), CD274 (programmed cell death-1 ligand [PD-L1]), CD273 (programmed cell death-2 ligand [PD-L2]), CD275 (inducible costimulator ligand [ICOS-L]), CD276 (B7-H3), B7-H4, and B7-H6. B7 ligands are expressed on both lymphoid and nonlymphoid tissues. The importance of the B7 family in regulating immune responses is clear from their demonstrated role in the development of immunodeficiency and autoimmune diseases. Manipulation of the signals delivered by B7 ligands shows great potential in the treatment of cancers including leukemias and lymphomas and in regulating allogeneic T-cell responses after stem cell transplantation. PMID:23223433

  4. Observing single enzyme molecules interconvert between activity states upon heating.

    PubMed

    Rojek, Marcin J; Walt, David R

    2014-01-01

    In this paper, we demonstrate that single enzyme molecules of β-galactosidase interconvert between different activity states upon exposure to short pulses of heat. We show that these changes in activity are the result of different enzyme conformations. Hundreds of single β-galactosidase molecules are trapped in femtoliter reaction chambers and the individual enzymes are subjected to short heating pulses. When heating pulses are introduced into the system, the enzyme molecules switch between different activity states. Furthermore, we observe that the changes in activity are random and do not correlate with the enzyme's original activity. This study demonstrates that different stable conformations play an important role in the static heterogeneity reported previously, resulting in distinct long-lived activity states of enzyme molecules in a population.

  5. We Are a Family: Children's Activities in Family Living.

    ERIC Educational Resources Information Center

    Matiella, Ana Consuelo

    This booklet is part of the "Children's Activity Series," a set of four supplemental teaching resources that promote awareness about health, family life, and cultural diversity for children in kindergarten through third grade. The booklet contains stories, drawings, and "sockpuppets" to help children in kindergarten through third grade develop a…

  6. Family gym: a model to promote physical activity for families with young children.

    PubMed

    Castaneda-Sceppa, Carmen; Hoffman, Jessica A; Thomas, Jordan; DuBois, Matthew; Agrawal, Tara; Griffin, Daphne; Bhaumik, Urmi; Healey, Christine Locke; Dickerson, Deborah; Nethersole, Shari; Wirth, Catherine

    2014-08-01

    This report describes Family Gym, a family-centered model that (1) provides free access to physical activity for low-income families in the inner city; (2) targets young children (3-8 years) and their families; (3) engages families together in physical activity; and (4) stimulates social interaction among families.

  7. Family caregivers and limitations in social activities.

    PubMed

    Miller, B; Montgomery, A

    1990-03-01

    Constriction of social and personal activities is one of the most frequently noted consequences of caring for a frail elder. This study analyzed the correlates of perceived limitations in social activities using data from a national sample of the frail elderly and their caregivers. Two research issues were addressed: 1) What differences in demographic, family, and caregiving attributes are associated with variation in perceived restricted social activities? and, 2) How does the process influencing restriction of social activities vary by family relationship of the caregiver? Higher levels of elder dependency and task demands were characteristic of those who reported social limitations, and daughters and wives were more likely to report such limitations than sons and husbands. Subjective assessment of time and task demands, however, were more important influences than objective caregiving activities for all family caregivers. PMID:2180010

  8. Family caregivers and limitations in social activities.

    PubMed

    Miller, B; Montgomery, A

    1990-03-01

    Constriction of social and personal activities is one of the most frequently noted consequences of caring for a frail elder. This study analyzed the correlates of perceived limitations in social activities using data from a national sample of the frail elderly and their caregivers. Two research issues were addressed: 1) What differences in demographic, family, and caregiving attributes are associated with variation in perceived restricted social activities? and, 2) How does the process influencing restriction of social activities vary by family relationship of the caregiver? Higher levels of elder dependency and task demands were characteristic of those who reported social limitations, and daughters and wives were more likely to report such limitations than sons and husbands. Subjective assessment of time and task demands, however, were more important influences than objective caregiving activities for all family caregivers.

  9. Trefoil peptides: a newly recognized family of epithelial mucin-associated molecules.

    PubMed

    Poulsom, R; Wright, N A

    1993-08-01

    Members of the trefoil family of peptides are generally small stable secreted molecules, structurally related by the presence of one, or up to six, compact 6-cysteine motifs. Several trefoil peptides are expressed in mammalian gut and Xenopus gut and skin, often in association with mucins. Chronic ulcerative conditions of the gut, such as Crohn's disease, result in the growth of glandular structures of the ulcer-associated cell lineage (UACL) that secrete epidermal growth factor/urogastrone, transforming growth factor-alpha, and at least three trefoil peptides [pS2, human spasmolytic polypeptide (hSP), and intestinal trefoil factor (hITF/hP1.B)]. Neuroendocrine and goblet cells near the UACL are "recruited" into expressing pS2 and hSP, but the purpose of this concerted expression is unclear. A role in mucosal healing has been proposed. Biological functions of trefoil peptides have been difficult to establish. Pancreatic spasmolytic polypeptide of porcine origin inhibits gastric acid secretion and smooth muscle contraction and is a growth factor for some cultured cells, but pS2, once thought to be breast cancer specific, is not a mitogen. Recombinant trefoil peptides have allowed localization of binding sites and will allow structure-activity relationships to be studied, once the functions are clear.

  10. Regulation of PI-3-Kinase and Akt Signaling in T Lymphocytes and Other Cells by TNFR Family Molecules

    PubMed Central

    So, Takanori; Croft, Michael

    2013-01-01

    Activation of phosphoinositide 3-kinase (PI3K) and Akt (protein kinase B) is a common response triggered by a range of membrane-bound receptors on many cell types. In T lymphocytes, the PI3K-Akt pathway promotes clonal expansion, differentiation, and survival of effector cells and suppresses the generation of regulatory T cells. PI3K activation is tightly controlled by signals through the T cell receptor (TCR) and the co-stimulatory receptor CD28, however sustained and periodic signals from additional co-receptors are now being recognized as critical contributors to the activation of this pathway. Accumulating evidence suggests that many members of the Tumor Necrosis Factor receptor (TNFR) superfamily, TNFR2 (TNFRSF1B), OX40 (TNFRSF4), 4-1BB (TNFRSF9), HVEM (TNFRSF14), and DR3 (TNFRSF25), that are constitutive or inducible on T cells, can directly or indirectly promote activity in the PI3K-Akt pathway. We discuss recent data which suggests that ligation of one TNFR family molecule organizes a signalosome, via TNFR-associated factor (TRAF) adapter proteins in T cell membrane lipid microdomains, that results in the subsequent accumulation of highly concentrated depots of PI3K and Akt in close proximity to TCR signaling units. We propose this may be a generalizable mechanism applicable to other TNFR family molecules that will result in a quantitative contribution of these signalosomes to enhancing and sustaining PI3K and Akt activation triggered by the TCR. We also review data that other TNFR molecules, such as CD40 (TNFRSF5), RANK (TNFRSF11A), FN14 (TNFRSF12A), TACI (TNFRSF13B), BAFFR (TNFRSF13C), and NGFR (TNFRSF16), contribute to the activation of this pathway in diverse cell types through a similar ability to recruit PI3K or Akt into their signaling complexes. PMID:23760533

  11. Expression and localization of the diacylglycerol kinase family and of phosphoinositide signaling molecules in adrenal gland.

    PubMed

    Hozumi, Yasukazu; Akimoto, Ryo; Suzuki, Akihito; Otani, Koichi; Watanabe, Masahiko; Goto, Kaoru

    2015-11-01

    Adrenal glands play a central role in the secretion of steroid hormones and catecholamines. Previous studies have revealed that molecules engaged in phosphoinositide (PI) turnover are expressed in the adrenal gland, suggesting the importance of PI signaling in adrenal signal transduction. Diacylglycerol kinase (DGK) catalyzes the phosphorylation of diacylglycerol (DG), a major second messenger in the PI signaling cascade. The DGK family is expressed in distinct patterns in endocrine organs at the mRNA and protein levels. Nevertheless, little is known about the characteristics and morphological aspects of DGKs in the adrenal gland. We have performed immunohistochemical analyses to investigate the expression and localization of DGK isozymes, together with PI signaling molecules, in the adrenal gland at the protein level. Our results show that the DGK family and a set of PI signaling molecules are expressed intensely in zona glomerulosa cells and medullary chromaffin cells in the adrenal gland. In adrenal cells, DGKγ localizes to the Golgi complex, DGKε to the plasma membrane, and DGKζ to the nucleus. These findings show the distinct expression and subcellular localization of DGK isozymes and PI signaling molecules in the adrenal gland, suggesting that each DGK isozyme has a role in signal transduction in adrenal cells, especially in the zona glomerulosa and medulla.

  12. Exploration of the spontaneous fluctuating activity of single enzyme molecules.

    PubMed

    Schwabe, Anne; Maarleveld, Timo R; Bruggeman, Frank J

    2013-09-01

    Single enzyme molecules display inevitable, stochastic fluctuations in their catalytic activity. In metabolism, for instance, the stochastic activity of individual enzymes is averaged out due to their high copy numbers per single cell. However, many processes inside cells rely on single enzyme activity, such as transcription, replication, translation, and histone modifications. Here we introduce the main theoretical concepts of stochastic single-enzyme activity starting from the Michaelis-Menten enzyme mechanism. Next, we discuss stochasticity of multi-substrate enzymes, of enzymes and receptors with multiple conformational states and finally, how fluctuations in receptor activity arise from fluctuations in signal concentration. This paper aims to introduce the exciting field of single-molecule enzyme kinetics and stochasticity to a wider audience of biochemists and systems biologists.

  13. Secrets of Lost Empires: Family Activity Book.

    ERIC Educational Resources Information Center

    Whitman, John D.; Gaffney, Dennis

    This family activity book features information on the background and filming of five stories. "Pharaoh's Obelisk" questions how ancient Egyptians transported and raised stone obelisks. "Roman Bath" studies how Romans built bathhouses and attempts to build a working Roman bath. "China Bridge" investigates the structure of the China Bridge and…

  14. Small Molecule Activators of the Heat Shock Response: Chemical Properties, Molecular Targets, and Therapeutic Promise

    PubMed Central

    West, James D.; Wang, Yanyu; Morano, Kevin A.

    2012-01-01

    All cells have developed various mechanisms to respond and adapt to a variety of environmental challenges, including stresses that damage cellular proteins. One such response, the heat shock response (HSR), leads to the transcriptional activation of a family of molecular chaperone proteins that promote proper folding or clearance of damaged proteins within the cytosol. In addition to its role in protection against acute insults, the HSR also regulates lifespan and protects against protein misfolding that is associated with degenerative diseases of aging. As a result, identifying pharmacological regulators of the HSR has become an active area of research in recent years. Here, we review progress made in identifying small molecule activators of the HSR, what cellular targets these compounds interact with to drive response activation, and how such molecules may ultimately be employed to delay or reverse protein misfolding events that contribute to a number of diseases. PMID:22799889

  15. Self-assembly of active colloidal molecules with dynamic function

    NASA Astrophysics Data System (ADS)

    Soto, Rodrigo; Golestanian, Ramin

    Catalytically active colloids maintain non-equilibrium conditions in which they produce and deplete chemicals at their surface. While individual colloids that are symmetrically coated do not exhibit dynamical activity, the concentration fields resulting from their chemical activity decay as 1/r and produce gradients that attract or repel other colloids depending on their surface chemistry and ambient variables. This results in a non-equilibrium analogue of ionic systems, but with the remarkable novel feature of action-reaction symmetry breaking. In dilute conditions these active colloids join up to form molecules via generalized ionic bonds. Colloids are found to join up to form self-assembled molecules that could be inert or have spontaneous activity in the form of net translational velocity and spin depending on their symmetry properties and their constituents. As the interactions do not satisfy detailed-balance, it is possible to achieve structures with time dependent functionality. We study a molecule that adopts spontaneous oscillations and another that exhibits a run-and-tumble dynamics similar to bacteria. Our study shows that catalytically active colloids could be used for designing self-assembled structures that posses dynamical functionalities.

  16. Structural basis of AMPK regulation by small molecule activators

    NASA Astrophysics Data System (ADS)

    Xiao, Bing; Sanders, Matthew J.; Carmena, David; Bright, Nicola J.; Haire, Lesley F.; Underwood, Elizabeth; Patel, Bhakti R.; Heath, Richard B.; Walker, Philip A.; Hallen, Stefan; Giordanetto, Fabrizio; Martin, Stephen R.; Carling, David; Gamblin, Steven J.

    2013-12-01

    AMP-activated protein kinase (AMPK) plays a major role in regulating cellular energy balance by sensing and responding to increases in AMP/ADP concentration relative to ATP. Binding of AMP causes allosteric activation of the enzyme and binding of either AMP or ADP promotes and maintains the phosphorylation of threonine 172 within the activation loop of the kinase. AMPK has attracted widespread interest as a potential therapeutic target for metabolic diseases including type 2 diabetes and, more recently, cancer. A number of direct AMPK activators have been reported as having beneficial effects in treating metabolic diseases, but there has been no structural basis for activator binding to AMPK. Here we present the crystal structure of human AMPK in complex with a small molecule activator that binds at a site between the kinase domain and the carbohydrate-binding module, stabilising the interaction between these two components. The nature of the activator-binding pocket suggests the involvement of an additional, as yet unidentified, metabolite in the physiological regulation of AMPK. Importantly, the structure offers new opportunities for the design of small molecule activators of AMPK for treatment of metabolic disorders.

  17. Anti-Ebola Activity of Diazachrysene Small Molecules.

    PubMed

    Selaković, Života; Soloveva, Veronica; Gharaibeh, Dima N; Wells, Jay; Šegan, Sandra; Panchal, Rekha G; Šolaja, Bogdan A

    2015-06-12

    Herein we report on a diazachrysene class of small molecules that exhibit potent antiviral activity against the Ebola (EBOV) virus. The antiviral compounds are easily synthesized, and the most active compounds have excellent in vitro activity (0.34-0.70 μM) and are significantly less lipophilic than their predecessors. The three most potent diazachrysene antivirals do not exhibit any toxicity in vivo and protected 70-90% of the mice at 10 mg/kg following EBOV challenge. Together, these studies suggest that diazachrysenes are a promising class of compounds for hit to lead optimization and as potential Ebola therapeutics. PMID:27622742

  18. Small molecules reveal an alternative mechanism of Bax activation

    PubMed Central

    Brahmbhatt, Hetal; Uehling, David; Al-awar, Rima; Leber, Brian; Andrews, David

    2016-01-01

    The pro-apoptotic protein Bax commits a cell to death by permeabilizing the mitochondrial outer membrane (MOM). To obtain small-molecule probes for elucidating the molecular mechanism(s) of Bax activation, we screened for compounds that induced Bax-mediated liposome permeabilization. We identified five structurally different small molecules that promoted both Bax targeting to and oligomerization at membranes. All five compounds initiated Bax oligomerization in the absence of membranes by a mechanism unlike Bax activation by Bcl-2 homology 3 domain (BH3) proteins. Some of the compounds induced Bax/Bak-dependent apoptosis in cells. Activation of Bax by the most active compound was poorly inhibited by the anti-apoptotic protein Bcl-XL and requires a cysteine residue at position 126 of Bax that is not required for activation by BH3 proteins. Our results reveal a novel pathway for Bax activation independent of pro-apoptotic BH3 proteins that may have important implications for the regulation of Bax activity in cells. PMID:26916338

  19. Studying p53 family proteins in yeast: Induction of autophagic cell death and modulation by interactors and small molecules

    SciTech Connect

    Leão, Mariana; Gomes, Sara; Bessa, Cláudia; Soares, Joana; Raimundo, Liliana; Monti, Paola; Fronza, Gilberto; Pereira, Clara; Saraiva, Lucília

    2015-01-01

    In this work, the yeast Saccharomyces cerevisiae was used to individually study human p53, p63 (full length and truncated forms) and p73. Using this cell system, the effect of these proteins on cell proliferation and death, and the influence of MDM2 and MDMX on their activities were analyzed. When expressed in yeast, wild-type p53, TAp63, ΔNp63 and TAp73 induced growth inhibition associated with S-phase cell cycle arrest. This growth inhibition was accompanied by reactive oxygen species production and autophagic cell death. Furthermore, they stimulated rapamycin-induced autophagy. On the contrary, none of the tested p53 family members induced apoptosis either per se or after apoptotic stimuli. As previously reported for p53, also TAp63, ΔNp63 and TAp73 increased actin expression levels and its depolarization, suggesting that ACT1 is also a p63 and p73 putative yeast target gene. Additionally, MDM2 and MDMX inhibited the activity of all tested p53 family members in yeast, although the effect was weaker on TAp63. Moreover, Nutlin-3a and SJ-172550 were identified as potential inhibitors of the p73 interaction with MDM2 and MDMX, respectively. Altogether, the yeast-based assays herein developed can be envisaged as a simplified cell system to study the involvement of p53 family members in autophagy, the modulation of their activities by specific interactors (MDM2 and MDMX), and the potential of new small molecules to modulate these interactions. - Highlights: • p53, p63 and p73 are individually studied in the yeast S. cerevisiae. • p53 family members induce ROS production, cell cycle arrest and autophagy in yeast. • p53 family members increase actin depolarization and expression levels in yeast. • MDM2 and MDMX inhibit the activity of p53 family members in yeast. • Yeast can be a useful tool to study the biology and drugability of p53, p63 and p73.

  20. Single-Molecule Electronic Monitoring of DNA Polymerase Activity

    NASA Astrophysics Data System (ADS)

    Marushchak, Denys O.; Pugliese, Kaitlin M.; Turvey, Mackenzie W.; Choi, Yongki; Gul, O. Tolga; Olsen, Tivoli J.; Rajapakse, Arith J.; Weiss, Gregory A.; Collins, Philip G.

    Single-molecule techniques can reveal new spatial and kinetic details of the conformational changes occurring during enzymatic catalysis. Here, we investigate the activity of DNA polymerases using an electronic single-molecule technique based on carbon nanotube transistors. Single molecules of the Klenow fragment (KF) of polymerase I were conjugated to the transistors and then monitored via fluctuations in electrical conductance. Continuous, long-term monitoring recorded single KF molecules incorporating up to 10,000 new bases into single-stranded DNA templates. The duration of individual incorporation events was invariant across all analog and native nucleotides, indicating that the precise structure of different base pairs has no impact on the timing of incorporation. Despite similar timings, however, the signal magnitudes generated by certain analogs reveal alternate conformational states that do not occur with native nucleotides. The differences induced by these analogs suggest that the electronic technique is sensing KF's O-helix as it tests the stability of nascent base pairs.

  1. Single molecule study of ClpP enzymatic activity

    NASA Astrophysics Data System (ADS)

    Mazouchi, Amir; Yu, Angela; Houry, Walid; Gradinaru, Claudiu

    2009-03-01

    Elementary processes that form the basis of biological activities pass through a number of short-lived intermediate states while progressing from initial state to final state. Single-molecule techniques, unlike ensemble averaging measurements, are often able to resolve these transient states. ClpP, a known target of antibacterial drugs like acydepsipeptides (ADEPs), is a classical representative of serine proteases, enzymes that cleave peptide bonds in proteins. We performed single-molecule fluorescence measurements including burst spectroscopy and fluorescence correlation spectroscopy (FCS) to address unknown aspects of this degradation process. Our study reveals important molecular details of protein degradation, such as the enzyme-substrate binding rate, the lifetime distribution of the conjugated state and the probability of substrate cleavage upon conjugation.

  2. Familial amyloid polyneuropathy: alanine-for-threonine substitution in the transthyretin (prealbumin) molecule.

    PubMed

    Koeppen, A H; Wallace, M R; Benson, M D; Altland, K

    1990-11-01

    A previously reported family with amyloid polyneuropathy (FAP) was reinvestigated to determine the type of mutation in the transthyretin (prealbumin) molecule. Transthyretin was isolated from amyloid-laden myocardium and serum, and tryptic peptides were resolved by high-performance liquid chromatography. Amino acid sequencing of an anomalous peptide revealed an alanine-for-threonine substitution corresponding to position No. 60 of the transthyretin monomer. Detection of the FAP gene in asymptomatic carriers was accomplished by hybrid isoelectric focusing of transthyretin in the presence of dithiothreitol and high concentrations of urea, and by Southern blotting of Pvull-digested leukocyte deoxyribonucleic acid. This type of FAP was found to be identical to the previously described Appalachian amyloid. Patients with FAP and their asymptomatic gene-carrying offspring had significantly reduced levels of serum transthyretin and retinol-binding protein.

  3. Single-Molecule Manipulation Studies of a Mechanically Activated Protein

    NASA Astrophysics Data System (ADS)

    Botello, Eric; Harris, Nolan; Choi, Huiwan; Bergeron, Angela; Dong, Jing-Fei; Kiang, Ching-Hwa

    2009-10-01

    Plasma von Willebrand factor (pVWF) is the largest multimeric adhesion ligand found in human blood and must be adhesively activated by exposure to shear stress, like at sites of vascular injury, to initiate blood clotting. Sheared pVWF (sVWF) will undergo a conformational change from a loose tangled coil to elongated strings forming adhesive fibers by binding with other sVWF. VWF's adhesion activity is also related to its length, with the ultra-large form of VWF (ULVWF) being hyper-actively adhesive without exposure to shear stress; it has also been shown to spontaneously form fibers. We used single molecule manipulation techniques with the AFM to stretch pVWF, sVWF and ULVWF and monitor the forces as a function of molecular extension. We showed a similar increase in resistance to unfolding for sVWF and ULVWF when compared to pVWF. This mechanical resistance to forced unfolding is reduced when other molecules known to disrupt their fibril formation are present. Our results show that sVWF and ULVWF domains unfold at higher forces than pVWF, which is consistent with the hypothesis that shear stress induces lateral association that alters adhesion activity of pVWF.

  4. Identification of Small Molecules That Suppress Ricin-Induced Stress-Activated Signaling Pathways

    PubMed Central

    Wahome, Paul G.; Ahlawat, Sarita; Mantis, Nicholas J.

    2012-01-01

    Ricin is a member of the ribosome-inactivating protein (RIP) family of plant and bacterial toxins. In this study we used a high-throughput, cell-based assay to screen more than 118,000 compounds from diverse chemical libraries for molecules that reduced ricin-induced cell death. We describe three compounds, PW66, PW69, and PW72 that at micromolar concentrations significantly delayed ricin-induced cell death. None of the compounds had any demonstrable effect on ricin's ability to arrest protein synthesis in cells or on ricin's enzymatic activity as assessed in vitro. Instead, all three compounds appear to function by blocking downstream stress-induced signaling pathways associated with the toxin-mediated apoptosis. PW66 virtually eliminated ricin-induced TNF-α secretion by J774A.1 macrophages and concomitantly blocked activation of the p38 MAPK and JNK signaling pathways. PW72 suppressed ricin-induced TNF-α secretion, but not p38 MAPK and JNK signaling. PW69 suppressed activity of the executioner caspases 3/7 in ricin toxin- and Shiga toxin 2-treated cells. While the actual molecular targets of the three compounds have yet to be identified, these data nevertheless underscore the potential of small molecules to down-regulate inflammatory signaling pathways associated with exposure to the RIP family of toxins. PMID:23133670

  5. A family of helminth molecules that modulate innate cell responses via molecular mimicry of host antimicrobial peptides.

    PubMed

    Robinson, Mark W; Donnelly, Sheila; Hutchinson, Andrew T; To, Joyce; Taylor, Nicole L; Norton, Raymond S; Perugini, Matthew A; Dalton, John P

    2011-05-01

    Over the last decade a significant number of studies have highlighted the central role of host antimicrobial (or defence) peptides in modulating the response of innate immune cells to pathogen-associated ligands. In humans, the most widely studied antimicrobial peptide is LL-37, a 37-residue peptide containing an amphipathic helix that is released via proteolytic cleavage of the precursor protein CAP18. Owing to its ability to protect against lethal endotoxaemia and clinically-relevant bacterial infections, LL-37 and its derivatives are seen as attractive candidates for anti-sepsis therapies. We have identified a novel family of molecules secreted by parasitic helminths (helminth defence molecules; HDMs) that exhibit similar biochemical and functional characteristics to human defence peptides, particularly CAP18. The HDM secreted by Fasciola hepatica (FhHDM-1) adopts a predominantly α-helical structure in solution. Processing of FhHDM-1 by F. hepatica cathepsin L1 releases a 34-residue C-terminal fragment containing a conserved amphipathic helix. This is analogous to the proteolytic processing of CAP18 to release LL-37, which modulates innate cell activation by classical toll-like receptor (TLR) ligands such as lipopolysaccharide (LPS). We show that full-length recombinant FhHDM-1 and a peptide analogue of the amphipathic C-terminus bind directly to LPS in a concentration-dependent manner, reducing its interaction with both LPS-binding protein (LBP) and the surface of macrophages. Furthermore, FhHDM-1 and the amphipathic C-terminal peptide protect mice against LPS-induced inflammation by significantly reducing the release of inflammatory mediators from macrophages. We propose that HDMs, by mimicking the function of host defence peptides, represent a novel family of innate cell modulators with therapeutic potential in anti-sepsis treatments and prevention of inflammation.

  6. A Family of Helminth Molecules that Modulate Innate Cell Responses via Molecular Mimicry of Host Antimicrobial Peptides

    PubMed Central

    Hutchinson, Andrew T.; To, Joyce; Taylor, Nicole L.; Norton, Raymond S.; Perugini, Matthew A.

    2011-01-01

    Over the last decade a significant number of studies have highlighted the central role of host antimicrobial (or defence) peptides in modulating the response of innate immune cells to pathogen-associated ligands. In humans, the most widely studied antimicrobial peptide is LL-37, a 37-residue peptide containing an amphipathic helix that is released via proteolytic cleavage of the precursor protein CAP18. Owing to its ability to protect against lethal endotoxaemia and clinically-relevant bacterial infections, LL-37 and its derivatives are seen as attractive candidates for anti-sepsis therapies. We have identified a novel family of molecules secreted by parasitic helminths (helminth defence molecules; HDMs) that exhibit similar biochemical and functional characteristics to human defence peptides, particularly CAP18. The HDM secreted by Fasciola hepatica (FhHDM-1) adopts a predominantly α-helical structure in solution. Processing of FhHDM-1 by F. hepatica cathepsin L1 releases a 34-residue C-terminal fragment containing a conserved amphipathic helix. This is analogous to the proteolytic processing of CAP18 to release LL-37, which modulates innate cell activation by classical toll-like receptor (TLR) ligands such as lipopolysaccharide (LPS). We show that full-length recombinant FhHDM-1 and a peptide analogue of the amphipathic C-terminus bind directly to LPS in a concentration-dependent manner, reducing its interaction with both LPS-binding protein (LBP) and the surface of macrophages. Furthermore, FhHDM-1 and the amphipathic C-terminal peptide protect mice against LPS-induced inflammation by significantly reducing the release of inflammatory mediators from macrophages. We propose that HDMs, by mimicking the function of host defence peptides, represent a novel family of innate cell modulators with therapeutic potential in anti-sepsis treatments and prevention of inflammation. PMID:21589904

  7. Rho family and Rap GTPase activation assays.

    PubMed

    Jennings, Richard T; Knaus, Ulla G

    2014-01-01

    The detection of Ras superfamily GTPase activity in innate immune cells is important when studying signaling events elicited by various ligands and cellular processes. The development of high-affinity probes detecting the activated, GTP-bound form of small GTPases has significantly enhanced our understanding of initiation and termination of GTPase-regulated signaling pathways. These probes are created by fusing a high-affinity GTPase-binding domain derived from a specific downstream effector protein to glutathione S-transferase (GST). Such domains bind preferentially to the GTP-bound form of the upstream Rho or Ras GTPase. Coupling these probes to beads enables extraction of the complex and subsequent quantification of the active GTP-binding protein by immunoblotting. Although effector domains that discriminate efficiently between GDP- and GTP-bound states and highly specific antibodies are not yet available for every small GTPase, analysis of certain members of the Rho and Ras GTPase family is now routinely performed. Here, we describe affinity-based pulldown assays for detection of Rho GTPase (Rac1/2, Cdc42, RhoA/B) and Rap1/2 activity in stimulated neutrophils or macrophages.

  8. Small Molecule Inhibitors Targeting Activator Protein 1 (AP-1)

    PubMed Central

    2015-01-01

    Activator protein 1 (AP-1) is a pivotal transcription factor that regulates a wide range of cellular processes including proliferation, apoptosis, differentiation, survival, cell migration, and transformation. Accumulating evidence supports that AP-1 plays an important role in several severe disorders including cancer, fibrosis, and organ injury, as well as inflammatory disorders such as asthma, psoriasis, and rheumatoid arthritis. AP-1 has emerged as an actively pursued drug discovery target over the past decade. Excitingly, a selective AP-1 inhibitor T-5224 (51) has been investigated in phase II human clinical trials. Nevertheless, no effective AP-1 inhibitors have yet been approved for clinical use. Despite significant advances achieved in understanding AP-1 biology and function, as well as the identification of small molecules modulating AP-1 associated signaling pathways, medicinal chemistry efforts remain an urgent need to yield selective and efficacious AP-1 inhibitors as a viable therapeutic strategy for human diseases. PMID:24831826

  9. Small molecule activators of pre-mRNA 3′ cleavage

    PubMed Central

    Ryan, Kevin; Khleborodova, Asya; Pan, Jingyi; Ryan, Xiaozhou P.

    2009-01-01

    3′ Cleavage and polyadenylation are obligatory steps in the biogenesis of most mammalian pre-mRNAs. In vitro reconstitution of the 3′ cleavage reaction from human cleavage factors requires high concentrations of creatine phosphate (CP), though how CP activates cleavage is not known. Previously, we proposed that CP might work by competitively inhibiting a cleavage-suppressing serine/threonine (S/T) phosphatase. Here we show that fluoride/EDTA, a general S/T phosphatase inhibitor, activates in vitro cleavage in place of CP. Subsequent testing of inhibitors specific for different S/T phosphatases showed that inhibitors of the PPM family of S/T phosphatases, which includes PP2C, but not the PPP family, which includes PP1, PP2A, and PP2B, activated 3′ cleavage in vitro. In particular, NCI 83633, an inhibitor of PP2C, activated extensive 3′ cleavage at a concentration 50-fold below that required by fluoride or CP. The testing of structural analogs led to the identification of a more potent compound that activated 3′ cleavage at 200 μM. While testing CP analogs to understand the origin of its cleavage activation effect, we found phosphocholine to be a more effective activator than CP. The minimal structural determinants of 3′ cleavage activation by phosphocholine were identified. Our results describe a much improved small molecule activator of in vitro pre-mRNA cleavage, identify the molecular determinants of cleavage activation by phosphoamines such as phosphocholine, and suggest that a PPM family phosphatase is involved in the negative regulation of mammalian pre-mRNA 3′ cleavage. PMID:19155323

  10. Myricetin: A Dietary Molecule with Diverse Biological Activities

    PubMed Central

    Semwal, Deepak Kumar; Semwal, Ruchi Badoni; Combrinck, Sandra; Viljoen, Alvaro

    2016-01-01

    Myricetin is a common plant-derived flavonoid and is well recognised for its nutraceuticals value. It is one of the key ingredients of various foods and beverages. The compound exhibits a wide range of activities that include strong anti-oxidant, anticancer, antidiabetic and anti-inflammatory activities. It displays several activities that are related to the central nervous system and numerous studies have suggested that the compound may be beneficial to protect against diseases such as Parkinson’s and Alzheimer’s. The use of myricetin as a preserving agent to extend the shelf life of foods containing oils and fats is attributed to the compound’s ability to protect lipids against oxidation. A detailed search of existing literature revealed that there is currently no comprehensive review available on this important molecule. Hence, the present work includes the history, synthesis, pharmaceutical applications and toxicity studies of myricetin. This report also highlights structure-activity relationships and mechanisms of action for various biological activities. PMID:26891321

  11. Active Microfluidic Devices for Single-Molecule Experiments

    NASA Astrophysics Data System (ADS)

    Chen, Hao; Meiners, Jens-Christian

    2003-03-01

    Microfluidic chips have become an increasingly powerful and versatile tool in the life sciences. Multilayer devices fabricated from soft silicone elastomers in a replication molding technique are especially promising, because they permit flexible integration of active elements such as valves and pumps. In addition, they are fairly easy and inexpensive to produce. In a wide range of applications, microfluidic chips are used in conjunction with optical detection and manipulation techniques. However their widespread use has been hampered due to problems with interconnect stability, optical accessibility, and ability to perform surface chemistry. We have developed a packaging technique that encapsulates the elastomer in an epoxy resin of high optical quality. This stabilizes the interconnects so that a chip can be repeatedly plugged in and out of a socket. Our technique also eliminates the need for a baking step that is conventionally used to attach a glass cover slip to the elastomer surface. This allows us to assemble devices that contain a cover slip coated with proteins, thereby permitting subsequent in situ attachment of DNA molecules to the bottom of the flow channels. We demonstrate the utility of our chips in single-molecule applications involving tethered-particles and optical tweezers. Support: NIH R01 GM065934 & Research Corporation

  12. Tyrosine phosphorylation at a site highly conserved in the L1 family of cell adhesion molecules abolishes ankyrin binding and increases lateral mobility of neurofascin.

    PubMed

    Garver, T D; Ren, Q; Tuvia, S; Bennett, V

    1997-05-01

    This paper presents evidence that a member of the L1 family of ankyrin-binding cell adhesion molecules is a substrate for protein tyrosine kinase(s) and phosphatase(s), identifies the highly conserved FIGQY tyrosine in the cytoplasmic domain as the principal site of phosphorylation, and demonstrates that phosphorylation of the FIGQY tyrosine abolishes ankyrin-binding activity. Neurofascin expressed in neuroblastoma cells is subject to tyrosine phosphorylation after activation of tyrosine kinases by NGF or bFGF or inactivation of tyrosine phosphatases with vanadate or dephostatin. Furthermore, both neurofascin and the related molecule Nr-CAM are tyrosine phosphorylated in a developmentally regulated pattern in rat brain. The FIGQY sequence is present in the cytoplasmic domains of all members of the L1 family of neural cell adhesion molecules. Phosphorylation of the FIGQY tyrosine abolishes ankyrin binding, as determined by coimmunoprecipitation of endogenous ankyrin and in vitro ankyrin-binding assays. Measurements of fluorescence recovery after photobleaching demonstrate that phosphorylation of the FIGQY tyrosine also increases lateral mobility of neurofascin expressed in neuroblastoma cells to the same extent as removal of the cytoplasmic domain. Ankyrin binding, therefore, appears to regulate the dynamic behavior of neurofascin and is the target for regulation by tyrosine phosphorylation in response to external signals. These findings suggest that tyrosine phosphorylation at the FIGQY site represents a highly conserved mechanism, used by the entire class of L1-related cell adhesion molecules, for regulation of ankyrin-dependent connections to the spectrin skeleton.

  13. Family Science Activities for Adult Basic and Literacy Education.

    ERIC Educational Resources Information Center

    Community Action Southwest, Waynesburg, PA.

    A staff development project created a series of family science activities to be used in adult basic and literacy education (ABLE) and family literacy programs and a training guide for staff and volunteers. The training guide provides background principles and concepts for science activities. The activities identify materials and indicate ways the…

  14. Ankyrin-binding activity of nervous system cell adhesion molecules expressed in adult brain.

    PubMed

    Davis, J Q; Bennett, V

    1993-01-01

    A family of ankyrin-binding glycoproteins have been identified in adult rat brain that include alternatively spliced products of the same pre-mRNA. A composite sequence of ankyrin-binding glycoprotein (ABGP) shares 72% amino acid sequence identity with chicken neurofascin, a membrane-spanning neural cell adhesion molecule in the Ig super-family expressed in embryonic brain. ABGP polypeptides and ankyrin associate as pure proteins in a 1:1 molar stoichiometry at a site located in the predicted cytoplasmic domain. ABGP polypeptides are expressed late in postnatal development to approximately the same levels as ankyrin, and comprise a significant fraction of brain membrane proteins. Immunofluorescence studies have shown that ABGP polypeptides are co-localized with ankyrinB. Major differences in developmental expression have been reported for neurofascin in embryos compared with the late postnatal expression of ABGP, suggesting that ABGP and neurofascin represent products of gene duplication events that have subsequently evolved in parallel with distinct roles. Predicted cytoplasmic domains of rat ABGP and chicken neurofascin are nearly identical to each other and closely related to a group of nervous system cell adhesion molecules with variable extracellular domains, including L1, Nr-CAM and Ng-CAM of vertebrates, and neuroglian of Drosophila. A hypothesis to be evaluated is that ankyrin-binding activity is shared by all of these proteins.

  15. Families on the Go: Active or Hyperactive?

    ERIC Educational Resources Information Center

    Villaire, Ted

    2003-01-01

    Discusses why families are so overscheduled, explaining that when children spend time by themselves doing nothing in particular, they are relaxing and learning how to be alone and enjoy their own company. When children do not have time to relax, there can be damaging stress-related consequences. Suggestions to help families spend more time…

  16. Manipulating lipid bilayer material properties using biologically active amphipathic molecules

    NASA Astrophysics Data System (ADS)

    Ashrafuzzaman, Md; Lampson, M. A.; Greathouse, D. V.; Koeppe, R. E., II; Andersen, O. S.

    2006-07-01

    Lipid bilayers are elastic bodies with properties that can be manipulated/controlled by the adsorption of amphipathic molecules. The resulting changes in bilayer elasticity have been shown to regulate integral membrane protein function. To further understand the amphiphile-induced modulation of bilayer material properties (thickness, intrinsic monolayer curvature and elastic moduli), we examined how an enantiomeric pair of viral anti-fusion peptides (AFPs)—Z-Gly-D-Phe and Z-Gly-Phe, where Z denotes a benzyloxycarbonyl group, as well as Z-Phe-Tyr and Z-D-Phe-Phe-Gly—alters the function of enantiomeric pairs of gramicidin channels of different lengths in planar bilayers. For both short and long channels, the channel lifetimes and appearance frequencies increase as linear functions of the aqueous AFP concentration, with no apparent effect on the single-channel conductance. These changes in channel function do not depend on the chirality of the channels or the AFPs. At pH 7.0, the relative changes in channel lifetimes do not vary when the channel length is varied, indicating that these compounds exert their effects primarily by causing a positive-going change in the intrinsic monolayer curvature. At pH 4.0, the AFPs are more potent than at pH 7.0 and have greater effects on the shorter channels, indicating that these compounds now change the bilayer elastic moduli. When AFPs of different anti-fusion potencies are compared, the rank order of the anti-fusion activity and the channel-modifying activity is similar, but the relative changes in anti-fusion potency are larger than the changes in channel-modifying activity. We conclude that gramicidin channels are useful as molecular force transducers to probe the influence of small amphiphiles upon lipid bilayer material properties.

  17. Application of Optical Biosensors in Small-Molecule Screening Activities

    PubMed Central

    Geschwindner, Stefan; Carlsson, Johan F.; Knecht, Wolfgang

    2012-01-01

    The last two decades have seen remarkable progress and improvements in optical biosensor systems such that those are currently seen as an important and value-adding component of modern drug screening activities. In particular the introduction of microplate-based biosensor systems holds the promise to match the required throughput without compromising on data quality thus representing a sought-after complement to traditional fluidic systems. This article aims to highlight the application of the two most prominent optical biosensor technologies, namely surface plasmon resonance (SPR) and optical waveguide grating (OWG), in small-molecule screening and will present, review and discuss the advantages and disadvantages of different assay formats on these platforms. A particular focus will be on the specific advantages of the inhibition in solution assay (ISA) format in contrast to traditional direct binding assays (DBA). Furthermore we will discuss different application areas for both fluidic as well as plate-based biosensor systems by considering the individual strength of the platforms. PMID:22666031

  18. Activating Molecules, Ions, and Solid Particles with Acoustic Cavitation

    PubMed Central

    Pflieger, Rachel; Chave, Tony; Virot, Matthieu; Nikitenko, Sergey I.

    2014-01-01

    The chemical and physical effects of ultrasound arise not from a direct interaction of molecules with sound waves, but rather from the acoustic cavitation: the nucleation, growth, and implosive collapse of microbubbles in liquids submitted to power ultrasound. The violent implosion of bubbles leads to the formation of chemically reactive species and to the emission of light, named sonoluminescence. In this manuscript, we describe the techniques allowing study of extreme intrabubble conditions and chemical reactivity of acoustic cavitation in solutions. The analysis of sonoluminescence spectra of water sparged with noble gases provides evidence for nonequilibrium plasma formation. The photons and the "hot" particles generated by cavitation bubbles enable to excite the non-volatile species in solutions increasing their chemical reactivity. For example the mechanism of ultrabright sonoluminescence of uranyl ions in acidic solutions varies with uranium concentration: sonophotoluminescence dominates in diluted solutions, and collisional excitation contributes at higher uranium concentration. Secondary sonochemical products may arise from chemically active species that are formed inside the bubble, but then diffuse into the liquid phase and react with solution precursors to form a variety of products. For instance, the sonochemical reduction of Pt(IV) in pure water provides an innovative synthetic route for monodispersed nanoparticles of metallic platinum without any templates or capping agents. Many studies reveal the advantages of ultrasound to activate the divided solids. In general, the mechanical effects of ultrasound strongly contribute in heterogeneous systems in addition to chemical effects. In particular, the sonolysis of PuO2 powder in pure water yields stable colloids of plutonium due to both effects. PMID:24747272

  19. Sustainable production of biologically active molecules of marine based origin.

    PubMed

    Murray, Patrick M; Moane, Siobhan; Collins, Catherine; Beletskaya, Tanya; Thomas, Olivier P; Duarte, Alysson W F; Nobre, Fernando S; Owoyemi, Ifeloju O; Pagnocca, Fernando C; Sette, L D; McHugh, Edward; Causse, Eric; Pérez-López, Paula; Feijoo, Gumersindo; Moreira, Ma T; Rubiolo, Juan; Leirós, Marta; Botana, Luis M; Pinteus, Susete; Alves, Celso; Horta, André; Pedrosa, Rui; Jeffryes, Clayton; Agathos, Spiros N; Allewaert, Celine; Verween, Annick; Vyverman, Wim; Laptev, Ivan; Sineoky, Sergei; Bisio, Angela; Manconi, Renata; Ledda, Fabio; Marchi, Mario; Pronzato, Roberto; Walsh, Daniel J

    2013-09-25

    The marine environment offers both economic and scientific potential which are relatively untapped from a biotechnological point of view. These environments whilst harsh are ironically fragile and dependent on a harmonious life form balance. Exploitation of natural resources by exhaustive wild harvesting has obvious negative environmental consequences. From a European industry perspective marine organisms are a largely underutilised resource. This is not due to lack of interest but due to a lack of choice the industry faces for cost competitive, sustainable and environmentally conscientious product alternatives. Knowledge of the biotechnological potential of marine organisms together with the development of sustainable systems for their cultivation, processing and utilisation are essential. In 2010, the European Commission recognised this need and funded a collaborative RTD/SME project under the Framework 7-Knowledge Based Bio-Economy (KBBE) Theme 2 Programme 'Sustainable culture of marine microorganisms, algae and/or invertebrates for high value added products'. The scope of that project entitled 'Sustainable Production of Biologically Active Molecules of Marine Based Origin' (BAMMBO) is outlined. Although the Union is a global leader in many technologies, it faces increasing competition from traditional rivals and emerging economies alike and must therefore improve its innovation performance. For this reason innovation is placed at the heart of a European Horizon 2020 Strategy wherein the challenge is to connect economic performance to eco performance. This article provides a synopsis of the research activities of the BAMMBO project as they fit within the wider scope of sustainable environmentally conscientious marine resource exploitation for high-value biomolecules.

  20. Discovery of Diverse Small Molecule Chemotypes with Cell-Based PKD1 Inhibitory Activity

    PubMed Central

    Sharlow, Elizabeth R.; Mustata Wilson, Gabriela; Close, David; Leimgruber, Stephanie; Tandon, Manuj; Reed, Robyn B.; Shun, Tong Ying; Wang, Q. Jane; Wipf, Peter; Lazo, John S.

    2011-01-01

    Protein kinase D (PKD) is a novel family of serine/threonine kinases regulated by diacylglycerol, which is involved in multiple cellular processes and various pathological conditions. The limited number of cell-active, selective inhibitors has historically restricted biochemical and pharmacological studies of PKD. We now markedly expand the PKD1 inhibitory chemotype inventory with eleven additional novel small molecule PKD1 inhibitors derived from our high throughput screening campaigns. The in vitro IC50s for these eleven compounds ranged in potency from 0.4 to 6.1 µM with all of the evaluated compounds being competitive with ATP. Three of the inhibitors (CID 1893668, (1Z)-1-(3-ethyl-5-methoxy-1,3-benzothiazol-2-ylidene)propan-2-one; CID 2011756, 5-(3-chlorophenyl)-N-[4-(morpholin-4-ylmethyl)phenyl]furan-2-carboxamide; CID 5389142, (6Z)-6-[4-(3-aminopropylamino)-6-methyl-1H-pyrimidin-2-ylidene]cyclohexa-2,4-dien-1-one) inhibited phorbol ester-induced endogenous PKD1 activation in LNCaP prostate cancer cells in a concentration-dependent manner. The specificity of these compounds for PKD1 inhibitory activity was supported by kinase assay counter screens as well as by bioinformatics searches. Moreover, computational analyses of these novel cell-active PKD1 inhibitors indicated that they were structurally distinct from the previously described cell-active PKD1 inhibitors while computational docking of the new cell-active compounds in a highly conserved ATP-binding cleft suggests opportunities for structural modification. In summary, we have discovered novel PKD1 inhibitors with in vitro and cell-based inhibitory activity, thus successfully expanding the structural diversity of small molecule inhibitors available for this important pharmacological target. PMID:21998636

  1. Familial adult onset hyperinsulinism due to an activating glucokinase mutation: Implications for pharmacological glucokinase activation

    PubMed Central

    Challis, Benjamin G.; Harris, Julie; Sleigh, Alison; Isaac, Iona; Orme, Steve M.; Seevaratnam, Nandini; Dhatariya, Ketan; Simpson, Helen L.; Semple, Robert K.

    2016-01-01

    Context Glucokinase (GCK) phosphorylates and thereby “traps” glucose in cells, thus serving as a gatekeeper for cellular glucose metabolism, particularly in hepatocytes and pancreatic beta cells. In humans, activating GCK mutations cause familial hyperinsulinaemic hypoglycaemia (GCK-HH), leading to keen interest in the potential of small molecule glucokinase activators (GKAs) as treatments for diabetes mellitus. Many such agents have been developed, however observation of side effects including hypertriglyceridaemia and hepatic steatosis have delayed their clinical development. Objective To describe the clinical presentation and metabolic profiles of affected family members in a kindred with familial hyperinsulinism of adult presentation due to a known activating mutation in GCK. Design Clinical, biochemical and metabolic assessment, and GCK sequencing in affected family members. Results In the 60 year-old female proband, hyperinsulinaemic hypoglycaemia (blood glucose 2.1mmol/mol, insulin 18pmol/l) was confirmed following 34 hours of fasting, however abdominal computed tomography (CT), pancreatic MRI, endoscopic ultrasound, octreotide scintigraphy and selective arterial calcium stimulation failed to localise an insulinoma. A prolonged OGTT revealed fasting hypoglycaemia that was exacerbated after glucose challenge, consistent with dysregulated glucose-stimulated insulin release. A heterozygous activating mutation, p.Val389Leu, in the glucokinase gene (GCK) was found in the proband and four other family members. Of these, two had been investigated elsewhere for recurrent hypoglycaemia in adulthood, while the other two adult relatives were asymptomatic despite profound hypoglycaemia. All three of the available family members with the p.Val389Leu mutation had normal serum lipid profiles, normal rates of fasting hepatic de novo lipogenesis and had hepatic triglyceride levels commensurate with their degree of adiposity. Conclusion Activating GCK mutations may

  2. Understanding Enzyme Activity Using Single Molecule Tracking (Poster)

    SciTech Connect

    Liu, Y.-S.; Zeng, Y.; Luo, Y.; Xu, Q.; Himmel, M.; Smith S.; Wei, H.; Ding, S.-Y.

    2009-06-01

    This poster describes single-molecule tracking and total internal reflection fluorescence microscopy. It discusses whether the carbohydrate-binding module (CBM) moves on cellulose, how the CBM binds to cellulose, and the mechanism of cellulosome assembly.

  3. A small molecule modulates Jumonji histone demethylase activity and selectively inhibits cancer growth

    PubMed Central

    Wang, Lei; Chang, Jianjun; Varghese, Diana; Dellinger, Michael; Kumar, Subodh; Best, Anne M.; Ruiz, Julio; Bruick, Richard; Peña-Llopis, Samuel; Xu, Junjie; Babinski, David J.; Frantz, Doug E.; Brekken, Rolf A.; Quinn, Amy M.; Simeonov, Anton; Easmon, Johnny; Martinez, Elisabeth D.

    2013-01-01

    The pharmacological inhibition of general transcriptional regulators has the potential to block growth through targeting multiple tumorigenic signaling pathways simultaneously. Here, using an innovative cell-based screen, we identify a structurally unique small molecule (named JIB-04) which specifically inhibits the activity of the Jumonji family of histone demethylases in vitro, in cancer cells, and in tumors in vivo. Unlike known inhibitors, JIB-04 is not a competitive inhibitor of α-ketoglutarate. In cancer but not in patient-matched normal cells, JIB-04 alters a subset of transcriptional pathways and blocks viability. In mice, JIB-04 reduces tumor burden and prolongs survival. Importantly, we find that patients with breast tumors that overexpress Jumonji demethylases have significantly lower survival. Thus JIB-04, a novel inhibitor of Jumonji demethylases in vitro and in vivo, constitutes a unique potential therapeutic and research tool against cancer, and validates the use of unbiased cellular screens to discover chemical modulators with disease relevance. PMID:23792809

  4. A family of starch-active polysaccharide monooxygenases.

    PubMed

    Vu, Van V; Beeson, William T; Span, Elise A; Farquhar, Erik R; Marletta, Michael A

    2014-09-23

    The recently discovered fungal and bacterial polysaccharide monooxygenases (PMOs) are capable of oxidatively cleaving chitin, cellulose, and hemicelluloses that contain β(1→4) linkages between glucose or substituted glucose units. They are also known collectively as lytic PMOs, or LPMOs, and individually as AA9 (formerly GH61), AA10 (formerly CBM33), and AA11 enzymes. PMOs share several conserved features, including a monocopper center coordinated by a bidentate N-terminal histidine residue and another histidine ligand. A bioinformatic analysis using these conserved features suggested several potential new PMO families in the fungus Neurospora crassa that are likely to be active on novel substrates. Herein, we report on NCU08746 that contains a C-terminal starch-binding domain and an N-terminal domain of previously unknown function. Biochemical studies showed that NCU08746 requires copper, oxygen, and a source of electrons to oxidize the C1 position of glycosidic bonds in starch substrates, but not in cellulose or chitin. Starch contains α(1→4) and α(1→6) linkages and exhibits higher order structures compared with chitin and cellulose. Cellobiose dehydrogenase, the biological redox partner of cellulose-active PMOs, can serve as the electron donor for NCU08746. NCU08746 contains one copper atom per protein molecule, which is likely coordinated by two histidine ligands as shown by X-ray absorption spectroscopy and sequence analysis. Results indicate that NCU08746 and homologs are starch-active PMOs, supporting the existence of a PMO superfamily with a much broader range of substrates. Starch-active PMOs provide an expanded perspective on studies of starch metabolism and may have potential in the food and starch-based biofuel industries.

  5. Signaling Active CD95 Receptor Molecules Trigger Co-translocation of Inactive CD95 Molecules into Lipid Rafts*

    PubMed Central

    Lang, Isabell; Fick, Andrea; Schäfer, Viktoria; Giner, Tina; Siegmund, Daniela; Wajant, Harald

    2012-01-01

    The capability of soluble CD95L trimers to trigger CD95-associated signaling pathways is drastically increased by oligomerization. The latter can be achieved, for example, by antibodies recognizing a N-terminal epitope tag in recombinant CD95L variants or by genetic engineering-enforced formation of hexamers. Using highly sensitive and accurate binding studies with recombinant CD95L variants equipped with a Gaussia princeps luciferase reporter domain, we found that oligomerization of CD95L has no major effect on CD95 occupancy. This indicates that the higher activity of oligomerized CD95L trimers is not related to an avidity-related increase in apparent affinity and points instead to a crucial role of aggregation of initially formed trimeric CD95L-CD95 complexes in CD95 activation. Furthermore, binding of soluble CD95L trimers was found to be insufficient to increase the association of CD95 with the lipid raft-containing membrane fraction. However, when Gaussia princeps luciferase-CD95L trimers were used as tracers to “mark” inactive CD95 molecules, increased association of these inactive receptors was observed upon activation of the remaining CD95 molecules by help of highly active hexameric Fc-CD95L or membrane CD95L. Moreover, in cells expressing endogenous CD95 and chimeric CD40-CD95 receptors, triggering of CD95 signaling via endogenous CD95 resulted in co-translocation of CD40-CD95 to the lipid raft fraction, whereas vice versa activation of CD95-associated pathways with Fc-CD40L via CD40-CD95 resulted in co-translocation of endogenous CD95. In sum, this shows that signaling-active CD95 molecules not only enhance their own association with the lipid raft-containing membrane fraction but also those of inactive CD95 molecules. PMID:22645131

  6. Family Therapy: A Phenomenological and Active Directive Approach

    ERIC Educational Resources Information Center

    Ellis, Albert

    1978-01-01

    A "third force" in family therapy is outlined in this paper, which combines a phenomenological-humanistic approach with a highly active-directive attempt to help family members surrender their misperceptions of themselves and others and to make profound philosophic changes in their intrapersonal and interpersonal attitudes and behaviors. (Author)

  7. Drosophila chaoptin, a member of the leucine-rich repeat family, is a photoreceptor cell-specific adhesion molecule.

    PubMed Central

    Krantz, D E; Zipursky, S L

    1990-01-01

    Drosophila chaoptin, required for photoreceptor cell morphogenesis, is a member of the leucine-rich repeat family of proteins. On the basis of biochemical and genetic analyses we previously proposed that chaoptin might function as a cell adhesion molecule. To test this hypothesis, chaoptin cDNA driven by the hsp 70 promoter was transfected into non-self-adherent Drosophila Schneider line 2 (S2) cells. Following heat shock induction of chaoptin expression, the transfected S2 cells formed multicellular aggregates. Mixing experiments of chaoptin expressing and non-expressing cells suggest that chaoptin expressing cells adhere homotypically. Previously it was shown that chaoptin is exclusively localized to photoreceptor cells. Thus, chaoptin is a cell-type-specific adhesion molecule. Biochemical analyses presented in this paper demonstrate that chaoptin is linked to the extracellular surface of the plasma membrane by covalent attachment to glycosyl-phosphatidylinositol. We propose that chaoptin and several other members of the leucine-rich repeat family of proteins define a new class of cell adhesion molecules. Images Fig. 1. Fig. 2. Fig. 3. Fig. 4. Fig. 5. Fig. 6. Fig. 8. PMID:2189727

  8. Homophilic Adhesion Mechanism of Neurofascin, a Member of the L1 Family of Neural Cell Adhesion Molecules

    SciTech Connect

    Liu, Heli; Focia, Pamela J.; He, Xiaolin

    2012-02-13

    The L1 family neural cell adhesion molecules play key roles in specifying the formation and remodeling of the neural network, but their homophilic interaction that mediates adhesion is not well understood. We report two crystal structures of a dimeric form of the headpiece of neurofascin, an L1 family member. The four N-terminal Ig-like domains of neurofascin form a horseshoe shape, akin to several other immunoglobulin superfamily cell adhesion molecules such as hemolin, axonin, and Dscam. The neurofascin dimer, captured in two crystal forms with independent packing patterns, reveals a pair of horseshoes in trans-synaptic adhesion mode. The adhesion interaction is mediated mostly by the second Ig-like domain, which features an intermolecular {beta}-sheet formed by the joining of two individual GFC {beta}-sheets and a large but loosely packed hydrophobic cluster. Mutagenesis combined with gel filtration assays suggested that the side chain hydrogen bonds at the intermolecular {beta}-sheet are essential for the homophilic interaction and that the residues at the hydrophobic cluster play supplementary roles. Our structures reveal a conserved homophilic adhesion mode for the L1 family and also shed light on how the pathological mutations of L1 affect its structure and function.

  9. Homophilic adhesion mechanism of neurofascin, a member of the L1 family of neural cell adhesion molecules.

    PubMed

    Liu, Heli; Focia, Pamela J; He, Xiaolin

    2011-01-01

    The L1 family neural cell adhesion molecules play key roles in specifying the formation and remodeling of the neural network, but their homophilic interaction that mediates adhesion is not well understood. We report two crystal structures of a dimeric form of the headpiece of neurofascin, an L1 family member. The four N-terminal Ig-like domains of neurofascin form a horseshoe shape, akin to several other immunoglobulin superfamily cell adhesion molecules such as hemolin, axonin, and Dscam. The neurofascin dimer, captured in two crystal forms with independent packing patterns, reveals a pair of horseshoes in trans-synaptic adhesion mode. The adhesion interaction is mediated mostly by the second Ig-like domain, which features an intermolecular β-sheet formed by the joining of two individual GFC β-sheets and a large but loosely packed hydrophobic cluster. Mutagenesis combined with gel filtration assays suggested that the side chain hydrogen bonds at the intermolecular β-sheet are essential for the homophilic interaction and that the residues at the hydrophobic cluster play supplementary roles. Our structures reveal a conserved homophilic adhesion mode for the L1 family and also shed light on how the pathological mutations of L1 affect its structure and function. PMID:21047790

  10. Persistently Active Microbial Molecules Prolong Innate Immune Tolerance In Vivo

    PubMed Central

    Lu, Mingfang; Varley, Alan W.; Munford, Robert S.

    2013-01-01

    Measures that bolster the resolution phase of infectious diseases may offer new opportunities for improving outcome. Here we show that inactivation of microbial lipopolysaccharides (LPS) can be required for animals to recover from the innate immune tolerance that follows exposure to Gram-negative bacteria. When wildtype mice are exposed to small parenteral doses of LPS or Gram-negative bacteria, their macrophages become reprogrammed (tolerant) for a few days before they resume normal function. Mice that are unable to inactivate LPS, in contrast, remain tolerant for several months; during this time they respond sluggishly to Gram-negative bacterial challenge, with high mortality. We show here that prolonged macrophage reprogramming is maintained in vivo by the persistence of stimulatory LPS molecules within the cells' in vivo environment, where naïve cells can acquire LPS via cell-cell contact or from the extracellular fluid. The findings provide strong evidence that inactivation of a stimulatory microbial molecule can be required for animals to regain immune homeostasis following parenteral exposure to bacteria. Measures that disable microbial molecules might enhance resolution of tissue inflammation and help restore innate defenses in individuals recovering from many different infectious diseases. PMID:23675296

  11. 5 Family Fitness Activities to Try Today | Smokefree.gov

    Cancer.gov

    No time to exercise and spend quality time with your kids? Feeling like there is no way to squeeze in exercise and make room for fun time with the family? Do both—together! Being active with your family can be a fun way to get moving. You’ll be a great role model for your kids, and it’s a good way to help the important people in your life live healthy, active lives.

  12. Activation of specific apoptotic caspases with an engineered small-molecule-activated protease.

    PubMed

    Gray, Daniel C; Mahrus, Sami; Wells, James A

    2010-08-20

    Apoptosis is a conserved cellular pathway that results in the activation of cysteine-aspartyl proteases, or caspases. To dissect the nonredundant roles of the executioner caspase-3, -6, and -7 in orchestrating apoptosis, we have developed an orthogonal protease to selectively activate each isoform in human cells. Our approach uses a split-tobacco etch virus (TEV) protease under small-molecule control, which we call the SNIPer, with caspase alleles containing genetically encoded TEV cleavage sites. These studies reveal that all three caspases are transiently activated but only activation of caspase-3 or -7 is sufficient to induce apoptosis. Proteomic analysis shown here and from others reveals that 20 of the 33 subunits of the 26S proteasome can be cut by caspases, and we demonstrate synergy between proteasome inhibition and dose-dependent caspase activation. We propose a model of proteolytic reciprocal negative regulation with mechanistic implications for the combined clinical use of proteasome inhibitors and proapoptotic drugs.

  13. Crystallographic structure of a small molecule SIRT1 activator-enzyme complex

    PubMed Central

    Dai, Han; Case, April W.; Riera, Thomas V.; Considine, Thomas; Lee, Jessica E.; Hamuro, Yoshitomo; Zhao, Huizhen; Jiang, Yong; Sweitzer, Sharon M.; Pietrak, Beth; Schwartz, Benjamin; Blum, Charles A.; Disch, Jeremy S.; Caldwell, Richard; Szczepankiewicz, Bruce; Oalmann, Christopher; Yee Ng, Pui; White, Brian H.; Casaubon, Rebecca; Narayan, Radha; Koppetsch, Karsten; Bourbonais, Francis; Wu, Bo; Wang, Junfeng; Qian, Dongming; Jiang, Fan; Mao, Cheney; Wang, Minghui; Hu, Erding; Wu, Joe C.; Perni, Robert B.; Vlasuk, George P.; Ellis, James L.

    2015-01-01

    SIRT1, the founding member of the mammalian family of seven NAD+-dependent sirtuins, is composed of 747 amino acids forming a catalytic domain and extended N- and C-terminal regions. We report the design and characterization of an engineered human SIRT1 construct (mini-hSIRT1) containing the minimal structural elements required for lysine deacetylation and catalytic activation by small molecule sirtuin-activating compounds (STACs). Using this construct, we solved the crystal structure of a mini-hSIRT1-STAC complex, which revealed the STAC-binding site within the N-terminal domain of hSIRT1. Together with hydrogen-deuterium exchange mass spectrometry (HDX-MS) and site-directed mutagenesis using full-length hSIRT1, these data establish a specific STAC-binding site and identify key intermolecular interactions with hSIRT1. The determination of the interface governing the binding of STACs with human SIRT1 facilitates greater understanding of STAC activation of this enzyme, which holds significant promise as a therapeutic target for multiple human diseases. PMID:26134520

  14. Crystallographic structure of a small molecule SIRT1 activator-enzyme complex.

    PubMed

    Dai, Han; Case, April W; Riera, Thomas V; Considine, Thomas; Lee, Jessica E; Hamuro, Yoshitomo; Zhao, Huizhen; Jiang, Yong; Sweitzer, Sharon M; Pietrak, Beth; Schwartz, Benjamin; Blum, Charles A; Disch, Jeremy S; Caldwell, Richard; Szczepankiewicz, Bruce; Oalmann, Christopher; Yee Ng, Pui; White, Brian H; Casaubon, Rebecca; Narayan, Radha; Koppetsch, Karsten; Bourbonais, Francis; Wu, Bo; Wang, Junfeng; Qian, Dongming; Jiang, Fan; Mao, Cheney; Wang, Minghui; Hu, Erding; Wu, Joe C; Perni, Robert B; Vlasuk, George P; Ellis, James L

    2015-07-02

    SIRT1, the founding member of the mammalian family of seven NAD(+)-dependent sirtuins, is composed of 747 amino acids forming a catalytic domain and extended N- and C-terminal regions. We report the design and characterization of an engineered human SIRT1 construct (mini-hSIRT1) containing the minimal structural elements required for lysine deacetylation and catalytic activation by small molecule sirtuin-activating compounds (STACs). Using this construct, we solved the crystal structure of a mini-hSIRT1-STAC complex, which revealed the STAC-binding site within the N-terminal domain of hSIRT1. Together with hydrogen-deuterium exchange mass spectrometry (HDX-MS) and site-directed mutagenesis using full-length hSIRT1, these data establish a specific STAC-binding site and identify key intermolecular interactions with hSIRT1. The determination of the interface governing the binding of STACs with human SIRT1 facilitates greater understanding of STAC activation of this enzyme, which holds significant promise as a therapeutic target for multiple human diseases.

  15. Crystallographic structure of a small molecule SIRT1 activator-enzyme complex

    NASA Astrophysics Data System (ADS)

    Dai, Han; Case, April W.; Riera, Thomas V.; Considine, Thomas; Lee, Jessica E.; Hamuro, Yoshitomo; Zhao, Huizhen; Jiang, Yong; Sweitzer, Sharon M.; Pietrak, Beth; Schwartz, Benjamin; Blum, Charles A.; Disch, Jeremy S.; Caldwell, Richard; Szczepankiewicz, Bruce; Oalmann, Christopher; Yee Ng, Pui; White, Brian H.; Casaubon, Rebecca; Narayan, Radha; Koppetsch, Karsten; Bourbonais, Francis; Wu, Bo; Wang, Junfeng; Qian, Dongming; Jiang, Fan; Mao, Cheney; Wang, Minghui; Hu, Erding; Wu, Joe C.; Perni, Robert B.; Vlasuk, George P.; Ellis, James L.

    2015-07-01

    SIRT1, the founding member of the mammalian family of seven NAD+-dependent sirtuins, is composed of 747 amino acids forming a catalytic domain and extended N- and C-terminal regions. We report the design and characterization of an engineered human SIRT1 construct (mini-hSIRT1) containing the minimal structural elements required for lysine deacetylation and catalytic activation by small molecule sirtuin-activating compounds (STACs). Using this construct, we solved the crystal structure of a mini-hSIRT1-STAC complex, which revealed the STAC-binding site within the N-terminal domain of hSIRT1. Together with hydrogen-deuterium exchange mass spectrometry (HDX-MS) and site-directed mutagenesis using full-length hSIRT1, these data establish a specific STAC-binding site and identify key intermolecular interactions with hSIRT1. The determination of the interface governing the binding of STACs with human SIRT1 facilitates greater understanding of STAC activation of this enzyme, which holds significant promise as a therapeutic target for multiple human diseases.

  16. Income-generating activities for family planning acceptors.

    PubMed

    1989-07-01

    The Income Generating Activities program for Family Planning Acceptors was introduced in Indonesia in 1979. Capital input by the Indonesian National Family Planning Coordination Board and the UN Fund for Population Activities was used to set up small businesses by family planning acceptors. In 2 years, when the businesses become self-sufficient, the loans are repaid, and the money is used to set up new family planning acceptors in business. The program strengthens family planning acceptance, improves the status of women, and enhances community self-reliance. The increase in household income generated by the program raises the standards of child nutrition, encourages reliance on the survival of children, and decreases the value of large families. Approximately 18,000 Family Planning-Income Generating Activities groups are now functioning all over Indonesia, with financial assistance from the central and local governments, the World Bank, the US Agency for International Development, the UN Population Fund, the Government of the Netherlands, and the Government of Australia through the Association of South East Asian Nations.

  17. A family of variable immunoglobulin and lect in domain containing molecules in the snail Biomphalaria glabrata

    PubMed Central

    Dheilly, Nolwenn M; Duval, David; Mouahid, Gabriel; Emans, Rémi; Allienne, Jean-François; Galinier, Richard; Genthon, Clémence; Dubois, Emeric; Pasquier, Louis Du; Adema, Coen M; Grunau, Christoph; Mitta, Guillaume; Gourbal, Benjamin

    2014-01-01

    Technical limitations have hindered comprehensive studies of highly variable immune response molecules that are thought to have evolved due to pathogen-mediated selection such as Fibrinogen-related proteins (FREPs) from Biomphalaria glabrata. FREPs combine upstream immunoglobulin superfamily (IgSF) domains with a C-terminal fibrinogen-related domain (FreD) and participate in reactions against trematode parasites. From RNAseq data we assembled a de novo reference transcriptome of B. glabrata to investigate the diversity of FREP transcripts. This study increased over two-fold the number of bonafide FREP subfamilies and revealed important sequence diversity within FREP12 subfamily. We also report the discovery of related molecules that feature one or two IgSF domains associated with different C-terminal lectin domains, named C-type lectin-related proteins (CREPs) and Galectin-related protein (GREP). Together, the highly similar FREPs, CREPs and GREP were designated VIgL (Variable Immunoglobulin and Lectin domain containing molecules). PMID:25451302

  18. Combined single channel and single molecule detection identifies subunit composition of STIM1-activated transient receptor potential canonical (TRPC) channels.

    PubMed

    Asanov, Alexander; Sampieri, Alicia; Moreno, Claudia; Pacheco, Jonathan; Salgado, Alfonso; Sherry, Ryan; Vaca, Luis

    2015-01-01

    Depletion of intracellular calcium ion stores initiates a rapid cascade of events culminating with the activation of the so-called Store-Operated Channels (SOC) at the plasma membrane. Calcium influx via SOC is essential in the initiation of calcium-dependent intracellular signaling and for the refilling of internal calcium stores, ensuring the regeneration of the signaling cascade. In spite of the significance of this evolutionary conserved mechanism, the molecular identity of SOC has been the center of a heated controversy spanning over the last 20 years. Initial studies positioned some members of the transient receptor potential canonical (TRPC) channel superfamily of channels (with the more robust evidence pointing to TRPC1) as a putative SOC. Recent evidence indicates that Stromal Interacting Molecule 1 (STIM1) activates some members from the TRPC family of channels. However, the exact subunit composition of TRPC channels remains undetermined to this date. To identify the subunit composition of STIM1-activated TRPC channels, we developed novel method, which combines single channel electrophysiological measurements based on the patch clamp technique with single molecule fluorescence imaging. We termed this method Single ion Channel Single Molecule Detection technique (SC-SMD). Using SC-SMD method, we have obtained direct evidence of the subunit composition of TRPC channels activated by STIM1. Furthermore, our electrophysiological-imaging SC-SMD method provides evidence at the molecular level of the mechanism by which STIM1 and calmodulin antagonize to modulate TRPC channel activity.

  19. STEM learning activity among home-educating families

    NASA Astrophysics Data System (ADS)

    Bachman, Jennifer

    2011-12-01

    Science, technology, engineering, and mathematics (STEM) learning was studied among families in a group of home-educators in the Pacific Northwest. Ethnographic methods recorded learning activity (video, audio, fieldnotes, and artifacts) which was analyzed using a unique combination of Cultural-Historical Activity Theory (CHAT) and Mediated Action (MA), enabling analysis of activity at multiple levels. Findings indicate that STEM learning activity is family-led, guided by parents' values and goals for learning, and negotiated with children to account for learner interests and differences, and available resources. Families' STEM education practice is dynamic, evolves, and influenced by larger societal STEM learning activity. Parents actively seek support and resources for STEM learning within their home-school community, working individually and collectively to share their funds of knowledge. Home-schoolers also access a wide variety of free-choice learning resources: web-based materials, museums, libraries, and community education opportunities (e.g. afterschool, weekend and summer programs, science clubs and classes, etc.). A lesson-heuristic, grounded in Mediated Action, represents and analyzes home STEM learning activity in terms of tensions between parental goals, roles, and lesson structure. One tension observed was between 'academic' goals or school-like activity and 'lifelong' goals or everyday learning activity. Theoretical and experiential learning was found in both activity, though parents with academic goals tended to focus more on theoretical learning and those with lifelong learning goals tended to be more experiential. Examples of the National Research Council's science learning strands (NRC, 2009) were observed in the STEM practices of all these families. Findings contribute to the small but growing body of empirical CHAT research in science education, specifically to the empirical base of family STEM learning practices at home. It also fills a

  20. Identification of small molecule agonists of human relaxin family receptor 1 (RXFP1) by utilizing a homogenous cell-based cAMP assay

    PubMed Central

    Chen, Catherine Z.; Southall, Noel; Xiao, Jingbo; Marugan, Juan J.; Ferrer, Marc; Hu, Xin; Jones, Raisa E.; Feng, Shu; Agoulnik, Irina U.

    2016-01-01

    The relaxin hormone is involved in a variety of biological functions including female reproduction and parturition, regulation of cardiovascular, renal, pulmonary, and hepatic functions. It regulates extracellular matrix remodeling, cell invasiveness, proliferation, differentiation, and overall tissue homeostasis. The G protein-coupled receptor (GPCR) RXFP1, relaxin family receptor 1, is a cognate relaxin receptor that mainly signals through cyclic AMP second messenger. While agonists of the receptor could have a wide range of pharmacological utility, up to date, there are no reported small molecule agonists for relaxin receptors. Here, we report the development of quantitative high-throughput platform for RXFP1 agonist screen based on homogenous cell-based HTRF cAMP assay technology. Two small molecules of similar structure were independently identified from a screen of more than 365,677 compounds. Neither compound showed activity in a counter screen with HEK293T cells transfected with an unrelated GPCR vasopressin 1b receptor. These small molecule agonists also demonstrated selectivity against the RXFP2 receptor, providing a basis for future medicinal chemistry optimization of selective relaxin receptor agonists. PMID:23212924

  1. High quality, small molecule-activity datasets for kinase research.

    PubMed

    Sharma, Rajan; Schürer, Stephan C; Muskal, Steven M

    2016-01-01

    Kinases regulate cell growth, movement, and death. Deregulated kinase activity is a frequent cause of disease. The therapeutic potential of kinase inhibitors has led to large amounts of published structure activity relationship (SAR) data. Bioactivity databases such as the Kinase Knowledgebase (KKB), WOMBAT, GOSTAR, and ChEMBL provide researchers with quantitative data characterizing the activity of compounds across many biological assays. The KKB, for example, contains over 1.8M kinase structure-activity data points reported in peer-reviewed journals and patents. In the spirit of fostering methods development and validation worldwide, we have extracted and have made available from the KKB 258K structure activity data points and 76K associated unique chemical structures across eight kinase targets. These data are freely available for download within this data note. PMID:27429748

  2. High quality, small molecule-activity datasets for kinase research

    PubMed Central

    Sharma, Rajan; Schürer, Stephan C.; Muskal, Steven M.

    2016-01-01

    Kinases regulate cell growth, movement, and death. Deregulated kinase activity is a frequent cause of disease. The therapeutic potential of kinase inhibitors has led to large amounts of published structure activity relationship (SAR) data. Bioactivity databases such as the Kinase Knowledgebase (KKB), WOMBAT, GOSTAR, and ChEMBL provide researchers with quantitative data characterizing the activity of compounds across many biological assays. The KKB, for example, contains over 1.8M kinase structure-activity data points reported in peer-reviewed journals and patents. In the spirit of fostering methods development and validation worldwide, we have extracted and have made available from the KKB 258K structure activity data points and 76K associated unique chemical structures across eight kinase targets. These data are freely available for download within this data note. PMID:27429748

  3. Selective inhibition of the Kir2 family of inward rectifier potassium channels by a small molecule probe: the discovery, SAR and pharmacological characterization of ML133

    PubMed Central

    Wang, Hao-Ran; Wu, Meng; Yu, Haibo; Long, Shunyou; Stevens, Amy; Engers, Darren W.; Sackin, Henry; Daniels, J. Scott; Dawson, Eric S.; Hopkins, Corey R.; Lindsley, Craig W.; Li, Min; McManus, Owen B

    2011-01-01

    The Kir inward rectifying potassium channels have a broad tissue distribution and are implicated in a variety of functional roles. At least seven classes (Kir1 – Kir7) of structurally related inward rectifier potassium channels are known, and there are no selective small molecule tools to study their function. In an effort to develop selective Kir2.1 inhibitors, we performed a high-throughput screen (HTS) of more than 300,000 small molecules within the MLPCN for modulators of Kir2.1 function. Here we report one potent Kir2.1 inhibitor, ML133, which inhibits Kir2.1 with IC50 of 1.8 μM at pH 7.4 and 290 nM at pH 8.5, but exhibits little selectivity against other members of Kir2.x family channels. However, ML133 has no effect on Kir1.1 (IC50 > 300 μM), and displays weak activity for Kir4.1 (76 μM) and Kir7.1 (33 μM), making ML133 the most selective small molecule inhibitor of the Kir family reported to date. Due to the high homology within the Kir family, the channels share a common design of a pore region flanked by two transmembrane domains, identification of site(s) critical for isoform specificity would be an important basis for future development of more specific and potent Kir inhibitors. Using chimeric channels between Kir2.1 and Kir1.1 and site-directed mutagenesis, we have identified D172 and I176 within M2 segment of Kir2.1 as molecular determinants critical for the potency of ML133 mediated inhibition. Double mutation of the corresponding residues of Kir1.1 to those of Kir2.1 (N171D and C175I) transplants ML133 inhibition to Kir1.1. Together, the combination of a potent, Kir2 family selective inhibitor and identification of molecular determinants for the specificity provides both a tool and a model system to enable further mechanistic studies of modulation of Kir2 inward rectifier potassium channels. PMID:21615117

  4. Methods for Detection of Families of Molecules in the Interstellar Medium

    NASA Astrophysics Data System (ADS)

    Langston, Glen

    2014-06-01

    We present a high velocity resolution (0.04 km/sec) molecular line survey of the Taurus Molecular Cloud in the frequency range 39 to 48 GHz with NSF's Robert C. Byrd Green Bank telescope (GBT). The observing method and data reduction process are outlined. We describe the method of obtaining the calibrated, averaged spectral line data online. The RMS survey sensitivity was slightly different for each 200MHz frequency band, and ranged from 0.02 to 0.15 K (T_B) for the different bands. A large number of molecular lines are detected, most of which have previously been associated with already known interstellar molecules. We present a summary processes to combine a number of lines of molecular species in order to identify new species.

  5. The L1 family of cell adhesion molecules: a sickening number of mutations and protein functions.

    PubMed

    Hortsch, Michael; Nagaraj, Kakanahalli; Mualla, Rula

    2014-01-01

    L1-type proteins are transmembrane cell adhesion molecules with an evolutionary well-conserved protein domain structure of usually six immunoglobulin and five fibronectin type III domains. By engaging in many different protein-protein interactions they are involved in a multitude of molecular functions and are important players during the formation and maintenance of metazoan nervous systems. As a result, mutations in L1-type genes cause a great variety of phenotypes, most of which are neurological in nature. In humans, mutations in the L1CAM gene are responsible for L1 syndrome and other L1-type genes have been implicated in conditions as varied as mental retardation, autism, schizophrenia, multiple sclerosis, and other disorders. Equally, the overexpression of L1-type proteins appears to have deleterious effects in various types of human tumor cells, where they generally contribute to an increase in cell mobility and metastatic potential. PMID:25300138

  6. Astronomy Outreach Activities for Special Needs Children and Their Families

    NASA Astrophysics Data System (ADS)

    Lubowich, Donald A.

    2008-05-01

    While there are outreach programs for the public and for children, there are few programs for special needs children. Here I describe two NASA-IDEAS/STSc funded outreach programs I created for special needs children using telescope observations (including remote and robotic observations) and hands-on astronomy activities. The target audience is seriously ill children and their families who are staying at the Ronald McDonald House of Long Island in conjunction the children's surgery and medical treatments or children hospitalized at the Hagedorn Pediatric Inpatient Center at Winthrop University Hospital. These educational activities help children and their families learn about astronomy while providing a diversion to take their minds off their illness during a stressful time. I have also conducted a similar program in camps for special needs children. These programs should be expanded so that special needs children and their families part of the IYA 2009 activities.

  7. Astronomy Outreach Activities for Special Needs Children and Their Families

    NASA Astrophysics Data System (ADS)

    Lubowich, D.

    2008-11-01

    While there are outreach programs for the public and for children, there are few programs for special needs children. Here I describe two NASA-IDEAS/STScI funded outreach programs I created for special needs children using telescope observations (including remote observations) and hands-on astronomy activities. The target audience is seriously ill children and their families who are staying at the Ronald McDonald House of Long Island in conjunction the children's medical treatments or children hospitalized at the Children's Medical Center at Winthrop University Hospital. These educational activities help children and their families learn about astronomy while providing a diversion to take their minds off their illness during a stressful time. I have also conducted a similar program in camps for special needs children. These programs should be expanded so that special needs children and their families are part of the IYA2009 activities.

  8. Association between Family Structure and Physical Activity of Chinese Adolescents

    PubMed Central

    Wang, Lijuan; Qi, Jing

    2016-01-01

    Background. This study examines the association between family structure and moderate-to-vigorous physical activity (MVPA) of adolescents in China. Methods. The participants included 612 adolescents (317 boys and 295 girls) from Shanghai with ages ranging from 10 to 16 years. Accelerometers were used to measure the duration of MVPA of adolescents, and questionnaires on family structure were completed by the parents of these adolescents. Results. Findings suggested that family structure significantly increased the likelihood of adolescents engaging in physical activity (PA) and explained 6% of MPVA variance. Adolescents living in single-parent households and step families were more physically active than those living in two-parent homes and with biological parents, respectively. However, adolescents residing with grandparents were less active than those living with neither grandparent. No significant difference was found in MVPA time between adolescents living with one sibling and those without siblings. Conclusion. Family environment may be considered in the development of PA interventions and policies, and adolescents living with their grandparents may be targeted in PA promotion. PMID:27123446

  9. Family Ecological Predictors of Physical Activity Parenting in Low-Income Families

    ERIC Educational Resources Information Center

    Lampard, Amy M.; Jurkowski, Janine M.; Lawson, Hal A.; Davison, Kirsten K.

    2013-01-01

    Physical activity (PA) parenting, or strategies parents use to promote PA in children, has been associated with increased PA in children of all ages, including preschool-aged children. However, little is known about the circumstances under which parents adopt such behaviors. This study examined family ecological factors associated with PA…

  10. Canine and feline trefoil factor family peptides: highly conserved molecules with some unique characteristics.

    PubMed

    Campbell, B G; Jabbes, M

    2008-08-01

    Trefoil factor family (TFF) peptides maintain and repair the gastrointestinal mucosa and are aberrantly expressed in human and rodent inflammatory bowel disease and carcinomas, diseases common in dogs and cats. Study objectives were to sequence and translate canine and feline tff cDNAs and define any unique residues that might influence their structure and/or function. After isolation and reverse transcription of canine and feline gastrointestinal mucosal RNA, TFF cDNAs were amplified, sequenced, and cloned. Dogs and cats had unique amino acids in several places that were highly or completely conserved in other mammals, including a hydrophobic area in the TFF1 functional site, loop 2 of each TFF2 trefoil domain, a TFF3 dimerization site, and the TFF2 C-terminus. By identifying conserved and unique characteristics of canine and feline TFFs, this study establishes a foundation for investigation of dog and cat models of TFF-related diseases in both human and veterinary medicine.

  11. Small molecule activation of NOTCH signaling inhibits acute myeloid leukemia.

    PubMed

    Ye, Qi; Jiang, Jue; Zhan, Guanqun; Yan, Wanyao; Huang, Liang; Hu, Yufeng; Su, Hexiu; Tong, Qingyi; Yue, Ming; Li, Hua; Yao, Guangmin; Zhang, Yonghui; Liu, Hudan

    2016-01-01

    Aberrant activation of the NOTCH signaling pathway is crucial for the onset and progression of T cell leukemia. Yet recent studies also suggest a tumor suppressive role of NOTCH signaling in acute myeloid leukemia (AML) and reactivation of this pathway offers an attractive opportunity for anti-AML therapies. N-methylhemeanthidine chloride (NMHC) is a novel Amaryllidaceae alkaloid that we previously isolated from Zephyranthes candida, exhibiting inhibitory activities in a variety of cancer cells, particularly those from AML. Here, we report NMHC not only selectively inhibits AML cell proliferation in vitro but also hampers tumor development in a human AML xenograft model. Genome-wide gene expression profiling reveals that NMHC activates the NOTCH signaling. Combination of NMHC and recombinant human NOTCH ligand DLL4 achieves a remarkable synergistic effect on NOTCH activation. Moreover, pre-inhibition of NOTCH by overexpression of dominant negative MAML alleviates NMHC-mediated cytotoxicity in AML. Further mechanistic analysis using structure-based molecular modeling as well as biochemical assays demonstrates that NMHC docks in the hydrophobic cavity within the NOTCH1 negative regulatory region (NRR), thus promoting NOTCH1 proteolytic cleavage. Our findings thus establish NMHC as a potential NOTCH agonist that holds great promises for future development as a novel agent beneficial to patients with AML. PMID:27211848

  12. Small molecule activation of NOTCH signaling inhibits acute myeloid leukemia

    PubMed Central

    Ye, Qi; Jiang, Jue; Zhan, Guanqun; Yan, Wanyao; Huang, Liang; Hu, Yufeng; Su, Hexiu; Tong, Qingyi; Yue, Ming; Li, Hua; Yao, Guangmin; Zhang, Yonghui; Liu, Hudan

    2016-01-01

    Aberrant activation of the NOTCH signaling pathway is crucial for the onset and progression of T cell leukemia. Yet recent studies also suggest a tumor suppressive role of NOTCH signaling in acute myeloid leukemia (AML) and reactivation of this pathway offers an attractive opportunity for anti-AML therapies. N-methylhemeanthidine chloride (NMHC) is a novel Amaryllidaceae alkaloid that we previously isolated from Zephyranthes candida, exhibiting inhibitory activities in a variety of cancer cells, particularly those from AML. Here, we report NMHC not only selectively inhibits AML cell proliferation in vitro but also hampers tumor development in a human AML xenograft model. Genome-wide gene expression profiling reveals that NMHC activates the NOTCH signaling. Combination of NMHC and recombinant human NOTCH ligand DLL4 achieves a remarkable synergistic effect on NOTCH activation. Moreover, pre-inhibition of NOTCH by overexpression of dominant negative MAML alleviates NMHC-mediated cytotoxicity in AML. Further mechanistic analysis using structure-based molecular modeling as well as biochemical assays demonstrates that NMHC docks in the hydrophobic cavity within the NOTCH1 negative regulatory region (NRR), thus promoting NOTCH1 proteolytic cleavage. Our findings thus establish NMHC as a potential NOTCH agonist that holds great promises for future development as a novel agent beneficial to patients with AML. PMID:27211848

  13. Small-molecule inhibition of MLL activity by disruption of its interaction with WDR5.

    PubMed

    Senisterra, Guillermo; Wu, Hong; Allali-Hassani, Abdellah; Wasney, Gregory A; Barsyte-Lovejoy, Dalia; Dombrovski, Ludmila; Dong, Aiping; Nguyen, Kong T; Smil, David; Bolshan, Yuri; Hajian, Taraneh; He, Hao; Seitova, Alma; Chau, Irene; Li, Fengling; Poda, Gennadiy; Couture, Jean-François; Brown, Peter J; Al-Awar, Rima; Schapira, Matthieu; Arrowsmith, Cheryl H; Vedadi, Masoud

    2013-01-01

    WDR5 (WD40 repeat protein 5) is an essential component of the human trithorax-like family of SET1 [Su(var)3-9 enhancer-of-zeste trithorax 1] methyltransferase complexes that carry out trimethylation of histone 3 Lys4 (H3K4me3), play key roles in development and are abnormally expressed in many cancers. In the present study, we show that the interaction between WDR5 and peptides from the catalytic domain of MLL (mixed-lineage leukaemia protein) (KMT2) can be antagonized with a small molecule. Structural and biophysical analysis show that this antagonist binds in the WDR5 peptide-binding pocket with a Kd of 450 nM and inhibits the catalytic activity of the MLL core complex in vitro. The degree of inhibition was enhanced at lower protein concentrations consistent with a role for WDR5 in directly stabilizing the MLL multiprotein complex. Our data demonstrate inhibition of an important protein-protein interaction and form the basis for further development of inhibitors of WDR5-dependent enzymes implicated in MLL-rearranged leukaemias or other cancers.

  14. Small-molecule inhibition of MLL activity by disruption of its interaction with WDR5

    PubMed Central

    Senisterra, Guillermo; Wu, Hong; Allali-Hassani, Abdellah; Wasney, Gregory A.; Barsyte-Lovejoy, Dalia; Dombrovski, Ludmila; Dong, Aiping; Nguyen, Kong T.; Smil, David; Bolshan, Yuri; Hajian, Taraneh; He, Hao; Seitova, Alma; Chau, Irene; Li, Fengling; Poda, Gennadiy; Couture, Jean-François; Brown, Peter J.; Al-Awar, Rima; Schapira, Matthieu; Arrowsmith, Cheryl H.; Vedadi, Masoud

    2012-01-01

    WDR5 (WD40 repeat protein 5) is an essential component of the human trithorax-like family of SET1 [Su(var)3–9 enhancer-of-zeste trithorax 1] methyltransferase complexes that carry out trimethylation of histone 3 Lys4 (H3K4me3), play key roles in development and are abnormally expressed in many cancers. In the present study, we show that the interaction between WDR5 and peptides from the catalytic domain of MLL (mixed-lineage leukaemia protein) (KMT2) can be antagonized with a small molecule. Structural and biophysical analysis show that this antagonist binds in the WDR5 peptide-binding pocket with a Kd of 450 nM and inhibits the catalytic activity of the MLL core complex in vitro. The degree of inhibition was enhanced at lower protein concentrations consistent with a role for WDR5 in directly stabilizing the MLL multiprotein complex. Our data demonstrate inhibition of an important protein–protein interaction and form the basis for further development of inhibitors of WDR5-dependent enzymes implicated in MLL-rearranged leukaemias or other cancers. PMID:22989411

  15. Cardenolides from the Apocynaceae family and their anticancer activity.

    PubMed

    Wen, Shiyuan; Chen, Yanyan; Lu, Yunfang; Wang, Yuefei; Ding, Liqin; Jiang, Miaomiao

    2016-07-01

    Cardenolides, as a group of natural products that can bind to Na(+)/K(+)-ATPase with an inhibiting activity, are traditionally used to treat congestive heart failure. Recent studies have demonstrated that the strong tumor cytotoxicities of cardenolides are mainly due to inducing the tumor cells apoptosis through different expression and cellular location of Na(+)/K(+)-ATPase α-subunits. The leaves, flesh, seeds and juices of numerous plants from the genera of Nerium, Thevetia, Cerbera, Apocynum and Strophanthus in Apocynaceae family, are the major sources of natural cardenolides. So far, 109 cardenolides have been isolated and identified from this family, and about a quarter of them are reported to exhibit the capability to regulate cancer cell survival and death through multiple signaling pathways. In this review, we compile the phytochemical characteristics and anticancer activity of the cardenolides from this family. PMID:27167183

  16. Features of the electronic structure of the active center of an HbS molecule

    NASA Astrophysics Data System (ADS)

    Novoselov, D. Yu.; Korotin, Dm. M.; Anisimov, V. I.

    2016-01-01

    Features of the electronic structure of the nonprotein part of the mutant form of the human hemoglobin molecule, HbS, are studied along with the magnetic state of the iron ion that is the "nucleus" of the active center of the molecule. It is found that the mutant form of the HbS molecule differs from a normal hemoglobin molecule by the distortion of the local environment of the iron ion, which changes the energy level splitting by a crystal field. As a result of ab initio calculations, the magnetic transition in the iron atom from the high-spin state to the low-spin state upon the addition of molecular oxygen to hemoglobin molecule is reproduced. It is established for the first time that a change in the crystal and electronic structure of the active center as a result of a mutation can lead to a substantial change in the energy of the bond between the active center of the hemoglobin molecule and an oxygen molecule.

  17. Interaction of metallic clusters with biologically active curcumin molecules

    NASA Astrophysics Data System (ADS)

    Gupta, Sanjeev K.; He, Haiying; Liu, Chunhui; Dutta, Ranu; Pandey, Ravindra

    2015-09-01

    We have investigated the interaction of subnano metallic Gd and Au clusters with curcumin, an important biomolecule having pharmacological activity. Gd clusters show different site preference to curcumin and much stronger interaction strength, in support of the successful synthesis of highly stable curcumin-coated Gd nanoparticles as reported recently. It can be attributed to significant charge transfer from the Gd cluster to curcumin together with a relatively strong hybridization of the Gd df-orbitals with curcumin p-orbitals. These results suggest that Gd nanoparticles can effectively be used as delivery carriers for curcumin at the cellular level for therapy and medical imaging applications.

  18. Ozone: A Multifaceted Molecule with Unexpected Therapeutic Activity.

    PubMed

    Zanardi, I; Borrelli, E; Valacchi, G; Travagli, V; Bocci, V

    2016-01-01

    A comprehensive outline for understanding and recommending the therapeutic use of ozone in combination with established therapy in diseases characterized by a chronic oxidative stress is currently available. The view of the absolute ozone toxicity is incorrect, because it has been based either on lung or on studies performed in artificial environments that do not correspond to the real antioxidant capacity of body compartments. In fact, ozone exerts either a potent toxic activity or it can stimulate biological responses of vital importance, analogously to gases with prospective therapeutic value such as NO, CO, H2S, H2, as well as O2 itself. Such a crucial difference has increasingly become evident during the last decade. The purpose of this review is to explain the aspects still poorly understood, highlighting the divergent activity of ozone on the various biological districts. It will be clarified that such a dual effect does not depend only upon the final gas concentration, but also on the particular biological system where ozone acts. The real significance of ozone as adjuvant therapeutic treatment concerns severe chronic pathologies among which are cardiovascular diseases, chronic obstructive pulmonary diseases, multiple sclerosis, and the dry form of age-related macular degeneration. It is time for a full insertion of ozone therapy within pharmaceutical sciences, responding to all the requirements of quality, efficacy and safety, rather than as either an alternative or an esoteric approach.

  19. Bacterial peptidoglycan-derived molecules activate Candida albicans hyphal growth.

    PubMed

    Wang, Yue; Xu, Xiao-Li

    2008-01-01

    Serum strongly induces the yeast-to-hypha growth transition in the human fungal pathogen Candida albicans, playing an important role in infection. However, identity of the serum inducer(s) and its sensor remain poorly defined. We used NMR to analyze the chromatographic serum fractionations enriched for the hypha-inducing activity and found structures resembling subunits of bacterial peptidoglycan (PGN). We then confirmed that several purified and synthetic muramyl dipeptides (MDPs), subunits of PGN, can indeed strongly promote C. albicans hyphal growth. Taking cue from the recognition of MDPs by the mammalian bacterial sensor Nod2 using its leucine-rich-repeat (LRR) domain, we discovered that MDPs activate the adenylyl cyclase Cyr1 by binding to its LRR domain. The cAMP/PKA signaling pathway is well known to control hyphal morphogenesis and other infection-related traits. Given the abundance of PGN at the large intestinal epithelial surface, a natural habitat and invasion site for C. albcians, our findings have important implications in the mechanisms of infection by this pathogen. PMID:19704871

  20. Bacterial peptidoglycan-derived molecules activate Candida albicans hyphal growth

    PubMed Central

    Xu, Xiao-Li

    2008-01-01

    Serum strongly induces the yeast-to-hypha growth transition in the human fungal pathogen Candida albicans, playing an important role in infection. However, identity of the serum inducer(s) and its sensor remain poorly defined. We used NMR to analyze the chromatographic serum fractionations enriched for the hypha-inducing activity and found structures resembling subunits of bacterial peptidoglycan (PGN). We then confirmed that several purified and synthetic muramyl dipeptides (MDPs), subunits of PGN, can indeed strongly promote C. albicans hyphal growth. Taking cue from the recognition of MDPs by the mammalian bacterial sensor Nod2 using its leucine-rich-repeat (LRR) domain, we discovered that MDPs activate the adenylyl cyclase Cyr1 by binding to its LRR domain. The cAMP/PKA signaling pathway is well known to control hyphal morphogenesis and other infection-related traits. Given the abundance of PGN at the large intestinal epithelial surface, a natural habitat and invasion site for C. albcians, our findings have important implications in the mechanisms of infection by this pathogen. PMID:19704871

  1. Religious Commitment, Peace Activism, and Marital Violence in Quaker Families.

    ERIC Educational Resources Information Center

    Brutz, Judith L.; Allen, Craig M.

    1986-01-01

    Religious commitment is found to differentiate levels of both communication and physical violence for both wives and husbands in Quaker families. High levels of peace activism are associated with low levels of marital violence for wives but with high levels for husbands, which suggests that commitment to Quaker principles is confounded with…

  2. Family Science Night: Fun Tips, Activities, and Ideas

    ERIC Educational Resources Information Center

    Connell, Shelley S.

    2013-01-01

    At last! A practical, readable guide for teachers, school leaders, and parent/teacher associations that shows how to plan fun, hands-on science nights! Get easy-to-implement, content-rich tips and ideas that will cultivate positive attitudes toward science! Learn how to involve and actively engage families in their children's science education.…

  3. Key Role of Active-Site Water Molecules in Bacteriorhodopsin Proton-Transfer Reactions

    SciTech Connect

    Bondar, A.N.; Baudry, Jerome Y; Suhai, Sandor; Fischer, S.; Smith, Jeremy C

    2008-10-01

    The functional mechanism of the light-driven proton pump protein bacteriorhodopsin depends on the location of water molecules in the active site at various stages of the photocycle and on their roles in the proton-transfer steps. Here, free energy computations indicate that electrostatic interactions favor the presence of a cytoplasmic-side water molecule hydrogen bonding to the retinal Schiff base in the state preceding proton transfer from the retinal Schiff base to Asp85. However, the nonequilibrium nature of the pumping process means that the probability of occupancy of a water molecule in a given site depends both on the free energies of insertion of the water molecule in this and other sites during the preceding photocycle steps and on the kinetic accessibility of these sites on the time scale of the reaction steps. The presence of the cytoplasmic-side water molecule has a dramatic effect on the mechanism of proton transfer: the proton is channeled on the Thr89 side of the retinal, whereas the transfer on the Asp212 side is hindered. Reaction-path simulations and molecular dynamics simulations indicate that the presence of the cytoplasmic-side water molecule permits a low-energy bacteriorhodopsin conformer in which the water molecule bridges the twisted retinal Schiff base and the proton acceptor Asp85. From this low-energy conformer, proton transfer occurs via a concerted mechanism in which the water molecule participates as an intermediate proton carrier.

  4. Small Molecule Activation by Constrained Phosphorus Compounds: Insights from Theory.

    PubMed

    Pal, Amrita; Vanka, Kumar

    2016-01-19

    An exciting new development in main group chemistry has been the use of a constrained, "flat", phosphorus-based complex to mediate in reactions such as the dehydrogenation of ammonia borane (AB), and the activation of the N-H bond in primary amines. Its importance is based on the fact that it shows that main group compounds, when properly designed, can be as effective as transition metal complexes for doing significant chemical transformations. What the current computational study, employing density functional theory (DFT), reveals is that a common, general mechanism exists that accounts for the behavior of the flat phosphorus compound in the different reactions that have been experimentally reported to date. This mechanism, which involves the mediation by a base as a proton transfer agent, is simpler and energetically more favorable than the previous mechanisms that have been proposed for the same reactions in the literature. It is likely that the knowledge gained from the current work about the chemical behavior of this phosphorus compound can be utilized to design new constrained phosphorus-based compounds. PMID:26700074

  5. Aryl-alkyl-lysines: Membrane-Active Small Molecules Active against Murine Model of Burn Infection.

    PubMed

    Ghosh, Chandradhish; Manjunath, Goutham B; Konai, Mohini M; Uppu, Divakara S S M; Paramanandham, Krishnamoorthy; Shome, Bibek R; Ravikumar, Raju; Haldar, Jayanta

    2016-02-12

    Infections caused by drug-resistant Gram-negative pathogens continue to be significant contributors to human morbidity. The recent advent of New Delhi metallo-β-lactamase-1 (blaNDM-1) producing pathogens, against which few drugs remain active, has aggravated the problem even further. This paper shows that aryl-alkyl-lysines, membrane-active small molecules, are effective in treating infections caused by Gram-negative pathogens. One of the compounds of the study was effective in killing planktonic cells as well as dispersing biofilms of Gram-negative pathogens. The compound was extremely effective in disrupting preformed biofilms and did not select resistant bacteria in multiple passages. The compound retained activity in different physiological conditions and did not induce any toxic effect in female Balb/c mice until concentrations of 17.5 mg/kg. In a murine model of Acinetobacter baumannii burn infection, the compound was able to bring the bacterial burden down significantly upon topical application for 7 days. PMID:27624962

  6. A new family of covalent inhibitors block nucleotide binding to the active site of pyruvate kinase

    PubMed Central

    Morgan, Hugh P.; Walsh, Martin J.; Blackburn, Elizabeth A.; Wear, Martin A.; Boxer, Matthew B.; Shen, Min; Mcnae, Iain W.; Nowicki, Matthew W.; Michels, Paul A. M.; Auld, Douglas S.; Fothergill-Gilmore, Linda A.; Walkinshaw, Malcolm D.

    2012-01-01

    SYNOPSIS Pyruvate kinase (PYK) plays a central role in the metabolism of many organisms and cell types, but the elucidation of the details of its function in a systems biology context has been hampered by the lack of specific high-affinity small molecule inhibitors. High-throughput screening has been used to identify a family of saccharin derivatives which inhibit Leishmania mexicana PYK (LmPYK) activity in a time- (and dose-) dependent manner; a characteristic of irreversible inhibition. The crystal structure of 4-[(1,1-dioxo-1,2-benzothiazol-3-yl)sulfanyl]benzoic acid (DBS) complexed with LmPYK shows that the saccharin moiety reacts with an active-site lysine residue (Lys335), forming a covalent bond and sterically hindering the binding of ADP/ATP. Mutation of the lysine residue to an arginine residue eliminated the effect of the inhibitor molecule, providing confirmation of the proposed inhibitor mechanism. This lysine residue is conserved in the active sites of the four human PYK isoenzymes, which were also found to be irreversibly inhibited by DBS. X-ray structures of PYK isoforms show structural differences at the DBS binding pocket, and this covalent inhibitor of PYK provides a chemical scaffold for the design of new families of potentially isoform-specific irreversible inhibitors. PMID:22906073

  7. On-chip silicon-based active photonic molecules by complete photonic bandgap light confinement

    NASA Astrophysics Data System (ADS)

    Qian, Bo; Chen, Kunji; Chen, San; Li, Wei; Zhang, Xiangao; Xu, Jun; Huang, Xinfan; Pavesi, Lorenzo; Jiang, Chunping

    2011-07-01

    We demonstrate an on-chip silicon-based active photonic molecule (PM) structures formed by two coupled photonic quantum dots with complete photonic bandgap (PBG) light confinement. The photonic quantum dots are grown by conformal deposition of amorphous silicon nitride multilayers on patterned substrates. A fine structure of the coupled optical modes in PMs has been observed which shows similarity to the electronic bonding (BN) and antibonding (ABN) states in a molecule.

  8. Activation of latent myostatin by the BMP-1/tolloid family of metalloproteinases.

    PubMed

    Wolfman, Neil M; McPherron, Alexandra C; Pappano, William N; Davies, Monique V; Song, Kening; Tomkinson, Kathleen N; Wright, Jill F; Zhao, Liz; Sebald, Suzanne M; Greenspan, Daniel S; Lee, Se-Jin

    2003-12-23

    Myostatin is a transforming growth factor beta family member that acts as a negative regulator of skeletal muscle growth. Myostatin circulates in the blood of adult mice in a noncovalently held complex with other proteins, including its propeptide, which maintain the C-terminal dimer in a latent, inactive state. This latent form of myostatin can be activated in vitro by treatment with acid; however, the mechanisms by which latent myostatin is activated in vivo are unknown. Here, we show that members of the bone morphogenetic protein-1/tolloid (BMP-1/TLD) family of metalloproteinases can cleave the myostatin propeptide in this complex and can thereby activate latent myostatin. Furthermore, we show that a mutant form of the propeptide resistant to cleavage by BMP-1/TLD proteinases can cause significant increases in muscle mass when injected into adult mice. These findings raise the possibility that members of the BMP-1/TLD family may be involved in activating latent myostatin in vivo and that molecules capable of inhibiting these proteinases may be effective agents for increasing muscle mass for both human therapeutic and agricultural applications.

  9. Activation of complement by an IgG molecule without a genetic hinge.

    PubMed

    Brekke, O H; Michaelsen, T E; Sandin, R; Sandlie, I

    1993-06-17

    The hinge region links the two Fab arms to the Fc portion of the IgG molecule. It mediates flexibility to the molecule and serves as a connecting structure between the two heavy chains. In addition it provides space between the Fab and Fc parts. All three properties have been proposed to be important for the ability of IgG to initiate complement activation leading to complement-mediated cell lysis (CML). Here we report the construction of a hinge-deleted mouse-human chimaeric IgG3 molecule with specificity for the hapten NIP (3-iodo-4-hydroxy-5-nitrophenacetyl), HM-1. HM-1 lacks the genetic hinge, but has an introduced cysteine between Ala 231 (EU numbering) and Pro 232 in the lower hinge encoded by the CH2 exon. The introduced cysteine forms a disulphide bond between the two heavy chains of the molecule. In CML, HM-1 shows a greater activity than IgG3 wild type. This is the first time an IgG molecule without a genetic hinge has been found to be active in CML. We conclude that the hinge functioning as a spacer is not a prerequisite for complement activation. Rather, its major role seems to be to connect the heavy chains to each other in the amino-terminal part of CH2. Because HM-1 is expected to have low Fab-Fc flexibility, this molecular feature is probably of no importance for complement activation.

  10. Structural basis for specificity of TGF[beta] family receptor small molecule inhibitors

    SciTech Connect

    Ogunjimi, Abiodun A.; Zeqiraj, Elton; Ceccarelli, Derek F.; Sicheri, Frank; Wrana, Jeffrey L.; David, Laurent

    2012-07-24

    Transforming growth factor-{beta} (TGF{beta}) receptor kinase inhibitors have a great therapeutic potential. SB431542 is one of the mainly used kinase inhibitors of the TGF{beta}/Activin pathway receptors, but needs improvement of its EC{sub 50} (EC{sub 50} = 1 {mu}M) to be translated to clinical use. A key feature of SB431542 is that it specifically targets receptors from the TGF{beta}/Activin pathway but not the closely related receptors from the bone morphogenic proteins (BMP) pathway. To understand the mechanisms of this selectivity, we solved the crystal structure of the TGF{beta} type I receptor (T{beta}RI) kinase domain in complex with SB431542. We mutated T{beta}RI residues coordinating SB431542 to their counterparts in activin-receptor like kinase 2 (ALK2), a BMP receptor kinase, and tested the kinase activity of mutated T{beta}RI. We discovered that a Ser280Thr mutation yielded a T{beta}RI variant that was resistant to SB431542 inhibition. Furthermore, the corresponding Thr283Ser mutation in ALK2 yielded a BMP receptor sensitive to SB431542. This demonstrated that Ser280 is the key determinant of selectivity for SB431542. This work provides a framework for optimising the SB431542 scaffold to more potent and selective inhibitors of the TGF{beta}/Activin pathway.

  11. Nox family NADPH oxidases: Molecular mechanisms of activation.

    PubMed

    Brandes, Ralf P; Weissmann, Norbert; Schröder, Katrin

    2014-11-01

    NADPH oxidases of the Nox family are important enzymatic sources of reactive oxygen species (ROS). Numerous homologue-specific mechanisms control the activity of this enzyme family involving calcium, free fatty acids, protein-protein interactions, intracellular trafficking, and posttranslational modifications such as phosphorylation, acetylation, or sumoylation. After a brief review on the classic pathways of Nox activation, this article will focus on novel mechanisms of homologue-specific activity control and on cell-specific aspects which govern Nox activity. From these findings of the recent years it must be concluded that the activity control of Nox enzymes is much more complex than anticipated. Moreover, depending on the cellular activity state, Nox enzymes are selectively activated or inactivated. The complex upstream signaling aspects of these events make the development of "intelligent" Nox inhibitors plausible, which selectively attenuate disease-related Nox-mediated ROS formation without altering physiological signaling ROS. This approach might be of relevance for Nox-mediated tissue injury in ischemia-reperfusion and inflammation and also for chronic Nox overactivation as present in cancer initiation and cardiovascular disease.

  12. Photo-activation of Single Molecule Magnet Behavior in a Manganese-based Complex.

    PubMed

    Fetoh, Ahmed; Cosquer, Goulven; Morimoto, Masakazu; Irie, Masahiro; El-Gammal, Ola; Abu El-Reash, Gaber; Breedlove, Brian K; Yamashita, Masahiro

    2016-01-01

    A major roadblock to fully realizing molecular electronic devices is the ability to control the properties of each molecule in the device. Herein we report the control of the magnetic properties of single-molecule magnets (SMMs), which can be used in memory devices, by using a photo-isomerizable diarthylenthene ligand. Photo-isomerization of the diarylethene ligand bridging two manganese salen complexes with visible light caused a significant change in the SMM behavior due to opening of the six-membered ring of diarylethene ligand, accompanied by reorganization of the entire molecule. The ring-opening activated the frequency-dependent magnetization of the complex. Our results are a major step towards the realization of molecular memory devices composed of SMMs because the SMM behaviour can be turned on and off simply by irradiating the molecule. PMID:27026506

  13. Target identification for biologically active small molecules using chemical biology approaches.

    PubMed

    Lee, Heesu; Lee, Jae Wook

    2016-09-01

    The identification and validation of the targets of biologically active molecules is an important step in the field of chemical biology. While recent advances in proteomic and genomic technology have accelerated this identification process, the discovery of small molecule targets remains the most challenging step. A general method for the identification of these small molecule targets has not yet been established. To overcome the difficulty in target identification, new technology derived from the fields of genomics, proteomics, and bioinformatics has been developed. To date, pull-down methods using small molecules immobilized on a solid support followed by mass spectrometry have been the most successful approach. Here, we discuss current procedures for target identification. We also review the most recent target identification approaches and present several examples that illustrate advanced target identification technology.

  14. Photo-activation of Single Molecule Magnet Behavior in a Manganese-based Complex

    PubMed Central

    Fetoh, Ahmed; Cosquer, Goulven; Morimoto, Masakazu; Irie, Masahiro; El-Gammal, Ola; El-Reash, Gaber Abu; Breedlove, Brian K.; Yamashita, Masahiro

    2016-01-01

    A major roadblock to fully realizing molecular electronic devices is the ability to control the properties of each molecule in the device. Herein we report the control of the magnetic properties of single-molecule magnets (SMMs), which can be used in memory devices, by using a photo-isomerizable diarthylenthene ligand. Photo-isomerization of the diarylethene ligand bridging two manganese salen complexes with visible light caused a significant change in the SMM behavior due to opening of the six-membered ring of diarylethene ligand, accompanied by reorganization of the entire molecule. The ring-opening activated the frequency-dependent magnetization of the complex. Our results are a major step towards the realization of molecular memory devices composed of SMMs because the SMM behaviour can be turned on and off simply by irradiating the molecule. PMID:27026506

  15. Implication of crystal water molecules in inhibitor binding at ALR2 active site.

    PubMed

    Hymavati; Kumar, Vivek; Sobhia, M Elizabeth

    2012-01-01

    Water molecules play a crucial role in mediating the interaction between a ligand and a macromolecule. The solvent environment around such biomolecule controls their structure and plays important role in protein-ligand interactions. An understanding of the nature and role of these water molecules in the active site of a protein could greatly increase the efficiency of rational drug design approaches. We have performed the comparative crystal structure analysis of aldose reductase to understand the role of crystal water in protein-ligand interaction. Molecular dynamics simulation has shown the versatile nature of water molecules in bridge H bonding during interaction. Occupancy and life time of water molecules depend on the type of cocrystallized ligand present in the structure. The information may be useful in rational approach to customize the ligand, and thereby longer occupancy and life time for bridge H-bonding. PMID:22649481

  16. Electro-optical parameters in excited states of some spectrally active molecules

    NASA Astrophysics Data System (ADS)

    Benchea, Andreea Celia; Closca, Valentina; Rusu, Cristina Marcela; Morosanu, Cezarina; Dorohoi, Dana Ortansa

    2014-08-01

    The spectral shifts measured in different solvents are expressed as functions of the solvent macroscopic parameters. The value of the correlation coefficient multiplying the functions of electric permittivity was determined by statistical means. The correlation coefficient depends on the electric dipole moment of the spectrally active molecules. The electro-optical parameters in the ground state of the solute molecules can be approximated by molecular modeling. The excited state parameters are usually estimated using the results obtained both by HyperChem Programme and solvatochromic study. The importance of this approximate method is that it offers information about of the excited state of solute molecule for which our measuring possibilities are very restrictive. The information about the excited electronic state is affected by the limits in which the theories of liquid solutions are developed. Our results refer to two molecules of vitamins from B class, namely B3 and B6.

  17. Astronomy Outreach Activities for Special Needs Children and Their Families

    NASA Astrophysics Data System (ADS)

    Lubowich, D.

    2010-08-01

    I present the results of two NASA-IDEAS/STScI sponsored astronomy outreach programs for seriously ill children and their families staying at the Ronald McDonald House of Long Island (New Hyde Park, NY) and for children hospitalized at the Children's Medical Center, Winthrop University Hospital (Mineola, NY). These programs are designed for children of all ages and include STSCi's Tonight's Sky (monthly guide to the sky); telescope observations of the Moon, Sun, planets, nebulae, and stars; and hands-on activities. During cloudy weather remote/robotic telescope observations are shown. Edible demonstrations using chocolate, marshmallows, and popcorn are used to stimulate interest. The staff at the Ronald McDonald House and Children's Medical Center are being trained to use the telescope and to do demonstrations. These educational activities help children and their families learn about astronomy while providing a diversion to take their minds off their illness during a stressful time.

  18. Small molecules that allosterically inhibit p21-activated kinase activity by binding to the regulatory p21-binding domain.

    PubMed

    Kim, Duk-Joong; Choi, Chang-Ki; Lee, Chan-Soo; Park, Mee-Hee; Tian, Xizhe; Kim, Nam Doo; Lee, Kee-In; Choi, Joong-Kwon; Ahn, Jin Hee; Shin, Eun-Young; Shin, Injae; Kim, Eung-Gook

    2016-01-01

    p21-activated kinases (PAKs) are key regulators of actin dynamics, cell proliferation and cell survival. Deregulation of PAK activity contributes to the pathogenesis of various human diseases, including cancer and neurological disorders. Using an ELISA-based screening protocol, we identified naphtho(hydro)quinone-based small molecules that allosterically inhibit PAK activity. These molecules interfere with the interactions between the p21-binding domain (PBD) of PAK1 and Rho GTPases by binding to the PBD. Importantly, they inhibit the activity of full-length PAKs and are selective for PAK1 and PAK3 in vitro and in living cells. These compounds may potentially be useful for determining the details of the PAK signaling pathway and may also be used as lead molecules in the development of more selective and potent PAK inhibitors. PMID:27126178

  19. Small molecules that allosterically inhibit p21-activated kinase activity by binding to the regulatory p21-binding domain

    PubMed Central

    Kim, Duk-Joong; Choi, Chang-Ki; Lee, Chan-Soo; Park, Mee-Hee; Tian, Xizhe; Kim, Nam Doo; Lee, Kee-In; Choi, Joong-Kwon; Ahn, Jin Hee; Shin, Eun-Young; Shin, Injae; Kim, Eung-Gook

    2016-01-01

    p21-activated kinases (PAKs) are key regulators of actin dynamics, cell proliferation and cell survival. Deregulation of PAK activity contributes to the pathogenesis of various human diseases, including cancer and neurological disorders. Using an ELISA-based screening protocol, we identified naphtho(hydro)quinone-based small molecules that allosterically inhibit PAK activity. These molecules interfere with the interactions between the p21-binding domain (PBD) of PAK1 and Rho GTPases by binding to the PBD. Importantly, they inhibit the activity of full-length PAKs and are selective for PAK1 and PAK3 in vitro and in living cells. These compounds may potentially be useful for determining the details of the PAK signaling pathway and may also be used as lead molecules in the development of more selective and potent PAK inhibitors. PMID:27126178

  20. Structures of human Bruton's tyrosine kinase in active and inactive conformations suggest a mechanism of activation for TEC family kinases

    SciTech Connect

    Marcotte, Douglas J.; Liu, Yu-Ting; Arduini, Robert M.; Hession, Catherine A.; Miatkowski, Konrad; Wildes, Craig P.; Cullen, Patrick F.; Hong, Victor; Hopkins, Brian T.; Mertsching, Elisabeth; Jenkins, Tracy J.; Romanowski, Michael J.; Baker, Darren P.; Silvian, Laura F.

    2010-11-15

    Bruton's tyrosine kinase (BTK), a member of the TEC family of kinases, plays a crucial role in B-cell maturation and mast cell activation. Although the structures of the unphosphorylated mouse BTK kinase domain and the unphosphorylated and phosphorylated kinase domains of human ITK are known, understanding the kinase selectivity profiles of BTK inhibitors has been hampered by the lack of availability of a high resolution, ligand-bound BTK structure. Here, we report the crystal structures of the human BTK kinase domain bound to either Dasatinib (BMS-354825) at 1.9 {angstrom} resolution or to 4-amino-5-(4-phenoxyphenyl)-7H-pyrrolospyrimidin- 7-yl-cyclopentane at 1.6 {angstrom} resolution. This data provides information relevant to the development of small molecule inhibitors targeting BTK and the TEC family of nonreceptor tyrosine kinases. Analysis of the structural differences between the TEC and Src families of kinases near the Trp-Glu-Ile motif in the N-terminal region of the kinase domain suggests a mechanism of regulation of the TEC family members.

  1. Ligand uptake in Mycobacterium tuberculosis truncated hemoglobins is controlled by both internal tunnels and active site water molecules

    PubMed Central

    Davidge, Kelly S; Singh, Sandip; Bowman, Lesley AH; Tinajero-Trejo, Mariana; Carballal, Sebastián; Radi, Rafael; Poole, Robert K; Dikshit, Kanak; Estrin, Dario A; Marti, Marcelo A; Boechi, Leonardo

    2015-01-01

    Mycobacterium tuberculosis, the causative agent of human tuberculosis, has two proteins belonging to the truncated hemoglobin (trHb) family. Mt-trHbN presents well-defined internal hydrophobic tunnels that allow O 2 and •NO to migrate easily from the solvent to the active site, whereas Mt-trHbO possesses tunnels that are partially blocked by a few bulky residues, particularly a tryptophan at position G8. Differential ligand migration rates allow Mt-trHbN to detoxify •NO, a crucial step for pathogen survival once under attack by the immune system, much more efficiently than Mt-trHbO. In order to investigate the differences between these proteins, we performed experimental kinetic measurements, •NO decomposition, as well as molecular dynamics simulations of the wild type Mt-trHbN and two mutants, VG8F and VG8W. These mutations introduce modifications in both tunnel topologies and affect the incoming ligand capacity to displace retained water molecules at the active site. We found that a single mutation allows Mt-trHbN to acquire ligand migration rates comparable to those observed for Mt-trHbO, confirming that ligand migration is regulated by the internal tunnel architecture as well as by water molecules stabilized in the active site. PMID:26478812

  2. Ligand uptake in Mycobacterium tuberculosis truncated hemoglobins is controlled by both internal tunnels and active site water molecules.

    PubMed

    Boron, Ignacio; Bustamante, Juan Pablo; Davidge, Kelly S; Singh, Sandip; Bowman, Lesley Ah; Tinajero-Trejo, Mariana; Carballal, Sebastián; Radi, Rafael; Poole, Robert K; Dikshit, Kanak; Estrin, Dario A; Marti, Marcelo A; Boechi, Leonardo

    2015-01-01

    Mycobacterium tuberculosis, the causative agent of human tuberculosis, has two proteins belonging to the truncated hemoglobin (trHb) family. Mt-trHbN presents well-defined internal hydrophobic tunnels that allow O 2 and (•)NO to migrate easily from the solvent to the active site, whereas Mt-trHbO possesses tunnels that are partially blocked by a few bulky residues, particularly a tryptophan at position G8. Differential ligand migration rates allow Mt-trHbN to detoxify (•)NO, a crucial step for pathogen survival once under attack by the immune system, much more efficiently than Mt-trHbO. In order to investigate the differences between these proteins, we performed experimental kinetic measurements, (•)NO decomposition, as well as molecular dynamics simulations of the wild type Mt-trHbN and two mutants, VG8F and VG8W. These mutations introduce modifications in both tunnel topologies and affect the incoming ligand capacity to displace retained water molecules at the active site. We found that a single mutation allows Mt-trHbN to acquire ligand migration rates comparable to those observed for Mt-trHbO, confirming that ligand migration is regulated by the internal tunnel architecture as well as by water molecules stabilized in the active site.

  3. Active Site and Laminarin Binding in Glycoside Hydrolase Family 55*

    PubMed Central

    Bianchetti, Christopher M.; Takasuka, Taichi E.; Deutsch, Sam; Udell, Hannah S.; Yik, Eric J.; Bergeman, Lai F.; Fox, Brian G.

    2015-01-01

    The Carbohydrate Active Enzyme (CAZy) database indicates that glycoside hydrolase family 55 (GH55) contains both endo- and exo-β-1,3-glucanases. The founding structure in the GH55 is PcLam55A from the white rot fungus Phanerochaete chrysosporium (Ishida, T., Fushinobu, S., Kawai, R., Kitaoka, M., Igarashi, K., and Samejima, M. (2009) Crystal structure of glycoside hydrolase family 55 β-1,3-glucanase from the basidiomycete Phanerochaete chrysosporium. J. Biol. Chem. 284, 10100–10109). Here, we present high resolution crystal structures of bacterial SacteLam55A from the highly cellulolytic Streptomyces sp. SirexAA-E with bound substrates and product. These structures, along with mutagenesis and kinetic studies, implicate Glu-502 as the catalytic acid (as proposed earlier for Glu-663 in PcLam55A) and a proton relay network of four residues in activating water as the nucleophile. Further, a set of conserved aromatic residues that define the active site apparently enforce an exo-glucanase reactivity as demonstrated by exhaustive hydrolysis reactions with purified laminarioligosaccharides. Two additional aromatic residues that line the substrate-binding channel show substrate-dependent conformational flexibility that may promote processive reactivity of the bound oligosaccharide in the bacterial enzymes. Gene synthesis carried out on ∼30% of the GH55 family gave 34 active enzymes (19% functional coverage of the nonredundant members of GH55). These active enzymes reacted with only laminarin from a panel of 10 different soluble and insoluble polysaccharides and displayed a broad range of specific activities and optima for pH and temperature. Application of this experimental method provides a new, systematic way to annotate glycoside hydrolase phylogenetic space for functional properties. PMID:25752603

  4. The protein tyrosine phosphatases PTPRZ and PTPRG bind to distinct members of the contactin family of neural recognition molecules

    SciTech Connect

    Bouyain, Samuel; Watkins, Dara J.

    2010-04-05

    The receptor protein tyrosine phosphatases gamma (PTPRG) and zeta (PTPRZ) are expressed primarily in the nervous system and mediate cell adhesion and signaling events during development. We report here the crystal structures of the carbonic anhydrase-like domains of PTPRZ and PTPRG and show that these domains interact directly with the second and third immunoglobulin repeats of the members of the contactin (CNTN) family of neural recognition molecules. Interestingly, these receptors exhibit distinct specificities: PTPRZ binds only to CNTN1, whereas PTPRG interacts with CNTN3, 4, 5, and 6. Furthermore, we present crystal structures of the four N-terminal immunoglobulin repeats of mouse CNTN4 both alone and in complex with the carbonic anhydrase-like domain of mouse PTPRG. In these structures, the N-terminal region of CNTN4 adopts a horseshoe-like conformation found also in CNTN2 and most likely in all CNTNs. This restrained conformation of the second and third immunoglobulin domains creates a binding site that is conserved among CNTN3, 4, 5, and 6. This site contacts a discrete region of PTPRG composed primarily of an extended {beta}-hairpin loop found in both PTPRG and PTPRZ. Overall, these findings implicate PTPRG, PTPRZ and CNTNs as a group of receptors and ligands involved in the manifold recognition events that underlie the construction of neural networks.

  5. Approach for targeting Ras with small molecules that activate SOS-mediated nucleotide exchange.

    PubMed

    Burns, Michael C; Sun, Qi; Daniels, R Nathan; Camper, DeMarco; Kennedy, J Phillip; Phan, Jason; Olejniczak, Edward T; Lee, Taekyu; Waterson, Alex G; Rossanese, Olivia W; Fesik, Stephen W

    2014-03-01

    Aberrant activation of the small GTPase Ras by oncogenic mutation or constitutively active upstream receptor tyrosine kinases results in the deregulation of cellular signals governing growth and survival in ∼30% of all human cancers. However, the discovery of potent inhibitors of Ras has been difficult to achieve. Here, we report the identification of small molecules that bind to a unique pocket on the Ras:Son of Sevenless (SOS):Ras complex, increase the rate of SOS-catalyzed nucleotide exchange in vitro, and modulate Ras signaling pathways in cells. X-ray crystallography of Ras:SOS:Ras in complex with these molecules reveals that the compounds bind in a hydrophobic pocket in the CDC25 domain of SOS adjacent to the Switch II region of Ras. The structure-activity relationships exhibited by these compounds can be rationalized on the basis of multiple X-ray cocrystal structures. Mutational analyses confirmed the functional relevance of this binding site and showed it to be essential for compound activity. These molecules increase Ras-GTP levels and disrupt MAPK and PI3K signaling in cells at low micromolar concentrations. These small molecules represent tools to study the acute activation of Ras and highlight a pocket on SOS that may be exploited to modulate Ras signaling.

  6. Investigating organic molecules responsible of auxin-like activity of humic acid fraction extracted from vermicompost.

    PubMed

    Scaglia, Barbara; Nunes, Ramom Rachide; Rezende, Maria Olímpia Oliveira; Tambone, Fulvia; Adani, Fabrizio

    2016-08-15

    This work studied the auxin-like activity of humic acids (HA) obtained from vermicomposts produced using leather wastes plus cattle dung at different maturation stages (fresh, stable and mature). Bioassays were performed by testing HA concentrations in the range of 100-6000mgcarbonL(-1). (13)C CPMAS-NMR and GC-MS instrumental methods were used to assess the effect of biological processes and starting organic mixtures on HA composition. Not all HAs showed IAA-like activity and in general, IAA-like activity increased with the length of the vermicomposting process. The presence of leather wastes was not necessary to produce the auxin-like activity of HA, since HA extracted from a mix of cattle manure and sawdust, where no leather waste was added, showed IAA-like activity as well. CPMAS (13)CNMR revealed that HAs were similar independently of the mix used and that the humification process involved the increasing concentration of pre-existing alkali soluble fractions in the biomass. GC/MS allowed the identification of the molecules involved in IAA-like effects: carboxylic acids and amino acids. The concentration of active molecules, rather than their simple presence in HA, determined the bio-stimulating effect, and a good linear regression between auxin-like activity and active stimulating molecules concentration was found (R(2)=-0.85; p<0.01, n=6). PMID:27100009

  7. Dense small molecule labeling enables activator-dependent STORM by proximity mapping.

    PubMed

    Chen, Ye; Gu, Min; Gunning, Peter W; Russell, Sarah M

    2016-09-01

    Stochastic optical reconstruction microscopy (STORM) enables high-resolution imaging, but multi-channel 3D imaging is problematic because of chromatic aberrations and alignment errors. The use of activator-dependent STORM in which spectrally distinct activators can be coupled with a single reporter can circumvent such issues. However, the standard approach of linking activators and reporters to a single antibody molecule is hampered by low labeling density and the large size of the antibody. We proposed that small molecule labels might enable activator-dependent STORM if the reporter or activator were linked to separate small molecules that bound within 3.5 nm of each other. This would greatly increase the labeling density and therefore improve resolution. We tested various mixtures of phalloidin- or mCling-conjugated fluorophore to demonstrate this feasibility. The specific activation was dependent on the choice of activator, its density, a matching activating laser and its power. In addition to providing an effective means of multi-channel 3D STORM imaging, this method also provides information about the local proximity between labels, potentially enabling super-resolved mapping of the conformation of the labeled structures. PMID:27246003

  8. Investigating organic molecules responsible of auxin-like activity of humic acid fraction extracted from vermicompost.

    PubMed

    Scaglia, Barbara; Nunes, Ramom Rachide; Rezende, Maria Olímpia Oliveira; Tambone, Fulvia; Adani, Fabrizio

    2016-08-15

    This work studied the auxin-like activity of humic acids (HA) obtained from vermicomposts produced using leather wastes plus cattle dung at different maturation stages (fresh, stable and mature). Bioassays were performed by testing HA concentrations in the range of 100-6000mgcarbonL(-1). (13)C CPMAS-NMR and GC-MS instrumental methods were used to assess the effect of biological processes and starting organic mixtures on HA composition. Not all HAs showed IAA-like activity and in general, IAA-like activity increased with the length of the vermicomposting process. The presence of leather wastes was not necessary to produce the auxin-like activity of HA, since HA extracted from a mix of cattle manure and sawdust, where no leather waste was added, showed IAA-like activity as well. CPMAS (13)CNMR revealed that HAs were similar independently of the mix used and that the humification process involved the increasing concentration of pre-existing alkali soluble fractions in the biomass. GC/MS allowed the identification of the molecules involved in IAA-like effects: carboxylic acids and amino acids. The concentration of active molecules, rather than their simple presence in HA, determined the bio-stimulating effect, and a good linear regression between auxin-like activity and active stimulating molecules concentration was found (R(2)=-0.85; p<0.01, n=6).

  9. Dense small molecule labeling enables activator-dependent STORM by proximity mapping.

    PubMed

    Chen, Ye; Gu, Min; Gunning, Peter W; Russell, Sarah M

    2016-09-01

    Stochastic optical reconstruction microscopy (STORM) enables high-resolution imaging, but multi-channel 3D imaging is problematic because of chromatic aberrations and alignment errors. The use of activator-dependent STORM in which spectrally distinct activators can be coupled with a single reporter can circumvent such issues. However, the standard approach of linking activators and reporters to a single antibody molecule is hampered by low labeling density and the large size of the antibody. We proposed that small molecule labels might enable activator-dependent STORM if the reporter or activator were linked to separate small molecules that bound within 3.5 nm of each other. This would greatly increase the labeling density and therefore improve resolution. We tested various mixtures of phalloidin- or mCling-conjugated fluorophore to demonstrate this feasibility. The specific activation was dependent on the choice of activator, its density, a matching activating laser and its power. In addition to providing an effective means of multi-channel 3D STORM imaging, this method also provides information about the local proximity between labels, potentially enabling super-resolved mapping of the conformation of the labeled structures.

  10. A large family of anti‐activators accompanying XylS/AraC family regulatory proteins

    PubMed Central

    Yan, Michael B.; Tran, Minh; Wright, Nathan; Luzader, Deborah H.; Kendall, Melissa M.; Ruiz‐Perez, Fernando; Nataro, James P.

    2016-01-01

    Summary AraC Negative Regulators (ANR) suppress virulence genes by directly down‐regulating AraC/XylS members in Gram‐negative bacteria. In this study, we sought to investigate the distribution and molecular mechanisms of regulatory function for ANRs among different bacterial pathogens. We identified more than 200 ANRs distributed in diverse clinically important gram negative pathogens, including Vibrio spp., Salmonella spp., Shigella spp., Yersinia spp., Citrobacter spp., enterotoxigenic (ETEC) and enteroaggregative E. coli (EAEC), and members of the Pasteurellaceae. By employing a bacterial two hybrid system, pull down assays and surface plasmon resonance (SPR) analysis, we demonstrate that Aar (AggR‐activated regulator), a prototype member of the ANR family in EAEC, binds with high affinity to the central linker domain of AraC‐like member AggR. ANR‐AggR binding disrupted AggR dimerization and prevented AggR‐DNA binding. ANR homologs of Vibrio cholerae, Citrobacter rodentium, Salmonella enterica and ETEC were capable of complementing Aar activity by repressing aggR expression in EAEC strain 042. ANR homologs of ETEC and Vibrio cholerae bound to AggR as well as to other members of the AraC family, including Rns and ToxT. The predicted proteins of all ANR members exhibit three highly conserved predicted α‐helices. Site‐directed mutagenesis studies suggest that at least predicted α‐helices 2 and 3 are required for Aar activity. In sum, our data strongly suggest that members of the novel ANR family act by directly binding to their cognate AraC partners. PMID:27038276

  11. Enhancer hijacking activates GFI1 family oncogenes in medulloblastoma.

    PubMed

    Northcott, Paul A; Lee, Catherine; Zichner, Thomas; Stütz, Adrian M; Erkek, Serap; Kawauchi, Daisuke; Shih, David J H; Hovestadt, Volker; Zapatka, Marc; Sturm, Dominik; Jones, David T W; Kool, Marcel; Remke, Marc; Cavalli, Florence M G; Zuyderduyn, Scott; Bader, Gary D; VandenBerg, Scott; Esparza, Lourdes Adriana; Ryzhova, Marina; Wang, Wei; Wittmann, Andrea; Stark, Sebastian; Sieber, Laura; Seker-Cin, Huriye; Linke, Linda; Kratochwil, Fabian; Jäger, Natalie; Buchhalter, Ivo; Imbusch, Charles D; Zipprich, Gideon; Raeder, Benjamin; Schmidt, Sabine; Diessl, Nicolle; Wolf, Stephan; Wiemann, Stefan; Brors, Benedikt; Lawerenz, Chris; Eils, Jürgen; Warnatz, Hans-Jörg; Risch, Thomas; Yaspo, Marie-Laure; Weber, Ursula D; Bartholomae, Cynthia C; von Kalle, Christof; Turányi, Eszter; Hauser, Peter; Sanden, Emma; Darabi, Anna; Siesjö, Peter; Sterba, Jaroslav; Zitterbart, Karel; Sumerauer, David; van Sluis, Peter; Versteeg, Rogier; Volckmann, Richard; Koster, Jan; Schuhmann, Martin U; Ebinger, Martin; Grimes, H Leighton; Robinson, Giles W; Gajjar, Amar; Mynarek, Martin; von Hoff, Katja; Rutkowski, Stefan; Pietsch, Torsten; Scheurlen, Wolfram; Felsberg, Jörg; Reifenberger, Guido; Kulozik, Andreas E; von Deimling, Andreas; Witt, Olaf; Eils, Roland; Gilbertson, Richard J; Korshunov, Andrey; Taylor, Michael D; Lichter, Peter; Korbel, Jan O; Wechsler-Reya, Robert J; Pfister, Stefan M

    2014-07-24

    Medulloblastoma is a highly malignant paediatric brain tumour currently treated with a combination of surgery, radiation and chemotherapy, posing a considerable burden of toxicity to the developing child. Genomics has illuminated the extensive intertumoral heterogeneity of medulloblastoma, identifying four distinct molecular subgroups. Group 3 and group 4 subgroup medulloblastomas account for most paediatric cases; yet, oncogenic drivers for these subtypes remain largely unidentified. Here we describe a series of prevalent, highly disparate genomic structural variants, restricted to groups 3 and 4, resulting in specific and mutually exclusive activation of the growth factor independent 1 family proto-oncogenes, GFI1 and GFI1B. Somatic structural variants juxtapose GFI1 or GFI1B coding sequences proximal to active enhancer elements, including super-enhancers, instigating oncogenic activity. Our results, supported by evidence from mouse models, identify GFI1 and GFI1B as prominent medulloblastoma oncogenes and implicate 'enhancer hijacking' as an efficient mechanism driving oncogene activation in a childhood cancer.

  12. Single-molecule imaging of DNA polymerase I (Klenow fragment) activity by atomic force microscopy

    NASA Astrophysics Data System (ADS)

    Chao, J.; Zhang, P.; Wang, Q.; Wu, N.; Zhang, F.; Hu, J.; Fan, C. H.; Li, B.

    2016-03-01

    We report a DNA origami-facilitated single-molecule platform that exploits atomic force microscopy to study DNA replication. We imaged several functional activities of the Klenow fragment of E. coli DNA polymerase I (KF) including binding, moving, and dissociation from the template DNA. Upon completion of these actions, a double-stranded DNA molecule was formed. Furthermore, the direction of KF activities was captured and then confirmed by shifting the KF binding sites on the template DNA.We report a DNA origami-facilitated single-molecule platform that exploits atomic force microscopy to study DNA replication. We imaged several functional activities of the Klenow fragment of E. coli DNA polymerase I (KF) including binding, moving, and dissociation from the template DNA. Upon completion of these actions, a double-stranded DNA molecule was formed. Furthermore, the direction of KF activities was captured and then confirmed by shifting the KF binding sites on the template DNA. Electronic supplementary information (ESI) available. See DOI: 10.1039/c5nr06544e

  13. Identification of intrinsic catalytic activity for electrochemical reduction of water molecules to generate hydrogen.

    PubMed

    Shinagawa, Tatsuya; Takanabe, Kazuhiro

    2015-06-21

    Insufficient hydronium ion activities at near-neutral pH and under unbuffered conditions induce diffusion-limited currents for hydrogen evolution, followed by a reaction with water molecules to generate hydrogen at elevated potentials. The observed constant current behaviors at near neutral pH reflect the intrinsic electrocatalytic reactivity of the metal electrodes for water reduction.

  14. Machine learning models identify molecules active against the Ebola virus in vitro.

    PubMed

    Ekins, Sean; Freundlich, Joel S; Clark, Alex M; Anantpadma, Manu; Davey, Robert A; Madrid, Peter

    2015-01-01

    The search for small molecule inhibitors of Ebola virus (EBOV) has led to several high throughput screens over the past 3 years. These have identified a range of FDA-approved active pharmaceutical ingredients (APIs) with anti-EBOV activity in vitro and several of which are also active in a mouse infection model. There are millions of additional commercially-available molecules that could be screened for potential activities as anti-EBOV compounds. One way to prioritize compounds for testing is to generate computational models based on the high throughput screening data and then virtually screen compound libraries. In the current study, we have generated Bayesian machine learning models with viral pseudotype entry assay and the EBOV replication assay data. We have validated the models internally and externally. We have also used these models to computationally score the MicroSource library of drugs to select those likely to be potential inhibitors. Three of the highest scoring molecules that were not in the model training sets, quinacrine, pyronaridine and tilorone, were tested in vitro and had EC 50 values of 350, 420 and 230 nM, respectively. Pyronaridine is a component of a combination therapy for malaria that was recently approved by the European Medicines Agency, which may make it more readily accessible for clinical testing. Like other known antimalarial drugs active against EBOV, it shares the 4-aminoquinoline scaffold. Tilorone, is an investigational antiviral agent that has shown a broad array of biological activities including cell growth inhibition in cancer cells, antifibrotic properties, α7 nicotinic receptor agonist activity, radioprotective activity and activation of hypoxia inducible factor-1. Quinacrine is an antimalarial but also has use as an anthelmintic. Our results suggest data sets with less than 1,000 molecules can produce validated machine learning models that can in turn be utilized to identify novel EBOV inhibitors in vitro. PMID:26834994

  15. Machine learning models identify molecules active against the Ebola virus in vitro

    PubMed Central

    Ekins, Sean; Freundlich, Joel S.; Clark, Alex M.; Anantpadma, Manu; Davey, Robert A.; Madrid, Peter

    2016-01-01

    The search for small molecule inhibitors of Ebola virus (EBOV) has led to several high throughput screens over the past 3 years. These have identified a range of FDA-approved active pharmaceutical ingredients (APIs) with anti-EBOV activity in vitro and several of which are also active in a mouse infection model. There are millions of additional commercially-available molecules that could be screened for potential activities as anti-EBOV compounds. One way to prioritize compounds for testing is to generate computational models based on the high throughput screening data and then virtually screen compound libraries. In the current study, we have generated Bayesian machine learning models with viral pseudotype entry assay and the EBOV replication assay data. We have validated the models internally and externally. We have also used these models to computationally score the MicroSource library of drugs to select those likely to be potential inhibitors. Three of the highest scoring molecules that were not in the model training sets, quinacrine, pyronaridine and tilorone, were tested in vitro and had EC 50 values of 350, 420 and 230 nM, respectively. Pyronaridine is a component of a combination therapy for malaria that was recently approved by the European Medicines Agency, which may make it more readily accessible for clinical testing. Like other known antimalarial drugs active against EBOV, it shares the 4-aminoquinoline scaffold. Tilorone, is an investigational antiviral agent that has shown a broad array of biological activities including cell growth inhibition in cancer cells, antifibrotic properties, α7 nicotinic receptor agonist activity, radioprotective activity and activation of hypoxia inducible factor-1. Quinacrine is an antimalarial but also has use as an anthelmintic. Our results suggest data sets with less than 1,000 molecules can produce validated machine learning models that can in turn be utilized to identify novel EBOV inhibitors in vitro. PMID:26834994

  16. Lessons from isolable nickel(I) precursor complexes for small molecule activation.

    PubMed

    Yao, Shenglai; Driess, Matthias

    2012-02-21

    Small-molecule activation by transition metals is essential to numerous organic transformations, both biological and industrial. Creating useful metal-mediated activation systems often depends on stabilizing the metal with uncommon low oxidation states and low coordination numbers. This provides a redox-active metal center with vacant coordination sites well suited for interacting with small molecules. Monovalent nickel species, with their d(9) electronic configuration, are moderately strong one-electron reducing agents that are synthetically attractive if they can be isolated. They represent suitable reagents for closing the knowledge gap in nickel-mediated activation of small molecules. Recently, the first strikingly stable dinuclear β-diketiminate nickel(I) precursor complexes were synthesized, proving to be suitable promoters for small-molecule binding and activation. They have led to many unprecedented nickel complexes bearing activated small molecules in different reduction stages. In this Account, we describe selected achievements in the activation of nitrous oxide (N(2)O), O(2), the heavier chalcogens (S, Se, and Te), and white phosphorus (P(4)) through this β-diketiminatonickel(I) precursor species. We emphasize the reductive activation of O(2), owing to its promise in oxidation processes. The one-electron-reduced O(2) activation product, that is, the corresponding β-diketiminato-supported Ni-O(2) complex, is a genuine superoxonickel(II) complex, representing an important intermediate in the early stages of O(2) activation. It selectively acts as an oxygen-atom transfer agent, hydrogen-atom scavenger, or both towards exogenous organic substrates to yield oxidation products. The one-electron reduction of the superoxonickel(II) moiety was examined by using elemental potassium, β-diketiminatozinc(II) chloride, and β-diketiminatoiron(I) complexes, affording the first heterobimetallic complexes featuring a [NiO(2)M] subunit (M is K, Zn, or Fe). Through

  17. Specific antiviral activity demonstrated by TGTP, a member of a new family of interferon-induced GTPases.

    PubMed

    Carlow, D A; Teh, S J; Teh, H S

    1998-09-01

    The GTPase superfamily includes a diversity of molecules whose functions are regulated through the binding and hydrolysis of GTP. This superfamily can be segregated into families of functionally related molecules that typically share amino acid sequence similarity within and around the nucleotide-binding domains. A new family of putative GTPases, including IRG-47, LRG-47, IGTP, and TGTP/Mg21, has recently emerged that share significant sequence identity (25-40%). Expression of these molecules has been shown to be selectively induced by IFN-gamma and in some cases by IFN-alpha beta or bacterial LPS. This induction pattern implicates these putative GTPases as part of the innate defense of cells to infection, but their role in such defense has not yet been defined. We have previously described the cloning of TGTP and now confirm its intrinsic activity as a GTPase. We found that TGTP is strongly induced by endogenous IFN-alpha beta produced in response to standard lipofection of plasmid DNA or polyinosinic polycytidylic acid. The ability of endogenously produced IFN-alpha beta to efficiently induce expression of TGTP under these conditions suggested that TGTP might participate in defense against viral infection. This proposal was borne out when TGTP-transfected L cells displayed relative resistance to plaque formation by vesicular stomatitis virus but not herpes simplex virus. This observation places TGTP among a small family of innate antiviral agents and has implications for the functions of other members of this family of GTPases.

  18. Unprecedented activation and CO2 capture properties of an elastic single-molecule trap.

    PubMed

    Wriedt, Mario; Sculley, Julian P; Verdegaal, Wolfgang M; Yakovenko, Andrey A; Zhou, Hong-Cai

    2013-10-25

    The activation and CO2 capture properties of a microporous metal-organic framework with elastic single-molecule traps were systematically investigated. This material shows a unique low-energy gas-purge activation capability, high CO2 adsorption selectivities over various gases and optimized working capacities per energy of 2.9 mmol kJ(-1) at 128 °C. PMID:24022838

  19. Spin state transition in the active center of the hemoglobin molecule: DFT + DMFT study

    NASA Astrophysics Data System (ADS)

    Novoselov, D.; Korotin, Dm. M.; Anisimov, V. I.

    2016-05-01

    An ab initio study of electronic and spin configurations of the iron ion in the active center of the human hemoglobin molecule is presented. With a combination of the Density Functional Theory (DFT) method and the Dynamical Mean Field Theory (DMFT) approach, the spin state transition description in the iron ion during the oxidation process is significantly improved in comparison with previous attempts. It was found that the origin of the iron ion local moment behavior both for the high-spin and for the low-spin states in the hemoglobin molecule is caused by the presence of a mixture of several atomic states with comparable statistical probability.

  20. Erythropoietin and interleukin-2 activate distinct JAK kinase family members.

    PubMed Central

    Barber, D L; D'Andrea, A D

    1994-01-01

    The erythropoietin (EPO) receptor and the interleukin-2 (IL-2) receptor beta-chain subunit are members of the cytokine receptor superfamily. They have conserved primary amino acid sequences in their cytoplasmic domains and activate phosphorylation of common substrates, suggesting common biochemical signaling mechanisms. We have generated a cell line, CTLL-EPO-R, that contains functional cell surface receptors for both EPO and IL-2. CTLL-EPO-R cells demonstrated similar growth kinetics in EPO and IL-2. Stimulation with EPO resulted in the rapid, dose-dependent tyrosine phosphorylation of JAK2. In contrast, stimulation with IL-2 or the related cytokine IL-4 resulted in the rapid, dose-dependent tyrosine phosphorylation of JAK1 and an additional 116-kDa protein. This 116-kDa protein was itself immunoreactive with a polyclonal antiserum raised against JAK2 and appears to be a novel member of the JAK kinase family. Immune complex kinase assays confirmed that IL-2 and IL-4 activated JAK1 and EPO activated JAK2. These results demonstrate that multiple biochemical pathways are capable of conferring a mitogenic signal in CTLL-EPO-R cells and that the EPO and IL-2 receptors interact with distinct JAK kinase family members within the same cellular background. Images PMID:7935373

  1. Cardiac-Specific SOCS3 Deletion Prevents In Vivo Myocardial Ischemia Reperfusion Injury through Sustained Activation of Cardioprotective Signaling Molecules

    PubMed Central

    Nagata, Takanobu; Yasukawa, Hideo; Kyogoku, Sachiko; Oba, Toyoharu; Takahashi, Jinya; Nohara, Shoichiro; Minami, Tomoko; Mawatari, Kazutoshi; Sugi, Yusuke; Shimozono, Koutatsu; Pradervand, Sylvain; Hoshijima, Masahiko; Aoki, Hiroki; Fukumoto, Yoshihiro; Imaizumi, Tsutomu

    2015-01-01

    Myocardial ischemia reperfusion injury (IRI) adversely affects cardiac performance and the prognosis of patients with acute myocardial infarction. Although myocardial signal transducer and activator of transcription (STAT) 3 is potently cardioprotective during IRI, the inhibitory mechanism responsible for its activation is largely unknown. The present study aimed to investigate the role of the myocardial suppressor of cytokine signaling (SOCS)-3, an intrinsic negative feedback regulator of the Janus kinase (JAK)-STAT signaling pathway, in the development of myocardial IRI. Myocardial IRI was induced in mice by ligating the left anterior descending coronary artery for 1 h, followed by different reperfusion times. One hour after reperfusion, the rapid expression of JAK-STAT–activating cytokines was observed. We precisely evaluated the phosphorylation of cardioprotective signaling molecules and the expression of SOCS3 during IRI and then induced myocardial IRI in wild-type and cardiac-specific SOCS3 knockout mice (SOCS3-CKO). The activation of STAT3, AKT, and ERK1/2 rapidly peaked and promptly decreased during IRI. This decrease correlated with the induction of SOCS3 expression up to 24 h after IRI in wild-type mice. The infarct size 24 h after reperfusion was significantly reduced in SOCS3-CKO compared with wild-type mice. In SOCS3-CKO mice, STAT3, AKT, and ERK1/2 phosphorylation was sustained, myocardial apoptosis was prevented, and the expression of anti-apoptotic Bcl-2 family member myeloid cell leukemia-1 (Mcl-1) was augmented. Cardiac-specific SOCS3 deletion led to the sustained activation of cardioprotective signaling molecules including and prevented myocardial apoptosis and injury during IRI. Our findings suggest that SOCS3 may represent a key factor that exacerbates the development of myocardial IRI. PMID:26010537

  2. Soluble Forms of Intercellular and Vascular Cell Adhesion Molecules Independently Predict Progression to Type 2 Diabetes in Mexican American Families

    PubMed Central

    Kulkarni, Hemant; Mamtani, Manju; Peralta, Juan; Almeida, Marcio; Dyer, Thomas D.; Goring, Harald H.; Johnson, Matthew P.; Duggirala, Ravindranath; Mahaney, Michael C.; Olvera, Rene L.; Almasy, Laura; Glahn, David C.; Williams-Blangero, Sarah; Curran, Joanne E.; Blangero, John

    2016-01-01

    Objective While the role of type 2 diabetes (T2D) in inducing endothelial dysfunction is fairly well-established the etiological role of endothelial dysfunction in the onset of T2D is still a matter of debate. In the light of conflicting evidence in this regard, we conducted a prospective study to determine the association of circulating levels of soluble intercellular adhesion molecule 1 (sICAM-1) and soluble vessel cell adhesion molecule 1 (sVCAM-1) with incident T2D. Methods Data from this study came from 1,269 Mexican Americans of whom 821 initially T2D-free individuals were longitudinally followed up in the San Antonio Family Heart Study. These individuals were followed for 9752.95 person-years for development of T2D. Prospective association of sICAM-1 and sVCAM-1 with incident T2D was studied using Kaplan-Meier survival plots and mixed effects Cox proportional hazards modeling to account for relatedness among study participants. Incremental value of adhesion molecule biomarkers was studied using integrated discrimination improvement (IDI) and net reclassification improvement (NRI) indexes. Results Decreasing median values for serum concentrations of sICAM-1 and sVCAM-1 were observed in the following groups in this order: individuals with T2D at baseline, individuals who developed T2D during follow-up, individuals with prediabetes at baseline and normal glucose tolerant (NGT) individuals who remained T2D-free during follow-up. Top quartiles for sICAM-1 and sVCAM-1 were strongly and significantly associated with homeostatic model of assessment—insulin resistance (HOMA-IR). Mixed effects Cox proportional hazards modeling revealed that after correcting for important clinical confounders, high sICAM-1 and sVCAM-1 concentrations were associated with 2.52 and 1.99 times faster progression to T2D as compared to low concentrations, respectively. Individuals with high concentrations for both sICAM-1 and sVCAM-1 progressed to T2D 3.42 times faster than those with low

  3. Chemical activation of molecules by metals: Experimental studies of electron distributions and bonding

    SciTech Connect

    Lichtenberger, D.L.

    1992-01-01

    Purpose of this research program is to obtain experimental information on the different fundamental ways metals bond and activate organic molecules. Our approach has been to directly probe the electronic interactions between metals and molecules through a wide variety of ionization spectroscopies and other techniques, and to investigate the relationships with bonding modes, structures, and chemical behavior. During this period, we have (1) characterized the electronic features of diphosphines and monophosphines in their coordination to metals, (2) carried out theoretical and experimental investigations of the bonding capabilities of C[sub 60] to transition metals, (3) developed techniques for the imaging of single molecules on gold substrates that emphasizes the electronic backbonding from the metal to the molecule, (4) obtained the high resolution photoelectron spectrum of pure C[sub 70] in the gas phase, (5) compared the bonding of [eta][sup 3]- acetylide ligands to the bonding of other small organic molecules with metals, and (6) reported the photoelectron spectra and bonding of [eta][sup 3]-cyclopropenyl groups to metals.

  4. CHEMICAL ACTIVATION OF MOLECULES BY METALS: EXPERIMENTAL STUDIES OF ELECTRON DISTRIBUTIONS AND BONDING

    SciTech Connect

    LICHTENBERGER, DENNIS L.

    2002-03-26

    This research program is directed at obtaining detailed experimental information on the electronic interactions between metals and organic molecules. These interactions provide low energy pathways for many important chemical and catalytic processes. A major feature of the program is the continued development and application of our special high-resolution valence photoelectron spectroscopy (UPS), and high-precision X-ray core photoelectron spectroscopy (XPS) instrumentation for study of organometallic molecules in the gas phase. The study involves a systematic approach towards understanding the interactions and activation of bound carbonyls, C-H bonds, methylenes, vinylidenes, acetylides, alkenes, alkynes, carbenes, carbynes, alkylidenes, alkylidynes, and others with various monometal, dimetal, and cluster metal species. Supporting ligands include -aryls, alkoxides, oxides, and phosphines. We are expanding our studies of both early and late transition metal species and electron-rich and electron-poor environments in order to more completely understand the electronic factors that serve to stabilize particular organic fragments and intermediates on metals. Additional new directions for this program are being taken in ultra-high vacuum surface UPS, XPS, scanning tunneling microscopy (STM) and atomic force microscopy (AFM) experiments on both physisorbed and chemisorbed organometallic thin films. The combination of these methods provides additional electronic structure information on surface-molecule and molecule-molecule interactions. A very important general result emerging from this program is the identification of a close relationship between the ionization energies of the species and the thermodynamics of the chemical and catalytic reactions of these systems.

  5. Two New FRET Imaging Measures: Linearly Proportional to and Highly Contrasting the Fraction of Active Molecules

    PubMed Central

    Yamao, Masataka; Aoki, Kazuhiro; Yukinawa, Naoto; Ishii, Shin; Matsuda, Michiyuki; Naoki, Honda

    2016-01-01

    We developed two new FRET imaging measures for intramolecular FRET biosensors, called linearly proportional (LP) and highly contrasting (HC) measures, which can be easily calculated by the fluorescence intensities of donor and acceptor as a ratio between their weighted sums. As an alternative to the conventional ratiometric measure, which non-linearly depends on the fraction of active molecule, we first developed the LP measure, which is linearly proportional to the fraction of active molecules. The LP measure inherently unmixes bleed-through signals and is robust against fluorescence noise. By extending the LP measure, we furthermore designed the HC measure, which provides highly contrasting images of the molecular activity, more than the ratiometric measure. In addition to their advantages, these measures are insensitive to the biosensor expression level, which is a fundamental property of the ratiometric measure. Using artificial data and FRET imaging data, we showed that the LP measure effectively represents the fraction of active molecules and that the HC measure improves visual interpretability by providing high contrast images of molecular activity. Therefore, the LP and HC measures allow us to gain more quantitative and qualitative insights from FRET imaging than the ratiometric measure. PMID:27780260

  6. Aqueous phase adsorption of different sized molecules on activated carbon fibers: Effect of textural properties.

    PubMed

    Prajapati, Yogendra N; Bhaduri, Bhaskar; Joshi, Harish C; Srivastava, Anurag; Verma, Nishith

    2016-07-01

    The effect that the textural properties of rayon-based activated carbon fibers (ACFs), such as the BET surface area and pore size distribution (PSD), have on the adsorption of differently sized molecules, namely, brilliant yellow (BY), methyl orange (MO) and phenol (PH), was investigated in the aqueous phase. ACF samples with different BET areas and PSDs were produced by steam-activating carbonized fibers for different activation times (0.25, 0.5, and 1 h). The samples activated for 0.25 h were predominantly microporous, whereas those activated for relatively longer times contained hierarchical micro-mesopores. The adsorption capacities of the ACFs for the adsorbate increased with increasing BET surface area and pore volume, and ranged from 51 to 1306 mg/g depending on the textural properties of the ACFs and adsorbate size. The adsorption capacities of the hierarchical ACF samples followed the order BY > MO > PH. Interestingly, the number of molecules adsorbed by the ACFs followed the reverse order: PH > MO > BY. This anomaly was attributed to the increasing molecular weight of the PH, MO and BY molecules. The equilibrium adsorption data were described using the Langmuir isotherm. This study shows that suitable textural modifications to ACFs are required for the efficient aqueous phase removal of an adsorbate. PMID:27107386

  7. Aqueous phase adsorption of different sized molecules on activated carbon fibers: Effect of textural properties.

    PubMed

    Prajapati, Yogendra N; Bhaduri, Bhaskar; Joshi, Harish C; Srivastava, Anurag; Verma, Nishith

    2016-07-01

    The effect that the textural properties of rayon-based activated carbon fibers (ACFs), such as the BET surface area and pore size distribution (PSD), have on the adsorption of differently sized molecules, namely, brilliant yellow (BY), methyl orange (MO) and phenol (PH), was investigated in the aqueous phase. ACF samples with different BET areas and PSDs were produced by steam-activating carbonized fibers for different activation times (0.25, 0.5, and 1 h). The samples activated for 0.25 h were predominantly microporous, whereas those activated for relatively longer times contained hierarchical micro-mesopores. The adsorption capacities of the ACFs for the adsorbate increased with increasing BET surface area and pore volume, and ranged from 51 to 1306 mg/g depending on the textural properties of the ACFs and adsorbate size. The adsorption capacities of the hierarchical ACF samples followed the order BY > MO > PH. Interestingly, the number of molecules adsorbed by the ACFs followed the reverse order: PH > MO > BY. This anomaly was attributed to the increasing molecular weight of the PH, MO and BY molecules. The equilibrium adsorption data were described using the Langmuir isotherm. This study shows that suitable textural modifications to ACFs are required for the efficient aqueous phase removal of an adsorbate.

  8. Family History Predicts Stress Fracture in Active Female Adolescents

    PubMed Central

    Loud, Keith J.; Micheli, Lyle J.; Bristol, Stephanie; Austin, S. Bryn; Gordon, Catherine M.

    2011-01-01

    OBJECTIVE Increased physical activity and menstrual irregularity have been associated with increased risk for stress fracture among adult women active in athletics. The purposes of this study were to determine whether menstrual irregularity is also a risk factor for stress fracture in active female adolescents and to estimate the quantity of exercise associated with an increased risk for this injury. PATIENTS AND METHODS A case-control study was conducted of 13- to 22-year-old females diagnosed with their first stress fracture, each matched prospectively on age and self-reported ethnicity with 2 controls. Patients with chronic illnesses or use of medications known to affect bone mineral density were excluded, including use of hormonal preparations that could alter menstrual cycles. The primary outcome, stress fracture in any extremity or the spine, was confirmed radiographically. Girls with stress fracture had bone mineral density measured at the lumbar spine by dual-energy x-ray absorptiometry. RESULTS The mean ± SD age of the 168 participants was 15.9 ± 2.1 years; 91.7% were postmenarchal, with a mean age at menarche of 13.1 ± 1.1 years. The prevalence of menstrual irregularity was similar among cases and controls. There was no significant difference in the mean hours per week of total physical activity between girls in this sample with stress fracture (8.2 hours/week) and those without (7.4 hours/week). In multivariate models, case subjects had nearly 3 times the odds of having a family member with osteoporosis or osteopenia. In secondary analyses, participants with stress fracture had a low mean spinal bone mineral density for their age. CONCLUSIONS Among highly active female adolescents, only family history was independently associated with stress fracture. The magnitude of this association suggests that further investigations of inheritable skeletal factors are warranted in this population, along with evaluation of bone mineral density in girls with stress

  9. Discovery of Small Molecules for Fluorescent Detection of Complement Activation Product C3d.

    PubMed

    Gorham, Ronald D; Nuñez, Vicente; Lin, Jung-Hsin; Rooijakkers, Suzan H M; Vullev, Valentine I; Morikis, Dimitrios

    2015-12-24

    Complement activation plays a major role in many acute and chronic inflammatory conditions. C3d, a terminal product of complement activation, remains covalently attached to cells and is an excellent biomarker of complement-mediated inflammation. We employed a virtual high-throughput screening protocol to identify molecules with predicted binding to complement C3d and with intrinsic fluorescence properties to enable detection. Pharmacophore models were developed based on known C3d-ligand interactions and information from computational analysis of structural and molecular dynamics data. Iterative pharmacophore-based virtual screening was performed to identify druglike molecules with physicochemical similarity to the natural C3d ligand CR2. Hits from the pharmacophore screens were docked to C3d and ranked based on predicted binding free energies. Top-ranked molecules were selected for experimental validation of binding affinity to C3d, using microscale thermophoresis, and for their suitability to become molecular imaging agents, using fluorescence spectroscopy. This work serves as a foundation for identifying additional fluorescent molecules with high-affinity for C3d that will subsequently be explored as noninvasive in vivo diagnostics of complement-mediated inflammation, for spatiotemporal monitoring of disease progression, and for targeting therapeutics to sites of inflammation.

  10. Optoporation of impermeable molecules and genes for visualization and activation of cells

    NASA Astrophysics Data System (ADS)

    Dhakal, Kamal; Batbyal, Subrata; Kim, Young-Tae; Mohanty, Samarendra

    2015-03-01

    Visualization, activation, and detection of the cell(s) and their electrical activity require delivery of exogenous impermeable molecules and targeted expression of genes encoding labeling proteins, ion-channels and voltage indicators. While genes can be delivered by viral vector to cells, delivery of other impermeable molecules into the cytoplasm of targeted cells requires microinjection by mechanical needle or microelectrodes, which pose significant challenge to the viability of the cells. Further, it will be useful to localize the expression of the targeted molecules not only in specific cell types, but to specific cells in restricted spatial regions. Here, we report use of focused near-infrared (NIR) femtosecond laser beam to transiently perforate targeted cell membrane to insert genes encoding blue light activatable channelrhodopsin-2 (ChR2) and red-shifted opsin (ReachR). Optoporation of nanomolar concentrations of rhodamine phalloidin (an impermeable dye molecule for staining filamentous actin) into targeted living mammalian cells (both HEK and primary cortical neurons) is also achieved allowing imaging of dynamics and intact morphology of cellular structures without requiring fixation.

  11. Discovery of Small Molecules for Fluorescent Detection of Complement Activation Product C3d.

    PubMed

    Gorham, Ronald D; Nuñez, Vicente; Lin, Jung-Hsin; Rooijakkers, Suzan H M; Vullev, Valentine I; Morikis, Dimitrios

    2015-12-24

    Complement activation plays a major role in many acute and chronic inflammatory conditions. C3d, a terminal product of complement activation, remains covalently attached to cells and is an excellent biomarker of complement-mediated inflammation. We employed a virtual high-throughput screening protocol to identify molecules with predicted binding to complement C3d and with intrinsic fluorescence properties to enable detection. Pharmacophore models were developed based on known C3d-ligand interactions and information from computational analysis of structural and molecular dynamics data. Iterative pharmacophore-based virtual screening was performed to identify druglike molecules with physicochemical similarity to the natural C3d ligand CR2. Hits from the pharmacophore screens were docked to C3d and ranked based on predicted binding free energies. Top-ranked molecules were selected for experimental validation of binding affinity to C3d, using microscale thermophoresis, and for their suitability to become molecular imaging agents, using fluorescence spectroscopy. This work serves as a foundation for identifying additional fluorescent molecules with high-affinity for C3d that will subsequently be explored as noninvasive in vivo diagnostics of complement-mediated inflammation, for spatiotemporal monitoring of disease progression, and for targeting therapeutics to sites of inflammation. PMID:26613117

  12. Visualizing repetitive diffusion activity of double-strand RNA binding proteins by single molecule fluorescence assays.

    PubMed

    Koh, Hye Ran; Wang, Xinlei; Myong, Sua

    2016-08-01

    TRBP, one of double strand RNA binding proteins (dsRBPs), is an essential cofactor of Dicer in the RNA interference pathway. Previously we reported that TRBP exhibits repetitive diffusion activity on double strand (ds)RNA in an ATP independent manner. In the TRBP-Dicer complex, the diffusion mobility of TRBP facilitates Dicer-mediated RNA cleavage. Such repetitive diffusion of dsRBPs on a nucleic acid at the nanometer scale can be appropriately captured by several single molecule detection techniques. Here, we provide a step-by-step guide to four different single molecule fluorescence assays by which the diffusion activity of dsRBPs on dsRNA can be detected. One color assay, termed protein induced fluorescence enhancement enables detection of unlabeled protein binding and diffusion on a singly labeled RNA. Two-color Fluorescence Resonance Energy Transfer (FRET) in which labeled dsRBPs is applied to labeled RNA, allows for probing the motion of protein along the RNA axis. Three color FRET reports on the diffusion movement of dsRBPs from one to the other end of RNA. The single molecule pull down assay provides an opportunity to collect dsRBPs from mammalian cells and examine the protein-RNA interaction at single molecule platform. PMID:27012177

  13. Evolution of the protease-activated receptor family in vertebrates

    PubMed Central

    JIN, MIN; YANG, HAI-WEI; TAO, AI-LIN; WEI, JI-FU

    2016-01-01

    Belonging to the G protein-coupled receptor (GPcr) family, the protease-activated receptors (Pars) consist of 4 members, PAR1-4. PARs mediate the activation of cells via thrombin, serine and other proteases. Such protease-triggered signaling events are thought to be critical for hemostasis, thrombosis and other normal pathological processes. In the present study, we examined the evolution of PARs by analyzing phylogenetic trees, chromosome location, selective pressure and functional divergence based on the 169 functional gene alignment sequences from 57 vertebrate gene sequences. We found that the 4 PARs originated from 4 invertebrate ancestors by phylogenetic trees analysis. The selective pressure results revealed that only PAR1 appeared by positive selection during its evolution, while the other PAR members did not. In addition, we noticed that although these PARs evolved separately, the results of functional divergence indicated that their evolutional rates were similar and their functions did not significantly diverge. The findings of our study provide valuable insight into the evolutionary history of the vertebrate PAR family. PMID:26820116

  14. Matrine inhibits the expression of adhesion molecules in activated vascular smooth muscle cells.

    PubMed

    Liu, Jun; Zhang, Lihua; Ren, Yingang; Gao, Yanli; Kang, Li; Lu, Shaoping

    2016-03-01

    Atherosclerosis is a chronic inflammatory disease associated with increased expression of adhesion molecules in vascular smooth muscle cells (VSMCs). Matrine is a main active ingredient of Sophora flavescens roots, which are used to treat inflammatory diseases. However, the effects of matrine on the expression of adhesion molecules in VSMCs have largely remained elusive. Therefore, the present study investigated the effects of matrine on the expression of adhesion molecules in tumor necrosis factor (TNF)‑α‑stimulated human aortic smooth muscle cells (HASMCs). The results showed that matrine inhibited the expression of vascular cell adhesion molecule‑1 (VCAM‑1) and intercellular adhesion molecule‑1 (ICAM‑1) in TNF‑α‑stimulated HASMCs. Matrine markedly inhibited the TNF‑α‑induced expression of nuclear factor (NF)‑κB p65 and prevented the TNF‑α‑caused degradation of inhibitor of NF‑κB; it also inhibited TNF‑α‑induced activation of mitogen‑activated protein kinases (MAPKs). Furthermore, matrine inhibited the production of intracellular reactive oxygen species (ROS) in TNF‑α‑stimulated HASMCs. In conclusion, the results of the present study demonstrated that matrine inhibited the expression of VCAM‑1 and ICAM‑1 in TNF‑α‑stimulated HASMCs via the suppression of ROS production as well as NF‑κB and MAPK pathway activation. Therefore, matrine may have a potential therapeutic use for preventing the advancement of atherosclerotic lesions.

  15. Single molecule microscopy reveals mechanistic insight into RNA polymerase II preinitiation complex assembly and transcriptional activity

    PubMed Central

    Horn, Abigail E.; Kugel, Jennifer F.; Goodrich, James A.

    2016-01-01

    Transcription by RNA polymerase II (Pol II) is a complex process that requires general transcription factors and Pol II to assemble on DNA into preinitiation complexes that can begin RNA synthesis upon binding of NTPs (nucleoside triphosphate). The pathways by which preinitiation complexes form, and how this impacts transcriptional activity are not completely clear. To address these issues, we developed a single molecule system using TIRF (total internal reflection fluorescence) microscopy and purified human transcription factors, which allows us to visualize transcriptional activity at individual template molecules. We see that stable interactions between polymerase II (Pol II) and a heteroduplex DNA template do not depend on general transcription factors; however, transcriptional activity is highly dependent upon TATA-binding protein, TFIIB and TFIIF. We also found that subsets of general transcription factors and Pol II can form stable complexes that are precursors for functional transcription complexes upon addition of the remaining factors and DNA. Ultimately we found that Pol II, TATA-binding protein, TFIIB and TFIIF can form a quaternary complex in the absence of promoter DNA, indicating that a stable network of interactions exists between these proteins independent of promoter DNA. Single molecule studies can be used to learn how different modes of preinitiation complex assembly impact transcriptional activity. PMID:27112574

  16. Structure Based Discovery of Small Molecules to Regulate the Activity of Human Insulin Degrading Enzyme

    PubMed Central

    Çakir, Bilal; Dağliyan, Onur; Dağyildiz, Ezgi; Bariş, İbrahim; Kavakli, Ibrahim Halil; Kizilel, Seda; Türkay, Metin

    2012-01-01

    Background Insulin-degrading enzyme (IDE) is an allosteric Zn+2 metalloprotease involved in the degradation of many peptides including amyloid-β, and insulin that play key roles in Alzheimer's disease (AD) and type 2 diabetes mellitus (T2DM), respectively. Therefore, the use of therapeutic agents that regulate the activity of IDE would be a viable approach towards generating pharmaceutical treatments for these diseases. Crystal structure of IDE revealed that N-terminal has an exosite which is ∼30 Å away from the catalytic region and serves as a regulation site by orientation of the substrates of IDE to the catalytic site. It is possible to find small molecules that bind to the exosite of IDE and enhance its proteolytic activity towards different substrates. Methodology/Principal Findings In this study, we applied structure based drug design method combined with experimental methods to discover four novel molecules that enhance the activity of human IDE. The novel compounds, designated as D3, D4, D6, and D10 enhanced IDE mediated proteolysis of substrate V, insulin and amyloid-β, while enhanced degradation profiles were obtained towards substrate V and insulin in the presence of D10 only. Conclusion/Significance This paper describes the first examples of a computer-aided discovery of IDE regulators, showing that in vitro and in vivo activation of this important enzyme with small molecules is possible. PMID:22355395

  17. Single-Molecule Nanocatalysis Reveals Catalytic Activation Energy of Single Nanocatalysts.

    PubMed

    Chen, Tao; Zhang, Yuwei; Xu, Weilin

    2016-09-28

    By monitoring the temperature-dependent catalytic activity of single Au nanocatalysts for a fluorogenic reaction, we derive the activation energies via multiple methods for two sequential catalytic steps (product formation and dissociation) on single nanocatalysts. The wide distributions of activation energies across multiple individual nanocatalysts indicate a huge static heterogeneity among the individual nanocatalysts. The compensation effect and isokinetic relationship of catalytic reactions are observed at the single particle level. This study exemplifies another function of single-molecule nanocatalysis and improves our understanding of heterogeneous catalysis.

  18. Analysis of Risk Alleles and Complement Activation Levels in Familial and Non-Familial Age-Related Macular Degeneration

    PubMed Central

    Saksens, Nicole T. M.; Lechanteur, Yara T. E.; Verbakel, Sanne K.; Groenewoud, Joannes M. M.; Daha, Mohamed R.; Schick, Tina; Fauser, Sascha; Boon, Camiel J. F.; Hoyng, Carel B.; den Hollander, Anneke I.

    2016-01-01

    Aims Age-related macular degeneration (AMD) is a multifactorial disease, in which complement-mediated inflammation plays a pivotal role. A positive family history is an important risk factor for developing AMD. Certain lifestyle factors are shown to be significantly associated with AMD in non-familial cases, but not in familial cases. This study aimed to investigate whether the contribution of common genetic variants and complement activation levels differs between familial and sporadic cases with AMD. Methods and Results 1216 AMD patients (281 familial and 935 sporadic) and 1043 controls (143 unaffected members with a family history of AMD and 900 unrelated controls without a family history of AMD) were included in this study. Ophthalmic examinations were performed, and lifestyle and family history were documented with a questionnaire. Nine single nucleotide polymorphisms (SNPs) known to be associated with AMD were genotyped, and serum concentrations of complement components C3 and C3d were measured. Associations were assessed in familial and sporadic individuals. The association with risk alleles of the age-related maculopathy susceptibility 2 (ARMS2) gene was significantly stronger in sporadic AMD patients compared to familial cases (p = 0.017 for all AMD stages and p = 0.003 for advanced AMD, respectively). ARMS2 risk alleles had the largest effect in sporadic cases but were not significantly associated with AMD in densely affected families. The C3d/C3 ratio was a significant risk factor for AMD in sporadic cases and may also be associated with familial cases. In patients with a densely affected family this effect was particularly strong with ORs of 5.37 and 4.99 for all AMD and advanced AMD respectively. Conclusion This study suggests that in familial AMD patients, the common genetic risk variant in ARMS2 is less important compared to sporadic AMD. In contrast, factors leading to increased complement activation appear to play a larger role in patients with a

  19. Families and School Personnel Involved in a Literacy and Physical Activity Partnership

    ERIC Educational Resources Information Center

    Richardson, James A.; Richardson, Maurine V.; Sacks, Mary Kathleen

    2006-01-01

    A traditional part of American education has been to include the families in the educational process of their children. The needs and complexities of today's families and classrooms have never been greater. Programs and activities used with families, and school interactions 10 to 15 years ago are not effective for today's complex families and…

  20. Children of Victims of Terrorist Activities: A Family Approach to Dealing with Tragedy.

    ERIC Educational Resources Information Center

    Dreman, Solly B.; Cohen, Esther C.

    1982-01-01

    Deals with the treatment of families of victims of terrorist activities. Discusses specific issues common to families affected by terrorism. Describes therapy concentrated on promoting family strengths and coping and deemphasizing individual pathology. Suggests family therapy is an effective treatment modality for dealing with tragic loss. (Author)

  1. Nanoscale charge transport in cytochrome c3/DNA network: Comparative studies between redox-active molecules

    NASA Astrophysics Data System (ADS)

    Yamaguchi, Harumasa; Che, Dock-Chil; Hirano, Yoshiaki; Suzuki, Masayuki; Higuchi, Yoshiki; Matsumoto, Takuya

    2015-09-01

    The redox-active molecule of a cytochrome c3/DNA network exhibits nonlinear current-voltage (I-V) characteristics with a threshold bias voltage at low temperature and zero-bias conductance at room temperature. I-V curves for the cytochrome c3/DNA network are well matched with the Coulomb blockade network model. Comparative studies of the Mn12 cluster, cytochrome c, and cytochrome c3, which have a wide variety of redox potentials, indicate no difference in charge transport, which suggests that the conduction mechanism is not directly related to the redox states. The charge transport mechanism has been discussed in terms of the newly-formed electronic energy states near the Fermi level, induced by the ionic interaction between redox-active molecules with the DNA network.

  2. Critical roles of co-activation receptor DNAX accessory molecule-1 in natural killer cell immunity

    PubMed Central

    Xiong, Peng; Sang, Hai-Wei; Zhu, Min

    2015-01-01

    Natural killer (NK) cells, which can exert early and powerful anti-tumour and anti-viral responses, are important components of the innate immune system. DNAX accessory molecule-1 (DNAM-1) is an activating receptor molecule expressed on the surface of NK cells. Recent findings suggest that DNAM-1 is a critical regulator of NK cell biology. DNAM-1 is involved in NK cell education and differentiation, and also plays a pivotal role in the development of cancer, viral infections and immune-related diseases. However, tumours and viruses have developed multiple mechanisms to evade the immune system. They are able to impair DNAM-1 activity by targeting the DNAM-1 receptor–ligand system. We have reviewed the roles of DNAM-1, and its biological functions, with respect to NK cell biology and DNAM-1 chimeric antigen receptor-based immunotherapy. PMID:26235210

  3. Investigations of electron helicity in optically active molecules using polarized beams of electrons and positrons

    NASA Technical Reports Server (NTRS)

    Gidley, D. W.; Rich, A.; Van House, J. C.; Zitzewitz, P. W.

    1981-01-01

    A positronium-formation experiment with a high sensitivity to a possible relation between the helicity of beta particles emitted in nuclear beta decay and the optical asymmetry of biological molecules is presented. The experiment is based on a mechanism in which the electrons in optically active molecules possess a helicity of less than 0.001, too weak to detect in radiolysis experiments, the sign of which depends on the chirality of the isomer. A helicity-dependent asymmetry is sought in the formation of the triplet ground state of positronium when a low-energy beam of polarized positrons of reversible helicity interacts with an optically active substance coating a channel electron multiplier. Asymmetries between positronium decays observed at positive and negative helicities for the same substance can thus be determined with a sensitivity of 0.0001, which represents a factor of 100 improvement over previous positronium experiments.

  4. Delivery of Molecules into Human Corneal Endothelial Cells by Carbon Nanoparticles Activated by Femtosecond Laser

    PubMed Central

    Jumelle, Clotilde; Mauclair, Cyril; Houzet, Julien; Bernard, Aurélien; He, Zhiguo; Forest, Fabien; Peoc’h, Michel; Acquart, Sophie; Gain, Philippe; Thuret, Gilles

    2015-01-01

    Corneal endothelial cells (CECs) form a monolayer at the innermost face of the cornea and are the engine of corneal transparency. Nevertheless, they are a vulnerable population incapable of regeneration in humans, and their diseases are responsible for one third of corneal grafts performed worldwide. Donor corneas are stored in eye banks for security and quality controls, then delivered to surgeons. This period could allow specific interventions to modify the characteristics of CECs in order to increase their proliferative capacity, increase their resistance to apoptosis, or release immunosuppressive molecules. Delivery of molecules specifically into CECs during storage would therefore open up new therapeutic perspectives. For clinical applications, physical methods have a more favorable individual and general benefit/risk ratio than most biological vectors, but are often less efficient. The delivery of molecules into cells by carbon nanoparticles activated by femtosecond laser pulses is a promising recent technique developed on non-adherent cells. The nanoparticles are partly consummated by the reaction releasing CO and H2 gas bubbles responsible for the shockwave at the origin of cell transient permeation. Our aim was to develop an experimental setting to deliver a small molecule (calcein) into the monolayer of adherent CECs. We confirmed that increased laser fluence and time exposure increased uptake efficiency while keeping cell mortality below 5%. We optimized the area covered by the laser beam by using a motorized stage allowing homogeneous scanning of the cell culture surface using a spiral path. Calcein uptake reached median efficiency of 54.5% (range 50.3–57.3) of CECs with low mortality (0.5%, range (0.55–1.0)). After sorting by flow cytometry, CECs having uptaken calcein remained viable and presented normal morphological characteristics. Delivery of molecules into CECs by carbon nanoparticles activated by femtosecond laser could prove useful for

  5. Delivery of Molecules into Human Corneal Endothelial Cells by Carbon Nanoparticles Activated by Femtosecond Laser.

    PubMed

    Jumelle, Clotilde; Mauclair, Cyril; Houzet, Julien; Bernard, Aurélien; He, Zhiguo; Forest, Fabien; Peoc'h, Michel; Acquart, Sophie; Gain, Philippe; Thuret, Gilles

    2015-01-01

    Corneal endothelial cells (CECs) form a monolayer at the innermost face of the cornea and are the engine of corneal transparency. Nevertheless, they are a vulnerable population incapable of regeneration in humans, and their diseases are responsible for one third of corneal grafts performed worldwide. Donor corneas are stored in eye banks for security and quality controls, then delivered to surgeons. This period could allow specific interventions to modify the characteristics of CECs in order to increase their proliferative capacity, increase their resistance to apoptosis, or release immunosuppressive molecules. Delivery of molecules specifically into CECs during storage would therefore open up new therapeutic perspectives. For clinical applications, physical methods have a more favorable individual and general benefit/risk ratio than most biological vectors, but are often less efficient. The delivery of molecules into cells by carbon nanoparticles activated by femtosecond laser pulses is a promising recent technique developed on non-adherent cells. The nanoparticles are partly consummated by the reaction releasing CO and H2 gas bubbles responsible for the shockwave at the origin of cell transient permeation. Our aim was to develop an experimental setting to deliver a small molecule (calcein) into the monolayer of adherent CECs. We confirmed that increased laser fluence and time exposure increased uptake efficiency while keeping cell mortality below 5%. We optimized the area covered by the laser beam by using a motorized stage allowing homogeneous scanning of the cell culture surface using a spiral path. Calcein uptake reached median efficiency of 54.5% (range 50.3-57.3) of CECs with low mortality (0.5%, range (0.55-1.0)). After sorting by flow cytometry, CECs having uptaken calcein remained viable and presented normal morphological characteristics. Delivery of molecules into CECs by carbon nanoparticles activated by femtosecond laser could prove useful for future

  6. Delivery of Molecules into Human Corneal Endothelial Cells by Carbon Nanoparticles Activated by Femtosecond Laser.

    PubMed

    Jumelle, Clotilde; Mauclair, Cyril; Houzet, Julien; Bernard, Aurélien; He, Zhiguo; Forest, Fabien; Peoc'h, Michel; Acquart, Sophie; Gain, Philippe; Thuret, Gilles

    2015-01-01

    Corneal endothelial cells (CECs) form a monolayer at the innermost face of the cornea and are the engine of corneal transparency. Nevertheless, they are a vulnerable population incapable of regeneration in humans, and their diseases are responsible for one third of corneal grafts performed worldwide. Donor corneas are stored in eye banks for security and quality controls, then delivered to surgeons. This period could allow specific interventions to modify the characteristics of CECs in order to increase their proliferative capacity, increase their resistance to apoptosis, or release immunosuppressive molecules. Delivery of molecules specifically into CECs during storage would therefore open up new therapeutic perspectives. For clinical applications, physical methods have a more favorable individual and general benefit/risk ratio than most biological vectors, but are often less efficient. The delivery of molecules into cells by carbon nanoparticles activated by femtosecond laser pulses is a promising recent technique developed on non-adherent cells. The nanoparticles are partly consummated by the reaction releasing CO and H2 gas bubbles responsible for the shockwave at the origin of cell transient permeation. Our aim was to develop an experimental setting to deliver a small molecule (calcein) into the monolayer of adherent CECs. We confirmed that increased laser fluence and time exposure increased uptake efficiency while keeping cell mortality below 5%. We optimized the area covered by the laser beam by using a motorized stage allowing homogeneous scanning of the cell culture surface using a spiral path. Calcein uptake reached median efficiency of 54.5% (range 50.3-57.3) of CECs with low mortality (0.5%, range (0.55-1.0)). After sorting by flow cytometry, CECs having uptaken calcein remained viable and presented normal morphological characteristics. Delivery of molecules into CECs by carbon nanoparticles activated by femtosecond laser could prove useful for future

  7. A Novel Class of Small Molecule Agonists with Preference for Human over Mouse TLR4 Activation

    PubMed Central

    Heeke, Darren S.; Rao, Eileen; Maynard, Sean K.; Hornigold, David; McCrae, Christopher; Fraser, Neil; Tovchigrechko, Andrey; Yu, Li; Williams, Nicola; King, Sarah; Cooper, Martin E.; Hajjar, Adeline M.; Woo, Jennifer C.

    2016-01-01

    The best-characterized Toll-like receptor 4 (TLR4) ligands are lipopolysaccharide (LPS) and its chemically modified and detoxified variant, monophosphoryl lipid A (MPL). Although both molecules are active for human TLR4, they demonstrate a potency preference for mouse TLR4 based on data from transfected cell lines and primary cells of both species. After a high throughput screening process of small molecule libraries, we have discovered a new class of TLR4 agonist with a species preference profile differing from MPL. Products of the 4-component Ugi synthesis reaction were demonstrated to potently trigger human TLR4-transfected HEK cells but not mouse TLR4, although inclusion of the human MD2 with mTLR4 was able to partially recover activity. Co-expression of CD14 was not required for optimal activity of Ugi compounds on transfected cells, as it is for LPS. The species preference profile for the panel of Ugi compounds was found to be strongly active for human and cynomolgus monkey primary cells, with reduced but still substantial activity for most Ugi compounds on guinea pig cells. Mouse, rat, rabbit, ferret, and cotton rat cells displayed little or no activity when exposed to Ugi compounds. However, engineering the human versions of TLR4 and MD2 to be expressed in mTLR4/MD2 deficient mice allowed for robust activity by Ugi compounds both in vitro and in vivo. These findings extend the range of compounds available for development as agonists of TLR4 and identify novel molecules which reverse the TLR4 triggering preference of MPL for mouse TLR4 over human TLR4. Such compounds may be amenable to formulation as more potent human-specific TLR4L-based adjuvants than typical MPL-based adjuvants. PMID:27736941

  8. Smart magnetic poly(N-isopropylacrylamide) to control the release of bio-active molecules.

    PubMed

    Dionigi, Chiara; Lungaro, Lisa; Goranov, Vitaly; Riminucci, Alberto; Piñeiro-Redondo, Yolanda; Bañobre-López, Manuel; Rivas, José; Dediu, Valentin

    2014-10-01

    Thermo switchable magnetic hydrogels undoubtedly have a great potential for medical applications since they can behave as smart carriers able to transport bioactive molecules to a chosen part of the body and release them on demand via magneto-thermal activation. We report on the ability to modify the lower critical solution temperature (LCST) of poly(N-isopropylacrylamide) (PNIPAM) on demand from 32 °C to LCST ≥ 37 °C. This was achieved by the absorption of controlled amounts of magnetite nanoparticles on the polymer chains. We show, through the effect on cell viability, that the resulting magnetic PNIPAM is able to trap and to release bio-active molecules, such as cell growth factors. The activities of the released bio molecule are tested on human umbilical vein endothelial cells culture. We demonstrate that the LCST of the magnetic PNIPAM can be reached remotely via inductive heating with an alternating magnetic field. This approach on magnetic PNIPAM clearly supports appealing applications in safe biomedicine.

  9. Smart magnetic poly(N-isopropylacrylamide) to control the release of bio-active molecules.

    PubMed

    Dionigi, Chiara; Lungaro, Lisa; Goranov, Vitaly; Riminucci, Alberto; Piñeiro-Redondo, Yolanda; Bañobre-López, Manuel; Rivas, José; Dediu, Valentin

    2014-10-01

    Thermo switchable magnetic hydrogels undoubtedly have a great potential for medical applications since they can behave as smart carriers able to transport bioactive molecules to a chosen part of the body and release them on demand via magneto-thermal activation. We report on the ability to modify the lower critical solution temperature (LCST) of poly(N-isopropylacrylamide) (PNIPAM) on demand from 32 °C to LCST ≥ 37 °C. This was achieved by the absorption of controlled amounts of magnetite nanoparticles on the polymer chains. We show, through the effect on cell viability, that the resulting magnetic PNIPAM is able to trap and to release bio-active molecules, such as cell growth factors. The activities of the released bio molecule are tested on human umbilical vein endothelial cells culture. We demonstrate that the LCST of the magnetic PNIPAM can be reached remotely via inductive heating with an alternating magnetic field. This approach on magnetic PNIPAM clearly supports appealing applications in safe biomedicine. PMID:24477874

  10. Enhancer hijacking activates GFI1 family oncogenes in medulloblastoma

    PubMed Central

    Northcott, Paul A; Lee, Catherine; Zichner, Thomas; Stütz, Adrian M; Erkek, Serap; Kawauchi, Daisuke; Shih, David JH; Hovestadt, Volker; Zapatka, Marc; Sturm, Dominik; Jones, David TW; Kool, Marcel; Remke, Marc; Cavalli, Florence; Zuyderduyn, Scott; Bader, Gary; VandenBerg, Scott; Esparza, Lourdes Adriana; Ryzhova, Marina; Wang, Wei; Wittmann, Andrea; Stark, Sebastian; Sieber, Laura; Seker-Cin, Huriye; Linke, Linda; Kratochwil, Fabian; Jäger, Natalie; Buchhalter, Ivo; Imbusch, Charles D; Zipprich, Gideon; Raeder, Benjamin; Schmidt, Sabine; Diessl, Nicolle; Wolf, Stephan; Wiemann, Stefan; Brors, Benedikt; Lawerenz, Chris; Eils, Jürgen; Warnatz, Hans-Jörg; Risch, Thomas; Yaspo, Marie-Laure; Weber, Ursula D; Bartholomae, Cynthia C; von Kalle, Christof; Turányi, Eszter; Hauser, Peter; Sanden, Emma; Darabi, Anna; Siesjö, Peter; Sterba, Jaroslav; Zitterbart, Karel; Sumerauer, David; van Sluis, Peter; Versteeg, Rogier; Volckmann, Richard; Koster, Jan; Schuhmann, Martin U; Ebinger, Martin; Grimes, H. Leighton; Robinson, Giles W; Gajjar, Amar; Mynarek, Martin; von Hoff, Katja; Rutkowski, Stefan; Pietsch, Torsten; Scheurlen, Wolfram; Felsberg, Jörg; Reifenberger, Guido; Kulozik, Andreas E; von Deimlmg, Andreas; Witt, Olaf; Eils, Roland; Gilbertson, Richard J; Korshunov, Andrey; Taylor, Michael D; Lichter, Peter; Korbel, Jan O; Wechsler-Reya, Robert J; Pfister, Stefan M

    2014-01-01

    Summary Paragraph Medulloblastoma is a highly malignant paediatric brain tumour currently treated with a combination of surgery, radiation, and chemotherapy, posing a considerable burden of toxicity to the developing child. Genomics has illuminated the extensive intertumoural heterogeneity of medulloblastoma, identifying four distinct molecular subgroups. Group 3 and Group 4 subgroup medulloblastomas account for the majority of paediatric cases; yet, oncogenic drivers for these subtypes remain largely unidentified. Here we describe a series of prevalent, highly disparate genomic structural variants, restricted to Groups 3 and 4, resulting in specific and mutually exclusive activation of the growth factor independent 1 family protooncogenes, GFI1 and GFI1B. Somatic structural variants juxtapose GFI1/GFI1B coding sequences proximal to active enhancer elements, including super-enhancers, instigating oncogenic activity. Our results, supported by evidence from mouse models, identify GFI1 and GFI1B as prominent medulloblastoma oncogenes and implicate ‘enhancer hijacking’ as an efficient mechanism driving oncogene activation in a childhood cancer. PMID:25043047

  11. Human immunodeficiency virus type 1 Nef associates with a member of the p21-activated kinase family.

    PubMed Central

    Nunn, M F; Marsh, J W

    1996-01-01

    Although human immunodeficiency virus (HIV) Nef is essential for the induction of AIDS, its biochemical function has remained an enigma. In this study, HIV Nef protein is shown to associate with a serine-threonine kinase that recognizes histone H4 as a substrate, is serologically related to rat p21-activated kinase (PAK), and is specifically activated by Rac and Cdc42. These characteristics define the Nef-associated kinase as belonging to the PAK family. PAKs initiate kinase cascades in response to environmental stimuli, and their identification as a target of Nef implicates these signaling molecules in HIV pathogenesis and provides a novel target for clinical intervention. PMID:8709241

  12. Early-Late Heterobimetallic Complexes Linked by Phosphinoamide Ligands. Tuning Redox Potentials and Small Molecule Activation

    SciTech Connect

    Thomas, Christine M.

    2015-08-01

    Recent attention in the chemical community has been focused on the energy efficient and environmentally benign conversion of abundant small molecules (CO2, H2O, etc.) to useful liquid fuels. This project addresses these goals by examining fundamental aspects of catalyst design to ultimately access small molecule activation processes under mild conditions. Specifically, Thomas and coworkers have targetted heterobimetallic complexes that feature metal centers with vastly different electronic properties, dictated both by their respective positions on the periodic table and their coordination environment. Unlike homobimetallic complexes featuring identical or similar metals, the bonds between metals in early/late heterobimetallics are more polarized, with the more electron-rich late metal center donating electron density to the more electron-deficient early metal center. While metal-metal bonds pose an interesting strategy for storing redox equivalents and stabilizing reactive metal fragments, the polar character of metal-metal bonds in heterobimetallic complexes renders these molecules ideally poised to react with small molecule substrates via cleavage of energy-rich single and double bonds. In addition, metal-metal interactions have been shown to dramatically affect redox potentials and promote multielectron redox activity, suggesting that metal-metal interactions may provide a mechanism to tune redox potentials and access substrate reduction/activation at mild overpotentials. This research project has provided a better fundamental understanding of how interactions between transition metals can be used as a strategy to promote and/or control chemical transformations related to the clean production of fuels. While this project focused on the study of homogeneous systems, it is anticipated that the broad conclusions drawn from these investigations will be applicable to heterogeneous catalysis as well, particularly on heterogeneous processes that occur at interfaces in

  13. Characterization of abandoned rocket body families for active removal

    NASA Astrophysics Data System (ADS)

    Pardini, Carmen; Anselmo, Luciano

    2016-09-01

    A new ranking index was developed and applied to a wide set of rocket body families, characterized by stage dry masses greater than 500 kg and by the presence of at least 5 stages abandoned in LEO. The upper stages selected accounted for more than 80% of the unclassified rocket bodies in LEO and nearly 95% of the associated dry mass. The detailed results obtained for 657 objects clearly identified the most critical altitude-inclination bands and stage models, to be targeted first if and when a debris remediation strategy including the active removal of intact abandoned objects were deemed necessary. Apart from the evaluation of the criticality regarding the long-term evolution of the debris environment, resulting in a priority listing for optimal active removal, the application of the new ranking index is not limited to debris remediation. In fact, if applied before launch to spacecraft and rocket bodies to be disposed in orbit, at the end of mission, it would provide an additional debris mitigation analysis tool for evaluating competing disposal options. Concerning the rocket bodies abandoned in LEO, 274 resulted to have a criticality equal or larger than the average intact object abandoned in an 800 km sun-synchronous orbit. Among them, 243 belonged to the Russian Federation and Ukraine, 25 to China, 5 to Europe and 1 to Japan. In addition to being concentrated in relatively few and narrow altitude-inclinations bands, the most numerous rocket body families often present a quite uniform distribution in right ascension of the ascending node, which is especially convenient for multiple target removal missions.

  14. Family.

    ERIC Educational Resources Information Center

    Hurst, Hunter, Ed.; And Others

    1985-01-01

    This document contains the fourth volume of "Today's Delinquent," an annual publication of the National Center for Juvenile Justice. This volume deals with the issue of the family and delinquency. "The Family and Delinquency" (LaMar T. Empey) systematically reviews and weighs the evidence to support prominent theories on the origins of…

  15. Single-molecule quantification of lipotoxic expression of activating transcription factor 3

    PubMed Central

    Wilson, Dennis W.; Rutledge, John C.

    2014-01-01

    Activating transcription factor 3 (ATF3) is a member of the mammalian activation transcription factor/cAMP, physiologically important in the regulation of pro- and anti-inflammatory target genes. We compared the induction of ATF3 protein as measured by Western blot analysis with single-molecule localization microscopy dSTORM to quantify the dynamics of accumulation of intranuclear ATF3 of triglyceride-rich (TGRL) lipolysis product-treated HAEC (Human Aortic Endothelial Cells). The ATF3 expression rate within the first three hours after treatment with TGRL lipolysis products is about 3500/h. After three hours we detected 33,090 ± 3,491 single-molecule localizations of ATF3. This was accompanied by significant structural changes in the F-actin network of the cells at ~3-fold increased localization precision compared to widefield microscopy after treatment. Additionally, we discovered a cluster size of approximately 384 nanometers of ATF3 molecules. We show for the first time the time course of ATF3 accumulation in the nucleus undergoing lipotoxic injury. Furthermore, we demonstrate ATF3 accumulation associated with increased concentrations of TGRL lipolysis products occurs in large aggregates. PMID:25189785

  16. Single-molecule kinetics under force: probing protein folding and enzymatic activity with optical tweezers

    NASA Astrophysics Data System (ADS)

    Wong, Wesley

    2010-03-01

    Weak non-covalent bonds between and within single molecules govern many aspects of biological structure and function (e.g. DNA base-paring, receptor-ligand binding, protein folding, etc.) In living systems, these interactions are often subject to mechanical forces, which can greatly alter their kinetics and activity. My group develops and applies novel single-molecule manipulation techniques to explore and quantify these force-dependent kinetics. Using optical tweezers, we have quantified the force-dependent unfolding and refolding kinetics of different proteins, including the cytoskeletal protein spectrin in collaboration with E. Evans's group [1], and the A2 domain of the von Willebrand factor blood clotting protein in collaboration with T. Springer's group [2]. Furthermore, we have studied the kinetics of the ADAMTS13 enzyme acting on a single A2 domain, and have shown that physiolgical forces in the circulation can act as a cofactor for enzymatic cleavage, regulating hemostatic activity [2]. References: 1. E. Evans, K. Halvorsen, K. Kinoshita, and W.P. Wong, Handbook of Single Molecule Biophysics, P. Hinterdorfer, ed., Springer (2009). 2. X. Zhang, K. Halvorsen, C.-Z. Zhang, W.P. Wong, and T.A. Springer, Science 324 (5932), 1330-1334 (2009).

  17. Proteasome Activation is a Mechanism for Pyrazolone Small Molecules Displaying Therapeutic Potential in Amyotrophic Lateral Sclerosis

    PubMed Central

    2015-01-01

    Amyotrophic lateral sclerosis (ALS) is a progressive and ultimately fatal neurodegenerative disease. Pyrazolone containing small molecules have shown significant disease attenuating efficacy in cellular and murine models of ALS. Pyrazolone based affinity probes were synthesized to identify high affinity binding partners and ascertain a potential biological mode of action. Probes were confirmed to be neuroprotective in PC12-SOD1G93A cells. PC12-SOD1G93A cell lysates were used for protein pull-down, affinity purification, and subsequent proteomic analysis using LC-MS/MS. Proteomics identified the 26S proteasome regulatory subunit 4 (PSMC1), 26S proteasome regulatory subunit 6B (PSMC4), and T-complex protein 1 (TCP-1) as putative protein targets. Coincubation with appropriate competitors confirmed the authenticity of the proteomics results. Activation of the proteasome by pyrazolones was demonstrated in the absence of exogenous proteasome inhibitor and by restoration of cellular protein degradation of a fluorogenic proteasome substrate in PC12-SOD1G93A cells. Importantly, supplementary studies indicated that these molecules do not induce a heat shock response. We propose that pyrazolones represent a rare class of molecules that enhance proteasomal activation in the absence of a heat shock response and may have therapeutic potential in ALS. PMID:25001311

  18. The Small Molecule IMR-1 Inhibits the Notch Transcriptional Activation Complex to Suppress Tumorigenesis.

    PubMed

    Astudillo, Luisana; Da Silva, Thiago G; Wang, Zhiqiang; Han, Xiaoqing; Jin, Ke; VanWye, Jeffrey; Zhu, Xiaoxia; Weaver, Kelly; Oashi, Taiji; Lopes, Pedro E M; Orton, Darren; Neitzel, Leif R; Lee, Ethan; Landgraf, Ralf; Robbins, David J; MacKerell, Alexander D; Capobianco, Anthony J

    2016-06-15

    In many cancers, aberrant Notch activity has been demonstrated to play a role in the initiation and maintenance of the neoplastic phenotype and in cancer stem cells, which may allude to its additional involvement in metastasis and resistance to therapy. Therefore, Notch is an exceedingly attractive therapeutic target in cancer, but the full range of potential targets within the pathway has been underexplored. To date, there are no small-molecule inhibitors that directly target the intracellular Notch pathway or the assembly of the transcriptional activation complex. Here, we describe an in vitro assay that quantitatively measures the assembly of the Notch transcriptional complex on DNA. Integrating this approach with computer-aided drug design, we explored potential ligand-binding sites and screened for compounds that could disrupt the assembly of the Notch transcriptional activation complex. We identified a small-molecule inhibitor, termed Inhibitor of Mastermind Recruitment-1 (IMR-1), that disrupted the recruitment of Mastermind-like 1 to the Notch transcriptional activation complex on chromatin, thereby attenuating Notch target gene transcription. Furthermore, IMR-1 inhibited the growth of Notch-dependent cell lines and significantly abrogated the growth of patient-derived tumor xenografts. Taken together, our findings suggest that a novel class of Notch inhibitors targeting the transcriptional activation complex may represent a new paradigm for Notch-based anticancer therapeutics, warranting further preclinical characterization. Cancer Res; 76(12); 3593-603. ©2016 AACR. PMID:27197169

  19. Ethosomes for the delivery of anti-HSV-1 molecules: preparation, characterization and in vitro activity.

    PubMed

    Cortesi, R; Ravani, L; Zaid, A N; Menegatti, E; Romagnoli, R; Drechsler, M; Esposito, E

    2010-10-01

    This paper describes the production, characterization and in vitro activity of ethosomes containing two molecules with antiviral activity, such as acyclovir (ACY) and N1-beta-D-ribofuranosyl-pyrazole [3,4d]pyridazin-7(6p-chlorine-phenyl)-one nucleoside (N1CP). Ethosomes were prepared and morphologically characterized by Cryo-TEM. The encapsulation efficiency was 92.3 +/- 2.5% for ACY and 94.2 +/- 2.8% for N1CP. The release of the drug from vesicles, determined by a Franz cell method, indicated that both drugs were released in a controlled manner. In order to possibly guarantee the stability during long-term storage ethosome suspensions was freeze-dried. It was found that the freeze-dried ethosomes' cakes were compact, glassy characterized by low density and quick re-hydration. However, the storage time slightly influences the percentage of drug encapsulation within ethosomes showing a drug leakage after re-hydration around 10%. The antiviral activity against HSV-1 of both drugs was tested by plaque reduction assay in monolayer cultures of Vero cells. Data showed that ethosomes allowed a reduction of the ED50 of N1CP evidencing an increase of its antiviral activity. However, ACY remains more active than N1CP. No differences are appreciable between drug-containing ethosomes before and after freeze-drying. Taken together these results, ethosomal formulation could be possibly proposed as mean for topical administration of anti-herpetic molecules.

  20. Single-Molecule Imaging Reveals the Activation Dynamics of Intracellular Protein Smad3 on Cell Membrane

    NASA Astrophysics Data System (ADS)

    Li, Nan; Yang, Yong; He, Kangmin; Zhang, Fayun; Zhao, Libo; Zhou, Wei; Yuan, Jinghe; Liang, Wei; Fang, Xiaohong

    2016-09-01

    Smad3 is an intracellular protein that plays a key role in propagating transforming growth factor β (TGF-β) signals from cell membrane to nucleus. However whether the transient process of Smad3 activation occurs on cell membrane and how it is regulated remains elusive. Using advanced live-cell single-molecule fluorescence microscopy to image and track fluorescent protein-labeled Smad3, we observed and quantified, for the first time, the dynamics of individual Smad3 molecules docking to and activation on the cell membrane. It was found that Smad3 docked to cell membrane in both unstimulated and stimulated cells, but with different diffusion rates and dissociation kinetics. The change in its membrane docking dynamics can be used to study the activation of Smad3. Our results reveal that Smad3 binds with type I TGF-β receptor (TRI) even in unstimulated cells. Its activation is regulated by TRI phosphorylation but independent of receptor endocytosis. This study offers new information on TGF-β/Smad signaling, as well as a new approach to investigate the activation of intracellular signaling proteins for a better understanding of their functions in signal transduction.

  1. Ethosomes for the delivery of anti-HSV-1 molecules: preparation, characterization and in vitro activity.

    PubMed

    Cortesi, R; Ravani, L; Zaid, A N; Menegatti, E; Romagnoli, R; Drechsler, M; Esposito, E

    2010-10-01

    This paper describes the production, characterization and in vitro activity of ethosomes containing two molecules with antiviral activity, such as acyclovir (ACY) and N1-beta-D-ribofuranosyl-pyrazole [3,4d]pyridazin-7(6p-chlorine-phenyl)-one nucleoside (N1CP). Ethosomes were prepared and morphologically characterized by Cryo-TEM. The encapsulation efficiency was 92.3 +/- 2.5% for ACY and 94.2 +/- 2.8% for N1CP. The release of the drug from vesicles, determined by a Franz cell method, indicated that both drugs were released in a controlled manner. In order to possibly guarantee the stability during long-term storage ethosome suspensions was freeze-dried. It was found that the freeze-dried ethosomes' cakes were compact, glassy characterized by low density and quick re-hydration. However, the storage time slightly influences the percentage of drug encapsulation within ethosomes showing a drug leakage after re-hydration around 10%. The antiviral activity against HSV-1 of both drugs was tested by plaque reduction assay in monolayer cultures of Vero cells. Data showed that ethosomes allowed a reduction of the ED50 of N1CP evidencing an increase of its antiviral activity. However, ACY remains more active than N1CP. No differences are appreciable between drug-containing ethosomes before and after freeze-drying. Taken together these results, ethosomal formulation could be possibly proposed as mean for topical administration of anti-herpetic molecules. PMID:21105576

  2. Single-Molecule Imaging Reveals the Activation Dynamics of Intracellular Protein Smad3 on Cell Membrane.

    PubMed

    Li, Nan; Yang, Yong; He, Kangmin; Zhang, Fayun; Zhao, Libo; Zhou, Wei; Yuan, Jinghe; Liang, Wei; Fang, Xiaohong

    2016-01-01

    Smad3 is an intracellular protein that plays a key role in propagating transforming growth factor β (TGF-β) signals from cell membrane to nucleus. However whether the transient process of Smad3 activation occurs on cell membrane and how it is regulated remains elusive. Using advanced live-cell single-molecule fluorescence microscopy to image and track fluorescent protein-labeled Smad3, we observed and quantified, for the first time, the dynamics of individual Smad3 molecules docking to and activation on the cell membrane. It was found that Smad3 docked to cell membrane in both unstimulated and stimulated cells, but with different diffusion rates and dissociation kinetics. The change in its membrane docking dynamics can be used to study the activation of Smad3. Our results reveal that Smad3 binds with type I TGF-β receptor (TRI) even in unstimulated cells. Its activation is regulated by TRI phosphorylation but independent of receptor endocytosis. This study offers new information on TGF-β/Smad signaling, as well as a new approach to investigate the activation of intracellular signaling proteins for a better understanding of their functions in signal transduction. PMID:27641076

  3. Single-Molecule Imaging Reveals the Activation Dynamics of Intracellular Protein Smad3 on Cell Membrane

    PubMed Central

    Li, Nan; Yang, Yong; He, Kangmin; Zhang, Fayun; Zhao, Libo; Zhou, Wei; Yuan, Jinghe; Liang, Wei; Fang, Xiaohong

    2016-01-01

    Smad3 is an intracellular protein that plays a key role in propagating transforming growth factor β (TGF-β) signals from cell membrane to nucleus. However whether the transient process of Smad3 activation occurs on cell membrane and how it is regulated remains elusive. Using advanced live-cell single-molecule fluorescence microscopy to image and track fluorescent protein-labeled Smad3, we observed and quantified, for the first time, the dynamics of individual Smad3 molecules docking to and activation on the cell membrane. It was found that Smad3 docked to cell membrane in both unstimulated and stimulated cells, but with different diffusion rates and dissociation kinetics. The change in its membrane docking dynamics can be used to study the activation of Smad3. Our results reveal that Smad3 binds with type I TGF-β receptor (TRI) even in unstimulated cells. Its activation is regulated by TRI phosphorylation but independent of receptor endocytosis. This study offers new information on TGF-β/Smad signaling, as well as a new approach to investigate the activation of intracellular signaling proteins for a better understanding of their functions in signal transduction. PMID:27641076

  4. A Family Literacy Class Develops a Learning Activity.

    ERIC Educational Resources Information Center

    EFF HOT Topics, 2002

    2002-01-01

    An instructor and her students used questions to plan, teach, and assess a lesson in a Family Literacy/General Educational Development (GED) program. The class identified that their families should lose weight and exercise more to have healthier hearts. They focused on the area of responsibility, called Promote Family Members' Growth and…

  5. Plasmonic enhancement of Raman optical activity in molecules near metal nanoshells.

    PubMed

    Acevedo, Ramiro; Lombardini, Richard; Halas, Naomi J; Johnson, Bruce R

    2009-11-26

    Surface-enhanced Raman optical activity (SEROA) is investigated theoretically for molecules near a metal nanoshell. For this purpose, induced molecular electric dipole, magnetic dipole, and electric quadrupole moments must all be included. The incident field and the induced multipole fields all scatter from the nanoshell, and the scattered waves can be calculated via extended Mie theory. It is straightforward in this framework to calculate the incident frequency dependence of SEROA intensities, i.e., SEROA excitation profiles. The differential Raman scattering is examined in detail for a simple chiroptical model that provides analytical forms for the relevant dynamical molecular response tensors. This allows a detailed investigation into circumstances that simultaneously provide strong enhancement of differential intensities and remain selective for molecules with chirality. PMID:19639972

  6. Microgravimetric Analysis Method for Activation-Energy Extraction from Trace-Amount Molecule Adsorption.

    PubMed

    Xu, Pengcheng; Yu, Haitao; Li, Xinxin

    2016-05-01

    Activation-energy (Ea) value for trace-amount adsorption of gas molecules on material is rapidly and inexpensively obtained, for the first time, from a microgravimetric analysis experiment. With the material loaded, a resonant microcantilever is used to record in real time the adsorption process at two temperatures. The kinetic parameter Ea is thereby extracted by solving the Arrhenius equation. As an example, two CO2 capture nanomaterials are examined by the Ea extracting method for evaluation/optimization and, thereby, demonstrating the applicability of the microgravimetric analysis method. The achievement helps to solve the absence in rapid quantitative characterization of sorption kinetics and opens a new route to investigate molecule adsorption processes and materials.

  7. Antithrombotic and antiplatelet activities of small-molecule alkaloids from Scolopendra subspinipes mutilans

    PubMed Central

    Lee, Wonhwa; Lee, JungIn; Kulkarni, Roshan; Kim, Mi-Ae; Hwang, Jae Sam; Na, MinKyun; Bae, Jong-Sup

    2016-01-01

    The aim of this study was to discover small-molecule anticoagulants from Scolopendra subspinipes mutilans (SSM). A new acylated polyamine (1) and a new sulfated quinoline alkaloid (2) were isolated from SSM. Treatment with the new alkaloids 1, 2, and indole acetic acid 4 prolonged the activated partial thromboplastin time and prothrombin time and inhibited the activity and production of thrombin and activated factor X. Furthermore, compounds 1, 2, and 4 inhibited thrombin-catalyzed fibrin polymerization and platelet aggregation. In accordance with these potential in vitro antiplatelet activities, compounds 1, 2, and 4 showed enhanced antithrombotic effects in an in vivo pulmonary embolism and arterial thrombosis model. Compounds 1, 2, and 4 also elicited anticoagulant effects in mice. Collectively, this study may serve as the groundwork for commercializing SSM or compounds 1, 2, and 4 as functional food components for the prevention and treatment of pathogenic conditions and serve as new scaffolds for the development of anticoagulants. PMID:26905699

  8. Structural Basis for Selective Small Molecule Kinase Inhibition of Activated c-Met

    SciTech Connect

    Rickert, Keith W.; Patel, Sangita B.; Allison, Timothy J.; Byrne, Noel J.; Darke, Paul L.; Ford, Rachael E.; Guerin, David J.; Hall, Dawn L.; Kornienko, Maria; Lu, Jun; Munshi, Sanjeev K.; Reid, John C.; Shipman, Jennifer M.; Stanton, Elizabeth F.; Wilson, Kevin J.; Young, Jonathon R.; Soisson, Stephen M.; Lumb, Kevin J.

    2012-03-15

    The receptor tyrosine kinase c-Met is implicated in oncogenesis and is the target for several small molecule and biologic agents in clinical trials for the treatment of cancer. Binding of the hepatocyte growth factor to the cell surface receptor of c-Met induces activation via autophosphorylation of the kinase domain. Here we describe the structural basis of c-Met activation upon autophosphorylation and the selective small molecule inhibiton of autophosphorylated c-Met. MK-2461 is a potent c-Met inhibitor that is selective for the phosphorylated state of the enzyme. Compound 1 is an MK-2461 analog with a 20-fold enthalpy-driven preference for the autophosphorylated over unphosphorylated c-Met kinase domain. The crystal structure of the unbound kinase domain phosphorylated at Tyr-1234 and Tyr-1235 shows that activation loop phosphorylation leads to the ejection and disorder of the activation loop and rearrangement of helix {alpha}C and the G loop to generate a viable active site. Helix {alpha}C adopts a orientation different from that seen in activation loop mutants. The crystal structure of the complex formed by the autophosphorylated c-Met kinase domain and compound 1 reveals a significant induced fit conformational change of the G loop and ordering of the activation loop, explaining the selectivity of compound 1 for the autophosphorylated state. The results highlight the role of structural plasticity within the kinase domain in imparting the specificity of ligand binding and provide the framework for structure-guided design of activated c-Met inhibitors.

  9. Small-molecule activation of SERCA2a SUMOylation for the treatment of heart failure

    PubMed Central

    Kho, Changwon; Lee, Ahyoung; Jeong, Dongtak; Oh, Jae Gyun; Gorski, Przemek A.; Fish, Kenneth; Sanchez, Roberto; DeVita, Robert J.; Christensen, Geir; Dahl, Russell; Hajjar, Roger J.

    2015-01-01

    Decreased activity and expression of the cardiac sarcoplasmic reticulum calcium ATPase (SERCA2a), a critical pump regulating calcium cycling in cardiomyocyte, are hallmarks of heart failure. We have previously described a role for the small ubiquitin-like modifier type 1 (SUMO-1) as a regulator of SERCA2a and have shown that gene transfer of SUMO-1 in rodents and large animal models of heart failure restores cardiac function. Here, we identify and characterize a small molecule, N106, which increases SUMOylation of SERCA2a. This compound directly activates the SUMO-activating enzyme, E1 ligase, and triggers intrinsic SUMOylation of SERCA2a. We identify a pocket on SUMO E1 likely to be responsible for N106's effect. N106 treatment increases contractile properties of cultured rat cardiomyocytes and significantly improves ventricular function in mice with heart failure. This first-in-class small-molecule activator targeting SERCA2a SUMOylation may serve as a potential therapeutic strategy for treatment of heart failure. PMID:26068603

  10. Families, Disability, and Empowerment: Active Coping Skills and Strategies for Family Interventions.

    ERIC Educational Resources Information Center

    Singer, George H. S., Ed.; Powers, Laurie E., Ed.

    This book presents strategies for building strong partnerships between service providers and the families of individuals with disabilities. Papers have the following titles and authors: "Contributing to Resilience in Families: An Overview" (George H. S. Singer and Laurie E. Powers); "Parent to Parent Programs: A Unique Form of Mutual Support for…

  11. Influence of the water molecules near surface of viral protein on virus activation process

    NASA Astrophysics Data System (ADS)

    Shepelenko, S. O.; Salnikov, A. S.; Rak, S. V.; Goncharova, E. P.; Ryzhikov, A. B.

    2009-06-01

    The infection of a cell with influenza virus comprises the stages of receptor binding to the cell membrane, endocytosis of virus particle, and fusion of the virus envelope and cell endosome membrane, which is determined by the conformational changes in hemagglutinin, a virus envelope protein, caused by pH decrease within the endosome. The pH value that induces conformation rearrangements of hemagglutinin molecule considerably varies for different influenza virus strains, first and foremost, due to the differences in amino acid structure of the corresponding proteins. The main goal of this study was to construct a model making it possible to assess the critical pH value characterizing the fusogenic activity of influenza virus hemagglutinin from the data on hemagglutinin structure and experimental verification of this model. Under this model, we assume that when the electrostatic force between interacting hemagglutinin molecules in the virus envelop exceeds a certain value, the hemagglutinin HA1 subunits are arranged so that they form a cavity sufficient for penetration of water molecules. This event leads to an irreversible hydration of the inner fragments of hemagglutinin molecule in a trimer and to the completion of conformational changes. The geometry of electrostatic field in hemagglutinin trimer was calculated taking into account the polarization effects near the interface of two dielectrics, aqueous medium and protein macromolecule. The critical pH values for the conformational changes in hemagglutinin were measured by the erythrocyte hemolysis induced by influenza virus particles when decreasing pH. The critical pH value conditionally separating the pH range into the regions with and without the conformational changes was calculated for several influenza virus H1N1 and H3N2 strains based on the data on the amino acid structure of the corresponding hemagglutinin molecules. Comparison of the theoretical and experimental values of critical pH values for

  12. Thermal response, catalytic activity, and color change of the first hybrid vanadate containing Bpe guest molecules.

    PubMed

    Fernández de Luis, Roberto; Urtiaga, M Karmele; Mesa, José L; Larrea, Edurne S; Iglesias, Marta; Rojo, Teófilo; Arriortua, María I

    2013-03-01

    Four isomorphic compounds with formula [{Co2(H2O)2(Bpe)2}(V4O12)]·4H2O·Bpe, CoBpe 1; [{CoNi(H2O)2(Bpe)2}(V4O12)]·4H2O·Bpe, CoNiBpe 2; [{Co0.6Ni1.4(H2O)2(Bpe)2}(V4O12)]·4H2O·Bpe, NiCoBpe 3; and [{Ni2(H2O)2(Bpe)2}(V4O12)]·4H2O·Bpe, NiBpe 4, have been obtained by hydrothermal synthesis. The crystal structures of CoBpe 1 and NiBpe 4 were determined by single-crystal X-ray diffraction (XRD). The Rietveld refinement of CoNiBpe 2 and NiCoBpe 3 XRD patterns confirms that those are isomorphic. The compounds crystallize in the P1̅ space group, exhibiting a crystal structure constructed from inorganic layers pillared by Bpe ligands. The crystal structure contains intralayer and interlayer channels, in which the crystallization water molecules and Bpe guest molecules, respectively, are located. The solvent molecules establish a hydrogen bonding network with the coordinated water molecules. Thermodiffractometric and thermogravimetric studies showed that the loss of crystallization and coordinated water molecules takes place at different temperatures, giving rise to crystal structure transformations that involve important reduction of the interlayer distance, and strong reduction of crystallinity. The IR, Raman, and UV-vis spectra of the as-synthesized and heated compounds confirm that the structural building blocks and octahedral coordination environment of the metal centers are maintained after the structural transformations. The color change and reversibility of the water molecules uptake/removal were tested showing that the initial color is not completely recovered when the compounds are heated at temperatures higher than 200 °C. The thermal evolution of the magnetic susceptibility indicates one-dimensional antiferromagnetic coupling of the metal centers at high temperatures. For NiCoBpe 3 and NiBpe 4 compounds magnetic ordering is established at low temperatures, as can be judged by the maxima observed in the magnetic susceptibilities. CoNiBpe 2 was proved as

  13. Thermal response, catalytic activity, and color change of the first hybrid vanadate containing Bpe guest molecules.

    PubMed

    Fernández de Luis, Roberto; Urtiaga, M Karmele; Mesa, José L; Larrea, Edurne S; Iglesias, Marta; Rojo, Teófilo; Arriortua, María I

    2013-03-01

    Four isomorphic compounds with formula [{Co2(H2O)2(Bpe)2}(V4O12)]·4H2O·Bpe, CoBpe 1; [{CoNi(H2O)2(Bpe)2}(V4O12)]·4H2O·Bpe, CoNiBpe 2; [{Co0.6Ni1.4(H2O)2(Bpe)2}(V4O12)]·4H2O·Bpe, NiCoBpe 3; and [{Ni2(H2O)2(Bpe)2}(V4O12)]·4H2O·Bpe, NiBpe 4, have been obtained by hydrothermal synthesis. The crystal structures of CoBpe 1 and NiBpe 4 were determined by single-crystal X-ray diffraction (XRD). The Rietveld refinement of CoNiBpe 2 and NiCoBpe 3 XRD patterns confirms that those are isomorphic. The compounds crystallize in the P1̅ space group, exhibiting a crystal structure constructed from inorganic layers pillared by Bpe ligands. The crystal structure contains intralayer and interlayer channels, in which the crystallization water molecules and Bpe guest molecules, respectively, are located. The solvent molecules establish a hydrogen bonding network with the coordinated water molecules. Thermodiffractometric and thermogravimetric studies showed that the loss of crystallization and coordinated water molecules takes place at different temperatures, giving rise to crystal structure transformations that involve important reduction of the interlayer distance, and strong reduction of crystallinity. The IR, Raman, and UV-vis spectra of the as-synthesized and heated compounds confirm that the structural building blocks and octahedral coordination environment of the metal centers are maintained after the structural transformations. The color change and reversibility of the water molecules uptake/removal were tested showing that the initial color is not completely recovered when the compounds are heated at temperatures higher than 200 °C. The thermal evolution of the magnetic susceptibility indicates one-dimensional antiferromagnetic coupling of the metal centers at high temperatures. For NiCoBpe 3 and NiBpe 4 compounds magnetic ordering is established at low temperatures, as can be judged by the maxima observed in the magnetic susceptibilities. CoNiBpe 2 was proved as

  14. In Vitro and In Vivo Activity of a Novel Antifungal Small Molecule against Candida Infections

    PubMed Central

    Yuen, Kwok Yong; Wang, Yu; Yang, Dan; Samaranayake, Lakshman Perera

    2014-01-01

    Candida is the most common fungal pathogen of humans worldwide and has become a major clinical problem because of the growing number of immunocompromised patients, who are susceptible to infection. Moreover, the number of available antifungals is limited, and antifungal-resistant Candida strains are emerging. New and effective antifungals are therefore urgently needed. Here, we discovered a small molecule with activity against Candida spp. both in vitro and in vivo. We screened a library of 50,240 small molecules for inhibitors of yeast-to-hypha transition, a major virulence attribute of Candida albicans. This screening identified 20 active compounds. Further examination of the in vitro antifungal and anti-biofilm properties of these compounds, using a range of Candida spp., led to the discovery of SM21, a highly potent antifungal molecule (minimum inhibitory concentration (MIC) 0.2 – 1.6 µg/ml). In vitro, SM21 was toxic to fungi but not to various human cell lines or bacterial species and was active against Candida isolates that are resistant to existing antifungal agents. Moreover, SM21 was relatively more effective against biofilms of Candida spp. than the current antifungal agents. In vivo, SM21 prevented the death of mice in a systemic candidiasis model and was also more effective than the common antifungal nystatin at reducing the extent of tongue lesions in a mouse model of oral candidiasis. Propidium iodide uptake assay showed that SM21 affected the integrity of the cell membrane. Taken together, our results indicate that SM21 has the potential to be developed as a novel antifungal agent for clinical use. PMID:24465737

  15. In vitro and in vivo activity of a novel antifungal small molecule against Candida infections.

    PubMed

    Wong, Sarah Sze Wah; Kao, Richard Yi Tsun; Yuen, Kwok Yong; Wang, Yu; Yang, Dan; Samaranayake, Lakshman Perera; Seneviratne, Chaminda Jayampath

    2014-01-01

    Candida is the most common fungal pathogen of humans worldwide and has become a major clinical problem because of the growing number of immunocompromised patients, who are susceptible to infection. Moreover, the number of available antifungals is limited, and antifungal-resistant Candida strains are emerging. New and effective antifungals are therefore urgently needed. Here, we discovered a small molecule with activity against Candida spp. both in vitro and in vivo. We screened a library of 50,240 small molecules for inhibitors of yeast-to-hypha transition, a major virulence attribute of Candida albicans. This screening identified 20 active compounds. Further examination of the in vitro antifungal and anti-biofilm properties of these compounds, using a range of Candida spp., led to the discovery of SM21, a highly potent antifungal molecule (minimum inhibitory concentration (MIC) 0.2-1.6 µg/ml). In vitro, SM21 was toxic to fungi but not to various human cell lines or bacterial species and was active against Candida isolates that are resistant to existing antifungal agents. Moreover, SM21 was relatively more effective against biofilms of Candida spp. than the current antifungal agents. In vivo, SM21 prevented the death of mice in a systemic candidiasis model and was also more effective than the common antifungal nystatin at reducing the extent of tongue lesions in a mouse model of oral candidiasis. Propidium iodide uptake assay showed that SM21 affected the integrity of the cell membrane. Taken together, our results indicate that SM21 has the potential to be developed as a novel antifungal agent for clinical use. PMID:24465737

  16. Small-Molecule Activators of Insulin-Degrading Enzyme Discovered through High-Throughput Compound Screening

    PubMed Central

    Cabrol, Christelle; Huzarska, Malwina A.; Dinolfo, Christopher; Rodriguez, Maria C.; Reinstatler, Lael; Ni, Jake; Yeh, Li-An; Cuny, Gregory D.; Stein, Ross L.; Selkoe, Dennis J.; Leissring, Malcolm A.

    2009-01-01

    Background Hypocatabolism of the amyloid β-protein (Aβ) by insulin-degrading enzyme (IDE) is implicated in the pathogenesis of Alzheimer disease (AD), making pharmacological activation of IDE an attractive therapeutic strategy. However, it has not been established whether the proteolytic activity of IDE can be enhanced by drug-like compounds. Methodology/Principal Findings Based on the finding that ATP and other nucleotide polyphosphates modulate IDE activity at physiological concentrations, we conducted parallel high-throughput screening campaigns in the absence or presence of ATP and identified two compounds—designated Ia1 and Ia2—that significantly stimulate IDE proteolytic activity. Both compounds were found to interfere with the crosslinking of a photoaffinity ATP analogue to IDE, suggesting that they interact with a bona fide ATP-binding domain within IDE. Unexpectedly, we observed highly synergistic activation effects when the activity of Ia1 or Ia2 was tested in the presence of ATP, a finding that has implications for the mechanisms underlying ATP-mediated activation of IDE. Notably, Ia1 and Ia2 activated the degradation of Aβ by ∼700% and ∼400%, respectively, albeit only when Aβ was presented in a mixture also containing shorter substrates. Conclusions/Significance This study describes the first examples of synthetic small-molecule activators of IDE, showing that pharmacological activation of this important protease with drug-like compounds is achievable. These novel activators help to establish the putative ATP-binding domain as a key modulator of IDE proteolytic activity and offer new insights into the modulatory action of ATP. Several larger lessons abstracted from this screen will help inform the design of future screening campaigns and facilitate the eventual development of IDE activators with therapeutic utility. PMID:19384407

  17. Single Molecule Characterization of UV-Activated Antibodies on Gold by Atomic Force Microscopy.

    PubMed

    Funari, R; Della Ventura, B; Altucci, C; Offenhäusser, A; Mayer, D; Velotta, R

    2016-08-16

    The interaction between proteins and solid surfaces can influence their conformation and therefore also their activity and affinity. These interactions are highly specific for the respective combination of proteins and solids. Consequently, it is desirable to investigate the conformation of proteins on technical surfaces, ideally at single molecule level, and to correlate the results with their activity. This is in particular true for biosensors where the conformation-dependent target affinity of an immobilized receptor determines the sensitivity of the sensor. Here, we investigate for the first time the immobilization and orientation of antibodies (Abs) photoactivated by a photonic immobilization technique (PIT), which has previously demonstrated to enhance binding capabilities of antibody receptors. The photoactivated immunoglobulins are immobilized on ultrasmooth template stripped gold films and investigated by atomic force microscopy (AFM) at the level of individual molecules. The observed protein orientations are compared with results of nonactivated antibodies adsorbed on similar gold films and mica reference samples. We find that the behavior of Abs is similar for mica and gold when the protein are not treated (physisorption), whereas smaller contact area and larger heights are measured when Abs are treated (PIT). This is explained by assuming that the activated antibodies tend to be more upright compared with nonirradiated ones, thereby providing a better exposure of the binding sites. This finding matches the observed enhancement of Abs binding efficiency when PIT is used to functionalize gold surface of QCM-based biosensors.

  18. Novel small molecules targeting ciliary transport of Smoothened and oncogenic Hedgehog pathway activation

    PubMed Central

    Jung, Bomi; Messias, Ana C.; Schorpp, Kenji; Geerlof, Arie; Schneider, Günter; Saur, Dieter; Hadian, Kamyar; Sattler, Michael; Wanker, Erich E.; Hasenöder, Stefan; Lickert, Heiko

    2016-01-01

    Trafficking of the G protein-coupled receptor (GPCR) Smoothened (Smo) to the primary cilium (PC) is a potential target to inhibit oncogenic Hh pathway activation in a large number of tumors. One drawback is the appearance of Smo mutations that resist drug treatment, which is a common reason for cancer treatment failure. Here, we undertook a high content screen with compounds in preclinical or clinical development and identified ten small molecules that prevent constitutive active mutant SmoM2 transport into PC for subsequent Hh pathway activation. Eight of the ten small molecules act through direct interference with the G protein-coupled receptor associated sorting protein 2 (Gprasp2)-SmoM2 ciliary targeting complex, whereas one antagonist of ionotropic receptors prevents intracellular trafficking of Smo to the PC. Together, these findings identify several compounds with the potential to treat drug-resistant SmoM2-driven cancer forms, but also reveal off-target effects of established drugs in the clinics. PMID:26931153

  19. Single Molecule Characterization of UV-Activated Antibodies on Gold by Atomic Force Microscopy.

    PubMed

    Funari, R; Della Ventura, B; Altucci, C; Offenhäusser, A; Mayer, D; Velotta, R

    2016-08-16

    The interaction between proteins and solid surfaces can influence their conformation and therefore also their activity and affinity. These interactions are highly specific for the respective combination of proteins and solids. Consequently, it is desirable to investigate the conformation of proteins on technical surfaces, ideally at single molecule level, and to correlate the results with their activity. This is in particular true for biosensors where the conformation-dependent target affinity of an immobilized receptor determines the sensitivity of the sensor. Here, we investigate for the first time the immobilization and orientation of antibodies (Abs) photoactivated by a photonic immobilization technique (PIT), which has previously demonstrated to enhance binding capabilities of antibody receptors. The photoactivated immunoglobulins are immobilized on ultrasmooth template stripped gold films and investigated by atomic force microscopy (AFM) at the level of individual molecules. The observed protein orientations are compared with results of nonactivated antibodies adsorbed on similar gold films and mica reference samples. We find that the behavior of Abs is similar for mica and gold when the protein are not treated (physisorption), whereas smaller contact area and larger heights are measured when Abs are treated (PIT). This is explained by assuming that the activated antibodies tend to be more upright compared with nonirradiated ones, thereby providing a better exposure of the binding sites. This finding matches the observed enhancement of Abs binding efficiency when PIT is used to functionalize gold surface of QCM-based biosensors. PMID:27444884

  20. Probe molecule studies: Active species in alcohol synthesis. Final report, July 1993--July 1994

    SciTech Connect

    Blackmond, D.G.; Wender, I.; Oukaci, R.; Wang, Jian

    1994-07-01

    The objectives of this project are to investigate the role(s) of cobalt and copper in constructing the active sites for the formation of higher alcohols from CO/H{sub 2} over the Co-Cu based catalysts by using different reduction treatments and applying selected characterization tools such as TPR, TPD, XRD and XPS as well as to generate mechanistic information on the reaction pathway(s) and key intermediate(s) of higher alcohol synthesis from CO/H{sub 2} over Co-Cu/ZnO catalysts by the approach of in-situ addition of a probe molecule (nitromethane).

  1. Associations between children's physical activities, sedentary behaviours and family structure: a sequential mixed methods approach.

    PubMed

    Quarmby, T; Dagkas, S; Bridge, M

    2011-02-01

    This mixed method paper explored the effect of family structure on children's physical activities and sedentary pursuits. It furthers the limited understanding of how family structure impacts on children's time in, and reasons behind engaging in, certain physical activities. Children from three inner city comprehensive schools in the Midlands, United Kingdom, participated through questionnaires (n = 381) and semi-structured interviews (n = 62). The results indicated that boys and girls from single parent families spent more time in sedentary activities during the week and at the weekend (P < 0.01) when compared with their intact couple family counterparts. It was identified that children in single-parent families received less parental support due to a lack of time, transport and additional parental responsibilities that created a family environment that encouraged sedentary pursuits. Moreover, the barriers that encouraged sedentary activities also prevented children in single parent families from engaging in lifetime activities during the week. Children from intact couple families recorded more time in lifetime activities than those in single parent families (P < 0.01). Finally, children in two-parent families had more opportunities to engage not only in these activities individually but also in joint activities with their parents, further reinforcing these behaviours.

  2. The associations between family history of coronary heart disease, physical activity, dietary intake and body size.

    PubMed

    Slattery, M L; Schumacher, M C; Hunt, S C; Williams, R R

    1993-02-01

    Physical activity has been associated with coronary heart disease (CHD) as well as several CHD risk factors. In this study, we examine the association of a positive family history of CHD and physical activity on dietary intake and body size indicators among 891 healthy young adults (18 to 39 years of age) and 471 older adults (40 to 83) observed between 1980 and 1986. Participants reported the number of times per week they walked and/or jogged one mile, biked three miles, participated in sports, or performed other intense activities. Older men with a family history of CHD reported more physical activity than men without a family history of CHD (60% compared to 28.6%; p = 0.002). Younger women without a family history of CHD reported more physical activity than women with a family history of CHD (30.2% compared to 15.9%; p = 0.004). Fruit and vegetable intake increased with increasing levels of physical activity in younger adults. The only dietary association with family history was higher levels of fatty foods reported among older women with a family history versus those without a family history (p = 0.03). Young women with a family history of CHD were more likely to have higher BMI levels at all levels of physical activity and a higher percent of their ideal body weight per unit of physical activity (p = 0.01). For instance, young women who were most active with a family history of CHD were at 115% of their ideal body weight, while those without a family history were at 110.2% of their ideal body weight. There were no significant interactions between physical activity and CHD family history in this population. These findings suggest that family history of CHD alone may not be adequate to stimulate one to adopt a more healthy lifestyle.

  3. Single molecule analysis reveals reversible and irreversible steps during spliceosome activation

    PubMed Central

    Hoskins, Aaron A; Rodgers, Margaret L; Friedman, Larry J; Gelles, Jeff; Moore, Melissa J

    2016-01-01

    The spliceosome is a complex machine composed of small nuclear ribonucleoproteins (snRNPs) and accessory proteins that excises introns from pre-mRNAs. After assembly the spliceosome is activated for catalysis by rearrangement of subunits to form an active site. How this rearrangement is coordinated is not well-understood. During activation, U4 must be released to allow U6 conformational change, while Prp19 complex (NTC) recruitment is essential for stabilizing the active site. We used multi-wavelength colocalization single molecule spectroscopy to directly observe the key events in Saccharomyces cerevisiae spliceosome activation. Following binding of the U4/U6.U5 tri-snRNP, the spliceosome either reverses assembly by discarding tri-snRNP or proceeds to activation by irreversible U4 loss. The major pathway for NTC recruitment occurs after U4 release. ATP stimulates both the competing U4 release and tri-snRNP discard processes. The data reveal the activation mechanism and show that overall splicing efficiency may be maintained through repeated rounds of disassembly and tri-snRNP reassociation. DOI: http://dx.doi.org/10.7554/eLife.14166.001 PMID:27244240

  4. Soluble Collectin-12 (CL-12) Is a Pattern Recognition Molecule Initiating Complement Activation via the Alternative Pathway.

    PubMed

    Ma, Ying Jie; Hein, Estrid; Munthe-Fog, Lea; Skjoedt, Mikkel-Ole; Bayarri-Olmos, Rafael; Romani, Luigina; Garred, Peter

    2015-10-01

    Soluble defense collagens including the collectins play important roles in innate immunity. Recently, a new member of the collectin family named collectin-12 (CL-12 or CL-P1) has been identified. CL-12 is highly expressed in umbilical cord vascular endothelial cells as a transmembrane receptor and may recognize certain bacteria and fungi, leading to opsonophagocytosis. However, based on its structural and functional similarities with soluble collectins, we hypothesized the existence of a fluid-phase analog of CL-12 released from cells, which may function as a soluble pattern-recognition molecule. Using recombinant CL-12 full length or CL-12 extracellular domain, we determined the occurrence of soluble CL-12 shed from in vitro cultured cells. Western blot showed that soluble recombinant CL-12 migrated with a band corresponding to ∼ 120 kDa under reducing conditions, whereas under nonreducing conditions it presented multimeric assembly forms. Immunoprecipitation and Western blot analysis of human umbilical cord plasma enabled identification of a natural soluble form of CL-12 having an electrophoretic mobility pattern close to that of shed soluble recombinant CL-12. Soluble CL-12 could recognize Aspergillus fumigatus partially through the carbohydrate-recognition domain in a Ca(2+)-independent manner. This led to activation of the alternative pathway of complement exclusively via association with properdin on A. fumigatus as validated by detection of C3b deposition and formation of the terminal complement complex. These results demonstrate the existence of CL-12 in a soluble form and indicate a novel mechanism by which the alternative pathway of complement may be triggered directly by a soluble pattern-recognition molecule.

  5. The drosomycin multigene family: three-disulfide variants from Drosophila takahashii possess antibacterial activity.

    PubMed

    Gao, Bin; Zhu, Shunyi

    2016-01-01

    Drosomycin (DRS) is a strictly antifungal peptide in Drosophila melanogaster, which contains four disulfide bridges (DBs) with three buried in molecular interior and one exposed on molecular surface to tie the amino- and carboxyl-termini of the molecule together (called wrapper disulfide bridge, WDB). Based on computational analysis of genomes of Drosophila species belonging to the Oriental lineage, we identified a new multigene family of DRS in Drosphila takahashii that includes a total of 11 DRS-encoding genes (termed DtDRS-1 to DtDRS-11) and a pseudogene. Phylogenetic tree and synteny analyses reveal orthologous relationship between DtDRSs and DRSs, indicating that orthologous genes of DRS-1, DRS-2, DRS-3 and DRS-6 have undergone duplication in D. takahashii and three amplifications (DtDRS-9 to DtDRS-11) of DRS-3 have lost WDB. Among the 11 genes, five are transcriptionally active in adult fruitflies. The ortholog of DRS (DtDRS-1) shows high structural and functional similarity to DRS while two WDB-deficient members display antibacterial activity accompanying complete loss or remarkable reduction of antifungal activity. To the best of our knowledge, this is the first report on the presence of three-disulfide antibacterial DRSs in a specific Drosophila species, suggesting a potential role of DB loss in neofunctionalization of a protein via structural adjustment. PMID:27562645

  6. The drosomycin multigene family: three-disulfide variants from Drosophila takahashii possess antibacterial activity

    PubMed Central

    Gao, Bin; Zhu, Shunyi

    2016-01-01

    Drosomycin (DRS) is a strictly antifungal peptide in Drosophila melanogaster, which contains four disulfide bridges (DBs) with three buried in molecular interior and one exposed on molecular surface to tie the amino- and carboxyl-termini of the molecule together (called wrapper disulfide bridge, WDB). Based on computational analysis of genomes of Drosophila species belonging to the Oriental lineage, we identified a new multigene family of DRS in Drosphila takahashii that includes a total of 11 DRS-encoding genes (termed DtDRS-1 to DtDRS-11) and a pseudogene. Phylogenetic tree and synteny analyses reveal orthologous relationship between DtDRSs and DRSs, indicating that orthologous genes of DRS-1, DRS-2, DRS-3 and DRS-6 have undergone duplication in D. takahashii and three amplifications (DtDRS-9 to DtDRS-11) of DRS-3 have lost WDB. Among the 11 genes, five are transcriptionally active in adult fruitflies. The ortholog of DRS (DtDRS-1) shows high structural and functional similarity to DRS while two WDB-deficient members display antibacterial activity accompanying complete loss or remarkable reduction of antifungal activity. To the best of our knowledge, this is the first report on the presence of three-disulfide antibacterial DRSs in a specific Drosophila species, suggesting a potential role of DB loss in neofunctionalization of a protein via structural adjustment. PMID:27562645

  7. Small-molecule CFTR activators increase tear secretion and prevent experimental dry eye disease.

    PubMed

    Flores, Alyssa M; Casey, Scott D; Felix, Christian M; Phuan, Puay W; Verkman, A S; Levin, Marc H

    2016-05-01

    Dry eye disorders, including Sjögren's syndrome, constitute a common problem in the aging population, with limited effective therapeutic options available. The cAMP-activated Cl(-) channel cystic fibrosis transmembrane conductance regulator (CFTR) is a major prosecretory channel at the ocular surface. We investigated whether compounds that target CFTR can correct the abnormal tear film in dry eye. Small-molecule activators of human wild-type CFTR identified by high-throughput screening were evaluated in cell culture and in vivo assays, to select compounds that stimulate Cl(-)-driven fluid secretion across the ocular surface in mice. An aminophenyl-1,3,5-triazine, CFTRact-K089, fully activated CFTR in cell cultures with EC50 ∼250 nM and produced an ∼8.5 mV hyperpolarization in ocular surface potential difference. When delivered topically, CFTRact-K089 doubled basal tear volume for 4 h and had no effect in CF mice. CFTRact-K089 showed sustained tear film bioavailability without detectable systemic absorption. In a mouse model of aqueous-deficient dry eye produced by lacrimal ablation, topical administration of 0.1 nmol CFTRact-K089 3 times daily restored tear volume to basal levels, preventing corneal epithelial disruption when initiated at the time of surgery and reversing it when started after development of dry eye. Our results support the potential utility of CFTR-targeted activators as a novel prosecretory treatment for dry eye.-Flores, A. M., Casey, S. D., Felix, C. M., Phuan, P. W., Verkman, A. S., Levin, M. H. Small-molecule CFTR activators increase tear secretion and prevent experimental dry eye disease.

  8. Family Time Activities and Adolescents' Emotional Well-Being

    ERIC Educational Resources Information Center

    Offer, Shira

    2013-01-01

    The literature is divided on the issue of what matters for adolescents' well-being, with one approach focusing on quality and the other on routine family time. Using the experience sampling method, a unique form of time diary, and survey data drawn from the 500 Family Study ("N" = 237 adolescents with 8,122 observations), this study examined the…

  9. Family Influence on Teenage Participation in School Activities.

    ERIC Educational Resources Information Center

    Cashion, Barbara G.; Dager, Edward Z.

    This paper is concerned with the relationship between social participation and family structure. A theory is developed in the framework of George Herbert Mead's analysis on the development of a consistent self in response to a generalized other. According to this theory, the influence of the family is implicated as one of the social-psychological…

  10. The microRNA-200 family: small molecules with novel roles in cancer development, progression and therapy

    PubMed Central

    Humphries, Brock; Yang, Chengfeng

    2015-01-01

    MicroRNAs (miRNAs) are a large family of small non-coding RNAs that negatively regulate protein-coding gene expression post-transcriptionally via base pairing between the 5′ seed region of a miRNA and the 3′ untranslated region (3′UTR) of a messenger RNA (mRNA). Recent evidence has supported the critical role that miRNAs play in many diseases including cancer. The miR-200 family consisting of 5 members (miR-200a, -200b, -200c, -141, -429) is an emerging miRNA family that has been shown to play crucial roles in cancer initiation and metastasis, and potentially be important for the diagnosis and treatment of cancer. While miR-200s were found to be critically involved in the metastatic colonization to the lungs in mouse mammary xenograft tumor models, a large number of studies demonstrated their strong suppressive effects on cell transformation, cancer cell proliferation, migration, invasion, tumor growth and metastasis. This review aims to discuss research findings about the role of the miR-200 family in cancer initiation, each step of cancer metastatic cascade, cancer diagnosis and treatment. A comprehensive summary of currently validated miR-200 targets is also presented. It is concluded that miR-200 family may serve as novel targets for the therapy of multiple types of cancer. PMID:25762624

  11. Conserved Active Site Residues Limit Inhibition of a Copper-Containing Nitrite By Small Molecules

    SciTech Connect

    Tocheva, E.I.; Eltis, L.D.; Murphy, M.E.P.

    2009-05-26

    The interaction of copper-containing dissimilatory nitrite reductase from Alcaligenes faecalis S-6 ( AfNiR) with each of five small molecules was studied using crystallography and steady-state kinetics. Structural studies revealed that each small molecule interacted with the oxidized catalytic type 2 copper of AfNiR. Three small molecules (formate, acetate and nitrate) mimic the substrate by having at least two oxygen atoms for bidentate coordination to the type 2 copper atom. These three anions bound to the copper ion in the same asymmetric, bidentate manner as nitrite. Consistent with their weak inhibition of the enzyme ( K i >50 mM), the Cu-O distances in these AfNiR-inhibitor complexes were approximately 0.15 A longer than that observed in the AfNiR-nitrite complex. The binding mode of each inhibitor is determined in part by steric interactions with the side chain of active site residue Ile257. Moreover, the side chain of Asp98, a conserved residue that hydrogen bonds to type 2 copper-bound nitrite and nitric oxide, was either disordered or pointed away from the inhibitors. Acetate and formate inhibited AfNiR in a mixed fashion, consistent with the occurrence of second acetate binding site in the AfNiR-acetate complex that occludes access to the type 2 copper. A fourth small molecule, nitrous oxide, bound to the oxidized metal in a side-on fashion reminiscent of nitric oxide to the reduced copper. Nevertheless, nitrous oxide bound at a farther distance from the metal. The fifth small molecule, azide, inhibited the reduction of nitrite by AfNiR most strongly ( K ic = 2.0 +/- 0.1 mM). This ligand bound to the type 2 copper center end-on with a Cu-N c distance of approximately 2 A, and was the only inhibitor to form a hydrogen bond with Asp98. Overall, the data substantiate the roles of Asp98 and Ile257 in discriminating substrate from other small anions.

  12. Analysis of the Rotationally-Resolved Spectra of the Vibronically-Active Molecules

    NASA Astrophysics Data System (ADS)

    Melnik, Dmitry G.; Miller, Terry A.

    2013-06-01

    Rotational structure of the vibronically coupled, and specifically, Jahn-Teller active molecules in isolated vibronic states has been studied for the decades, and the corresponding Hamiltonian and relationship of its parameters to the molecular properties are well-established, at least for the e vibronic states. However, in many cases an isolated state approach, both for the ground and vibronically excited levels, does not produce satisfactory results either because the experimentally obtained parameters of such model are not physically transparent, or the model fails to predict the observed spectrum to the experimental accuracy. To circumvent these problems, we develop, from the molecular symmetry standpoint, an effective coupled state rotational Hamiltonian directly accounting for the interactions within the appropriate subset of the interacting vibronic states. This approach is expected to be useful for the analysis of the rotational level structure of the closely-spaced vibronic levels such as those occurring in the vibrationally excited manifolds of the open-shell molecules. The application of this approach to the spectra of the nitrate radical, NO_3, in the Jahn-Teller active ˜{A}^2E'' state, will be discussed. D. G. Melnik, T. A. Miller and J. Liu, TI15, 67^th Molecular Spectroscopy Symposium, Columbus, 2012 M. Roudjane, T. J. Codd and T. A. Miller, TI03, 67^th Molecular Spectroscopy Symposium, Columbus, 2012

  13. Incorporation and characterization of biological molecules in droplet-interface bilayer networks for novel active systems

    NASA Astrophysics Data System (ADS)

    Sarles, Stephen A.; Ghanbari Bavarsad, Pegah; Leo, Donald J.

    2009-03-01

    Biological molecules including phospholipids and proteins offer scientists and engineers a diverse selection of materials to develop new types of active materials and smart systems based on ion conduction. The inherent energy-coupling abilities of these components create novel kinds of transduction elements. Networks formed from droplet-interface bilayers (DIB) are a promising construct for creating cell mimics that allow for the assembly and study of these active biological molecules. The current-voltage relationship of symmetric, "lipid-in" dropletinterface bilayers are characterized using electrical impedance spectroscopy (EIS) and cyclic voltammetry (CV). "Lipid-in" diphytanoyl phosphatidylcholine (DPhPC) droplet-interface bilayers have specific resistances of nearly 10MΩ•cm2 and rupture at applied potentials greater than 300mV, indicating the "lipid-in" approach produces higher quality interfacial membranes than created using the original "lipid-out" method. The incorporation of phospholipids into the droplet interior allows for faster monolayer formation but does not inhibit the selfinsertion of transmembrane proteins into bilayer interfaces that separate adjacent droplets. Alamethicin proteins inserted into single and multi-DIB networks produce a voltage-dependent membrane conductance and current measurements on bilayers containing this type of protein exhibit a reversible, 3-4 order-of-magnitude conductance increase upon application of voltage.

  14. Single-molecule imaging at high fluorophore concentrations by local activation of dye

    DOE PAGES

    Geertsema, Hylkje J.; Mangel, Walter F.; Schulte, Aartje C.; Spenkelink, Lisanne M.; McGrath, William J.; Morrone, Seamus R.; Sohn, Jungsan; Robinson, Andrew; van Oijen, Antoine M.

    2015-02-17

    Single-molecule fluorescence microscopy is a powerful approach to observe biomolecular interactions with high spatial and temporal resolution. Detecting fluorescent signals from individual, labeled proteins above high levels of background fluorescence remains challenging, however. For this reason, the concentrations of labeled proteins in in vitro assays are often kept low compared to their in vivo concentrations. Here, we present a new fluorescence imaging technique by which single fluorescent molecules can be observed in real time at high, physiologically relevant concentrations. The technique requires a protein and its macromolecular substrate to be labeled each with a different fluorophore. Then, making use ofmore » short-distance energy-transfer mechanisms, the fluorescence from only those proteins bound to their substrate are selectively activated. This approach is demonstrated by labeling a DNA substrate with an intercalating stain, exciting the stain, and using energy transfer from the stain to activate the fluorescence of only those labeled DNA-binding proteins bound to the DNA. Such an experimental design allowed us to observe the sequence-independent interaction of Cy5-labeled interferon-inducible protein 16 (IFI16) with DNA and the sliding via one-dimensional diffusion of Cy5-labeled adenovirus protease (pVIc-AVP) on DNA in the presence of a background of hundreds of nM Cy5 fluorophore.« less

  15. Arrayed lipid bilayer chambers allow single-molecule analysis of membrane transporter activity.

    PubMed

    Watanabe, Rikiya; Soga, Naoki; Fujita, Daishi; Tabata, Kazuhito V; Yamauchi, Lisa; Hyeon Kim, Soo; Asanuma, Daisuke; Kamiya, Mako; Urano, Yasuteru; Suga, Hiroaki; Noji, Hiroyuki

    2014-07-24

    Nano- to micron-size reaction chamber arrays (femtolitre chamber arrays) have facilitated the development of sensitive and quantitative biological assays, such as single-molecule enzymatic assays, digital PCR and digital ELISA. However, the versatility of femtolitre chamber arrays is limited to reactions that occur in aqueous solutions. Here we report an arrayed lipid bilayer chamber system (ALBiC) that contains sub-million femtolitre chambers, each sealed with a stable 4-μm-diameter lipid bilayer membrane. When reconstituted with a limiting amount of the membrane transporter proteins α-hemolysin or F0F1-ATP synthase, the chambers within the ALBiC exhibit stochastic and quantized transporting activities. This demonstrates that the single-molecule analysis of passive and active membrane transport is achievable with the ALBiC system. This new platform broadens the versatility of femtolitre chamber arrays and paves the way for novel applications aimed at furthering our mechanistic understanding of membrane proteins' function.

  16. Tungsten polyoxometalate molecules as active nodes for dynamic carrier exchange in hybrid molecular/semiconductor capacitors

    SciTech Connect

    Balliou, A.; Douvas, A. M.; Normand, P.; Argitis, P.; Glezos, N.; Tsikritzis, D.; Kennou, S.

    2014-10-14

    In this work we study the utilization of molecular transition metal oxides known as polyoxometalates (POMs), in particular the Keggin structure anions of the formula PW₁₂O₄₀³⁻, as active nodes for potential switching and/or fast writing memory applications. The active molecules are being integrated in hybrid Metal-Insulator/POM molecules-Semiconductor capacitors, which serve as prototypes allowing investigation of critical performance characteristics towards the design of more sophisticated devices. The charging ability as well as the electronic structure of the molecular layer is probed by means of electrical characterization, namely, capacitance-voltage and current-voltage measurements, as well as transient capacitance measurements, C (t), under step voltage polarization. It is argued that the transient current peaks observed are manifestations of dynamic carrier exchange between the gate electrode and specific molecular levels, while the transient C (t) curves under conditions of molecular charging can supply information for the rate of change of the charge that is being trapped and de-trapped within the molecular layer. Structural characterization via surface and cross sectional scanning electron microscopy as well as atomic force microscopy, spectroscopic ellipsometry, UV and Fourier-transform IR spectroscopies, UPS, and XPS contribute to the extraction of accurate electronic structure characteristics and open the path for the design of new devices with on-demand tuning of their interfacial properties via the controlled preparation of the POM layer.

  17. Exchange and redistribution dynamics of the cytoskeleton of the active zone molecule bassoon.

    PubMed

    Tsuriel, Shlomo; Fisher, Arava; Wittenmayer, Nina; Dresbach, Thomas; Garner, Craig C; Ziv, Noam E

    2009-01-14

    Presynaptic sites typically appear as varicosities (boutons) distributed along axons. Ultrastructurally, presynaptic boutons lack obvious physical barriers that separate them from the axon proper, yet activity-related and constitutive dynamics continuously promote the "reshuffling" of presynaptic components and even their dispersal into flanking axonal segments. How presynaptic sites manage to maintain their organization and individual characteristics over long durations is thus unclear. Conceivably, presynaptic tenacity might depend on the active zone (AZ), an electron-dense specialization of the presynaptic membrane, and particularly on the cytoskeletal matrix associated with the AZ (CAZ) that could act as a relatively stable "core scaffold" that conserves and dictates presynaptic organization. At present, however, little is known on the molecular dynamics of CAZ molecules, and thus, the factual basis for this hypothesis remains unclear. To examine the stability of the CAZ, we studied the molecular dynamics of the major CAZ molecule Bassoon in cultured hippocampal neurons. Fluorescence recovery after photobleaching and photoactivation experiments revealed that exchange rates of green fluorescent protein and photoactivatable green fluorescent protein-tagged Bassoon at individual presynaptic sites are very low (tau > 8 h). Exchange rates varied between boutons and were only slightly accelerated by stimulation. Interestingly, photoactivation experiments revealed that Bassoon lost from one synapse was occasionally assimilated into neighboring presynaptic sites. Our findings indicate that Bassoon is engaged in relatively stable associations within the CAZ and thus support the notion that the CAZ or some of its components might constitute a relatively stable presynaptic core scaffold.

  18. Single-molecule imaging at high fluorophore concentrations by local activation of dye

    SciTech Connect

    Geertsema, Hylkje J.; Mangel, Walter F.; Schulte, Aartje C.; Spenkelink, Lisanne M.; McGrath, William J.; Morrone, Seamus R.; Sohn, Jungsan; Robinson, Andrew; van Oijen, Antoine M.

    2015-02-17

    Single-molecule fluorescence microscopy is a powerful approach to observe biomolecular interactions with high spatial and temporal resolution. Detecting fluorescent signals from individual, labeled proteins above high levels of background fluorescence remains challenging, however. For this reason, the concentrations of labeled proteins in in vitro assays are often kept low compared to their in vivo concentrations. Here, we present a new fluorescence imaging technique by which single fluorescent molecules can be observed in real time at high, physiologically relevant concentrations. The technique requires a protein and its macromolecular substrate to be labeled each with a different fluorophore. Then, making use of short-distance energy-transfer mechanisms, the fluorescence from only those proteins bound to their substrate are selectively activated. This approach is demonstrated by labeling a DNA substrate with an intercalating stain, exciting the stain, and using energy transfer from the stain to activate the fluorescence of only those labeled DNA-binding proteins bound to the DNA. Such an experimental design allowed us to observe the sequence-independent interaction of Cy5-labeled interferon-inducible protein 16 (IFI16) with DNA and the sliding via one-dimensional diffusion of Cy5-labeled adenovirus protease (pVIc-AVP) on DNA in the presence of a background of hundreds of nM Cy5 fluorophore.

  19. Arrayed lipid bilayer chambers allow single-molecule analysis of membrane transporter activity

    PubMed Central

    Watanabe, Rikiya; Soga, Naoki; Fujita, Daishi; Tabata, Kazuhito V.; Yamauchi, Lisa; Hyeon Kim, Soo; Asanuma, Daisuke; Kamiya, Mako; Urano, Yasuteru; Suga, Hiroaki; Noji, Hiroyuki

    2014-01-01

    Nano- to micron-size reaction chamber arrays (femtolitre chamber arrays) have facilitated the development of sensitive and quantitative biological assays, such as single-molecule enzymatic assays, digital PCR and digital ELISA. However, the versatility of femtolitre chamber arrays is limited to reactions that occur in aqueous solutions. Here we report an arrayed lipid bilayer chamber system (ALBiC) that contains sub-million femtolitre chambers, each sealed with a stable 4-μm-diameter lipid bilayer membrane. When reconstituted with a limiting amount of the membrane transporter proteins α-hemolysin or F0F1-ATP synthase, the chambers within the ALBiC exhibit stochastic and quantized transporting activities. This demonstrates that the single-molecule analysis of passive and active membrane transport is achievable with the ALBiC system. This new platform broadens the versatility of femtolitre chamber arrays and paves the way for novel applications aimed at furthering our mechanistic understanding of membrane proteins’ function. PMID:25058452

  20. p53 Small Molecule Inhibitor Enhances Temozolomide Cytotoxic Activity against Intracranial Glioblastoma Xenografts

    PubMed Central

    Dinca, Eduard B.; Lu, Kan V.; Sarkaria, Jann N.; Pieper, Russell O.; Prados, Michael D.; Haas-Kogan, Daphne A.; VandenBerg, Scott R.; Berger, Mitchel S.; James, C. David

    2010-01-01

    In this study we investigated corresponding precursor and active forms of a p53 small molecule inhibitor for effect on temozolomide (TMZ) anti-tumor activity against glioblastoma (GBM), using both in vitro and in vivo experimental approaches. Results from in vitro cell viability analysis showed that the cytotoxic activity of TMZ was substantially increased when GBMs with wild-type p53 were co-treated with the active form of p53 inhibitor, and this heightened cytotoxic response was accompanied by increased PARP cleavage as well as elevated cellular phospho-H2AX. Analysis of the same series of GBMs, as intracranial xenografts in athymic mice, and administering corresponding p53 inhibitor precursor, that is converted to the active compound in vivo, yielded results consistent with the in vitro analyses: i.e., TMZ + p53 inhibitor precursor co-treatment, of three distinct wild-type p53 GBM xenografts, resulted in significant enhancement of TMZ anti-tumor effect relative to treatment with TMZ alone, as indicated by serial bioluminescence monitoring as well as survival analysis (p < 0.001 for co-treatment survival benefit in each case). Mice receiving intracranial injection with p53 null GBM showed similar survival benefit from TMZ treatment regardless of the presence or absence of p53 inhibitor precursor. In total, our results indicate that the p53 active and precursor inhibitor pair enhance TMZ cytotoxicity in vitro and in vivo, respectively, and do so in a p53-dependent manner. PMID:19074867

  1. p53 Small-molecule inhibitor enhances temozolomide cytotoxic activity against intracranial glioblastoma xenografts.

    PubMed

    Dinca, Eduard B; Lu, Kan V; Sarkaria, Jann N; Pieper, Russell O; Prados, Michael D; Haas-Kogan, Daphne A; Vandenberg, Scott R; Berger, Mitchel S; James, C David

    2008-12-15

    In this study, we investigated the precursor and active forms of a p53 small-molecule inhibitor for their effects on temozolomide (TMZ) antitumor activity against glioblastoma (GBM), using both in vitro and in vivo experimental approaches. Results from in vitro cell viability analysis showed that the cytotoxic activity of TMZ was substantially increased when p53 wild-type (p53(wt)) GBMs were cotreated with the active form of p53 inhibitor, and this heightened cytotoxic response was accompanied by increased poly(ADP-ribose) polymerase cleavage as well as elevated cellular phospho-H2AX. Analysis of the same series of GBMs, as intracranial xenografts in athymic mice, and administering corresponding p53 inhibitor precursor, which is converted to the active compound in vivo, yielded results consistent with the in vitro analyses: TMZ + p53 inhibitor precursor cotreatment of three distinct p53(wt) GBM xenografts resulted in significant enhancement of TMZ antitumor effect relative to treatment with TMZ alone, as indicated by serial bioluminescence monitoring as well as survival analysis (P < 0.001 for cotreatment survival benefit in each case). Mice receiving intracranial injection with p53(null) GBM showed similar survival benefit from TMZ treatment regardless of the presence or absence of p53 inhibitor precursor. In total, our results indicate that the p53 active and precursor inhibitor pair enhances TMZ cytotoxicity in vitro and in vivo, respectively, and do so in a p53-dependent manner.

  2. Platelet-Rich Plasma: The Choice of Activation Method Affects the Release of Bioactive Molecules

    PubMed Central

    Cavallo, Carola; Mariani, Erminia; Pratelli, Loredana; Merli, Giulia; Marcacci, Maurilio

    2016-01-01

    Platelet-Rich Plasma (PRP) is a low-cost procedure to deliver high concentrations of autologous growth factors (GFs). Platelet activation is a crucial step that might influence the availability of bioactive molecules and therefore tissue healing. Activation of PRP from ten voluntary healthy males was performed by adding 10% of CaCl2, 10% of autologous thrombin, 10% of a mixture of CaCl2 + thrombin, and 10% of collagen type I. Blood derivatives were incubated for 15 and 30 minutes and 1, 2, and 24 hours and samples were evaluated for the release of VEGF, TGF-β1, PDGF-AB, IL-1β, and TNF-α. PRP activated with CaCl2, thrombin, and CaCl2/thrombin formed clots detected from the 15-minute evaluation, whereas in collagen-type-I-activated samples no clot formation was noticed. Collagen type I produced an overall lower GF release. Thrombin, CaCl2/thrombin, and collagen type I activated PRPs showed an immediate release of PDGF and TGF-β1 that remained stable over time, whereas VEGF showed an increasing trend from 15 minutes up to 24 hours. CaCl2 induced a progressive release of GFs from 15 minutes and increasing up to 24 hours. The method chosen to activate PRP influences both its physical form and the releasate in terms of GF amount and release kinetic.

  3. Platelet-Rich Plasma: The Choice of Activation Method Affects the Release of Bioactive Molecules

    PubMed Central

    Cavallo, Carola; Mariani, Erminia; Pratelli, Loredana; Merli, Giulia; Marcacci, Maurilio

    2016-01-01

    Platelet-Rich Plasma (PRP) is a low-cost procedure to deliver high concentrations of autologous growth factors (GFs). Platelet activation is a crucial step that might influence the availability of bioactive molecules and therefore tissue healing. Activation of PRP from ten voluntary healthy males was performed by adding 10% of CaCl2, 10% of autologous thrombin, 10% of a mixture of CaCl2 + thrombin, and 10% of collagen type I. Blood derivatives were incubated for 15 and 30 minutes and 1, 2, and 24 hours and samples were evaluated for the release of VEGF, TGF-β1, PDGF-AB, IL-1β, and TNF-α. PRP activated with CaCl2, thrombin, and CaCl2/thrombin formed clots detected from the 15-minute evaluation, whereas in collagen-type-I-activated samples no clot formation was noticed. Collagen type I produced an overall lower GF release. Thrombin, CaCl2/thrombin, and collagen type I activated PRPs showed an immediate release of PDGF and TGF-β1 that remained stable over time, whereas VEGF showed an increasing trend from 15 minutes up to 24 hours. CaCl2 induced a progressive release of GFs from 15 minutes and increasing up to 24 hours. The method chosen to activate PRP influences both its physical form and the releasate in terms of GF amount and release kinetic. PMID:27672658

  4. Small-Molecule Inhibition and Activation-Loop Trans-Phosphorylation of the IGF1 Receptor

    SciTech Connect

    Wu,J.; Li, W.; Craddock, B.; Foreman, K.; Mulvihill, M.; Ji, Q.; Miller, W.; Hubbard, S.

    2008-01-01

    The insulin-like growth factor-1 receptor (IGF1R) is a receptor tyrosine kinase (RTK) that has a critical role in mitogenic signalling during embryogenesis and an antiapoptotic role in the survival and progression of many human tumours. Here, we present the crystal structure of the tyrosine kinase domain of IGF1R (IGF1RK), in its unphosphorylated state, in complex with a novel compound, cis-3-[3-(4-methyl-piperazin-l-yl)-cyclobutyl]-1-(2-phenyl-quinolin-7-yl)-imidazo[1, 5-a]pyrazin-8-ylamine (PQIP), which we show is a potent inhibitor of both the unphosphorylated (basal) and phosphorylated (activated) states of the kinase. PQIP interacts with residues in the ATP-binding pocket and in the activation loop, which confers specificity for IGF1RK and the highly related insulin receptor (IR) kinase. In this crystal structure, the IGF1RK active site is occupied by Tyr1135 from the activation loop of an symmetry (two-fold)-related molecule. This dimeric arrangement affords, for the first time, a visualization of the initial trans-phosphorylation event in the activation loop of an RTK, and provides a molecular rationale for a naturally occurring mutation in the activation loop of the IR that causes type II diabetes mellitus.

  5. Novel small-molecule AMPK activator orally exerts beneficial effects on diabetic db/db mice

    SciTech Connect

    Li, Yuan-Yuan; Yu, Li-Fang; Zhang, Li-Na; Qiu, Bei-Ying; Su, Ming-Bo; Wu, Fang; Chen, Da-Kai; Pang, Tao; Gu, Min; Zhang, Wei; Ma, Wei-Ping; Jiang, Hao-Wen; Li, Jing-Ya Nan, Fa-Jun Li, Jia

    2013-12-01

    AMP-activated protein kinase (AMPK), which is a pivotal guardian of whole-body energy metabolism, has become an attractive therapeutic target for metabolic syndrome. Previously, using a homogeneous scintillation proximity assay, we identified the small-molecule AMPK activator C24 from an optimization based on the original allosteric activator PT1. In this paper, the AMPK activation mechanism of C24 and its potential beneficial effects on glucose and lipid metabolism on db/db mice were investigated. C24 allosterically stimulated inactive AMPK α subunit truncations and activated AMPK heterotrimers by antagonizing autoinhibition. In primary hepatocytes, C24 increased the phosphorylation of AMPK downstream target acetyl-CoA carboxylase dose-dependently without changing intracellular AMP/ATP ratio, indicating its allosteric activation in cells. Through activating AMPK, C24 decreased glucose output by down-regulating mRNA levels of phosphoenolpyruvate carboxykinase (PEPCK) and glucose-6-phosphatase (G6Pase) in primary hepatocytes. C24 also decreased the triglyceride and cholesterol contents in HepG2 cells. Due to its improved bioavailability, chronic oral treatment with multiple doses of C24 significantly reduced blood glucose and lipid levels in plasma, and improved the glucose tolerance of diabetic db/db mice. The hepatic transcriptional levels of PEPCK and G6Pase were reduced. These results demonstrate that this orally effective activator of AMPK represents a novel approach to the treatment of metabolic syndrome. - Highlights: • C24 activates AMPK through antagonizing autoinhibition within α subunit. • C24 activates AMPK in hepatocytes and decreases glucose output via AMPK. • C24 exerts beneficial effects on diabetic db/db mice. • C24 represents a novel therapeutic for treatment of metabolic syndrome.

  6. Accelerating the Discovery of Biologically Active Small Molecules Using a High-Throughput Yeast Halo Assay#

    PubMed Central

    Gassner, Nadine C.; Tamble, Craig M.; Bock, Jonathan E.; Cotton, Naomi; White, Kimberly N.; Tenney, Karen; St. Onge, Robert P.; Proctor, Michael J.; Giaever, Guri; Davis, Ronald W.; Crews, Phillip; Holman, Theodore R.; Lokey, R. Scott

    2008-01-01

    The budding yeast Saccharomyces cerevisiae, a powerful model system for the study of basic eukaryotic cell biology, has been used increasingly as a screening tool for the identification of bioactive small molecules. We have developed a novel yeast toxicity screen that is easily automated and compatible with high-throughput screening robotics. The new screen is quantitative and allows inhibitory potencies to be determined, since the diffusion of the sample provides a concentration gradient and a corresponding toxicity halo. The efficacy of this new screen was illustrated by testing materials including 3,104 compounds from the NCI libraries, 167 marine sponge crude extracts, and 149 crude marine-derived fungal extracts. There were 46 active compounds among the NCI set. One very active extract was selected for bioactivity-guided fractionation resulting in the identification of crambescidin 800 as a potent antifungal agent. PMID:17291044

  7. VAMP4 Is an Essential Cargo Molecule for Activity-Dependent Bulk Endocytosis.

    PubMed

    Nicholson-Fish, Jessica C; Kokotos, Alexandros C; Gillingwater, Thomas H; Smillie, Karen J; Cousin, Michael A

    2015-12-01

    The accurate formation of synaptic vesicles (SVs) and incorporation of their protein cargo during endocytosis is critical for the maintenance of neurotransmission. During intense neuronal activity, a transient and acute accumulation of SV cargo occurs at the plasma membrane. Activity-dependent bulk endocytosis (ADBE) is the dominant SV endocytosis mode under these conditions; however, it is currently unknown how ADBE mediates cargo retrieval. We examined the retrieval of different SV cargo molecules during intense stimulation using a series of genetically encoded pH-sensitive reporters in neuronal cultures. The retrieval of only one reporter, VAMP4-pHluorin, was perturbed by inhibiting ADBE. This selective recovery was confirmed by the enrichment of endogenous VAMP4 in purified bulk endosomes formed by ADBE. VAMP4 was also essential for ADBE, with a cytoplasmic di-leucine motif being critical for this role. Therefore, VAMP4 is the first identified ADBE cargo and is essential for this endocytosis mode to proceed.

  8. Small Molecule-Induced Allosteric Activation of the Vibrio Cholerae RTX Cysteine Protease Domain

    SciTech Connect

    Lupardus, P.J.; Shen, A.; Bogyo, M.; Garcia, K.C.

    2009-05-19

    Vibrio cholerae RTX (repeats in toxin) is an actin-disrupting toxin that is autoprocessed by an internal cysteine protease domain (CPD). The RTX CPD is efficiently activated by the eukaryote-specific small molecule inositol hexakisphosphate (InsP{sub 6}), and we present the 2.1 angstrom structure of the RTX CPD in complex with InsP{sub 6}. InsP{sub 6} binds to a conserved basic cleft that is distant from the protease active site. Biochemical and kinetic analyses of CPD mutants indicate that InsP{sub 6} binding induces an allosteric switch that leads to the autoprocessing and intracellular release of toxin-effector domains.

  9. The Integration of a Family Systems Approach for Understanding Youth Obesity, Physical Activity, and Dietary Programs

    PubMed Central

    Wilson, Dawn K.; St. George, Sara M.; Lawman, Hannah; Segal, Michelle; Fairchild, Amanda

    2012-01-01

    Rates of overweight in youth have reached epidemic proportions and are associated with adverse health outcomes. Family-based programs have been widely used to treat overweight in youth. However, few programs incorporate a theoretical framework for studying a family systems approach in relation to youth health behavior change. Therefore, this review provides a family systems theory framework for evaluating family-level variables in weight loss, physical activity, and dietary approaches in youth. Studies were reviewed and effect sizes were calculated for interventions that manipulated the family system, including components that targeted parenting styles, parenting skills, or family functioning, or which had novel approaches for including the family. Twenty-one weight loss interventions were identified, and 25 interventions related to physical activity and/or diet were identified. Overall, family-based treatment programs that incorporated training for authoritative parenting styles, parenting skills, or child management, and family functioning had positive effects on youth weight loss. Programs to improve physical activity and dietary behaviors that targeted the family system also demonstrated improvements in youth health behaviors; however, direct effects of parent-targeted programming is not clear. Both treatment and prevention programs would benefit from evaluating family functioning and parenting styles as possible mediators of intervention outcomes. Recommendations are provided to guide the development of future family-based obesity prevention and treatment programs for youth. PMID:20689989

  10. The integration of a family systems approach for understanding youth obesity, physical activity, and dietary programs.

    PubMed

    Kitzman-Ulrich, Heather; Wilson, Dawn K; St George, Sara M; Lawman, Hannah; Segal, Michelle; Fairchild, Amanda

    2010-09-01

    Rates of overweight in youth have reached epidemic proportions and are associated with adverse health outcomes. Family-based programs have been widely used to treat overweight in youth. However, few programs incorporate a theoretical framework for studying a family systems approach in relation to youth health behavior change. Therefore, this review provides a family systems theory framework for evaluating family-level variables in weight loss, physical activity, and dietary approaches in youth. Studies were reviewed and effect sizes were calculated for interventions that manipulated the family system, including components that targeted parenting styles, parenting skills, or family functioning, or which had novel approaches for including the family. Twenty-one weight loss interventions were identified, and 25 interventions related to physical activity and/or diet were identified. Overall, family-based treatment programs that incorporated training for authoritative parenting styles, parenting skills, or child management, and family functioning had positive effects on youth weight loss. Programs to improve physical activity and dietary behaviors that targeted the family system also demonstrated improvements in youth health behaviors; however, direct effects of parent-targeted programming is not clear. Both treatment and prevention programs would benefit from evaluating family functioning and parenting styles as possible mediators of intervention outcomes. Recommendations are provided to guide the development of future family-based obesity prevention and treatment programs for youth.

  11. Antitumor activity of a small-molecule inhibitor of the histone kinase Haspin

    PubMed Central

    Huertas, D; Soler, M; Moreto, J; Villanueva, A; Martinez, A; Vidal, A; Charlton, M; Moffat, D; Patel, S; McDermott, J; Owen, J; Brotherton, D; Krige, D; Cuthill, S; Esteller, M

    2012-01-01

    The approval of histone deacetylase inhibitors for treatment of lymphoma subtypes has positioned histone modifications as potential targets for the development of new classes of anticancer drugs. Histones also undergo phosphorylation events, and Haspin is a protein kinase the only known target of which is phosphorylation of histone H3 at Thr3 residue (H3T3ph), which is necessary for mitosis progression. Mitotic kinases can be blocked by small drugs and several clinical trials are underway with these agents. As occurs with Aurora kinase inhibitors, Haspin might be an optimal candidate for the pharmacological development of these compounds. A high-throughput screening for Haspin inhibitors identified the CHR-6494 compound as being one promising such agent. We demonstrate that CHR-6494 reduces H3T3ph levels in a dose-dependent manner and causes a mitotic catastrophe characterized by metaphase misalignment, spindle abnormalities and centrosome amplification. From the cellular standpoint, the identified small-molecule Haspin inhibitor causes arrest in G2/M and subsequently apoptosis. Importantly, ex vivo assays also demonstrate its anti-angiogenetic features; in vivo, it shows antitumor potential in xenografted nude mice without any observed toxicity. Thus, CHR-6494 is a first-in-class Haspin inhibitor with a wide spectrum of anticancer effects that merits further preclinical research as a new member of the family of mitotic kinase inhibitors. PMID:21804608

  12. Peptidomimetic Small Molecules Disrupt Type IV Secretion System Activity in Diverse Bacterial Pathogens

    PubMed Central

    Shaffer, Carrie L.; Good, James A. D.; Kumar, Santosh; Krishnan, K. Syam; Gaddy, Jennifer A.; Loh, John T.; Chappell, Joseph; Almqvist, Fredrik

    2016-01-01

    ABSTRACT Bacteria utilize complex type IV secretion systems (T4SSs) to translocate diverse effector proteins or DNA into target cells. Despite the importance of T4SSs in bacterial pathogenesis, the mechanism by which these translocation machineries deliver cargo across the bacterial envelope remains poorly understood, and very few studies have investigated the use of synthetic molecules to disrupt T4SS-mediated transport. Here, we describe two synthetic small molecules (C10 and KSK85) that disrupt T4SS-dependent processes in multiple bacterial pathogens. Helicobacter pylori exploits a pilus appendage associated with the cag T4SS to inject an oncogenic effector protein (CagA) and peptidoglycan into gastric epithelial cells. In H. pylori, KSK85 impedes biogenesis of the pilus appendage associated with the cag T4SS, while C10 disrupts cag T4SS activity without perturbing pilus assembly. In addition to the effects in H. pylori, we demonstrate that these compounds disrupt interbacterial DNA transfer by conjugative T4SSs in Escherichia coli and impede vir T4SS-mediated DNA delivery by Agrobacterium tumefaciens in a plant model of infection. Of note, C10 effectively disarmed dissemination of a derepressed IncF plasmid into a recipient bacterial population, thus demonstrating the potential of these compounds in mitigating the spread of antibiotic resistance determinants driven by conjugation. To our knowledge, this study is the first report of synthetic small molecules that impair delivery of both effector protein and DNA cargos by diverse T4SSs. PMID:27118587

  13. Polyphosphate, an active molecule derived from probiotic Lactobacillus brevis, improves the fibrosis in murine colitis.

    PubMed

    Kashima, Shin; Fujiya, Mikihiro; Konishi, Hiroaki; Ueno, Nobuhiro; Inaba, Yuhei; Moriichi, Kentaro; Tanabe, Hiroki; Ikuta, Katsuya; Ohtake, Takaaki; Kohgo, Yutaka

    2015-08-01

    Inflammatory bowel disease frequently causes intestinal obstruction because of extensive fibrosis. This study investigated whether polyphosphate (poly P), an active molecule derived from Lactobacillus brevis, could improve the fibrosis in a model of chronic colitis. In this study, dextran sodium sulfate (DSS)-induced chronic colitis models and trinitrobenzene sulfonic acid (TNBS)-induced colitis models were used as models of fibrosis. To clarify the mechanism responsible for the observed effects, Caco-2/brush border epithelial (BBE) and naive T helper lymphocyte (THP)-1 cells were treated with lipopolysaccharide (LPS) to induce inflammation. Non-cancer human colon fibroblast (CCD-18) cells were treated with transforming growth factor beta 1 (TGF-β1) to induce fibrosis. The expression levels of fibrosis- and inflammation-associated molecules were evaluated by both a Western blotting analysis and reverse transcriptase-polymerase chain reaction (RT-PCR). The histologic inflammation and fibrosis were significantly improved in the group administered poly P in both the DSS and TNBS colitis models. The levels of interleukin 1β (IL-1β) and tumor necrosis factor α (TNF-α) were significantly decreased by poly P treatment. The expression levels of TGF-β1 and collagens in the colitis mice were decreased by poly P. The LPS-induced expressions of IL-1β and TGF-β1 in Caco-2/BBE cells and of TNF-α in THP-1 cells were reduced by poly P treatment. Poly P did not affect the expression of collagens and connective tissue growth factor in the CCD-18 cells. In conclusion, poly P suppresses intestinal inflammation and fibrosis by downregulating the expression of inflammation- and fibrosis-associated molecules in the intestinal epithelium. The administration of poly P is therefore a novel option to treat fibrosis because of chronic intestinal inflammation. PMID:25766132

  14. Dendrimers and Polyamino-Phenolic Ligands: Activity of New Molecules Against Legionella pneumophila Biofilms

    PubMed Central

    Andreozzi, Elisa; Barbieri, Federica; Ottaviani, Maria F.; Giorgi, Luca; Bruscolini, Francesca; Manti, Anita; Battistelli, Michela; Sabatini, Luigia; Pianetti, Anna

    2016-01-01

    Legionnaires’ disease is a potentially fatal pneumonia caused by Legionella pneumophila, an aquatic bacterium often found within the biofilm niche. In man-made water systems microbial biofilms increase the resistance of legionella to disinfection, posing a significant threat to public health. Disinfection methods currently used in water systems have been shown to be ineffective against legionella over the long-term, allowing recolonization by the biofilm-protected microorganisms. In this study, the anti-biofilm activity of previously fabricated polyamino-phenolic ligands and polyamidoamine dendrimers was investigated against legionella mono-species and multi-species biofilms formed by L. pneumophila in association with other bacteria that can be found in tap water (Aeromonas hydrophila, Pseudomonas aeruginosa, Escherichia coli, Klebsiella pneumoniae). Bacterial ability to form biofilms was verified using a crystal violet colorimetric assay and testing cell viability by real-time quantitative PCR and Plate Count assay. The concentration of the chemicals tested as anti-biofilm agents was chosen based on cytotoxicity assays: the highest non-cytotoxic chemical concentration was used for biofilm inhibition assays, with dendrimer concentration 10-fold higher than polyamino-phenolic ligands. While Macrophen and Double Macrophen were the most active substances among polyamino-phenolic ligands, dendrimers were overall twofold more effective than all other compounds with a reduction up to 85 and 73% of legionella and multi-species biofilms, respectively. Chemical interaction with matrix molecules is hypothesized, based on SEM images and considering the low or absent anti-microbial activity on planktonic bacteria showed by flow cytometry. These data suggest that the studied compounds, especially dendrimers, could be considered as novel molecules in the design of research projects aimed at the development of efficacious anti-biofilm disinfection treatments of water systems

  15. Post-Spaceflight (STS-135) Mouse Splenocytes Demonstrate Altered Activation Properties and Surface Molecule Expression.

    PubMed

    Hwang, Shen-An; Crucian, Brian; Sams, Clarence; Actor, Jeffrey K

    2015-01-01

    Alterations in immune function have been documented during or post-spaceflight and in ground based models of microgravity. Identification of immune parameters that are dysregulated during spaceflight is an important step in mitigating crew health risks during deep space missions. The in vitro analysis of leukocyte activity post-spaceflight in both human and animal species is primarily focused on lymphocytic function. This report completes a broader spectrum analysis of mouse lymphocyte and monocyte changes post 13 days orbital flight (mission STS-135). Analysis includes an examination in surface markers for cell activation, and antigen presentation and co-stimulatory molecules. Cytokine production was measured after stimulation with T-cell mitogen or TLR-2, TLR-4, or TLR-5 agonists. Splenocyte surface marker analysis immediate post-spaceflight and after in vitro culture demonstrated unique changes in phenotypic populations between the flight mice and matched treatment ground controls. Post-spaceflight splenocytes (flight splenocytes) had lower expression intensity of CD4+CD25+ and CD8+CD25+ cells, lower percentage of CD11c+MHC II+ cells, and higher percentage of CD11c+MHC I+ populations compared to ground controls. The flight splenocytes demonstrated an increase in phagocytic activity. Stimulation with ConA led to decrease in CD4+ population but increased CD4+CD25+ cells compared to ground controls. Culturing with TLR agonists led to a decrease in CD11c+ population in splenocytes isolated from flight mice compared to ground controls. Consequently, flight splenocytes with or without TLR-agonist stimulation showed a decrease in CD11c+MHC I+, CD11c+MHC II+, and CD11c+CD86+ cells compared to ground controls. Production of IFN-γ was decreased and IL-2 was increased from ConA stimulated flight splenocytes. This study demonstrated that expression of surface molecules can be affected by conditions of spaceflight and impaired responsiveness persists under culture

  16. Post-Spaceflight (STS-135) Mouse Splenocytes Demonstrate Altered Activation Properties and Surface Molecule Expression

    PubMed Central

    Crucian, Brian; Sams, Clarence

    2015-01-01

    Alterations in immune function have been documented during or post-spaceflight and in ground based models of microgravity. Identification of immune parameters that are dysregulated during spaceflight is an important step in mitigating crew health risks during deep space missions. The in vitro analysis of leukocyte activity post-spaceflight in both human and animal species is primarily focused on lymphocytic function. This report completes a broader spectrum analysis of mouse lymphocyte and monocyte changes post 13 days orbital flight (mission STS-135). Analysis includes an examination in surface markers for cell activation, and antigen presentation and co-stimulatory molecules. Cytokine production was measured after stimulation with T-cell mitogen or TLR-2, TLR-4, or TLR-5 agonists. Splenocyte surface marker analysis immediate post-spaceflight and after in vitro culture demonstrated unique changes in phenotypic populations between the flight mice and matched treatment ground controls. Post-spaceflight splenocytes (flight splenocytes) had lower expression intensity of CD4+CD25+ and CD8+CD25+ cells, lower percentage of CD11c+MHC II+ cells, and higher percentage of CD11c+MHC I+ populations compared to ground controls. The flight splenocytes demonstrated an increase in phagocytic activity. Stimulation with ConA led to decrease in CD4+ population but increased CD4+CD25+ cells compared to ground controls. Culturing with TLR agonists led to a decrease in CD11c+ population in splenocytes isolated from flight mice compared to ground controls. Consequently, flight splenocytes with or without TLR-agonist stimulation showed a decrease in CD11c+MHC I+, CD11c+MHC II+, and CD11c+CD86+ cells compared to ground controls. Production of IFN-γ was decreased and IL-2 was increased from ConA stimulated flight splenocytes. This study demonstrated that expression of surface molecules can be affected by conditions of spaceflight and impaired responsiveness persists under culture

  17. Dendrimers and Polyamino-Phenolic Ligands: Activity of New Molecules Against Legionella pneumophila Biofilms.

    PubMed

    Andreozzi, Elisa; Barbieri, Federica; Ottaviani, Maria F; Giorgi, Luca; Bruscolini, Francesca; Manti, Anita; Battistelli, Michela; Sabatini, Luigia; Pianetti, Anna

    2016-01-01

    Legionnaires' disease is a potentially fatal pneumonia caused by Legionella pneumophila, an aquatic bacterium often found within the biofilm niche. In man-made water systems microbial biofilms increase the resistance of legionella to disinfection, posing a significant threat to public health. Disinfection methods currently used in water systems have been shown to be ineffective against legionella over the long-term, allowing recolonization by the biofilm-protected microorganisms. In this study, the anti-biofilm activity of previously fabricated polyamino-phenolic ligands and polyamidoamine dendrimers was investigated against legionella mono-species and multi-species biofilms formed by L. pneumophila in association with other bacteria that can be found in tap water (Aeromonas hydrophila, Pseudomonas aeruginosa, Escherichia coli, Klebsiella pneumoniae). Bacterial ability to form biofilms was verified using a crystal violet colorimetric assay and testing cell viability by real-time quantitative PCR and Plate Count assay. The concentration of the chemicals tested as anti-biofilm agents was chosen based on cytotoxicity assays: the highest non-cytotoxic chemical concentration was used for biofilm inhibition assays, with dendrimer concentration 10-fold higher than polyamino-phenolic ligands. While Macrophen and Double Macrophen were the most active substances among polyamino-phenolic ligands, dendrimers were overall twofold more effective than all other compounds with a reduction up to 85 and 73% of legionella and multi-species biofilms, respectively. Chemical interaction with matrix molecules is hypothesized, based on SEM images and considering the low or absent anti-microbial activity on planktonic bacteria showed by flow cytometry. These data suggest that the studied compounds, especially dendrimers, could be considered as novel molecules in the design of research projects aimed at the development of efficacious anti-biofilm disinfection treatments of water systems in

  18. Screening of Pharmacologically Active Small Molecule Compounds Identifies Antifungal Agents Against Candida Biofilms

    PubMed Central

    Watamoto, Takao; Egusa, Hiroshi; Sawase, Takashi; Yatani, Hirofumi

    2015-01-01

    Candida species have emerged as important and common opportunistic human pathogens, particularly in immunocompromised individuals. The current antifungal therapies either have toxic side effects or are insufficiently effect. The aim of this study is develop new small-molecule antifungal compounds by library screening methods using Candida albicans, and to evaluate their antifungal effects on Candida biofilms and cytotoxic effects on human cells. Wild-type C. albicans strain SC5314 was used in library screening. To identify antifungal compounds, we screened a small-molecule library of 1,280 pharmacologically active compounds (LOPAC1280TM) using an antifungal susceptibility test (AST). To investigate the antifungal effects of the hit compounds, ASTs were conducted using Candida strains in various growth modes, including biofilms. We tested the cytotoxicity of the hit compounds using human gingival fibroblast (hGF) cells to evaluate their clinical safety. Only 35 compounds were identified by screening, which inhibited the metabolic activity of C. albicans by >50%. Of these, 26 compounds had fungistatic effects and nine compounds had fungicidal effects on C. albicans. Five compounds, BAY11-7082, BAY11-7085, sanguinarine chloride hydrate, ellipticine and CV-3988, had strong fungicidal effects and could inhibit the metabolic activity of Candida biofilms. However, BAY11-7082, BAY11-7085, sanguinarine chloride hydrate and ellipticine were cytotoxic to hGF cells at low concentrations. CV-3988 showed no cytotoxicity at a fungicidal concentration. Four of the compounds identified, BAY11-7082, BAY11-7085, sanguinarine chloride hydrate and ellipticine, had toxic effects on Candida strains and hGF cells. In contrast, CV-3988 had fungicidal effects on Candida strains, but low cytotoxic effects on hGF cells. Therefore, this screening reveals agent, CV-3988 that was previously unknown to be antifungal agent, which could be a novel therapies for superficial mucosal candidiasis. PMID

  19. Signal Transducers and Activators of Transcription (STAT) Family Members in Helminth Infections

    PubMed Central

    Becerra-Díaz, Mireya; Valderrama-Carvajal, Héctor; Terrazas, Luis I.

    2011-01-01

    Helminth parasites are a diverse group of multicellular organisms. Despite their heterogeneity, helminths share many common characteristics, such as the modulation of the immune system of their hosts towards a permissive state that favors their development. They induce strong Th2-like responses with high levels of IL-4, IL-5 and IL-13 cytokines, and decreased production of proinflammatory cytokines such as IFN-γ. IL-4, IFN-γ and other cytokines bind with their specific cytokine receptors to trigger an immediate signaling pathway in which different tyrosine kinases (e.g. Janus kinases) are involved. Furthermore, a seven-member family of transcription factors named Signal Transducers and Activators of Transcription (STAT) that initiate the transcriptional activation of different genes are also involved and regulate downstream the JAK/STAT signaling pathway. However, how helminths avoid and modulate immune responses remains unclear; moreover, information concerning STAT-mediated immune regulation during helminth infections is scarce. Here, we review the research on mice deficient in STAT molecules, highlighting the importance of the JAK/STAT signaling pathway in regulating susceptibility and/or resistance in these infections. PMID:22110388

  20. Identification of small molecules that selectively inhibit diacylglycerol lipase-α activity.

    PubMed

    Appiah, Kingsley K; Blat, Yuval; Robertson, Barbara J; Pearce, Bradley C; Pedicord, Donna L; Gentles, Robert G; Yu, Xuan-Chuan; Mseeh, Faika; Nguyen, Nghi; Swaffield, Jonathan C; Harden, David G; Westphal, Ryan S; Banks, Martyn N; O'Connell, Jonathan C

    2014-04-01

    Recent genetic evidence suggests that the diacylglycerol lipase (DAGL-α) isoform is the major biosynthetic enzyme for the most abundant endocannabinoid, 2-arachidonoyl-glycerol (2-AG), in the central nervous system. Revelation of its essential role in regulating retrograde synaptic plasticity and adult neurogenesis has made it an attractive therapeutic target. Therefore, it has become apparent that selective inhibition of DAGL-α enzyme activity with a small molecule could be a strategy for the development of novel therapies for the treatment of disease indications such as depression, anxiety, pain, and cognition. In this report, the authors present the identification of small-molecule inhibitor chemotypes of DAGL-α, which were selective (≥10-fold) against two other lipases, pancreatic lipase and monoacylglycerol lipase, via high-throughput screening of a diverse compound collection. Seven chemotypes of interest from a list of 185 structural clusters, which included 132 singletons, were initially selected for evaluation and characterization. Selection was based on potency, selectivity, and chemical tractability. One of the chemotypes, the glycine sulfonamide series, was prioritized as an initial lead for further medicinal chemistry optimization. PMID:24241710

  1. Activation-induced deoxycytidine deaminase (AID) co-transcriptional scanning at single-molecule resolution

    NASA Astrophysics Data System (ADS)

    Senavirathne, Gayan; Bertram, Jeffrey G.; Jaszczur, Malgorzata; Chaurasiya, Kathy R.; Pham, Phuong; Mak, Chi H.; Goodman, Myron F.; Rueda, David

    2015-12-01

    Activation-induced deoxycytidine deaminase (AID) generates antibody diversity in B cells by initiating somatic hypermutation (SHM) and class-switch recombination (CSR) during transcription of immunoglobulin variable (IgV) and switch region (IgS) DNA. Using single-molecule FRET, we show that AID binds to transcribed dsDNA and translocates unidirectionally in concert with RNA polymerase (RNAP) on moving transcription bubbles, while increasing the fraction of stalled bubbles. AID scans randomly when constrained in an 8 nt model bubble. When unconstrained on single-stranded (ss) DNA, AID moves in random bidirectional short slides/hops over the entire molecule while remaining bound for ~5 min. Our analysis distinguishes dynamic scanning from static ssDNA creasing. That AID alone can track along with RNAP during transcription and scan within stalled transcription bubbles suggests a mechanism by which AID can initiate SHM and CSR when properly regulated, yet when unregulated can access non-Ig genes and cause cancer.

  2. Small-molecule inhibitors of lethal factor protease activity protect against anthrax infection.

    PubMed

    Moayeri, Mahtab; Crown, Devorah; Jiao, Guan-Sheng; Kim, Seongjin; Johnson, Alan; Leysath, Clinton; Leppla, Stephen H

    2013-09-01

    Bacillus anthracis, the causative agent of anthrax, manifests its pathogenesis through the action of two secreted toxins. The bipartite lethal and edema toxins, a combination of lethal factor or edema factor with the protein protective antigen, are important virulence factors for this bacterium. We previously developed small-molecule inhibitors of lethal factor proteolytic activity (LFIs) and demonstrated their in vivo efficacy in a rat lethal toxin challenge model. In this work, we show that these LFIs protect against lethality caused by anthrax infection in mice when combined with subprotective doses of either antibiotics or neutralizing monoclonal antibodies that target edema factor. Significantly, these inhibitors provided protection against lethal infection when administered as a monotherapy. As little as two doses (10 mg/kg) administered at 2 h and 8 h after spore infection was sufficient to provide a significant survival benefit in infected mice. Administration of LFIs early in the infection was found to inhibit dissemination of vegetative bacteria to the organs in the first 32 h following infection. In addition, neutralizing antibodies against edema factor also inhibited bacterial dissemination with similar efficacy. Together, our findings confirm the important roles that both anthrax toxins play in establishing anthrax infection and demonstrate the potential for small-molecule therapeutics targeting these proteins.

  3. Activation-induced deoxycytidine deaminase (AID) co-transcriptional scanning at single-molecule resolution.

    PubMed

    Senavirathne, Gayan; Bertram, Jeffrey G; Jaszczur, Malgorzata; Chaurasiya, Kathy R; Pham, Phuong; Mak, Chi H; Goodman, Myron F; Rueda, David

    2015-01-01

    Activation-induced deoxycytidine deaminase (AID) generates antibody diversity in B cells by initiating somatic hypermutation (SHM) and class-switch recombination (CSR) during transcription of immunoglobulin variable (IgV) and switch region (IgS) DNA. Using single-molecule FRET, we show that AID binds to transcribed dsDNA and translocates unidirectionally in concert with RNA polymerase (RNAP) on moving transcription bubbles, while increasing the fraction of stalled bubbles. AID scans randomly when constrained in an 8 nt model bubble. When unconstrained on single-stranded (ss) DNA, AID moves in random bidirectional short slides/hops over the entire molecule while remaining bound for ∼ 5 min. Our analysis distinguishes dynamic scanning from static ssDNA creasing. That AID alone can track along with RNAP during transcription and scan within stalled transcription bubbles suggests a mechanism by which AID can initiate SHM and CSR when properly regulated, yet when unregulated can access non-Ig genes and cause cancer. PMID:26681117

  4. Neural cell adhesion molecule modulates mesenchymal stromal cell migration via activation of MAPK/ERK signaling.

    PubMed

    Shi, Yu; Xia, Yin-Yan; Wang, Lei; Liu, Rui; Khoo, King-Shung; Feng, Zhi-Wei

    2012-10-15

    Mesenchymal Stromal Cells (MSCs) represent promising tools for cellular therapy owing to their multipotentiality and ability to localize to injured, inflamed sites and tumor. Various approaches to manipulate expression of MSC surface markers, including adhesion molecules and chemokine receptors, have been explored to enhance homing of MSCs. Recently, Neural Cell Adhesion Molecule (NCAM) has been found to be expressed on MSCs yet its function remains largely elusive. Herein, we show that bone marrow-derived MSCs from NCAM deficient mice exhibit defective migratory ability and significantly impaired adipogenic and osteogenic differentiation potential. We further explore the mechanism governing NCAM mediated migration of MSCs by showing the interplay between NCAM and Fibroblast Growth Factor Receptor (FGFR) induces activation of MAPK/ERK signaling, thereby the migration of MSCs. In addition, re-expression of NCAM180, but not NCAM140, could restore the defective MAPK/ERK signaling thereby the migration of NCAM deficient MSCs. Finally, we demonstrate that NCAM180 expression level could be manipulated by pro-inflammatory cytokine Tumor Necrosis Factor (TNF)-α treatment. Overall, our data reveal the vital function of NCAM in MSCs migration and differentiation thus raising the possibility of manipulating NCAM expression to enhance homing and therapeutic potential of MSCs in cellular therapy.

  5. Antioxidant Content, Antioxidant Activity, and Antibacterial Activity of Five Plants from the Commelinaceae Family.

    PubMed

    Tan, Joash Ban Lee; Yap, Wei Jin; Tan, Shen Yeng; Lim, Yau Yan; Lee, Sui Mae

    2014-01-01

    Commelinaceae is a family of herbaceous flowering plants with many species used in ethnobotany, particularly in South America. However, thus far reports of their bioactivity are few and far between. The primary aim of this study was to quantify the antioxidant and antibacterial activity of five Commelinaceae methanolic leaf extracts. The antioxidant content was evaluated by the total phenolic content (TPC), total tannin content (TTC), and total flavonoid content (TFC) assays. The antioxidant activities measured were DPPH free radical scavenging (FRS), ferric reducing power (FRP), and ferrous ion chelating (FIC); of the five plants, the methanolic leaf extract of Tradescantia zebrina showed the highest antioxidant content and activity, and exhibited antibacterial activity against six species of Gram-positive and two species of Gram-negative bacteria in a range of 5-10 mg/mL based on the broth microdilution method. PMID:26785239

  6. Antioxidant Content, Antioxidant Activity, and Antibacterial Activity of Five Plants from the Commelinaceae Family

    PubMed Central

    Tan, Joash Ban Lee; Yap, Wei Jin; Tan, Shen Yeng; Lim, Yau Yan; Lee, Sui Mae

    2014-01-01

    Commelinaceae is a family of herbaceous flowering plants with many species used in ethnobotany, particularly in South America. However, thus far reports of their bioactivity are few and far between. The primary aim of this study was to quantify the antioxidant and antibacterial activity of five Commelinaceae methanolic leaf extracts. The antioxidant content was evaluated by the total phenolic content (TPC), total tannin content (TTC), and total flavonoid content (TFC) assays. The antioxidant activities measured were DPPH free radical scavenging (FRS), ferric reducing power (FRP), and ferrous ion chelating (FIC); of the five plants, the methanolic leaf extract of Tradescantia zebrina showed the highest antioxidant content and activity, and exhibited antibacterial activity against six species of Gram-positive and two species of Gram-negative bacteria in a range of 5–10 mg/mL based on the broth microdilution method. PMID:26785239

  7. Dissecting allosteric effects of activator-coactivator complexes using a covalent small molecule ligand.

    PubMed

    Wang, Ningkun; Lodge, Jean M; Fierke, Carol A; Mapp, Anna K

    2014-08-19

    Allosteric binding events play a critical role in the formation and stability of transcriptional activator-coactivator complexes, perhaps in part due to the often intrinsically disordered nature of one or more of the constituent partners. The kinase-inducible domain interacting (KIX) domain of the master coactivator CREB binding protein/p300 is a conformationally dynamic domain that complexes with transcriptional activators at two discrete binding sites in allosteric communication. The complexation of KIX with the transcriptional activation domain of mixed-lineage leukemia protein leads to an enhancement of binding by the activation domain of CREB (phosphorylated kinase-inducible domain of CREB) to the second site. A transient kinetic analysis of the ternary complex formation aided by small molecule ligands that induce positive or negative cooperative binding reveals that positive cooperativity is largely governed by stabilization of the bound complex as indicated by a decrease in koff. Thus, this suggests the increased binding affinity for the second ligand is not due to an allosteric creation of a more favorable binding interface by the first ligand. This is consistent with data from us and from others indicating that the on rates of conformationally dynamic proteins approach the limits of diffusion. In contrast, negative cooperativity is manifested by alterations in both kon and koff, suggesting stabilization of the binary complex.

  8. Topical Anti-inflammatory Activity of New Hybrid Molecules of Terpenes and Synthetic Drugs.

    PubMed

    Theoduloz, Cristina; Delporte, Carla; Valenzuela-Barra, Gabriela; Silva, Ximena; Cádiz, Solange; Bustamante, Fernanda; Pertino, Mariano Walter; Schmeda-Hirschmann, Guillermo

    2015-06-18

    The aim of the study was to assess changes in the activity of anti-inflammatory terpenes from Chilean medicinal plants after the formation of derivatives incorporating synthetic anti-inflammatory agents. Ten new hybrid molecules were synthesized combining terpenes (ferruginol (1), imbricatolic acid (2) and oleanolic acid (3)) with ibuprofen (4) or naproxen (5). The topical anti-inflammatory activity of the compounds was assessed in mice by the arachidonic acid (AA) and 12-O-tetradecanoyl phorbol 13-acetate (TPA) induced ear edema assays. Basal cytotoxicity was determined towards human lung fibroblasts, gastric epithelial cells and hepatocytes. At 1.4 µmol/mouse, a strong anti-inflammatory effect in the TPA assay was observed for oleanoyl ibuprofenate 12 (79.9%) and oleanoyl ibuprofenate methyl ester 15 (80.0%). In the AA assay, the best activity was observed for 12 at 3.2 µmol/mouse, with 56.8% reduction of inflammation, in the same range as nimesulide (48.9%). All the terpenyl-synthetic anti-inflammatory hybrids showed better effects in the TPA assay, with best activity for 6, 12 and 15. The cytotoxicity of the compounds 8 and 10 with a free COOH, was higher than that of 2. The derivatives from 3 were less toxic than the triterpene. Several of the new compounds presented better anti-inflammatory effect and lower cytotoxicity than the parent terpenes.

  9. Topical Anti-inflammatory Activity of New Hybrid Molecules of Terpenes and Synthetic Drugs.

    PubMed

    Theoduloz, Cristina; Delporte, Carla; Valenzuela-Barra, Gabriela; Silva, Ximena; Cádiz, Solange; Bustamante, Fernanda; Pertino, Mariano Walter; Schmeda-Hirschmann, Guillermo

    2015-01-01

    The aim of the study was to assess changes in the activity of anti-inflammatory terpenes from Chilean medicinal plants after the formation of derivatives incorporating synthetic anti-inflammatory agents. Ten new hybrid molecules were synthesized combining terpenes (ferruginol (1), imbricatolic acid (2) and oleanolic acid (3)) with ibuprofen (4) or naproxen (5). The topical anti-inflammatory activity of the compounds was assessed in mice by the arachidonic acid (AA) and 12-O-tetradecanoyl phorbol 13-acetate (TPA) induced ear edema assays. Basal cytotoxicity was determined towards human lung fibroblasts, gastric epithelial cells and hepatocytes. At 1.4 µmol/mouse, a strong anti-inflammatory effect in the TPA assay was observed for oleanoyl ibuprofenate 12 (79.9%) and oleanoyl ibuprofenate methyl ester 15 (80.0%). In the AA assay, the best activity was observed for 12 at 3.2 µmol/mouse, with 56.8% reduction of inflammation, in the same range as nimesulide (48.9%). All the terpenyl-synthetic anti-inflammatory hybrids showed better effects in the TPA assay, with best activity for 6, 12 and 15. The cytotoxicity of the compounds 8 and 10 with a free COOH, was higher than that of 2. The derivatives from 3 were less toxic than the triterpene. Several of the new compounds presented better anti-inflammatory effect and lower cytotoxicity than the parent terpenes. PMID:26096431

  10. A Pipeline for Screening Small Molecules with Growth Inhibitory Activity against Burkholderia cenocepacia

    PubMed Central

    Selin, Carrie; Stietz, Maria S.; Blanchard, Jan E.; Hall, Dennis G.; Brown, Eric D.; Cardona, Silvia T.

    2015-01-01

    Infections with the bacteria Burkholderia cepacia complex (Bcc) are very difficult to eradicate in cystic fibrosis patients due the intrinsic resistance of Bcc to most available antibiotics and the emergence of multiple antibiotic resistant strains during antibiotic treatment. In this work, we used a whole-cell based assay to screen a diverse collection of small molecules for growth inhibitors of a relevant strain of Bcc, B. cenocepacia K56-2. The primary screen used bacterial growth in 96-well plate format and identified 206 primary actives among 30,259 compounds. From 100 compounds with no previous record of antibacterial activity secondary screening and data mining selected a total of Bce bioactives that were further analyzed. An experimental pipeline, evaluating in vitro antibacterial and antibiofilm activity, toxicity and in vivo antibacterial activity using C. elegans was used for prioritizing compounds with better chances to be further investigated as potential Bcc antibacterial drugs. This high throughput screen, along with the in vitro and in vivo analysis highlights the utility of this experimental method to quickly identify bioactives as a starting point of antibacterial drug discovery. PMID:26053039

  11. Screening and characterization of molecules that modulate the biological activity of IFNs-I.

    PubMed

    Bürgi, Milagros; Zapol'skii, Viktor A; Hinkelmann, Bettina; Köster, Mario; Kaufmann, Dieter E; Sasse, Florenz; Hauser, Hansjörg; Etcheverrigaray, Marina; Kratje, Ricardo; Bollati-Fogolín, Mariela; Oggero, Marcos

    2016-09-10

    Type I Interferons (IFNs-I) are species-specific glycoproteins which play an important role as primary defence against viral infections and that can also modulate the adaptive immune system. In some autoimmune diseases, interferons (IFNs) are over-produced. IFNs are widely used as biopharmaceuticals for a variety of cancer indications, chronic viral diseases, and for their immuno-modulatory action in patients with multiple sclerosis; therefore, increasing their therapeutic efficiency and decreasing their side effects is of high clinical value. In this sense, it is interesting to find molecules that can modulate the activity of IFNs. In order to achieve that, it was necessary to establish a simple, fast and robust assay to analyze numerous compounds simultaneously. We developed four reporter gene assays (RGAs) to identify IFN activity modulator compounds by using WISH-Mx2/EGFP, HeLa-Mx2/EGFP, A549-Mx2/EGFP, and HEp2-Mx2/EGFP reporter cell lines (RCLs). All of them present a Z' factor higher than 0.7. By using these RGAs, natural and synthetic compounds were analyzed simultaneously. A total of 442 compounds were studied by the Low Throughput Screening (LTS) assay using the four RCLs to discriminate between their inhibitory or enhancing effects on IFN activity. Some of them were characterized and 15 leads were identified. Finally, one promising candidate with enhancing effect on IFN-α/-β activity and five compounds with inhibitory effect were described.

  12. A Pipeline for Screening Small Molecules with Growth Inhibitory Activity against Burkholderia cenocepacia.

    PubMed

    Selin, Carrie; Stietz, Maria S; Blanchard, Jan E; Gehrke, Sebastian S; Bernard, Sylvain; Hall, Dennis G; Brown, Eric D; Cardona, Silvia T

    2015-01-01

    Infections with the bacteria Burkholderia cepacia complex (Bcc) are very difficult to eradicate in cystic fibrosis patients due the intrinsic resistance of Bcc to most available antibiotics and the emergence of multiple antibiotic resistant strains during antibiotic treatment. In this work, we used a whole-cell based assay to screen a diverse collection of small molecules for growth inhibitors of a relevant strain of Bcc, B. cenocepacia K56-2. The primary screen used bacterial growth in 96-well plate format and identified 206 primary actives among 30,259 compounds. From 100 compounds with no previous record of antibacterial activity secondary screening and data mining selected a total of Bce bioactives that were further analyzed. An experimental pipeline, evaluating in vitro antibacterial and antibiofilm activity, toxicity and in vivo antibacterial activity using C. elegans was used for prioritizing compounds with better chances to be further investigated as potential Bcc antibacterial drugs. This high throughput screen, along with the in vitro and in vivo analysis highlights the utility of this experimental method to quickly identify bioactives as a starting point of antibacterial drug discovery. PMID:26053039

  13. Family and Community Involvement in the Comprehensive School Physical Activity Program

    ERIC Educational Resources Information Center

    Cipriani, Kristin; Richardson, Cheryl; Roberts, Georgi

    2012-01-01

    Engaging families and communities in physical activities for the benefit of children is an extension of the role of a physical education instructor. Although it is possible for a physical educator to generate ideas that encourage families and communities to move, a certified director of physical activity (C-DPA) would be better trained to…

  14. Project CRAFT: Culturally Responsive and Family Focused Training: A Learning Activities Guide.

    ERIC Educational Resources Information Center

    Chen, Deborah; Chan, Sam; Brekken, Linda; Lynch, Eleanor W.; Valverde, Aracelly

    This learning activities guide was developed as part of Project CRAFT (Culturally Responsive and Family Focused Training), an effort to train service providers in California in ways of working with young children (particularly those with disabilities) and their families from diverse cultural backgrounds. The learning activities are intended to be…

  15. Associations between Children's Physical Activities, Sedentary Behaviours and Family Structure: A Sequential Mixed Methods Approach

    ERIC Educational Resources Information Center

    Quarmby, T.; Dagkas, S.; Bridge, M.

    2011-01-01

    This mixed method paper explored the effect of family structure on children's physical activities and sedentary pursuits. It furthers the limited understanding of how family structure impacts on children's time in, and reasons behind engaging in, certain physical activities. Children from three inner city comprehensive schools in the Midlands,…

  16. Anger expression and natural killer cell activity in family caregivers participating in a physical activity trial.

    PubMed

    Wilcox, S; King, A C; Vitaliano, P P; Brassington, G S

    2000-07-01

    Associations between psychological functioning and natural killer cell activity (NKA) were examined in 23 older (62.2 ± 7.5 years) family caregivers randomized to a moderate intensity four-month exercise program or to a wait-list control condition. At baseline, although NKA was related to anger-control (r = -.42; trend p < .06) and anger-out (r = .50; p < .03), it was not related to depression, anxiety, perceived stress, or caregiver burden. After controlling for baseline NKA, changes in anger-control explained 14 percent of the variance in NKA four months later. Decreases in anger-control predicted increases in NKA. Group assignment (exercise vs control) was unrelated to changes in NKA over the four-month period; however, the study was not powered to detect this effect. These results are consistent with reported relationships of anger expression with other physiological measures, and extend the importance of anger expression to immune functioning in older family caregivers.

  17. Tetrandrine identified in a small molecule screen to activate mesenchymal stem cells for enhanced immunomodulation.

    PubMed

    Yang, Zijiang; Concannon, John; Ng, Kelvin S; Seyb, Kathleen; Mortensen, Luke J; Ranganath, Sudhir; Gu, Fangqi; Levy, Oren; Tong, Zhixiang; Martyn, Keir; Zhao, Weian; Lin, Charles P; Glicksman, Marcie A; Karp, Jeffrey M

    2016-07-26

    Pre-treatment or priming of mesenchymal stem cells (MSC) prior to transplantation can significantly augment the immunosuppressive effect of MSC-based therapies. In this study, we screened a library of 1402 FDA-approved bioactive compounds to prime MSC. We identified tetrandrine as a potential hit that activates the secretion of prostaglandin E2 (PGE2), a potent immunosuppressive agent, by MSC. Tetrandrine increased MSC PGE2 secretion through the NF-κB/COX-2 signaling pathway. When co-cultured with mouse macrophages (RAW264.7), tetrandrine-primed MSC attenuated the level of TNF-α secreted by RAW264.7. Furthermore, systemic transplantation of primed MSC into a mouse ear skin inflammation model significantly reduced the level of TNF-α in the inflamed ear, compared to unprimed cells. Screening of small molecules to pre-condition cells prior to transplantation represents a promising strategy to boost the therapeutic potential of cell therapy.

  18. Tetrandrine identified in a small molecule screen to activate mesenchymal stem cells for enhanced immunomodulation

    PubMed Central

    Yang, Zijiang; Concannon, John; Ng, Kelvin S.; Seyb, Kathleen; Mortensen, Luke J.; Ranganath, Sudhir; Gu, Fangqi; Levy, Oren; Tong, Zhixiang; Martyn, Keir; Zhao, Weian; Lin, Charles P.; Glicksman, Marcie A.; Karp, Jeffrey M.

    2016-01-01

    Pre-treatment or priming of mesenchymal stem cells (MSC) prior to transplantation can significantly augment the immunosuppressive effect of MSC-based therapies. In this study, we screened a library of 1402 FDA-approved bioactive compounds to prime MSC. We identified tetrandrine as a potential hit that activates the secretion of prostaglandin E2 (PGE2), a potent immunosuppressive agent, by MSC. Tetrandrine increased MSC PGE2 secretion through the NF-κB/COX-2 signaling pathway. When co-cultured with mouse macrophages (RAW264.7), tetrandrine-primed MSC attenuated the level of TNF-α secreted by RAW264.7. Furthermore, systemic transplantation of primed MSC into a mouse ear skin inflammation model significantly reduced the level of TNF-α in the inflamed ear, compared to unprimed cells. Screening of small molecules to pre-condition cells prior to transplantation represents a promising strategy to boost the therapeutic potential of cell therapy. PMID:27457881

  19. Tetrandrine identified in a small molecule screen to activate mesenchymal stem cells for enhanced immunomodulation.

    PubMed

    Yang, Zijiang; Concannon, John; Ng, Kelvin S; Seyb, Kathleen; Mortensen, Luke J; Ranganath, Sudhir; Gu, Fangqi; Levy, Oren; Tong, Zhixiang; Martyn, Keir; Zhao, Weian; Lin, Charles P; Glicksman, Marcie A; Karp, Jeffrey M

    2016-01-01

    Pre-treatment or priming of mesenchymal stem cells (MSC) prior to transplantation can significantly augment the immunosuppressive effect of MSC-based therapies. In this study, we screened a library of 1402 FDA-approved bioactive compounds to prime MSC. We identified tetrandrine as a potential hit that activates the secretion of prostaglandin E2 (PGE2), a potent immunosuppressive agent, by MSC. Tetrandrine increased MSC PGE2 secretion through the NF-κB/COX-2 signaling pathway. When co-cultured with mouse macrophages (RAW264.7), tetrandrine-primed MSC attenuated the level of TNF-α secreted by RAW264.7. Furthermore, systemic transplantation of primed MSC into a mouse ear skin inflammation model significantly reduced the level of TNF-α in the inflamed ear, compared to unprimed cells. Screening of small molecules to pre-condition cells prior to transplantation represents a promising strategy to boost the therapeutic potential of cell therapy. PMID:27457881

  20. Osteogenic Activity of Locally Applied Small Molecule Drugs in a Rat Femur Defect Model

    PubMed Central

    Cottrell, Jessica A.; Vales, Francis M.; Schachter, Deborah; Wadsworth, Scott; Gundlapalli, Rama; Kapadia, Rasesh; O'Connor, J. Patrick

    2010-01-01

    The long-term success of arthroplastic joints is dependent on the stabilization of the implant within the skeletal site. Movement of the arthroplastic implant within the bone can stimulate osteolysis, and therefore methods which promote rigid fixation or bone growth are expected to enhance implant stability and the long-term success of joint arthroplasty. In the present study, we used a simple bilateral bone defect model to analyze the osteogenic activity of three small-molecule drug implants via microcomputerized tomography (micro-CT) and histomorphometry. In this study, we show that local delivery of alendronate, but not lovastatin or omeprazole, led to significant new bone formation at the defect site. Since alendronate impedes osteoclast-development, it is theorized that alendronate treatment results in a net increase in bone formation by preventing osteoclast mediated remodeling of the newly formed bone and upregulating osteoblasts. PMID:20625499

  1. Developing novel organocatalyzed aldol reactions for the enantioselective synthesis of biologically active molecules

    PubMed Central

    Bhanushali, Mayur; Zhao, Cong-Gui

    2011-01-01

    Aldol reaction is one of the most important methods for the formation of carbon-carbon bonds. Because of its significance and usefulness, asymmetric versions of this reaction have been realized with different approaches in the past. Over the last decade, the area of organocatalysis has made significant progresses. As one of most studied reactions in organocatalyses, organocatalyzed aldol reaction has emerged as a powerful tool for the synthesis of a large number of useful products in optically enriched forms. In this review, we summarize our efforts on the development of novel organocatalyzed aldol reactions for the enantioselective synthesis of biological active molecules. Literatures closely related to our studies are also covered. PMID:21918584

  2. Broadband standoff detection of large molecules by mid-infrared active coherent laser spectrometry.

    PubMed

    Macleod, Neil A; Molero, Francisco; Weidmann, Damien

    2015-01-26

    A widely tunable active coherent laser spectrometer (ACLaS) has been demonstrated for standoff detection of broadband absorbers in the 1280 to 1318 cm-1 spectral region using an external cavity quantum cascade laser as a mid-infrared source. The broad tuning range allows detection and quantification of vapor phase molecules, such as dichloroethane, ethylene glycol dinitrate, and tetrafluoroethane. The level of confidence in molecular mixing ratios retrieved from interfering spectral measurements is assessed in a quantitative manner. A first qualitative demonstration of condensed phase chemical detection on nitroacetanilide has also been conducted. Detection performances of the broadband ACLaS have been placed in the context of explosive detection and compared to that obtained using distributed feedback quantum cascade lasers.

  3. Single-Molecule Observation of a Mechanically Activated Cis-to-Trans Cyclopropane Isomerization.

    PubMed

    Wang, Junpeng; Kouznetsova, Tatiana B; Craig, Stephen L

    2016-08-24

    The mechanochemical activation of cis-gem-difluorocyclopropane (cis-gDFC) mechanophore in toluene was characterized with single-molecule force spectroscopy. Unlike previously reported behavior in methyl benzoate (MB), two transitions are observed in the force vs extension curves of cis-gDFC polymers in toluene. The first transition occurs at the same force of ∼1300 pN observed previously in MB, but a second transition is observed at forces of ∼1800 pN that reveal the partial formation of the trans-gDFC isomer. The behavior is attributed to competing reactions of the cis-gDFC at the 1300 pN plateau: addition of oxygen to a ring-opened diradicaloid intermediate, and isomerization of cis-gDFC to its trans isomer. PMID:27500711

  4. Interactions of water, methanol and diethyl ether molecules with the surface of oxidized activated carbon

    NASA Astrophysics Data System (ADS)

    Salame, Issa I.; Bandosz, Teresa J.

    Two samples of oxidized activated carbon of wood origin were used as adsorbents of water, methanol, and diethyl ether. Structural and chemical characteristics of the samples' surfaces were obtained using adsorption of nitrogen and Boehm titration. The adsorption isotherms of water and methanol were measured using a volumetric apparatus whereas the adsorption of diethyl ether was studied by means of inverse gas chromatography at finite concentration. Then the isotherms at three different temperatures were used to calculate the isosteric heats of adsorption. The results showed that the strength of interaction depends on the porosity of the sample and its surface chemistry. The effect of surface chemistry and the presence of oxygenated groups are predominant in the case of water and the least important in the case of diethyl ether. This is the result of the chemical nature of the molecules, their sizes, and the relative strengths of the dispersive interactions in small pores in comparison with hydrogen bonding to surface functional groups.

  5. Expression of the bitter receptor T2R38 in pancreatic cancer: localization in lipid droplets and activation by a bacteria-derived quorum-sensing molecule

    PubMed Central

    Gaida, Matthias M.; Mayer, Christine; Dapunt, Ulrike; Stegmaier, Sabine; Schirmacher, Peter; Wabnitz, Guido H.; Hänsch, G. Maria

    2016-01-01

    T2R38 belongs to the family of bitter receptors and was initially detected in cells of the oral cavity. We now describe expression of T2R38 in tumor cells in patients with pancreatic cancer and in tumor-derived cell lines. T2R38 is localized predominantly intracellular in association with lipid droplets, particularly with the lipid droplet membrane. The receptor can be activated by the bona fide ligand for T2R38, phenylthiourea (PTU), and by N-acetyl-dodecanoyl homoserine (AHL-12), a quorum sensing molecule of Pseudomonas aeruginosa, the latter is the only known natural ligand for T2R38. In response to PTU or AHL-12, key transcription factors are activated including phosphorylation of the MAP kinases p38 and ERK1/2, and upregulation of NFATc1. Moreover, we found increased expression of the multi-drug resistance protein 1 (also known as ABCB1), a transmembrane transporter molecule, participating in shuttling of a plethora of drugs, such as chemotherapeutics or antibiotics. In conclusion, our data indicate a new, additional function of the taste receptor T2R38 beyond sensing ‘bitter’. Moreover, because T2R38 can be stimulated by a bacteria-derived signaling molecule the receptor could link microbiota and cancer. PMID:26862855

  6. Expression of the bitter receptor T2R38 in pancreatic cancer: localization in lipid droplets and activation by a bacteria-derived quorum-sensing molecule.

    PubMed

    Gaida, Matthias M; Mayer, Christine; Dapunt, Ulrike; Stegmaier, Sabine; Schirmacher, Peter; Wabnitz, Guido H; Hänsch, G Maria

    2016-03-15

    T2R38 belongs to the family of bitter receptors and was initially detected in cells of the oral cavity. We now describe expression of T2R38 in tumor cells in patients with pancreatic cancer and in tumor-derived cell lines. T2R38 is localized predominantly intracellular in association with lipid droplets, particularly with the lipid droplet membrane. The receptor can be activated by the bona fide ligand for T2R38, phenylthiourea (PTU), and by N-acetyl-dodecanoyl homoserine (AHL-12), a quorum sensing molecule of Pseudomonas aeruginosa, the latter is the only known natural ligand for T2R38. In response to PTU or AHL-12, key transcription factors are activated including phosphorylation of the MAP kinases p38 and ERK1/2, and upregulation of NFATc1. Moreover, we found increased expression of the multi-drug resistance protein 1 (also known as ABCB1), a transmembrane transporter molecule, participating in shuttling of a plethora of drugs, such as chemotherapeutics or antibiotics. In conclusion, our data indicate a new, additional function of the taste receptor T2R38 beyond sensing "bitter". Moreover, because T2R38 can be stimulated by a bacteria-derived signaling molecule the receptor could link microbiota and cancer. PMID:26862855

  7. The Y’s that bind: negative regulators of Src family kinase activity in platelets

    PubMed Central

    NEWMAN, D. K.

    2015-01-01

    Summary Members of the Src family of protein tyrosine kinases play important roles in platelet adhesion, activation, and aggregation. The purpose of this review is to summarize current knowledge regarding how Src family kinase activity is regulated in general, to describe what is known about mechanisms underlying SFK activation in platelets, and to discuss platelet proteins that contribute to SFK inactivation, particularly those that use phosphotyrosine-containing sequences to recruit phosphatases and kinases to sites of SFK activity. PMID:19630799

  8. Screening of an OBOC combinatorial library for beta-actin identifies molecules active toward Ramos B-lymphoma cells

    PubMed Central

    Miyamoto, Suzanne; Liu, Ruiwu; Hung, Susan; Wang, Xiaobing; Lam, Kit S.

    2009-01-01

    The search for small molecules that specifically recognize protein targets is a laborious process if conducted in a one protein – one compound manner. A high throughput antibody based screening of "one-bead one-compound" (OBOC) combinatorial small molecule libraries is described here, whereby libraries contain thousands of different small molecule ligands are synthesized on individual TentaGel beads and simultaneously screened for protein binding to individual beads, each with a different compound. The use of "OBOC" libraries greatly facilitates this simultaneous screening of thousands of compounds. Now, through the use of monoclonal or affinity purified antibodies, we identified small molecules that bind a particular protein contained in a complex mixture of biological molecules. This method identified small molecule ligands that bound beta-actin present in cytoplasmic cell extracts of Ramos B-lymphoma cells. These small molecule ligands were resynthesized in immobilized and soluble forms and tested for binding of beta-actin present in Ramos B-cell extracts and for activity against Ramos lymphoma cells. This high throughput screening immunoassay method has great promise for improving our ability to find relevant, bioactive small molecules that target a specific native protein in a complex protein mixture without purification of the protein. PMID:18023409

  9. Small-Molecule Inhibitors That Target Protein–Protein Interactions in the RAD51 Family of Recombinases

    PubMed Central

    Scott, Duncan E; Coyne, Anthony G; Venkitaraman, Ashok; Blundell, Tom L; Abell, Chris; Hyvönen, Marko

    2015-01-01

    The development of small molecules that inhibit protein–protein interactions continues to be a challenge in chemical biology and drug discovery. Herein we report the development of indole-based fragments that bind in a shallow surface pocket of a humanised surrogate of RAD51. RAD51 is an ATP-dependent recombinase that plays a key role in the repair of double-strand DNA breaks. It both self-associates, forming filament structures with DNA, and interacts with the BRCA2 protein through a common “FxxA” tetrapeptide motif. We elaborated previously identified fragment hits that target the FxxA motif site and developed small-molecule inhibitors that are approximately 500-fold more potent than the initial fragments. The lead compounds were shown to compete with the BRCA2-derived Ac-FHTA-NH2 peptide and the self-association peptide of RAD51, but they had no effect on ATP binding. This study is the first reported elaboration of small-molecular-weight fragments against this challenging target. PMID:25470112

  10. Computer simulation of adsorption of a Stockmayer molecule chlorodifluoromethane in activated carbon slit pores

    NASA Astrophysics Data System (ADS)

    Jin, Wenzheng; Wang, Wenchuan

    2001-06-01

    The adsorption recovery of HCFC-22 is an urgent task for environment protection. In this work we use the grand canonical Monte Carlo (GCMC) method to simulate the adsorption of HCFC-22 in terms of a slit-like activated carbon adsorbent. In our simulation the molecule of HCFC-22 is modeled by using the effective Stockmayer potential developed by this group. Heterogeneous activated sites with three different densities are imposed on the carbon walls. Three types of simulations are carried out: (1) The Gibbs ensemble MC method is used to test the Stockmayer potential parameters recommended here. (2) The Widom test particle method is used for determining the relationship of the chemical potential and the bulk phase pressure. (3) The GCMC method is used for adsorption simulations. Simulation results suggest that the optimum carbon slit pore is of width 1.75 nm and the activated site density is 0.8 sites/nm2, when the adsorption is conducted at ambient temperature and pressure, and the exhaustion pressure is 0.011 MPa. In this case, the maximum amount of HCFC-22 would be recovered.

  11. Signaling lymphocytic activation molecule (CDw150) is homophilic but self-associates with very low affinity.

    PubMed

    Mavaddat, N; Mason, D W; Atkinson, P D; Evans, E J; Gilbert, R J; Stuart, D I; Fennelly, J A; Barclay, A N; Davis, S J; Brown, M H

    2000-09-01

    Signaling lymphocytic activating molecule ((SLAM) CDw150) is a glycoprotein that belongs to the CD2 subset of the immunoglobulin superfamily and is expressed on the surface of activated T- and B-cells. It has been proposed that SLAM is homophilic and required for bidirectional signaling during T- and B-cell activation. Previous work has suggested that the affinity of SLAM self-association might be unusually high, undermining the concept that protein interactions mediating transient cell-cell contacts, such as those involving leukocytes, have to be weak in order that such contacts are readily reversible. Using surface plasmon resonance-based methods and analytical ultracentrifugation (AUC), we confirm that SLAM is homophilic. However, we also establish a new theoretical treatment of surface plasmon resonance-derived homophilic binding data, which indicates that SLAM-SLAM interactions (solution K(d) approximately 200 micrometer) are in fact considerably weaker than most other well characterized protein-protein interactions at the cell surface (solution K(d) approximately 0.4-20 micrometer), a conclusion that is supported by the AUC analysis. Whereas further analysis of the AUC data imply that SLAM could form "head to head" dimers spanning adjacent cells, the very low affinity raises important questions regarding the physiological role and/or properties of such interactions. PMID:10831600

  12. Force and twist dependence of RepC nicking activity on torsionally-constrained DNA molecules

    PubMed Central

    Pastrana, Cesar L.; Carrasco, Carolina; Akhtar, Parvez; Leuba, Sanford H.; Khan, Saleem A.; Moreno-Herrero, Fernando

    2016-01-01

    Many bacterial plasmids replicate by an asymmetric rolling-circle mechanism that requires sequence-specific recognition for initiation, nicking of one of the template DNA strands and unwinding of the duplex prior to subsequent leading strand DNA synthesis. Nicking is performed by a replication-initiation protein (Rep) that directly binds to the plasmid double-stranded origin and remains covalently bound to its substrate 5′-end via a phosphotyrosine linkage. It has been proposed that the inverted DNA sequences at the nick site form a cruciform structure that facilitates DNA cleavage. However, the role of Rep proteins in the formation of this cruciform and the implication for its nicking and religation functions is unclear. Here, we have used magnetic tweezers to directly measure the DNA nicking and religation activities of RepC, the replication initiator protein of plasmid pT181, in plasmid sized and torsionally-constrained linear DNA molecules. Nicking by RepC occurred only in negatively supercoiled DNA and was force- and twist-dependent. Comparison with a type IB topoisomerase in similar experiments highlighted a relatively inefficient religation activity of RepC. Based on the structural modeling of RepC and on our experimental evidence, we propose a model where RepC nicking activity is passive and dependent upon the supercoiling degree of the DNA substrate. PMID:27488190

  13. Small-Molecule Procaspase-3 Activation Sensitizes Cancer to Treatment with Diverse Chemotherapeutics

    PubMed Central

    2016-01-01

    Conventional chemotherapeutics remain essential treatments for most cancers, but their combination with other anticancer drugs (including targeted therapeutics) is often complicated by unpredictable synergies and multiplicative toxicities. As cytotoxic anticancer chemotherapeutics generally function through induction of apoptosis, we hypothesized that a molecularly targeted small molecule capable of facilitating a central and defining step in the apoptotic cascade, the activation of procaspase-3 to caspase-3, would broadly and predictably enhance activity of cytotoxic drugs. Here we show that procaspase-activating compound 1 (PAC-1) enhances cancer cell death induced by 15 different FDA-approved chemotherapeutics, across many cancer types and chemotherapeutic targets. In particular, the promising combination of PAC-1 and doxorubicin induces a synergistic reduction in tumor burden and enhances survival in murine tumor models of osteosarcoma and lymphoma. This PAC-1/doxorubicin combination was evaluated in 10 pet dogs with naturally occurring metastatic osteosarcoma or lymphoma, eliciting a biologic response in 3 of 6 osteosarcoma patients and 4 of 4 lymphoma patients. Importantly, in both mice and dogs, coadministration of PAC-1 with doxorubicin resulted in no additional toxicity. On the basis of the mode of action of PAC-1 and the high expression of procaspase-3 in many cancers, these results suggest the combination of PAC-1 with cytotoxic anticancer drugs as a potent and general strategy to enhance therapeutic response.

  14. Small-Molecule Procaspase-3 Activation Sensitizes Cancer to Treatment with Diverse Chemotherapeutics

    PubMed Central

    2016-01-01

    Conventional chemotherapeutics remain essential treatments for most cancers, but their combination with other anticancer drugs (including targeted therapeutics) is often complicated by unpredictable synergies and multiplicative toxicities. As cytotoxic anticancer chemotherapeutics generally function through induction of apoptosis, we hypothesized that a molecularly targeted small molecule capable of facilitating a central and defining step in the apoptotic cascade, the activation of procaspase-3 to caspase-3, would broadly and predictably enhance activity of cytotoxic drugs. Here we show that procaspase-activating compound 1 (PAC-1) enhances cancer cell death induced by 15 different FDA-approved chemotherapeutics, across many cancer types and chemotherapeutic targets. In particular, the promising combination of PAC-1 and doxorubicin induces a synergistic reduction in tumor burden and enhances survival in murine tumor models of osteosarcoma and lymphoma. This PAC-1/doxorubicin combination was evaluated in 10 pet dogs with naturally occurring metastatic osteosarcoma or lymphoma, eliciting a biologic response in 3 of 6 osteosarcoma patients and 4 of 4 lymphoma patients. Importantly, in both mice and dogs, coadministration of PAC-1 with doxorubicin resulted in no additional toxicity. On the basis of the mode of action of PAC-1 and the high expression of procaspase-3 in many cancers, these results suggest the combination of PAC-1 with cytotoxic anticancer drugs as a potent and general strategy to enhance therapeutic response. PMID:27610416

  15. Small-Molecule Procaspase-3 Activation Sensitizes Cancer to Treatment with Diverse Chemotherapeutics.

    PubMed

    Botham, Rachel C; Roth, Howard S; Book, Alison P; Roady, Patrick J; Fan, Timothy M; Hergenrother, Paul J

    2016-08-24

    Conventional chemotherapeutics remain essential treatments for most cancers, but their combination with other anticancer drugs (including targeted therapeutics) is often complicated by unpredictable synergies and multiplicative toxicities. As cytotoxic anticancer chemotherapeutics generally function through induction of apoptosis, we hypothesized that a molecularly targeted small molecule capable of facilitating a central and defining step in the apoptotic cascade, the activation of procaspase-3 to caspase-3, would broadly and predictably enhance activity of cytotoxic drugs. Here we show that procaspase-activating compound 1 (PAC-1) enhances cancer cell death induced by 15 different FDA-approved chemotherapeutics, across many cancer types and chemotherapeutic targets. In particular, the promising combination of PAC-1 and doxorubicin induces a synergistic reduction in tumor burden and enhances survival in murine tumor models of osteosarcoma and lymphoma. This PAC-1/doxorubicin combination was evaluated in 10 pet dogs with naturally occurring metastatic osteosarcoma or lymphoma, eliciting a biologic response in 3 of 6 osteosarcoma patients and 4 of 4 lymphoma patients. Importantly, in both mice and dogs, coadministration of PAC-1 with doxorubicin resulted in no additional toxicity. On the basis of the mode of action of PAC-1 and the high expression of procaspase-3 in many cancers, these results suggest the combination of PAC-1 with cytotoxic anticancer drugs as a potent and general strategy to enhance therapeutic response. PMID:27610416

  16. High pressure chemistry of red phosphorus by photo-activated simple molecules

    NASA Astrophysics Data System (ADS)

    Ceppatelli, M.; Fanetti, S.; Bini, R.; Caporali, M.; Peruzzini, M.

    2014-05-01

    High pressure (HP) is very effective in reducing intermolecular distances and inducing unexpected chemical reactions. In addition the photo-activation of the reactants in HP conditions can lead to very efficient and selective processes. The chemistry of phosphorus is currently based on the white molecular form. The red polymeric allotrope, despite more stable and much less toxic, has not attracted much attention so far. However, switching from the white to the red form would benefit any industrial procedure, especially from an environmental point of view. On the other side, water and ethanol are renewable, environmental friendly and largely available molecules, usable as reactants and photo-activators in HP conditions. Here we report a study on the HP photo-induced reactivity of red phosphorus with water and ethanol, showing the possibility of very efficient and selective processes, leading to molecular hydrogen and valuable phosphorus compounds. The reactions have been studied by means of FTIR and Raman spectroscopy and pressure has been generated using membrane Diamond (DAC) and Sapphire (SAC) anvil cells. HP reactivity has been activated by the two-photon absorption of near-UV wavelengths and occurred in total absence of solvents, catalysts and radical initiators, at room T and mild pressure conditions (0.2-1.5 GPa).

  17. Antiproliferation activity of a small molecule repressor of liver receptor homolog 1.

    PubMed

    Corzo, Cesar A; Mari, Yelenis; Chang, Mi Ra; Khan, Tanya; Kuruvilla, Dana; Nuhant, Philippe; Kumar, Naresh; West, Graham M; Duckett, Derek R; Roush, William R; Griffin, Patrick R

    2015-02-01

    The orphan nuclear receptor liver receptor homolog 1 (LRH-1; NR5A2) is a potent regulator of cholesterol metabolism and bile acid homeostasis. Recently, LRH-1 has been shown to play an important role in intestinal inflammation and in the progression of estrogen receptor positive and negative breast cancers and pancreatic cancer. Structural studies have revealed that LRH-1 can bind phospholipids and the dietary phospholipid dilauroylphosphatidylcholine activates LRH-1 activity in rodents. Here we characterize the activity of a novel synthetic nonphospholipid small molecule repressor of LRH-1, SR1848 (6-[4-(3-chlorophenyl)piperazin-1-yl]-3-cyclohexyl-1H-pyrimidine-2,4-dione). In cotransfection studies, SR1848 reduced LRH-1-dependent expression of a reporter gene and in cells that endogenously express LRH-1 dose dependently reduced the expression of cyclin-D1 and -E1, resulting in inhibition of cell proliferation. The cellular effects of SR1848 treatment are recapitulated after transfection of cells with small-interfering RNA targeting LRH-1. Immunocytochemistry analysis shows that SR1848 induces rapid translocation of nuclear LRH-1 to the cytoplasm. Combined, these results suggest that SR1848 is a functional repressor of LRH-1 that impacts expression of genes involved in proliferation in LRH-1-expressing cancers. Thus, SR1848 represents a novel chemical scaffold for the development of therapies targeting malignancies driven by LRH-1.

  18. Antiproliferation Activity of a Small Molecule Repressor of Liver Receptor Homolog 1

    PubMed Central

    Corzo, Cesar A.; Mari, Yelenis; Chang, Mi Ra; Khan, Tanya; Kuruvilla, Dana; Nuhant, Philippe; Kumar, Naresh; West, Graham M.; Duckett, Derek R.; Roush, William R.

    2015-01-01

    The orphan nuclear receptor liver receptor homolog 1 (LRH-1; NR5A2) is a potent regulator of cholesterol metabolism and bile acid homeostasis. Recently, LRH-1 has been shown to play an important role in intestinal inflammation and in the progression of estrogen receptor positive and negative breast cancers and pancreatic cancer. Structural studies have revealed that LRH-1 can bind phospholipids and the dietary phospholipid dilauroylphosphatidylcholine activates LRH-1 activity in rodents. Here we characterize the activity of a novel synthetic nonphospholipid small molecule repressor of LRH-1, SR1848 (6-[4-(3-chlorophenyl)piperazin-1-yl]-3-cyclohexyl-1H-pyrimidine-2,4-dione). In cotransfection studies, SR1848 reduced LRH-1-dependent expression of a reporter gene and in cells that endogenously express LRH-1 dose dependently reduced the expression of cyclin-D1 and -E1, resulting in inhibition of cell proliferation. The cellular effects of SR1848 treatment are recapitulated after transfection of cells with small-interfering RNA targeting LRH-1. Immunocytochemistry analysis shows that SR1848 induces rapid translocation of nuclear LRH-1 to the cytoplasm. Combined, these results suggest that SR1848 is a functional repressor of LRH-1 that impacts expression of genes involved in proliferation in LRH-1–expressing cancers. Thus, SR1848 represents a novel chemical scaffold for the development of therapies targeting malignancies driven by LRH-1. PMID:25473120

  19. Identification of a small molecule with activity against drug-resistant and persistent tuberculosis

    PubMed Central

    Wang, Feng; Sambandan, Dhinakaran; Halder, Rajkumar; Wang, Jianing; Batt, Sarah M.; Weinrick, Brian; Ahmad, Insha; Yang, Pengyu; Zhang, Yong; Kim, John; Hassani, Morad; Huszar, Stanislav; Trefzer, Claudia; Ma, Zhenkun; Kaneko, Takushi; Mdluli, Khisi E.; Franzblau, Scott; Chatterjee, Arnab K.; Johnsson, Kai; Mikusova, Katarina; Besra, Gurdyal S.; Fütterer, Klaus; Robbins, Scott H.; Barnes, S. Whitney; Walker, John R.; Jacobs, William R.; Schultz, Peter G.

    2013-01-01

    A cell-based phenotypic screen for inhibitors of biofilm formation in mycobacteria identified the small molecule TCA1, which has bactericidal activity against both drug-susceptible and -resistant Mycobacterium tuberculosis (Mtb) and sterilizes Mtb in vitro combined with rifampicin or isoniazid. In addition, TCA1 has bactericidal activity against nonreplicating Mtb in vitro and is efficacious in acute and chronic Mtb infection mouse models both alone and combined with rifampicin or isoniazid. Transcriptional analysis revealed that TCA1 down-regulates genes known to be involved in Mtb persistence. Genetic and affinity-based methods identified decaprenyl-phosphoryl-β-D-ribofuranose oxidoreductase DprE1 and MoeW, enzymes involved in cell wall and molybdenum cofactor biosynthesis, respectively, as targets responsible for the activity of TCA1. These in vitro and in vivo results indicate that this compound functions by a unique mechanism and suggest that TCA1 may lead to the development of a class of antituberculosis agents. PMID:23776209

  20. Signaling lymphocytic activation molecule (CDw150) is homophilic but self-associates with very low affinity.

    PubMed

    Mavaddat, N; Mason, D W; Atkinson, P D; Evans, E J; Gilbert, R J; Stuart, D I; Fennelly, J A; Barclay, A N; Davis, S J; Brown, M H

    2000-09-01

    Signaling lymphocytic activating molecule ((SLAM) CDw150) is a glycoprotein that belongs to the CD2 subset of the immunoglobulin superfamily and is expressed on the surface of activated T- and B-cells. It has been proposed that SLAM is homophilic and required for bidirectional signaling during T- and B-cell activation. Previous work has suggested that the affinity of SLAM self-association might be unusually high, undermining the concept that protein interactions mediating transient cell-cell contacts, such as those involving leukocytes, have to be weak in order that such contacts are readily reversible. Using surface plasmon resonance-based methods and analytical ultracentrifugation (AUC), we confirm that SLAM is homophilic. However, we also establish a new theoretical treatment of surface plasmon resonance-derived homophilic binding data, which indicates that SLAM-SLAM interactions (solution K(d) approximately 200 micrometer) are in fact considerably weaker than most other well characterized protein-protein interactions at the cell surface (solution K(d) approximately 0.4-20 micrometer), a conclusion that is supported by the AUC analysis. Whereas further analysis of the AUC data imply that SLAM could form "head to head" dimers spanning adjacent cells, the very low affinity raises important questions regarding the physiological role and/or properties of such interactions.

  1. Lysophosphatidylcholine and lysophosphatidylinosiol--novel promissing signaling molecules and their possible therapeutic activity.

    PubMed

    Drzazga, Anna; Sowińska, Agata; Koziołkiewicz, Maria

    2014-01-01

    For many years the role of lysophospholipids (LPLs) was associated only with structural and storage components of the cell without any informational function. Today, based on many research projects performed during the last decades, it is clear that some of the LPLs act as hormone-like signaling molecules and thus are very important inter- and intracellular lipid mediators. They can activate specific membrane receptors and/or nuclear receptors regulating many crucial physiological and pathophysiological processes. The LPLs were iden- tified as involved in a majority of cellular processes, including modulation of disease-related mechanisms observed, for instance, in case of diabetes, obesity, atherosclerosis and cancer. Among LPLs, lysophosphatidylcholine (LPC) and lysophosphatidylinositol (LPI) are becoming attractive research topics. Their recently revealed activities as novel ligands of orphan G protein-coupled receptors (i.e., GPR55 and GPR119) involved in modulation of tumor physiology and insulin secretion seem to be one of the most interesting aspects of these compounds. Moreover, the most recent scientific reports emphasize the significance of the acyl chain structure bound to the glycerol basis of LPL, as it entails different biological properties and activities of the compounds. PMID:25745761

  2. Single molecule analysis of B cell receptor motion during signaling activation

    NASA Astrophysics Data System (ADS)

    Rey Suarez, Ivan; Koo, Peter; Mochrie, Simon; Song, Wenxia; Upadhyaya, Arpita

    B cells are an essential part of the adaptive immune system. They patrol the body looking for signs of infection in the form of antigen on the surface of antigen presenting cells. The binding of the B cell receptor (BCR) to antigen induces signaling cascades that lead to B cell activation and eventual production of high affinity antibodies. During activation, BCR organize into signaling microclusters, which are platforms for signal amplification. The physical processes underlying receptor movement and aggregation are not well understood. Here we study the dynamics of single BCRs on activated murine primary B cells using TIRF imaging and single particle tracking. The tracks obtained are analyzed using perturbation expectation-maximization (pEM) a systems-level analysis that allows the identification of different short-time diffusive states from a set of single particle tracks. We identified five different diffusive states on wild type cells, which correspond to different molecular states of the BCR. By using actin polymerization inhibitors and mutant cells lacking important actin regulators we were able to identify the BCR molecule configuration associated with each diffusive state.

  3. Small molecule-mediated refolding and activation of myosin motor function

    PubMed Central

    Radke, Michael B; Taft, Manuel H; Stapel, Britta; Hilfiker-Kleiner, Denise; Preller, Matthias; Manstein, Dietmar J

    2014-01-01

    The small molecule EMD 57033 has been shown to stimulate the actomyosin ATPase activity and contractility of myofilaments. Here, we show that EMD 57033 binds to an allosteric pocket in the myosin motor domain. EMD 57033-binding protects myosin against heat stress and thermal denaturation. In the presence of EMD 57033, ATP hydrolysis, coupling between actin and nucleotide binding sites, and actin affinity in the presence of ATP are increased more than 10-fold. Addition of EMD 57033 to heat-inactivated β-cardiac myosin is followed by refolding and reactivation of ATPase and motile activities. In heat-stressed cardiomyocytes expression of the stress-marker atrial natriuretic peptide is suppressed by EMD 57033. Thus, EMD 57033 displays a much wider spectrum of activities than those previously associated with small, drug-like compounds. Allosteric effectors that mediate refolding and enhance enzymatic function have the potential to improve the treatment of heart failure, myopathies, and protein misfolding diseases. DOI: http://dx.doi.org/10.7554/eLife.01603.001 PMID:24520162

  4. Mutagenesis and crystallographic studies of the catalytic residues of the papain family protease bleomycin hydrolase: new insights into active-site structure

    PubMed Central

    O'Farrell, Paul A.; Joshua-Tor, Leemor

    2006-01-01

    Bleomycin hydrolase (BH) is a hexameric papain family cysteine protease which is involved in preparing peptides for antigen presentation and has been implicated in tumour cell resistance to bleomycin chemotherapy. Structures of active-site mutants of yeast BH yielded unexpected results. Replacement of the active-site asparagine with alanine, valine or leucine results in the destabilization of the histidine side chain, demonstrating unambiguously the role of the asparagine residue in correctly positioning the histidine for catalysis. Replacement of the histidine with alanine or leucine destabilizes the asparagine position, indicating a delicate arrangement of the active-site residues. In all of the mutants, the C-terminus of the protein, which lies in the active site, protrudes further into the active site. All mutants were compromised in their catalytic activity. The structures also revealed the importance of a tightly bound water molecule which stabilizes a loop near the active site and which is conserved throughout the papain family. It is displaced in a number of the mutants, causing destabilization of this loop and a nearby loop, resulting in a large movement of the active-site cysteine. The results imply that this water molecule plays a key structural role in this family of enzymes. PMID:17007609

  5. Circulating soluble adhesion molecules in patients with giant cell arteritis. Correlation between soluble intercellular adhesion molecule-1 (sICAM-1) concentrations and disease activity

    PubMed Central

    Coll-Vinent, B.; Vilardell, C.; Font, C.; Oristrell, J.; Hernandez-Rodrigu..., J.; Yague, J.; Urbano-Marquez, A.; Grau, J.; Cid, M.

    1999-01-01

    OBJECTIVE—To evaluate whether changes in concentrations of circulating adhesion molecules are related to disease activity in patients with giant cell arteritis (GCA).
METHODS—A sandwich ELISA was used to measure soluble intercellular adhesion molecule-1 (sICAM-1), sICAM-3, vascular cell adhesion molecule-1 (sVCAM-1), E-selectin (sE-selectin), and L-selectin (sL-selectin) in serum and plasma samples from patients with GCA. A cross sectional study was performed on 64 GCA patients at different activity stages and on 35 age and sex matched healthy donors. Thirteen of these patients were evaluated at the time of diagnosis and serially during follow up.
RESULTS—At the time of diagnosis, sICAM-1 concentrations were significantly higher in active GCA patients than in controls (mean (SD) 360.55 (129.78) ng/ml versus 243.25 (47.43) ng/ml, p<0.001). In contrast, sICAM-3, sVCAM-1, sE-selectin, and sL-selectin values did not differ from those obtained in normal donors. With corticosteroid administration, a decrease in sICAM-1 concentrations was observed, reaching normal values when clinical remission was achieved (263.18 (92.7) ng/ml globally, 293.59 (108.39) ng/ml in the group of patients in recent remission, and 236.83 (70.02) ng/ml in those in long term remission). In the 13 patients followed up longitudinally, sICAM-1 values also normalised with clinical remission (225.87 (64.25) ng/ml in patients in recent remission, and 256.29 (75.15) ng/ml in those in long term remission).
CONCLUSIONS—Circulating sICAM-1 concentrations clearly correlate with clinically apparent disease activity in GCA patients. Differences with results previously found in patients with other vasculitides may indicate that different pathogenic mechanisms contribute to vascular inflammation in different disorders.

 Keywords: adhesion molecules; giant cell arteritis; inflammation PMID:10364919

  6. Inhibition of Both Hsp70 Activity and Tau Aggregation in Vitro Best Predicts Tau Lowering Activity of Small Molecules.

    PubMed

    Martin, Mackenzie D; Baker, Jeremy D; Suntharalingam, Amirthaa; Nordhues, Bryce A; Shelton, Lindsey B; Zheng, Dali; Sabbagh, Jonathan J; Haystead, Timothy A J; Gestwicki, Jason E; Dickey, Chad A

    2016-07-15

    Three scaffolds with inhibitory activity against the heat shock protein 70 (Hsp70) family of chaperones have been found to enhance the degradation of the microtubule associated protein tau in cells, neurons, and brain tissue. This is important because tau accumulation is linked to neurodegenerative diseases including Alzheimer's disease (AD) and chronic traumatic encephalopathy (CTE). Here, we expanded upon this study to investigate the anti-tau efficacy of additional scaffolds with Hsp70 inhibitory activity. Five of the nine scaffolds tested lowered tau levels, with the rhodacyanine and phenothiazine scaffolds exhibiting the highest potency as previously described. Because phenothiazines also inhibit tau aggregation in vitro, we suspected that this activity might be a more accurate predictor of tau lowering. Interestingly, the rhodacyanines did inhibit in vitro tau aggregation to a similar degree as phenothiazines, correlating well with tau-lowering efficacy in cells and ex vivo slices. Moreover, other Hsp70 inhibitor scaffolds with weaker tau-lowering activity in cells inhibited tau aggregation in vitro, albeit at lower potencies. When we tested six well-characterized tau aggregation inhibitors, we determined that this mechanism of action was not a better predictor of tau-lowering than Hsp70 inhibition. Instead, we found that compounds possessing both activities were the most effective at promoting tau clearance. Moreover, cytotoxicity and PAINS activity are critical factors that can lead to false-positive lead identification. Strategies designed around these principles will likely yield more efficacious tau-lowering compounds. PMID:27177119

  7. Relationship of lipoprotein(a) levels to physical activity and family history of coronary heart disease.

    PubMed Central

    Martín, S; Elosua, R; Covas, M I; Pavesi, M; Vila, J; Marrugat, J

    1999-01-01

    OBJECTIVES: This study evaluated the association of physical activity with serum lipoprotein(a) [La(a)] levels in individuals according to whether they had a family history of coronary heart disease (CHD). METHODS: Lp(a) levels in 332 healthy Spanish men aged 20 to 60 years were measured. Physical activity and family history of CHD were assessed. RESULTS: For men with a family history of CHD, the odds ratio for Lp(a) levels above the median value was 0.13 (95% confidence interval = 0.03, 0.50) in very active men (energy expended in physical activity > 300 kcal/day) compared with active men (energy expended in physical activity < 300 kcal/day). CONCLUSIONS: Regular daily physical activity in individuals with a family history of CHD could be useful for controlling Lp(a) levels. PMID:10076490

  8. The TNF Family Molecules LIGHT and Lymphotoxin αβ Induce a Distinct Steroid-Resistant Inflammatory Phenotype in Human Lung Epithelial Cells.

    PubMed

    da Silva Antunes, Ricardo; Madge, Lisa; Soroosh, Pejman; Tocker, Joel; Croft, Michael

    2015-09-01

    Lung epithelial cells are considered important sources of inflammatory molecules and extracellular matrix proteins that contribute to diseases such as asthma. Understanding the factors that stimulate epithelial cells may lead to new insights into controlling lung inflammation. This study sought to investigate the responsiveness of human lung epithelial cells to the TNF family molecules LIGHT and lymphotoxin αβ (LTαβ). Bronchial and alveolar epithelial cell lines, and primary human bronchial epithelial cells, were stimulated with LIGHT and LTαβ, and expression of inflammatory cytokines and chemokines and markers of epithelial-mesenchymal transition and fibrosis/remodeling was measured. LTβ receptor, the receptor shared by LIGHT and LTαβ, was constitutively expressed on all epithelial cells. Correspondingly, LIGHT and LTαβ strongly induced a limited but highly distinct set of inflammatory genes in all epithelial cells tested, namely the adhesion molecules ICAM-1 and VCAM-1; the chemokines CCL5, CCL20, CXCL1, CXCL3, CXCL5, and CXCL11; the cytokines IL-6, activin A and GM-CSF; and metalloproteinases matrix metalloproteinase-9 and a disintegrin and metalloproteinase domain-8. Importantly, induction of the majority of these inflammatory molecules was insensitive to the suppressive effects of the corticosteroid budesonide. LIGHT and LTαβ also moderately downregulated E-cadherin, a protein associated with maintaining epithelial integrity, but did not significantly drive production of extracellular matrix proteins or α-smooth muscle actin. Thus, LIGHT and LTαβ induce a distinct steroid-resistant inflammatory signature in airway epithelial cells via constitutively expressed LTβ receptor. These findings support our prior murine studies that suggested the receptors for LIGHT and LTαβ contribute to development of lung inflammation characteristic of asthma and idiopathic pulmonary fibrosis.

  9. Midkine and pleiotrophin have bactericidal properties: preserved antibacterial activity in a family of heparin-binding growth factors during evolution.

    PubMed

    Svensson, Sara L; Pasupuleti, Mukesh; Walse, Björn; Malmsten, Martin; Mörgelin, Matthias; Sjögren, Camilla; Olin, Anders I; Collin, Mattias; Schmidtchen, Artur; Palmer, Ruth; Egesten, Arne

    2010-05-21

    Antibacterial peptides of the innate immune system combat pathogenic microbes, but often have additional roles in promoting inflammation and as growth factors during tissue repair. Midkine (MK) and pleiotrophin (PTN) are the only two members of a family of heparin-binding growth factors. They show restricted expression during embryogenesis and are up-regulated in neoplasia. In addition, MK shows constitutive and inflammation-dependent expression in some non-transformed tissues of the adult. In the present study, we show that both MK and PTN display strong antibacterial activity, present at physiological salt concentrations. Electron microscopy of bacteria and experiments using artificial lipid bilayers suggest that MK and PTN exert their antibacterial action via a membrane disruption mechanism. The predicted structure of PTN, employing the previously solved MK structure as a template, indicates that both molecules consist of two domains, each containing three antiparallel beta-sheets. The antibacterial activity was mapped to the unordered C-terminal tails of both molecules and the last beta-sheets of the N-terminals. Analysis of the highly conserved MK and PTN orthologues from the amphibian Xenopus laevis and the fish Danio rerio suggests that they also harbor antibacterial activity in the corresponding domains. In support of an evolutionary conserved function it was found that the more distant orthologue, insect Miple2 from Drosophila melanogaster, also displays strong antibacterial activity. Taken together, the findings suggest that MK and PTN, in addition to their earlier described activities, may have previously unrealized important roles as innate antibiotics.

  10. Soluble adhesion molecules correlate with surface expression in an in vitro model of endothelial activation.

    PubMed

    Kjaergaard, Anders G; Dige, Anders; Krog, Jan; Tønnesen, Else; Wogensen, Lise

    2013-10-01

    Endothelial activation is a pivotal event in the development and progression of inflammation. Central to endothelial activation is the up-regulation of cellular adhesion molecules (CAMs) including E-selectin (CD62E), ICAM-1 (CD54), VCAM-1 (CD106) and PECAM-1 (CD31). These CAMs are also found in soluble forms (sCAMs). In this in vitro study of endothelial activation, we examined whether the levels of sCAMs correlate with the endothelial surface expression of CAMs in a dose-dependent and time-dependent manner. Such a correlation would support the use of sCAMs as surrogate markers for endothelial activation in inflammatory conditions. Human umbilical vein endothelial cells (HUVEC) were cultured with various concentrations of TNF-α for 8 hr and at a fixed concentration of TNF-α for various durations. The levels of soluble and surface-bound E-selectin, ICAM-1, VCAM-1 and PECAM-1 were quantified by flow cytometry. TNF-α stimulation increased CAM and sCAM expression in a dose-dependent and time-dependent manner. There was a significant positive correlation between the levels of ICAM-1 and sICAM-1 and between the levels of VCAM and sVCAM-1 in both the dose-response and time-response experiments. A positive correlation between the levels of E-selectin and sE-selectin was observed in the time-response experiment. This study supports the use of sCAMs as potential biomarkers of endothelial activation. In particular, the use of sICAM-1, sVCAM-1 and sE-selectin seems promising.

  11. Conversations about Science across Activities in Mexican-Descent Families

    ERIC Educational Resources Information Center

    Siegel, Deborah R.; Esterly, Jennifer; Callanan, Maureen A.; Wright, Ramser; Navarro, Rocio

    2007-01-01

    Parent-child "everyday" conversations have been suggested as a source of children's early science learning. If such conversations are important, then it would be pertinent to know whether children from different family backgrounds have different experiences talking about science in informal settings. We focus on the relation between parents'…

  12. Mechanism for attenuation of DNA binding by MarR family transcriptional regulators by small molecule ligands.

    PubMed

    Perera, Inoka C; Lee, Yong-Hwan; Wilkinson, Steven P; Grove, Anne

    2009-07-31

    Members of the multiple antibiotic resistance regulator (MarR) family control gene expression in a variety of metabolic processes in bacteria and archaea. Hypothetical uricase regulator (HucR), which belongs to the ligand-responsive branch of the MarR family, regulates uricase expression in Deinococcus radiodurans by binding a shared promoter region between uricase and HucR genes. We show here that HucR responds only to urate and, to a lesser extent, to xanthine by attenuated DNA binding, compared to other intermediates of purine degradation. Using molecular-dynamics-guided mutational analysis, we identified the ligand-binding site in HucR. Electrophoretic mobility shift assays and intrinsic Trp fluorescence have identified W20 from the N-terminal helix and R80 from helix 3, which serves as a scaffold for the DNA recognition helix, as being essential for ligand binding. Using structural data combined with in silico and in vitro analyses, we propose a mechanism for the attenuation of DNA binding in which a conformational change initiated by charge repulsion due to a bound ligand propagates to DNA recognition helices. This mechanism may apply generally to MarR homologs that bind anionic phenolic ligands. PMID:19501097

  13. Family Functioning: Associations with Weight Status, Eating Behaviors, and Physical Activity in Adolescents

    PubMed Central

    Berge, Jerica M.; Wall, Melanie; Larson, Nicole; Loth, Katie A.; Neumark-Sztainer, Dianne

    2012-01-01

    Purpose This paper examines the relationship between family functioning (e.g. communication, closeness, problem solving, behavioral control) and adolescent weight status and relevant eating and physical activity behaviors. Methods Data are from EAT 2010 (Eating and Activity in Teens), a population-based study that assessed eating and activity among socioeconomically and racially/ethnically diverse youth (n = 2,793). Adolescents (46.8% boys, 53.2% girls) completed anthropometric assessments and surveys at school in 2009–2010. Multiple linear regression was used to test the relationship between family functioning and adolescent weight, dietary intake, family meal patterns, and physical activity. Additional regression models were fit to test for interactions by race/ethnicity. Results For adolescent girls, higher family functioning was associated with lower body mass index z-score and percent overweight, less sedentary behavior, higher intake of fruits and vegetables, and more frequent family meals and breakfast consumption. For adolescent boys, higher family functioning was associated with more physical activity, less sedentary behavior, less fast food consumption, and more frequent family meals and breakfast consumption. There was one significant interaction by race/ethnicity for family meals; the association between higher family functioning and more frequent family meals was stronger for non-white boys compared to white boys. Overall, strengths of associations tended to be small with effect sizes ranging from - 0.07 to 0.31 for statistically significant associations. Conclusions Findings suggest that family functioning may be protective for adolescent weight and weight-related health behaviors across all race/ethnicities, although assumptions regarding family functioning in the homes of overweight children should be avoided given small effect sizes. PMID:23299010

  14. Conformation-activity relationship of sweet molecules. Comparison of aspartame and naphthimidazolesulfonic acids.

    PubMed

    Castiglione-Morelli, M A; Lelj, F; Naider, F; Tallon, M; Tancredi, T; Temussi, P A

    1990-02-01

    The shape of the active site of the receptor for sweet molecules was previously defined on the basis of a combination of both rigid (saccharins) and flexible (aspartame) molds. In this paper, the sweetness receptor is refined with use of the shapes of 3-anilino-2-styryl-3H-naphtho[1,2-d]imidazolesulfonate (sweet) and of 3-anilino-2-phenyl-3H-naphtho[1,2-d]imidazolesulfonate (tasteless), two large and almost completely rigid tastants. The minimum-energy conformations of the flexible portions of these tastants have been determined by using a detailed conformational analysis based on ab initio calculations. The refined receptor site is still consistent with all previously examined sweet molecules. In order to unequivocally assign the prochiral beta-CH2 protons of the Phe moiety of aspartame, (2S,3S)-[2H]-alpha-L-Asp-L-PheOMe was synthesized and examined by 500-MHz 1H NMR spectroscopy. The results indicate that the minimum-energy conformation for aspartame in water, DMSO-d6, and CDCl3 (as a crown ether complex) is different from that originally proposed (FIIDII instead of FIDII, according to a notation referred to the side chains). Although this conformation is not directly consistent with the shape of the sweet receptor, the interconversion of FIIDII to FIDII was found to require only 1 kcal/mol. Furthermore, a 120-ps molecular dynamics simulation in vacuo confirms the high flexibility of aspartame and the accessibility of the FIDII conformer whose topology is fully consistent with our model. PMID:2299622

  15. Novel lead structures and activation mechanisms for CO-releasing molecules (CORMs)

    PubMed Central

    Schatzschneider, U

    2015-01-01

    Carbon monoxide (CO) is an endogenous small signalling molecule in the human body, produced by the action of haem oxygenase on haem. Since it is very difficult to apply safely as a gas, solid storage and delivery forms for CO are now explored. Most of these CO-releasing molecules (CORMs) are based on the inactivation of the CO by coordinating it to a transition metal centre in a prodrug approach. After a brief look at the potential cellular target structures of CO, an overview of the design principles and activation mechanisms for CO release from a metal coordination sphere is given. Endogenous and exogenous triggers discussed include ligand exchange reactions with medium, enzymatically-induced CO release and photoactivated liberation of CO. Furthermore, the attachment of CORMs to hard and soft nanomaterials to confer additional target specificity to such systems is critically assessed. A survey of analytical methods for the study of the stoichiometry and kinetics of CO release, as well as the tracking of CO in living systems by using fluorescent probes, concludes this review. CORMs are very valuable tools for studying CO bioactivity and might lead to new drug candidates; however, in the design of future generations of CORMs, particular attention has to be paid to their drug-likeness and the tuning of the peripheral ‘drug sphere’ for specific biomedical applications. Further progress in this field will thus critically depend on a close interaction between synthetic chemists and researchers exploring the physiological effects and therapeutic applications of CO. Linked Articles This article is part of a themed section on Pharmacology of the Gasotransmitters. To view the other articles in this section visit http://dx.doi.org/10.1111/bph.2015.172.issue-6 PMID:24628281

  16. Endothelial juxtaposition of distinct adult stem cells activates angiogenesis signaling molecules in endothelial cells.

    PubMed

    Mohammadi, Elham; Nassiri, Seyed Mahdi; Rahbarghazi, Reza; Siavashi, Vahid; Araghi, Atefeh

    2015-12-01

    Efficacy of therapeutic angiogenesis needs a comprehensive understanding of endothelial cell (EC) function and biological factors and cells that interplay with ECs. Stem cells are considered the key components of pro- and anti-angiogenic milieu in a wide variety of physiopathological states, and interactions of EC-stem cells have been the subject of controversy in recent years. In this study, the potential effects of three tissue-specific adult stem cells, namely rat marrow-derived mesenchymal stem cells (rBMSCs), rat adipose-derived stem cells (rADSCs) and rat muscle-derived satellite cells (rSCs), on the endothelial activation of key angiogenic signaling molecules, including VEGF, Ang-2, VEGFR-2, Tie-2, and Tie2-pho, were investigated. Human umbilical vein endothelial cells (HUVECs) and rat lung microvascular endothelial cells (RLMECs) were cocultured with the stem cells or incubated with the stem cell-derived conditioned media on Matrigel. Following HUVEC-stem cell coculture, CD31-positive ECs were flow sorted and subjected to western blotting to analyze potential changes in the expression of the pro-angiogenic signaling molecules. Elongation and co-alignment of the stem cells were seen along the EC tubes in the EC-stem cell cocultures on Matrigel, with cell-to-cell dye communication in the EC-rBMSC cocultures. Moreover, rBMSCs and rADSCs significantly improved endothelial tubulogenesis in both juxtacrine and paracrine manners. These two latter stem cells dynamically up-regulated VEGF, Ang-2, VREGR-2, and Tie-2 but down-regulated Tie2-pho and the Tie2-pho/Tie-2 ratio in HUVECs. Induction of pro-angiogenic signaling in ECs by marrow- and adipose-derived MSCs further indicates the significance of stem cell milieu in angiogenesis dynamics. PMID:26068799

  17. Conformation-activity relationship of sweet molecules. Comparison of aspartame and naphthimidazolesulfonic acids.

    PubMed

    Castiglione-Morelli, M A; Lelj, F; Naider, F; Tallon, M; Tancredi, T; Temussi, P A

    1990-02-01

    The shape of the active site of the receptor for sweet molecules was previously defined on the basis of a combination of both rigid (saccharins) and flexible (aspartame) molds. In this paper, the sweetness receptor is refined with use of the shapes of 3-anilino-2-styryl-3H-naphtho[1,2-d]imidazolesulfonate (sweet) and of 3-anilino-2-phenyl-3H-naphtho[1,2-d]imidazolesulfonate (tasteless), two large and almost completely rigid tastants. The minimum-energy conformations of the flexible portions of these tastants have been determined by using a detailed conformational analysis based on ab initio calculations. The refined receptor site is still consistent with all previously examined sweet molecules. In order to unequivocally assign the prochiral beta-CH2 protons of the Phe moiety of aspartame, (2S,3S)-[2H]-alpha-L-Asp-L-PheOMe was synthesized and examined by 500-MHz 1H NMR spectroscopy. The results indicate that the minimum-energy conformation for aspartame in water, DMSO-d6, and CDCl3 (as a crown ether complex) is different from that originally proposed (FIIDII instead of FIDII, according to a notation referred to the side chains). Although this conformation is not directly consistent with the shape of the sweet receptor, the interconversion of FIIDII to FIDII was found to require only 1 kcal/mol. Furthermore, a 120-ps molecular dynamics simulation in vacuo confirms the high flexibility of aspartame and the accessibility of the FIDII conformer whose topology is fully consistent with our model.

  18. Cloning and characterization of the 2B4 gene encoding a molecule associated with non-MHC-restricted killing mediated by activated natural killer cells and T cells

    SciTech Connect

    Mathew, P.A.; Garni-Wagner, B.A.; Land, K.; Takashima, A.; Stoneman, E.; Bennett, M.; Kumar, V. )

    1993-11-15

    The authors have recently described a signal transducing molecule, 2B4, expressed on all NK and T cells that mediate non-MHC-restricted killing. The gene encoding this molecule was cloned and its nucleotide sequence determined. The encoded protein of 398 amino acids has a leader peptide of 18 amino acids and a transmembrane region of 24 amino acids. The predicted protein has eight N-linked glycosylation sites, suggesting that it is highly glycosylated. Comparison of 2B4 with sequences in the databanks indicates that 2B4 is a member of the Ig supergene family, and it shows homology to murine and rat CD48 and human LFA-3. Northern blot analysis has shown at least three transcripts for 2B4 in adherent lymphokine-activated killer cells of several mouse strains and TCR-[gamma]/[delta] dendritic epidermal T cell lines but not in allospecific T cell clones. These three mRNA are the products of differential splicing of heterogeneous nuclear RNA. Southern blot analysis of genomic DNA from several mouse strains revealed that 2B4 belongs to a family of closely related genes. The 2B4 gene has been mapped to mouse chromosome 1 by analysis of 2B4 expression in recombinant inbred mouse strains. 48 refs., 6 figs., 2 tabs.

  19. Therapeutic potential of an orally effective small molecule inhibitor of plasminogen activator inhibitor for asthma.

    PubMed

    Liu, Rui-Ming; Eldridge, Stephanie; Watanabe, Nobuo; Deshane, Jessy; Kuo, Hui-Chien; Jiang, Chunsun; Wang, Yong; Liu, Gang; Schwiebert, Lisa; Miyata, Toshio; Thannickal, Victor J

    2016-02-15

    Asthma is one of the most common respiratory diseases. Although progress has been made in our understanding of airway pathology and many drugs are available to relieve asthma symptoms, there is no cure for chronic asthma. Plasminogen activator inhibitor 1 (PAI-1), a primary inhibitor of tissue-type and urokinase-type plasminogen activators, has pleiotropic functions besides suppression of fibrinolysis. In this study, we show that administration of TM5275, an orally effective small-molecule PAI-1 inhibitor, 25 days after ovalbumin (OVA) sensitization-challenge, significantly ameliorated airway hyperresponsiveness in an OVA-induced chronic asthma model. Furthermore, we show that TM5275 administration significantly attenuated OVA-induced infiltration of inflammatory cells (neutrophils, eosinophils, and monocytes), the increase in the levels of OVA-specific IgE and Th2 cytokines (IL-4 and IL-5), the production of mucin in the airways, and airway subepithelial fibrosis. Together, the results suggest that the PAI-1 inhibitor TM5275 may have therapeutic potential for asthma through suppressing eosinophilic allergic response and ameliorating airway remodeling. PMID:26702150

  20. Abnormal high-energy phosphate molecule metabolism during regional brain activation in patients with bipolar disorder.

    PubMed

    Yuksel, C; Du, F; Ravichandran, C; Goldbach, J R; Thida, T; Lin, P; Dora, B; Gelda, J; O'Connor, L; Sehovic, S; Gruber, S; Ongur, D; Cohen, B M

    2015-09-01

    Converging evidence suggests bioenergetic abnormalities in bipolar disorder (BD). In the brain, phosphocreatine (PCr) acts a reservoir of high-energy phosphate (HEP) bonds, and creatine kinases (CK) catalyze the transfer of HEP from adenosine triphosphate (ATP) to PCr and from PCr back to ATP, at times of increased need. This study examined the activity of this mechanism in BD by measuring the levels of HEP molecules during a stimulus paradigm that increased local energy demand. Twenty-three patients diagnosed with BD-I and 22 healthy controls (HC) were included. Levels of phosphorus metabolites were measured at baseline and during visual stimulation in the occipital lobe using (31)P magnetic resonance spectroscopy at 4T. Changes in metabolite levels showed different patterns between the groups. During stimulation, HC had significant reductions in PCr but not in ATP, as expected. In contrast, BD patients had significant reductions in ATP but not in PCr. In addition, PCr/ATP ratio was lower at baseline in patients, and there was a higher change in this measure during stimulation. This pattern suggests a disease-related failure to replenish ATP from PCr through CK enzyme catalysis during tissue activation. Further studies measuring the CK flux in BD are required to confirm and extend this finding.

  1. Concise Synthesis of Spergualin-Inspired Molecules With Broad-Spectrum Antibiotic Activity

    PubMed Central

    Assimon, Victoria A.; Shao, Hao; Garneau-Tsodikova, Sylvie

    2016-01-01

    There is a growing need to identify new, broad-spectrum antibiotics. The natural product spergualin was previously shown to have promising anti-bacterial activity and a privileged structure, but its challenging synthesis had limited further exploration. For example, syntheses of spergualin and its analogs have been reported in approximately 10 linear steps, with overall yields between 0.3 and 18%. Using the Ugi multi-component reaction, we assembled spergualin-inspired molecules in a single step, dramatically improving the overall yield (20% to 96%). Using this strategy, we generated 43 new analogs and tested them for anti-bacterial activity against two Gram-negative and four Gram-positive strains. We found that the most potent analogue, compound 6, had MIC values between 4 and 32 μg/mL against the six strains, which is a significant improvement on spergualin (MIC ∼ 6.25 to 50 μg/mL). These studies provide a concise route to a broad-spectrum antibiotic with a novel chemical scaffold. PMID:27087913

  2. Inhibition of Streptococcus mutans polysaccharide synthesis by molecules targeting glycosyltransferase activity

    PubMed Central

    Ren, Zhi; Chen, Lulu; Li, Jiyao; Li, Yuqing

    2016-01-01

    Glycosyltransferase (Gtf) is one of the crucial virulence factors of Streptococcus mutans, a major etiological pathogen of dental caries. All the available evidence indicates that extracellular polysaccharide, particularly glucans produced by S. mutans Gtfs, contribute to the cariogenicity of dental biofilms. Therefore, inhibition of Gtf activity and the consequential polysaccharide synthesis may impair the virulence of cariogenic biofilms, which could be an alternative strategy to prevent the biofilm-related disease. Up to now, many Gtf inhibitors have been recognized in natural products, which remain the major and largely unexplored source of Gtf inhibitors. These include catechin-based polyphenols, flavonoids, proanthocyanidin oligomers, polymeric polyphenols, and some other plant-derived compounds. Metal ions, oxidizing agents, and some other synthetic compounds represent another source of Gtf inhibitors, with some novel molecules either discovered by structure-based virtual screening or synthesized based on key structures of known inhibitors as templates. Antibodies that inhibit one or more Gtfs have also been developed as topical agents. Although many agents have been shown to possess potent inhibitory activity against glucan synthesis by Gtfs, bacterial cell adherence, and caries development in animal models, much research remains to be performed to find out their mechanism of action, biological safety, cariostatic efficacies, and overall influence on the entire oral community. As a strategy to inhibit the virulence of cariogenic microbes rather than eradicate them from the microbial community, Gtf inhibition represents an approach of great potential to prevent dental caries. PMID:27105419

  3. Therapeutic potential of an orally effective small molecule inhibitor of plasminogen activator inhibitor for asthma.

    PubMed

    Liu, Rui-Ming; Eldridge, Stephanie; Watanabe, Nobuo; Deshane, Jessy; Kuo, Hui-Chien; Jiang, Chunsun; Wang, Yong; Liu, Gang; Schwiebert, Lisa; Miyata, Toshio; Thannickal, Victor J

    2016-02-15

    Asthma is one of the most common respiratory diseases. Although progress has been made in our understanding of airway pathology and many drugs are available to relieve asthma symptoms, there is no cure for chronic asthma. Plasminogen activator inhibitor 1 (PAI-1), a primary inhibitor of tissue-type and urokinase-type plasminogen activators, has pleiotropic functions besides suppression of fibrinolysis. In this study, we show that administration of TM5275, an orally effective small-molecule PAI-1 inhibitor, 25 days after ovalbumin (OVA) sensitization-challenge, significantly ameliorated airway hyperresponsiveness in an OVA-induced chronic asthma model. Furthermore, we show that TM5275 administration significantly attenuated OVA-induced infiltration of inflammatory cells (neutrophils, eosinophils, and monocytes), the increase in the levels of OVA-specific IgE and Th2 cytokines (IL-4 and IL-5), the production of mucin in the airways, and airway subepithelial fibrosis. Together, the results suggest that the PAI-1 inhibitor TM5275 may have therapeutic potential for asthma through suppressing eosinophilic allergic response and ameliorating airway remodeling.

  4. Discovery of Novel Small Molecules that Activate Satellite Cell Proliferation and Enhance Repair of Damaged Muscle.

    PubMed

    Billin, Andrew N; Bantscheff, Marcus; Drewes, Gerard; Ghidelli-Disse, Sonja; Holt, Jason A; Kramer, Henning F; McDougal, Alan J; Smalley, Terry L; Wells, Carrow I; Zuercher, William J; Henke, Brad R

    2016-02-19

    Skeletal muscle progenitor stem cells (referred to as satellite cells) represent the primary pool of stem cells in adult skeletal muscle responsible for the generation of new skeletal muscle in response to injury. Satellite cells derived from aged muscle display a significant reduction in regenerative capacity to form functional muscle. This decrease in functional recovery has been attributed to a decrease in proliferative capacity of satellite cells. Hence, agents that enhance the proliferative abilities of satellite cells may hold promise as therapies for a variety of pathological settings, including repair of injured muscle and age- or disease-associated muscle wasting. Through phenotypic screening of isolated murine satellite cells, we identified a series of 2,4-diaminopyrimidines (e.g., 2) that increased satellite cell proliferation. Importantly, compound 2 was effective in accelerating repair of damaged skeletal muscle in an in vivo mouse model of skeletal muscle injury. While these compounds were originally prepared as c-Jun N-terminal kinase 1 (JNK-1) inhibitors, structure-activity analyses indicated JNK-1 inhibition does not correlate with satellite cell activity. Screening against a broad panel of kinases did not result in identification of an obvious molecular target, so we conducted cell-based proteomics experiments in an attempt to identify the molecular target(s) responsible for the potentiation of the satellite cell proliferation. These data provide the foundation for future efforts to design improved small molecules as potential therapeutics for muscle repair and regeneration.

  5. VAMP4 Is an Essential Cargo Molecule for Activity-Dependent Bulk Endocytosis

    PubMed Central

    Nicholson-Fish, Jessica C.; Kokotos, Alexandros C.; Gillingwater, Thomas H.; Smillie, Karen J.; Cousin, Michael A.

    2015-01-01

    Summary The accurate formation of synaptic vesicles (SVs) and incorporation of their protein cargo during endocytosis is critical for the maintenance of neurotransmission. During intense neuronal activity, a transient and acute accumulation of SV cargo occurs at the plasma membrane. Activity-dependent bulk endocytosis (ADBE) is the dominant SV endocytosis mode under these conditions; however, it is currently unknown how ADBE mediates cargo retrieval. We examined the retrieval of different SV cargo molecules during intense stimulation using a series of genetically encoded pH-sensitive reporters in neuronal cultures. The retrieval of only one reporter, VAMP4-pHluorin, was perturbed by inhibiting ADBE. This selective recovery was confirmed by the enrichment of endogenous VAMP4 in purified bulk endosomes formed by ADBE. VAMP4 was also essential for ADBE, with a cytoplasmic di-leucine motif being critical for this role. Therefore, VAMP4 is the first identified ADBE cargo and is essential for this endocytosis mode to proceed. PMID:26607000

  6. Small-molecule nociceptin receptor agonist ameliorates mast cell activation and pain in sickle mice

    PubMed Central

    Vang, Derek; Paul, Jinny A.; Nguyen, Julia; Tran, Huy; Vincent, Lucile; Yasuda, Dennis; Zaveri, Nurulain T.; Gupta, Kalpna

    2015-01-01

    Treatment of pain with morphine and its congeners in sickle cell anemia is suboptimal, warranting the need for analgesics devoid of side effects, addiction and tolerance liability. Small-molecule nociceptin opioid receptor ligands show analgesic efficacy in acute and chronic pain models. We show that AT-200, a high affinity nociceptin opioid receptor agonist with low efficacy at the mu opioid receptor, ameliorated chronic and hypoxia/reoxygenation-induced mechanical, thermal and deep tissue/musculoskeletal hyperalgesia in HbSS-BERK sickle mice. The antinociceptive effect of AT-200 was antagonized by SB-612111, a nociceptin opioid receptor antagonist, but not naloxone, a non-selective mu opioid receptor antagonist. Daily 7-day treatment with AT-200 did not develop tolerance and showed a sustained anti-nociceptive effect, which improved over time and led to reduced plasma serum amyloid protein, neuropeptides, inflammatory cytokines and mast cell activation in the periphery. These data suggest that AT-200 ameliorates pain in sickle mice via the nociceptin opioid receptor by reducing inflammation and mast cell activation without causing tolerance. Thus, nociceptin opioid receptor agonists are promising drugs for treating pain in sickle cell anemia. PMID:26294734

  7. Inhibiting NF-κB Activation by Small Molecules As a Therapeutic Strategy

    PubMed Central

    Gupta, Subash C; Sundaram, Chitra; Reuter, Simone; Aggarwal, Bharat B

    2010-01-01

    Because nuclear factor-κB (NF-κB) is a ubiquitously expressed proinflammatory transcription factor that regulates the expression of over 500 genes involved in cellular transformation, survival, proliferation, invasion, angiogenesis, metastasis, and inflammation, the NF-κB signaling pathway has become a potential target for pharmacological intervention. A wide variety of agents can activate NF-κB through canonical and noncanonical pathways. Canonical pathway involves various steps including the phosphorylation, ubiquitnation, and degradation of the inhibitor of NF-κB (IκBα), which leads to the nuclear translocation of the p50- p65 subunits of NF-κB followed by p65 phosphorylation, acetylation and methylation, DNA binding, and gene transcription. Thus, agents that can inhibit protein kinases, protein phosphatases, proteasomes, ubiquitnation, acetylation, methylation, and DNA binding steps have been identified as NF-κB inhibitors. Here, we review the small molecules that suppress NF-κB activation and thus may have therapeutic potential. PMID:20493977

  8. A novel molecule with notable activity against multi-drug resistant tuberculosis.

    PubMed

    Nair, Vasu; Okello, Maurice O; Mangu, Naveen K; Seo, Byung I; Gund, Machhindra G

    2015-03-15

    Multi-drug resistant tuberculosis (MDR-TB) is emerging as a serious global health problem, which has been elevated through co-infection involving HIV and MDR-Mtb. The discovery of new compounds with anti-MDR TB efficacy and favorable metabolism profiles is an important scientific challenge. Using computational biology and ligand docking data, we have conceived a multifunctional molecule, 2, as a potential anti-MDR TB agent. This compound was produced through a multi-step synthesis. It exhibited significant in vitro activity against MDR-TB (MIC 1.56μg/mL) and its half-life (t1/2) in human liver microsomes was 14.4h. The metabolic profiles of compound 2 with respect to human cytochrome P450 (CYP) and uridine 5'-diphospho-glucuronosyltransferase (UGT) isozymes were favorable. Compound 2 also had relatively low in vitro cytotoxicity in uninfected macrophages. It displayed synergistic behavior against MDR-TB in combination with PA-824. Interestingly, compound 2 also displayed in vitro anti-HIV activity. PMID:25677656

  9. Fasciola hepatica Kunitz Type Molecule Decreases Dendritic Cell Activation and Their Ability to Induce Inflammatory Responses

    PubMed Central

    Falcón, Cristian R.; Masih, Diana; Gatti, Gerardo; Sanchez, María Cecilia; Motrán, Claudia C.; Cervi, Laura

    2014-01-01

    The complete repertoire of proteins with immunomodulatory activity in Fasciola hepatica (Fh) has not yet been fully described. Here, we demonstrated that Fh total extract (TE) reduced LPS-induced DC maturation, and the DC ability to induce allogeneic responses. After TE fractionating, a fraction lower than 10 kDa (F<10 kDa) was able to maintain the TE properties to modulate the DC pro- and anti-inflammatory cytokine production induced by LPS. In addition, TE or F<10 kDa treatment decreased the ability of immature DC to stimulate the allogeneic responses and induced a novo allogeneic CD4+CD25+Foxp3+ T cells. In contrast, treatment of DC with T/L or F<10 kDa plus LPS (F<10/L) induced a regulatory IL-27 dependent mechanism that diminished the proliferative and Th1 and Th17 allogeneic responses. Finally, we showed that a Kunitz type molecule (Fh-KTM), present in F<10 kDa, was responsible for suppressing pro-inflammatory cytokine production in LPS-activated DC, by printing tolerogenic features on DC that impaired their ability to induce inflammatory responses. These results suggest a modulatory role for this protein, which may be involved in the immune evasion mechanisms of the parasite. PMID:25486609

  10. Activation of trimeric P2X2 receptors by fewer than three ATP molecules.

    PubMed

    Stelmashenko, Olga; Lalo, Ulyana; Yang, Yue; Bragg, Laricia; North, R Alan; Compan, Vincent

    2012-10-01

    P2X receptors are trimeric membrane proteins. When they bind extracellular ATP, a conformational change occurs that opens a transmembrane ion channel. The ATP-binding pocket is formed in a cleft between two subunits, and a critical amino acid residue for ATP contact is Lys⁶⁹ (P2X2 numbering). In the present work, we sought to determine whether the binding of fewer than three ATP molecules could open the ion channel. We expressed eight concatenated cDNAs in human embryonic kidney cells, which encoded three serially joined, epitope-tagged, subunits with either Lys or Ala at position 69 (denoted as KKK, KKA, KAK, AKK, KAA, AKA, AAK, and AAA). Western blotting of surface-biotinylated proteins indicated that breakdown of concatemers to individual subunits was minimal. Recording of membrane currents in response to ATP (whole cell and excised outside-out patch) showed that all formed functional channels except AAK, AKA, and AAA. There was no difference in the kinetics of activation and deactivation among KKK, KKA, KAK, and AKK channels, and amplitude of the unitary conductances was in all cases not different from that found after expression of a single wild-type subunit. Currents through KKA and KAK receptors were larger than those observed for AKK receptors. The results indicate that trimeric P2X receptors containing only two intact binding sites can be readily activated by ATP.

  11. Measles Virus Entry Through the Signaling Lymphocyte Activation Molecule Governs Efficacy of Mantle Cell Lymphoma Radiovirotherapy

    PubMed Central

    Miest, Tanner S; Frenzke, Marie; Cattaneo, Roberto

    2013-01-01

    We developed here a vaccine-identical measles virus (MV) as an oncolytic agent against mantle cell lymphoma (MCL), an aggressive B-cell non-Hodgkin's lymphoma that is difficult to cure but radiosensitive. We armed the virus with the sodium-iodide symporter, which concentrates iodide within infected cells enabling noninvasive imaging and combination radiovirotherapy. Through high-resolution in vivo and ex vivo imaging, we visualized the spread of infections in primary and metastatic tumors for over 2 weeks after therapy, documenting homogeneous virus seeding and spread restricted to perfused tissue. Infection of metastases was more rapid and intense than primary tumors, achieving isotope uptake within about threefold the efficiency of the thyroid. Virotherapy combined with systemic 131I resulted in more rapid disease regression than either therapy alone. In addition to ubiquitous CD46, vaccine MV retains cell entry through its immune cell-specific receptor signaling lymphocytic activation molecule (SLAM). We asked whether both receptors could sustain effective oncolysis of MCL. Strikingly, only SLAM-dependent entry sustained efficient viral spread, tumor regression, and prolonged survival. These observations shift the focus of future clinical trials to SLAM-expressing hematologic malignancies and suggest that oncolytic vectors may depend on tissue-specific receptors for both cell entry and activation of responses assisting their replication. PMID:23913184

  12. Small molecule regulation of self-association and catalytic activity in a supramolecular coordination complex.

    PubMed

    McGuirk, C Michael; Stern, Charlotte L; Mirkin, Chad A

    2014-03-26

    Herein, we report the synthesis and characterization of the first weak-link approach (WLA) supramolecular construct that employs the small molecule regulation of intermolecular hydrogen bonding interactions for the in situ control of catalytic activity. A biaryl urea group, prone to self-aggregation, was functionalized with a phosphinoalkyl thioether (P,S) hemilabile moiety and incorporated into a homoligated Pt(II) tweezer WLA complex. This urea-containing construct, which has been characterized by a single crystal X-ray diffraction study, can be switched in situ from a rigid fully closed state to a flexible semiopen state via Cl(-) induced changes in the coordination mode at the Pt(II) structural node. FT-IR and (1)H NMR spectroscopy studies were used to demonstrate that while extensive urea self-association persists in the flexible semiopen complex, these interactions are deterred in the rigid, fully closed complex because of geometric and steric restraints. Consequently, the urea moieties in the fully closed complex are able to catalyze a Diels-Alder reaction between cyclopentadiene and methyl vinyl ketone to generate 2-acetyl-5-norbornene. The free urea ligand and the semiopen complex show no such activity. The successful incorporation and regulation of a hydrogen bond donating catalyst in a WLA construct open the doors to a vast and rapidly growing catalogue of allosteric catalysts for applications in the detection and amplification of organic analytes.

  13. Small-molecule nociceptin receptor agonist ameliorates mast cell activation and pain in sickle mice.

    PubMed

    Vang, Derek; Paul, Jinny A; Nguyen, Julia; Tran, Huy; Vincent, Lucile; Yasuda, Dennis; Zaveri, Nurulain T; Gupta, Kalpna

    2015-12-01

    Treatment of pain with morphine and its congeners in sickle cell anemia is suboptimal, warranting the need for analgesics devoid of side effects, addiction and tolerance liability. Small-molecule nociceptin opioid receptor ligands show analgesic efficacy in acute and chronic pain models. We show that AT-200, a high affinity nociceptin opioid receptor agonist with low efficacy at the mu opioid receptor, ameliorated chronic and hypoxia/reoxygenation-induced mechanical, thermal and deep tissue/musculoskeletal hyperalgesia in HbSS-BERK sickle mice. The antinociceptive effect of AT-200 was antagonized by SB-612111, a nociceptin opioid receptor antagonist, but not naloxone, a non-selective mu opioid receptor antagonist. Daily 7-day treatment with AT-200 did not develop tolerance and showed a sustained anti-nociceptive effect, which improved over time and led to reduced plasma serum amyloid protein, neuropeptides, inflammatory cytokines and mast cell activation in the periphery. These data suggest that AT-200 ameliorates pain in sickle mice via the nociceptin opioid receptor by reducing inflammation and mast cell activation without causing tolerance. Thus, nociceptin opioid receptor agonists are promising drugs for treating pain in sickle cell anemia.

  14. Measles virus entry through the signaling lymphocyte activation molecule governs efficacy of mantle cell lymphoma radiovirotherapy.

    PubMed

    Miest, Tanner S; Frenzke, Marie; Cattaneo, Roberto

    2013-11-01

    We developed here a vaccine-identical measles virus (MV) as an oncolytic agent against mantle cell lymphoma (MCL), an aggressive B-cell non-Hodgkin's lymphoma that is difficult to cure but radiosensitive. We armed the virus with the sodium-iodide symporter, which concentrates iodide within infected cells enabling noninvasive imaging and combination radiovirotherapy. Through high-resolution in vivo and ex vivo imaging, we visualized the spread of infections in primary and metastatic tumors for over 2 weeks after therapy, documenting homogeneous virus seeding and spread restricted to perfused tissue. Infection of metastases was more rapid and intense than primary tumors, achieving isotope uptake within about threefold the efficiency of the thyroid. Virotherapy combined with systemic (131)I resulted in more rapid disease regression than either therapy alone. In addition to ubiquitous CD46, vaccine MV retains cell entry through its immune cell-specific receptor signaling lymphocytic activation molecule (SLAM). We asked whether both receptors could sustain effective oncolysis of MCL. Strikingly, only SLAM-dependent entry sustained efficient viral spread, tumor regression, and prolonged survival. These observations shift the focus of future clinical trials to SLAM-expressing hematologic malignancies and suggest that oncolytic vectors may depend on tissue-specific receptors for both cell entry and activation of responses assisting their replication.

  15. L1 cell adhesion molecule induces melanoma cell motility by activation of mitogen-activated protein kinase pathways.

    PubMed

    Yi, Young-Su; Baek, Kwang-Soo; Cho, Jae Youl

    2014-06-01

    L1 cell adhesion molecule (L1CAM) is highly expressed in various types of cancer cells and has been implicated in the control of cell proliferation and motility. Recently, L1CAM was reported to induce the motility of melanoma cells, but the mechanism of this induction remains poorly understood. In this study, we investigated the molecular mechanisms by which L1CAM induces the motility of melanoma cells. Unlike other types of cancer cells, B16F10 melanoma cells highly expressed L1CAM at both the RNA and protein levels, and the expression of L1CAM induced AP-1 activity. In accordance to AP-1 activation, MAPK signaling pathways were activated by L1CAM. Inhibition of L1CAM expression by L1CAM-specific siRNA suppressed the activation of MAPKs such as ERK and p38. However, no significant change was observed in JNK activation. As expected, upstream MAP2K, MKK3/6, MAP3K, and TAK1 were also deactivated by the inhibition of L1CAM expression. L1CAM induced the motility of B16F10 cells. Inhibition of L1CAM expression suppressed migration and invasion of B16F10 cells, but no suppressive effect was observed on their proliferation and anti-apoptotic resistance. Treatment of B16F10 cells with U0126, an ERK inhibitor, or SB203580, a p38 inhibitor, suppressed the migration and invasion abilities of B16F10 cells. Taken together, our results suggest that L1CAM induces the motility of B16F10 melanoma cells via the activation of MAPK pathways. This finding provides a more detailed molecular mechanism of L1CAM-mediated induction of melanoma cell motility. PMID:24974583

  16. Cotton Rat (Sigmodon hispidus) Signaling Lymphocyte Activation Molecule (CD150) Is an Entry Receptor for Measles Virus

    PubMed Central

    Carsillo, Thomas; Huey, Devra; Levinsky, Amy; Obojes, Karola; Schneider-Schaulies, Jürgen; Niewiesk, Stefan

    2014-01-01

    Cotton rats (Sigmodon hispidus) replicate measles virus (MV) after intranasal infection in the respiratory tract and lymphoid tissue. We have cloned the cotton rat signaling lymphocytic activation molecule (CD150, SLAM) in order to investigate its role as a potential receptor for MV. Cotton rat CD150 displays 58% and 78% amino acid homology with human and mouse CD150, respectively. By staining with a newly generated cotton rat CD150 specific monoclonal antibody expression of CD150 was confirmed in cotton rat lymphoid cells and in tissues with a pattern of expression similar to mouse and humans. Previously, binding of MV hemagglutinin has been shown to be dependent on amino acids 60, 61 and 63 in the V region of CD150. The human molecule contains isoleucine, histidine and valine at these positions and binds to MV-H whereas the mouse molecule contains valine, arginine and leucine and does not function as a receptor for MV. In the cotton rat molecule, amino acids 61 and 63 are identical with the mouse molecule and amino acid 60 with the human molecule. After transfection with cotton rat CD150 HEK 293 T cells became susceptible to infection with single cycle VSV pseudotype virus expressing wild type MV glycoproteins and with a MV wildtype virus. After infection, cells expressing cotton rat CD150 replicated virus to lower levels than cells expressing the human molecule and formed smaller plaques. These data might explain why the cotton rat is a semipermissive model for measles virus infection. PMID:25295727

  17. Expression of intercellular adhesion molecule-1 on macrophages in vitro as a marker of activation.

    PubMed

    Bernatchez, S F; Atkinson, M R; Parks, P J

    1997-10-01

    Macrophage activation is a major component of wound healing. It also determines the extent of inflammatory reactions and the response of the body to implanted materials. We have previously shown, using an in vitro model, that the extent of spreading of macrophages on different materials is a marker of activation, and that a soluble inducer has a dose-response effect on the secretion of cytokines in the culture medium. This work investigates the expression of three different cell surface markers [macrophages MAC-1, MAC-3 and intercellular adhesion molecule-1 (ICAM-1)] on macrophages in vitro using confocal microscopy and shows that ICAM-1 is also a marker of macrophage activation in this model. We observed increased amounts of ICAM-1 on activated macrophages compared to unactivated macrophages, whereas MAC-1 and MAC-3 were either expressed constitutively or demonstrated no quantitative change in expression after activation under the same experimental conditions. We also tested the expression of ICAM-1 with various concentrations of soluble inducers (lipopolysaccharide, 0.001, 0.01, 0.1, 1 and 10 micrograms ml-1. S-27609, 0.1, 0.25, 0.5, 1, 2 and 3 micrograms ml-1 and on a sheet of polylactic acid alone or in combination with soluble inducers. All doses of soluble inducers induced the expression of ICAM-1 on cells grown in glass chamber slides. The induction was not dose related but seemed to work rather in an on-off manner. There was no effect of material on ICAM-1 expression on the cell surface when no soluble inducer was added. This was similar to cytokine secretion, which was not induced by our material alone. When either lipopolysaccharide or S-27609 was used in combination with the material, there was an increase in the average measured intensity of ICAM-1. In this in vitro model, ICAM-1 staining as measured by confocal microscopy is a marker for macrophage activation. Our results suggest that the extent of macrophage activation as measured by ICAM-1 and by

  18. Reactive oxygen species (ROS)--a family of fate deciding molecules pivotal in constructive inflammation and wound healing.

    PubMed

    Bryan, Nicholas; Ahswin, Helen; Smart, Neil; Bayon, Yves; Wohlert, Stephen; Hunt, John A

    2012-01-01

    Wound healing requires a fine balance between the positive and deleterious effects of reactive oxygen species (ROS); a group of extremely potent molecules, rate limiting in successful tissue regeneration. A balanced ROS response will debride and disinfect a tissue and stimulate healthy tissue turnover; suppressed ROS will result in infection and an elevation in ROS will destroy otherwise healthy stromal tissue. Understanding and anticipating the ROS niche within a tissue will greatly enhance the potential to exogenously augment and manipulate healing. Tissue engineering solutions to augment successful healing and remodelling of wounded or diseased tissue rely on a controlled balance between the constructive and destructive capacity of the leukocyte secretome, including ROS. This review comprehensively considers leukocyte derived ROS in tissue repair with particular interest in surgical intervention with inclusion of a biomaterial. The article considers ROS fundamental chemistry, formation, stimulation and clearance before applying this to discuss the implications of ROS in healing tissue with and without a biomaterial. We also systematically discuss ROS in leukocyte signalling and compare and contrast experimental means of measuring ROS. PMID:23007910

  19. Discovery of small molecule inhibitors of xyloglucan endotransglucosylase (XET) activity by high-throughput screening

    PubMed Central

    Chormova, Dimitra; Franková, Lenka; Defries, Andrew; Cutler, Sean R.; Fry, Stephen C.

    2015-01-01

    Small molecules (xenobiotics) that inhibit cell-wall-localised enzymes are valuable for elucidating the enzymes’ biological roles. We applied a high-throughput fluorescent dot-blot screen to search for inhibitors of Petroselinum xyloglucan endotransglucosylase (XET) activity in vitro. Of 4216 xenobiotics tested, with cellulose-bound xyloglucan as donor-substrate, 18 inhibited XET activity and 18 promoted it (especially anthraquinones and flavonoids). No compounds promoted XET in quantitative assays with (cellulose-free) soluble xyloglucan as substrate, suggesting that promotion was dependent on enzyme–cellulose interactions. With cellulose-free xyloglucan as substrate, we found 22 XET-inhibitors – especially compounds that generate singlet oxygen (1O2) e.g., riboflavin (IC50 29 μM), retinoic acid, eosin (IC50 27 μM) and erythrosin (IC50 36 μM). The riboflavin effect was light-dependent, supporting 1O2 involvement. Other inhibitors included tannins, sulphydryl reagents and triphenylmethanes. Some inhibitors (vulpinic acid and brilliant blue G) were relatively specific to XET, affecting only two or three, respectively, of nine other wall-enzyme activities tested; others [e.g. (−)-epigallocatechin gallate and riboflavin] were non-specific. In vivo, out of eight XET-inhibitors bioassayed, erythrosin (1 μM) inhibited cell expansion in Rosa and Zea cell-suspension cultures, and 40 μM mycophenolic acid and (−)-epigallocatechin gallate inhibited Zea culture growth. Our work showcases a general high-throughput strategy for discovering wall-enzyme inhibitors, some being plant growth inhibitors potentially valuable as physiological tools or herbicide leads. PMID:26093490

  20. Direct Measurements of Local Coupling between Myosin Molecules Are Consistent with a Model of Muscle Activation

    PubMed Central

    Walcott, Sam; Kad, Neil M.

    2015-01-01

    Muscle contracts due to ATP-dependent interactions of myosin motors with thin filaments composed of the proteins actin, troponin, and tropomyosin. Contraction is initiated when calcium binds to troponin, which changes conformation and displaces tropomyosin, a filamentous protein that wraps around the actin filament, thereby exposing myosin binding sites on actin. Myosin motors interact with each other indirectly via tropomyosin, since myosin binding to actin locally displaces tropomyosin and thereby facilitates binding of nearby myosin. Defining and modeling this local coupling between myosin motors is an open problem in muscle modeling and, more broadly, a requirement to understanding the connection between muscle contraction at the molecular and macro scale. It is challenging to directly observe this coupling, and such measurements have only recently been made. Analysis of these data suggests that two myosin heads are required to activate the thin filament. This result contrasts with a theoretical model, which reproduces several indirect measurements of coupling between myosin, that assumes a single myosin head can activate the thin filament. To understand this apparent discrepancy, we incorporated the model into stochastic simulations of the experiments, which generated simulated data that were then analyzed identically to the experimental measurements. By varying a single parameter, good agreement between simulation and experiment was established. The conclusion that two myosin molecules are required to activate the thin filament arises from an assumption, made during data analysis, that the intensity of the fluorescent tags attached to myosin varies depending on experimental condition. We provide an alternative explanation that reconciles theory and experiment without assuming that the intensity of the fluorescent tags varies. PMID:26536123

  1. Discovery of small molecule inhibitors of xyloglucan endotransglucosylase (XET) activity by high-throughput screening.

    PubMed

    Chormova, Dimitra; Franková, Lenka; Defries, Andrew; Cutler, Sean R; Fry, Stephen C

    2015-09-01

    Small molecules (xenobiotics) that inhibit cell-wall-localised enzymes are valuable for elucidating the enzymes' biological roles. We applied a high-throughput fluorescent dot-blot screen to search for inhibitors of Petroselinum xyloglucan endotransglucosylase (XET) activity in vitro. Of 4216 xenobiotics tested, with cellulose-bound xyloglucan as donor-substrate, 18 inhibited XET activity and 18 promoted it (especially anthraquinones and flavonoids). No compounds promoted XET in quantitative assays with (cellulose-free) soluble xyloglucan as substrate, suggesting that promotion was dependent on enzyme-cellulose interactions. With cellulose-free xyloglucan as substrate, we found 22 XET-inhibitors - especially compounds that generate singlet oxygen ((1)O2) e.g., riboflavin (IC50 29 μM), retinoic acid, eosin (IC50 27 μM) and erythrosin (IC50 36 μM). The riboflavin effect was light-dependent, supporting (1)O2 involvement. Other inhibitors included tannins, sulphydryl reagents and triphenylmethanes. Some inhibitors (vulpinic acid and brilliant blue G) were relatively specific to XET, affecting only two or three, respectively, of nine other wall-enzyme activities tested; others [e.g. (-)-epigallocatechin gallate and riboflavin] were non-specific. In vivo, out of eight XET-inhibitors bioassayed, erythrosin (1 μM) inhibited cell expansion in Rosa and Zea cell-suspension cultures, and 40 μM mycophenolic acid and (-)-epigallocatechin gallate inhibited Zea culture growth. Our work showcases a general high-throughput strategy for discovering wall-enzyme inhibitors, some being plant growth inhibitors potentially valuable as physiological tools or herbicide leads. PMID:26093490

  2. Family Outing Activities and Achievement among Fourth Graders in Compensatory Education Funded Schools.

    ERIC Educational Resources Information Center

    Griswold, Philip A.

    This study sought to identify the relationship between operationally-defined family outing activities and measures of achievement among fourth grade students from educationally disadvantaged backgrounds. The results, collected for four successive years from 1715 students in urban Los Angeles, indicated that: (1) participation in family activities…

  3. Activation of human ether-a-go-go related gene (hERG) potassium channels by small molecules

    PubMed Central

    Zhou, Ping-zheng; Babcock, Joseph; Liu, Lian-qing; Li, Min; Gao, Zhao-bing

    2011-01-01

    Human ether-a-go-go related gene (hERG) potassium (K+) channels play a critical role in cardiac action potential repolarization. Mutations that reduce hERG conductance or surface expression may cause congenital long QT syndrome (LQTS). However, the channels can be inhibited by structurally diverse small molecules, resulting in an acquired form of LQTS. Consequently, small molecules that increase the hERG current may be of value for treatment for LQTS. So far, nine hERG activators have been reported. The aim of this review is to discuss recent advances concerning the identification and action mechanism of hERG activators. PMID:21623390

  4. Morbilliviruses Use Signaling Lymphocyte Activation Molecules (CD150) as Cellular Receptors

    PubMed Central

    Tatsuo, Hironobu; Ono, Nobuyuki; Yanagi, Yusuke

    2001-01-01

    Morbilliviruses comprise measles virus, canine distemper virus, rinderpest virus, and several other viruses that cause devastating human and animal diseases accompanied by severe immunosuppression and lymphopenia. Recently, we have shown that human signaling lymphocyte activation molecule (SLAM) is a cellular receptor for measles virus. In this study, we examined whether canine distemper and rinderpest viruses also use canine and bovine SLAMs, respectively, as cellular receptors. The Onderstepoort vaccine strain and two B95a (marmoset B cell line)-isolated strains of canine distemper virus caused extensive cytopathic effects in normally resistant CHO (Chinese hamster ovary) cells after expression of canine SLAM. The Ako vaccine strain of rinderpest virus produced strong cytopathic effects in bovine SLAM-expressing CHO cells. The data on entry with vesicular stomatitis virus pseudotypes bearing measles, canine distemper, or rinderpest virus envelope proteins were consistent with development of cytopathic effects in SLAM-expressing CHO cell clones after infection with the respective viruses, confirming that SLAM acts at the virus entry step (as a cellular receptor). Furthermore, most measles, canine distemper, and rinderpest virus strains examined could any use of the human, canine, and bovine SLAMs to infect cells. Our findings suggest that the use of SLAM as a cellular receptor may be a property common to most, if not all, morbilliviruses and explain the lymphotropism and immunosuppressive nature of morbilliviruses. PMID:11390585

  5. Structure–activity exploration of a small-molecule Lipid II inhibitor

    PubMed Central

    Fletcher, Steven; Yu, Wenbo; Huang, Jing; Kwasny, Steven M; Chauhan, Jay; Opperman, Timothy J; MacKerell, Alexander D; de Leeuw, Erik PH

    2015-01-01

    We have recently identified low-molecular weight compounds that act as inhibitors of Lipid II, an essential precursor of bacterial cell wall biosynthesis. Lipid II comprises specialized lipid (bactoprenol) linked to a hydrophilic head group consisting of a peptidoglycan subunit (N-acetyl glucosamine [GlcNAc]–N-acetyl muramic acid [MurNAc] disaccharide coupled to a short pentapeptide moiety) via a pyrophosphate. One of our lead compounds, a diphenyl-trimethyl indolene pyrylium, termed BAS00127538, interacts with the MurNAc moiety and the isoprenyl tail of Lipid II. Here, we report on the structure–activity relationship of BAS00127538 derivatives obtained by in silico analyses and de novo chemical synthesis. Our results indicate that Lipid II binding and bacterial killing are related to three features: the diphenyl moiety, the indolene moiety, and the positive charge of the pyrylium. Replacement of the pyrylium moiety with an N-methyl pyridinium, which may have importance in stability of the molecule, did not alter Lipid II binding or antibacterial potency. PMID:25987836

  6. NK cells, displaying early activation, cytotoxicity and adhesion molecules, are associated with mild dengue disease

    PubMed Central

    Azeredo, E L; De Oliveira-Pinto, L M; Zagne, S M; Cerqueira, D I S; Nogueira, R M R; Kubelka, C F

    2006-01-01

    During the innate immune response against infections, Natural Killer (NK) cells are as important effector cells as are Cytotoxic T lymphocytes (CTL) generated after antigenic stimulation in the adaptative response. NK cells increase in numbers, after viral infection or vaccination. We investigated the NK cell and CD8 T lymphocyte status in 55 dengue infected patients. The NK (CD56+CD3−) and CD56+ T cell (CD56+CD3+) rates rise during the acute phase of disease. The majority of NK cells from dengue patients display early markers for activation (CD69, HLA-DR, and CD38) and cell adhesion molecules (CD44, CD11a) during the acute phase of disease. The intracellular cytotoxic granule, TIA-1, is also up-regulated early in NK cells. Most of these markers appear also on CD8+ T lymphocytes but during the late acute phase. Circulating IL-15 is elevated in a significant number of patients during early acute infection and its values were statistically correlated with NK frequencies and cytotoxic markers on NKs. We have therefore shown that dengue virus infection is very likely stimulating a cytotoxic response that may be efficient in controlling the virus in synergism with CD8+ T lymphocytes. Interestingly, the heightened CD56+CD3−, CD56+CD3+, CD56+TIA-1+ and CD56+CD11a+ cell rates are associated with mild dengue clinical manifestations and might indicate a good prognosis of the disease. PMID:16412060

  7. Discovery of Small-Molecule Nonfluorescent Inhibitors of Fluorogen-Fluorogen Activating Protein Binding Pair.

    PubMed

    Wu, Yang; Stauffer, Shaun R; Stanfield, Robyn L; Tapia, Phillip H; Ursu, Oleg; Fisher, Gregory W; Szent-Gyorgyi, Christopher; Evangelisti, Annette; Waller, Anna; Strouse, J Jacob; Carter, Mark B; Bologa, Cristian; Gouveia, Kristine; Poslusney, Mike; Waggoner, Alan S; Lindsley, Craig W; Jarvik, Jonathan W; Sklar, Larry A

    2016-01-01

    A new class of biosensors, fluorogen activating proteins (FAPs), has been successfully used to track receptor trafficking in live cells. Unlike the traditional fluorescent proteins (FPs), FAPs do not fluoresce unless bound to their specific small-molecule fluorogens, and thus FAP-based assays are highly sensitive. Application of the FAP-based assay for protein trafficking in high-throughput flow cytometry resulted in the discovery of a new class of compounds that interferes with the binding between fluorogens and FAP, thus blocking the fluorescence signal. These compounds are high-affinity, nonfluorescent analogs of fluorogens with little or no toxicity to the tested cells and no apparent interference with the normal function of FAP-tagged receptors. The most potent compound among these, N,4-dimethyl-N-(2-oxo-2-(4-(pyridin-2-yl)piperazin-1-yl)ethyl)benzenesulfonamide (ML342), has been investigated in detail. X-ray crystallographic analysis revealed that ML342 competes with the fluorogen, sulfonated thiazole orange coupled to diethylene glycol diamine (TO1-2p), for the same binding site on a FAP, AM2.2. Kinetic analysis shows that the FAP-fluorogen interaction is more complex than a homogeneous one-site binding process, with multiple conformational states of the fluorogen and/or the FAP, and possible dimerization of the FAP moiety involved in the process.

  8. The new generation drug candidate molecules: Spectral, electrochemical, DNA-binding and anticancer activity properties

    NASA Astrophysics Data System (ADS)

    Gölcü, Ayşegül; Muslu, Harun; Kılıçaslan, Derya; Çeşme, Mustafa; Eren, Özge; Ataş, Fatma; Demirtaş, İbrahim

    2016-09-01

    The new generation drug candidate molecules [Cu(5-Fu)2Cl2H2O] (NGDCM1) and [Zn(5-Fu)2(CH3COO)2] (NGDCM2) were obtained from the reaction of copper(II) and zinc(II) salts with the anticancer drug 5-fluoracil (5-Fu). These compounds have been characterized by spectroscopic and analytical techniques. Thermal behavior of the compounds were also investigated. The electrochemical properties of the compounds have been investigated by cyclic voltammetry (CV) using glassy carbon electrode. The biological activity of the NGDCM1 and NGDCM2 has been evaluated by examining their ability to bind to fish sperm double strand DNA (FSdsDNA) with UV spectroscopy. UV studies of the interaction of the 5-Fu and metal derivatives with FSdsDNA have shown that these compounds can bind to FSdsDNA. The binding constants of the compounds with FSdsDNA have also been calculated. Thermal decomposition of the compounds lead to the formation of CuO and ZnO as final products. The effect of proliferation 5-Fu, NGDCM1 and NGDCM2 were examined on the HeLa cells using real-time cell analyzer with three different concentrations.

  9. Endoplasmic reticulum stress-independent activation of unfolded protein response kinases by a small molecule ATP-mimic

    PubMed Central

    Mendez, Aaron S; Alfaro, Jennifer; Morales-Soto, Marisol A; Dar, Arvin C; McCullagh, Emma; Gotthardt, Katja; Li, Han; Acosta-Alvear, Diego; Sidrauski, Carmela; Korennykh, Alexei V; Bernales, Sebastian; Shokat, Kevan M; Walter, Peter

    2015-01-01

    Two ER membrane-resident transmembrane kinases, IRE1 and PERK, function as stress sensors in the unfolded protein response. IRE1 also has an endoribonuclease activity, which initiates a non-conventional mRNA splicing reaction, while PERK phosphorylates eIF2α. We engineered a potent small molecule, IPA, that binds to IRE1's ATP-binding pocket and predisposes the kinase domain to oligomerization, activating its RNase. IPA also inhibits PERK but, paradoxically, activates it at low concentrations, resulting in a bell-shaped activation profile. We reconstituted IPA-activation of PERK-mediated eIF2α phosphorylation from purified components. We estimate that under conditions of maximal activation less than 15% of PERK molecules in the reaction are occupied by IPA. We propose that IPA binding biases the PERK kinase towards its active conformation, which trans-activates apo-PERK molecules. The mechanism by which partial occupancy with an inhibitor can activate kinases may be wide-spread and carries major implications for design and therapeutic application of kinase inhibitors. DOI: http://dx.doi.org/10.7554/eLife.05434.001 PMID:25986605

  10. A simulation study on the hydrogen storage properties of fullerene family molecules Cx(x = 56,60,70) and their hydrides

    NASA Astrophysics Data System (ADS)

    Dai, Wei; Xiao, Ming; Chen, Mu-Qing; Xu, Jia-Jing; Tang, Yong-Jian

    2016-08-01

    Hydrogen storage is a key factor for the application of hydrogen energy. From first principle calculation, we have acquired the energy barrier for hydrogen molecules to pass through the hexagonal rings and pentagonal rings of the fullerene. Then the absorption energy and energy barrier are used to analyze the hydrogen adsorption capacity of the fullerene family and their hydrides. We have also studied the hydrogen storage properties of the fullerene family and their hydrides by grand canonical Monte Carlo method. It is found that the weight density of hydrogen storage at ambient temperature and pressure can reach 7.71 wt.%. The results show that it is difficult for hydrogen to get into the carbon cage of the fullerene because of the high energy barrier, while it is beneficial to destroy the fullerene structure for the processes of absorption and desorption. Meanwhile, fullerene hydrogenation is an effective method to improve the hydrogen storage properties. Our study facilitates the design and synthesis of hydrogen storage materials, and provides theoretical support to improve the hydrogen storage capability for materials.

  11. A simulation study on the hydrogen storage properties of fullerene family molecules Cx(x = 56,60,70) and their hydrides

    NASA Astrophysics Data System (ADS)

    Dai, Wei; Xiao, Ming; Chen, Mu-Qing; Xu, Jia-Jing; Tang, Yong-Jian

    2016-08-01

    Hydrogen storage is a key factor for the application of hydrogen energy. From first principle calculation, we have acquired the energy barrier for hydrogen molecules to pass through the hexagonal rings and pentagonal rings of the fullerene. Then the absorption energy and energy barrier are used to analyze the hydrogen adsorption capacity of the fullerene family and their hydrides. We have also studied the hydrogen storage properties of the fullerene family and their hydrides by grand canonical Monte Carlo method. It is found that the weight density of hydrogen storage at ambient temperature and pressure can reach 7.71 wt.%. The results show that it is difficult for hydrogen to get into the carbon cage of the fullerene because of the high energy barrier, while it is beneficial to destroy the fullerene structure for the processes of absorption and desorption. Meanwhile, fullerene hydrogenation is an effective method to improve the hydrogen storage properties. Our study facilitates the design and synthesis of hydrogen storage materials, and provides theoretical support to improve the hydrogen storage capability for materials.

  12. All members in the sphingomyelin synthase gene family have ceramide phosphoethanolamine synthase activity[S

    PubMed Central

    Ding, Tingbo; Kabir, Inamul; Li, Yue; Lou, Caixia; Yazdanyar, Amirfarbod; Xu, Jiachen; Dong, Jibin; Zhou, Hongwen; Park, Taesik; Boutjdir, Mohamed; Li, Zhiqiang; Jiang, Xian-Cheng

    2015-01-01

    Sphingomyelin synthase-related protein (SMSr) synthesizes the sphingomyelin analog ceramide phosphoethanolamine (CPE) in cells. Previous cell studies indicated that SMSr is involved in ceramide homeostasis and is crucial for cell function. To further examine SMSr function in vivo, we generated Smsr KO mice that were fertile and had no obvious phenotypic alterations. Quantitative MS analyses of plasma, liver, and macrophages from the KO mice revealed only marginal changes in CPE and ceramide as well as other sphingolipid levels. Because SMS2 also has CPE synthase activity, we prepared Smsr/Sms2 double KO mice. We found that CPE levels were not significantly changed in macrophages, suggesting that CPE levels are not exclusively dependent on SMSr and SMS2 activities. We then measured CPE levels in Sms1 KO mice and found that Sms1 deficiency also reduced plasma CPE levels. Importantly, we found that expression of Sms1 or Sms2 in SF9 insect cells significantly increased not only SM but also CPE formation, indicating that SMS1 also has CPE synthase activity. Moreover, we measured CPE synthase Km and Vmax for SMS1, SMS2, and SMSr using different NBD ceramides. Our study reveals that all mouse SMS family members (SMSr, SMS1, and SMS2) have CPE synthase activity. However, neither CPE nor SMSr appears to be a critical regulator of ceramide levels in vivo. PMID:25605874

  13. Determination of the Absolute Number of Cytokine mRNA Molecules within Individual Activated Human T Cells

    NASA Technical Reports Server (NTRS)

    Karr, Laurel J.; Marshall, Gwen; Hockett, Richard D.; Bucy, R. Pat; Curreri, Peter A. (Technical Monitor)

    2002-01-01

    A primary function of activated T cells is the expression and subsequent secretion of cytokines, which orchestrate the differentiation of other lymphocytes, modulate antigen presenting cell activity, and alter vascular endothelium to mediate an immune response. Since many features of immune regulation probably result from modest alterations of endogenous rates of multiple interacting processes, quantitative analysis of the frequency and specific activity of individual T cells is critically important. Using a coordinated set of quantitative methods, the absolute number of molecules of several key cytokine mRNA species in individual T cells has been determined. The frequency of human blood T cells activated in vitro by mitogens and recall protein antigens was determined by intracellular cytokine protein staining, in situ hybridization for cytokine mRNA, and by limiting dilution analysis for cytokine mRNA+ cells. The absolute number of mRNA molecules was simultaneously determined in both homogenates of the entire population of cells and in individual cells obtained by limiting dilution, using a quantitative, competitive RT-PCR assay. The absolute numbers of mRNA molecules in a population of cells divided by the frequency of individual positive cells, yielded essentially the same number of mRNA molecules per cell as direct analysis of individual cells by limiting dilution analysis. Mean numbers of mRNA per positive cell from both mitogen and antigen activated T cells, using these stimulation conditions, were 6000 for IL-2, 6300 for IFN-gamma, and 1600 for IL-4.

  14. Suppression of complement regulatory protein C1 inhibitor in vascular endothelial activation by inhibiting vascular cell adhesion molecule-1 action

    SciTech Connect

    Zhang, Haimou; Qin, Gangjian; Liang, Gang; Li, Jinan; Chiu, Isaac; Barrington, Robert A.; Liu, Dongxu . E-mail: dxliu001@yahoo.com

    2007-07-13

    Increased expression of adhesion molecules by activated endothelium is a critical feature of vascular inflammation associated with the several diseases such as endotoxin shock and sepsis/septic shock. Our data demonstrated complement regulatory protein C1 inhibitor (C1INH) prevents endothelial cell injury. We hypothesized that C1INH has the ability of an anti-endothelial activation associated with suppression of expression of adhesion molecule(s). C1INH blocked leukocyte adhesion to endothelial cell monolayer in both static assay and flow conditions. In inflammatory condition, C1INH reduced vascular cell adhesion molecule (VCAM-1) expression associated with its cytoplasmic mRNA destabilization and nuclear transcription level. Studies exploring the underlying mechanism of C1INH-mediated suppression in VCAM-1 expression were related to reduction of NF-{kappa}B activation and nuclear translocation in an I{kappa}B{alpha}-dependent manner. The inhibitory effects were associated with reduction of inhibitor I{kappa}B kinase activity and stabilization of the NF-{kappa}B inhibitor I{kappa}B. These findings indicate a novel role for C1INH in inhibition of vascular endothelial activation. These observations could provide the basis for new therapeutic application of C1INH to target inflammatory processes in different pathologic situations.

  15. Cross Talk between Neuroregulatory Molecule and Monocyte: Nerve Growth Factor Activates the Inflammasome

    PubMed Central

    Datta-Mitra, Ananya; Kundu-Raychaudhuri, Smriti; Mitra, Anupam; Raychaudhuri, Siba P.

    2015-01-01

    Background Increasing evidence points to a role for the extra-neuronal nerve growth factor (NGF) in acquired immune responses. However, very little information is available about its role and underlying mechanism in innate immunity. The role of innate immunity in autoimmune diseases is becoming increasingly important. In this study, we explored the contribution of pleiotropic NGF in the innate immune response along with its underlying molecular mechanism with respect to IL-1β secretion. Methods Human monocytes, null and NLRP3 deficient THP-1 cell lines were used for this purpose. We determined the effect of NGF on secretion of IL-1β at the protein and mRNA levels. To determine the underlying molecular mechanism, the effect of NGF on NLRP1/NLRP3 inflammasomes and its downstream key protein, activated caspase-1, were evaluated by ELISA, immunoflorescence, flow cytometry, and real-time PCR. Results In human monocytes and null THP-1 cell line, NGF significantly upregulates IL-1β at protein and mRNA levels in a caspase-1 dependent manner through its receptor, TrkA. Furthermore, we observed that NGF induces caspase-1 activation through NLRP1/NLRP3 inflammasomes, and it is dependent on the master transcription factor, NF-κB. Conclusions To best of our knowledge, this is the first report shedding light on the mechanistic aspect of a neuroregulatory molecule, NGF, in innate immune response, and thus enriches our understanding regarding its pathogenic role in inflammation. These observations add further evidence in favor of anti-NGF therapy in autoimmune diseases and also unlock a new area of research about the role of NGF in IL-1β mediated diseases. PMID:25876154

  16. Moving around the molecule: relationship between chemical structure and in vivo activity of synthetic cannabinoids.

    PubMed

    Wiley, Jenny L; Marusich, Julie A; Huffman, John W

    2014-02-27

    Originally synthesized for research purposes, indole- and pyrrole-derived synthetic cannabinoids are the most common psychoactive compounds contained in abused products marketed as "spice" or "herbal incense." While CB1 and CB2 receptor affinities are available for most of these research chemicals, in vivo pharmacological data are sparse. In mice, cannabinoids produce a characteristic profile of dose-dependent effects: antinociception, hypothermia, catalepsy and suppression of locomotion. In combination with receptor binding data, this tetrad battery has been useful in evaluation of the relationship between the structural features of synthetic cannabinoids and their in vivo cannabimimetic activity. Here, published tetrad studies are reviewed and additional in vivo data on synthetic cannabinoids are presented. Overall, the best predictor of likely cannabimimetic effects in the tetrad tests was good CB1 receptor affinity. Further, retention of good CB1 affinity and in vivo activity was observed across a wide array of structural manipulations of substituents of the prototypic aminoalkylindole molecule WIN55,212-2, including substitution of an alkyl for the morpholino group, replacement of an indole core with a pyrrole or phenylpyrrole, substitution of a phenylacetyl or tetramethylcyclopropyl group for JWH-018's naphthoyl, and halogenation of the naphthoyl group. This flexibility of cannabinoid ligand-receptor interactions has been a particular challenge for forensic scientists who have struggled to identify and regulate each new compound as it has appeared on the drug market. One of the most pressing future research needs is determination of the extent to which the pharmacology of these synthetic cannabinoids may differ from those of classical cannabinoids.

  17. Moving around the molecule: Relationship between chemical structure and in vivo activity of synthetic cannabinoids

    PubMed Central

    Wiley, Jenny L.; Marusich, Julie A.; Huffman, John W.

    2013-01-01

    Originally synthesized for research purposes, indole- and pyrrole-derived synthetic cannabinoids are the most common psychoactive compounds contained in abused products marketed as “spice” or “herbal incense.” While CB1 and CB2 receptor affinities are available for most of these research chemicals, in vivo pharmacological data are sparse. In mice, cannabinoids produce a characteristic profile of dose-dependent effects: antinociception, hypothermia, catalepsy and suppression of locomotion. In combination with receptor binding data, this tetrad battery has been useful in evaluation of the relationship between the structural features of synthetic cannabinoids and their in vivo cannabimimetic activity. Here, published tetrad studies are reviewed and additional in vivo data on synthetic cannabinoids are presented. Overall, the best predictor of likely cannabimimetic effects in the tetrad tests was good CB1 receptor affinity. Further, retention of good CB1 affinity and in vivo activity was observed across a wide array of structural manipulations of substituents of the prototypic aminoalkylindole molecule WIN55,212-2, including substitution of an alkyl for the morpholino group, replacement of an indole core with a pyrrole or phenylpyrrole, substitution of a phenylacetyl or tetramethylcyclopropyl group for JWH-018’s naphthoyl, and halogenation of the naphthoyl group. This flexibility of cannabinoid ligand-receptor interactions has been a particular challenge for forensic scientists who have struggled to identify and regulate each new compound as it has appeared on the drug market. One of the most pressing future research needs is determination of the extent to which the pharmacology of these synthetic cannabinoids may differ from those of classical cannabinoids. PMID:24071522

  18. Inhibition of Inducible Nitric Oxide Synthase Expression by a Novel Small Molecule Activator of the Unfolded Protein Response

    PubMed Central

    Symons, Kent T; Massari, Mark E; Dozier, Sara J; Nguyen, Phan M; Jenkins, David; Herbert, Mark; Gahman, Timothy C; Noble, Stewart A; Rozenkrants, Natasha; Zhang, Yan; Rao, Tadimeti S; Shiau, Andrew K; Hassig, Christian A

    2008-01-01

    The transcription of inducible nitric oxide synthase (iNOS) is activated by a network of proinflammatory signaling pathways. Here we describe the identification of a small molecule that downregulates the expression of iNOS mRNA and protein in cytokine-activated cells and suppresses nitric oxide production in vivo. Mechanistic analysis suggests that this small molecule, erstressin, also activates the unfolded protein response (UPR), a signaling pathway triggered by endoplasmic reticulum stress. Erstressin induces rapid phosphorylation of eIF2α and the alternative splicing of XBP-1, hallmark initiating events of the UPR. Further, erstressin activates the transcription of multiple genes involved in the UPR. These data suggest an inverse relationship between UPR activation and iNOS mRNA and protein expression under proinflammatory conditions. PMID:20161838

  19. Actions of Rho family small G proteins and p21-activated protein kinases on mitogen-activated protein kinase family members.

    PubMed Central

    Frost, J A; Xu, S; Hutchison, M R; Marcus, S; Cobb, M H

    1996-01-01

    The mitogen-activated protein (MAP) kinases are a family of serine/threonine kinases that are regulated by distinct extracellular stimuli. The currently known members include extracellular signal-regulated protein kinase 1 (ERK1), ERK2, the c-Jun N-terminal kinase/stress-activated protein kinases (JNK/SAPKs), and p38 MAP kinases. We find that overexpression of the Ste20-related enzymes p21-activated kinase 1 (PAK1) and PAK2 in 293 cells is sufficient to activate JNK/SAPK and to a lesser extent p38 MAP kinase but not ERK2. Rat MAP/ERK kinase kinase 1 can stimulate the activity of each of these MAP kinases. Although neither activated Rac nor the PAKs stimulate ERK2 activity, overexpression of either dominant negative Rac2 or the N-terminal regulatory domain of PAK1 inhibits Ras-mediated activation of ERK2, suggesting a permissive role for Rac in the control of the ERK pathway. Furthermore, constitutively active Rac2, Cdc42hs, and RhoA synergize with an activated form of Raf to increase ERK2 activity. These findings reveal a previously unrecognized connection between Rho family small G proteins and the ERK pathway. PMID:8668187

  20. Active Learning in a Family Day Care Setting.

    ERIC Educational Resources Information Center

    McCartney, Susan

    Practical tips for improving the quality of child care are offered in this guide. It presents early childhood research findings in everyday language and suggests ways to apply these findings with active learning experiences for children. Developmentally appropriate, holistic activities are presented for key areas. The first five parts of the book…

  1. Handbook of Family Activities for Parents of Learning Disabled Children.

    ERIC Educational Resources Information Center

    Nicholson, Georgia

    Intended for parents, the handbook describes characteristics of learning disabled (LD) children and offers activities that the child can perform in the home to build skill proficiency. It is explained that the activities are designed to relieve the parent and child of constant awareness of the disability, to avoid use of special materials and…

  2. Un dia en la vida: The Everyday Activities of Young Children from Central American Immigrant Families

    ERIC Educational Resources Information Center

    Denmark, Nicole; Jones Harden, Brenda

    2012-01-01

    The aim of this article was to explore the everyday activities of young children from low-income Central American (CA) immigrant families. From the perspective that everyday activities propel children's development of culturally and contextually valued behaviours and skills, 48 mothers were interviewed regarding the activities that are available…

  3. Family leadership styles and adolescent dietary and physical activity behaviors: a cross-sectional study

    PubMed Central

    2012-01-01

    Background Transformational leadership is conceptualized as a set of behaviors designed to inspire, energize and motivate others to achieve higher levels of functioning, and is associated with salient health-related outcomes in organizational settings. Given (a) the similarities that exist between leadership within organizational settings and parenting within families, and (b) the importance of the family environment in the promotion of adolescent health-enhancing behaviors, the purpose of this exploratory study was to examine the cross-sectional relationships between parents’ transformational leadership behaviors and adolescent dietary and physical activity behaviors. Methods 857 adolescents (aged 13–15, mean age = 14.70 yrs) completed measures of transformational parenting behaviors, healthful dietary intake and leisure-time physical activity. Regression analyses were conducted to examine relationships between family transformational leadership and adolescent health outcomes. A further ‘extreme group analysis’ was conducted by clustering families based on quartile splits. A MANCOVA (controlling for child gender) was conducted to examine differences between families displaying (a) HIGH levels of transformational parenting (consistent HIGH TP), (b) LOW levels of transformational parenting (consistent LOW TP), and (c) inconsistent levels of transformational parenting (inconsistent HIGH-LOW TP). Results Results revealed that adolescents’ perceptions of family transformational parenting were associated with both healthy dietary intake and physical activity. Adolescents who perceived their families to display the highest levels of transformational parenting (HIGH TP group) displayed greater healthy eating and physical activity behaviors than adolescents who perceived their families to display the lowest levels of transformational parenting behaviors (LOW TP group). Adolescents who perceived their families to display inconsistent levels of transformational

  4. Cell adhesion molecules as a marker reflecting the reduction of endothelial activation induced by glucocorticoids.

    PubMed

    Leone, Marc; Boutière-Albanèse, Brigitte; Valette, Sarah; Camoin-Jau, Laurence; Barrau, Karine; Albanèse, Jacques; Martin, Claude; Dignat-George, Françoise

    2004-04-01

    In vitro, steroids down-regulate the expression of cell adhesion molecules (CAMs) in endothelial cells stimulated by lipopolysaccharide. Low-dose hydrocortisone is a new treatment of patients with septic shock, a state that is characterized by an endothelial injury. The aim of the present study was to investigate whether the plasma levels of soluble CAMs, reflecting in vivo endothelial activation, could be modulated in patients with septic shock treated by hydrocortisone. This was a prospective and observational study conducted in the intensive care unit at a university hospital. The subjects included 40 patients with septic shock (American College of Chest Physicians Consensus Conference/Society of Critical Care Medicine definition); 45 healthy blood donors served as controls. The patients receiving the standard care ("reference group") during the first 6 months were compared with the patients receiving the hydrocortisone therapy ("hydrocortisone group") for the next 6 months. Measurements of sCAMs were performed on days 1 and 3 of the disease. On day 1, sE-selectin, sP-selectin, sVCAM-1, and sICAM-1 were significantly elevated in patients with septic shock compared with healthy donors. sE-selectin levels significantly decreased between days 1 and 3 in the "hydrocortisone group," whereas there was no significant change in the "reference group". Surprisingly, sICAM-1 levels significantly increased between days 1 and 3 only in patients treated by hydrocortisone. No significant changes were observed for sP-selectin and sVCAM-1 levels in the two groups. In patients with septic shock, glucocorticoids differently affected the pattern of evolution of sCAMs, with sE-selectin being decreased and sICAM-1 being increased. Expression of sP-selectin and sVCAM-1 was not affected.

  5. Endothelial activation by hydrogen peroxide. Selective increases of intercellular adhesion molecule-1 and major histocompatibility complex class I.

    PubMed Central

    Bradley, J. R.; Johnson, D. R.; Pober, J. S.

    1993-01-01

    Products of activated leukocytes may alter vascular endothelial cell (EC) function. For example, ECs respond to leukocyte-derived cytokines, such as tumor necrosis factor (TNF) or interleukin-1, by reversibly altering levels of expression of specific gene products that promote inflammation. In contrast, hydrogen peroxide, a product of TNF-activated neutrophils, can produce irreversible EC injury and death. In this study, we have investigated the effects of subinjurious concentrations of hydrogen peroxide on EC inflammatory functions. Treatment with 50 to 100 mumol/L hydrogen peroxide selectively increases surface expression of intercellular adhesion molecule-1 and major histocompatibility complex class I, but not endothelial leukocyte adhesion molecule-1 (also known as E-selectin), vascular cell adhesion molecule-1, or gp96, a constitutively expressed EC surface protein. Increased major histocompatibility complex class I and intercellular adhesion molecule-1 surface expression is associated with specifically increased messenger RNA levels, suggesting selective endothelial gene activation. Hydrogen peroxide does not activate the transcription factor Nuclear Factor kappa B, an important mediator of TNF-induced gene expression. Co-treatment with hydrogen peroxide inhibits TNF-induced gene expression at 4 hours, an effect which can be attributed to reversible inhibition of TNF binding to EC surface receptors. Hydrogen peroxide also antagonizes the actions of interleukin-1. At 24 hours, TNF and hydrogen peroxide produce, at most, additive increases in intercellular adhesion molecule-1 and major histocompatibility complex class I. These results suggest that subinjurious concentrations of hydrogen peroxide can activate endothelium and that the effects of hydrogen peroxide on ECs differ from those of inflammatory cytokines. Images Figure 3 Figure 4 Figure 5 PMID:8098585

  6. Interplay of solvent additive concentration and active layer thickness on the performance of small molecule solar cells.

    PubMed

    Love, John A; Collins, Samuel D; Nagao, Ikuhiro; Mukherjee, Subhrangsu; Ade, Harald; Bazan, Guillermo C; Nguyen, Thuc-Quyen

    2014-11-19

    A relationship between solvent additive concentration and active layer thickness in small-molecule solar cells is investigated. Specifically, the additive concentration must scale with the amount of semiconductor material and not as absolute concentration in solution. Devices with a wide range of active layers with thickness up to 200 nm can readily achieve efficiencies close to 6% when the right concentration of additive is used.

  7. [Endothelial cell adhesion molecules].

    PubMed

    Ivanov, A N; Norkin, I A; Puchin'ian, D M; Shirokov, V Iu; Zhdanova, O Iu

    2014-01-01

    The review presents current data concerning the functional role of endothelial cell adhesion molecules belonging to different structural families: integrins, selectins, cadherins, and the immunoglobulin super-family. In this manuscript the regulatory mechanisms and factors of adhesion molecules expression and distribution on the surface of endothelial cells are discussed. The data presented reveal the importance of adhesion molecules in the regulation of structural and functional state of endothelial cells in normal conditions and in pathology. Particular attention is paid to the importance of these molecules in the processes of physiological and pathological angiogenesis, regulation of permeability of the endothelial barrier and cell transmigration.

  8. Anti-biofilm activity of pseudoalteromonas haloplanktis tac125 against staphylococcus epidermidis biofilm: Evidence of a signal molecule involvement?

    PubMed

    Parrilli, E; Papa, R; Carillo, S; Tilotta, M; Casillo, A; Sannino, F; Cellini, A; Artini, M; Selan, L; Corsaro, M M; Tutino, M L

    2015-03-01

    Staphylococcus epidermidis is recognized as cause of biofilm-associated infections and interest in the development of new approaches for S. epidermidis biofilm treatment has increased. In a previous paper we reported that the supernatant of Antarctic bacterium Pseudoalteromonas haloplanktis TAC125 presents an anti-biofilm activity against S. epidermidis and preliminary physico-chemical characterization of the supernatant suggested that this activity is due to a polysaccharide. In this work we further investigated the chemical nature of the anti-biofilm P. haloplanktis TAC125 molecule. The production of the molecule was evaluated in different conditions, and reported data demonstrated that it is produced in all P. haloplanktis TAC125 biofilm growth stages, also in minimal medium and at different temperatures. By using a surface coating assay, the surfactant nature of the anti-biofilm compound was excluded. Moreover, a purification procedure was set up and the analysis of an enriched fraction demonstrated that the anti-biofilm activity is not due to a polysaccharide molecule but that it is due to small hydrophobic molecules that likely work as signal. The enriched fraction was also used to evaluate the effect on S. epidermidis biofilm formation in dynamic condition by BioFlux system.

  9. Phosphate Activation via Reduced Oxidation State Phosphorus (P). Mild Routes to Condensed-P Energy Currency Molecules

    PubMed Central

    Kee, Terence P.; Bryant, David E.; Herschy, Barry; Marriott, Katie E. R.; Cosgrove, Nichola E.; Pasek, Matthew A.; Atlas, Zachary D.; Cousins, Claire R.

    2013-01-01

    The emergence of mechanisms for phosphorylating organic and inorganic molecules is a key step en route to the earliest living systems. At the heart of all contemporary biochemical systems reside reactive phosphorus (P) molecules (such as adenosine triphosphate, ATP) as energy currency molecules to drive endergonic metabolic processes and it has been proposed that a predecessor of such molecules could have been pyrophosphate [P2O74−; PPi(V)]. Arguably the most geologically plausible route to PPi(V) is dehydration of orthophosphate, Pi(V), normally a highly endergonic process in the absence of mechanisms for activating Pi(V). One possible solution to this problem recognizes the presence of reactive-P containing mineral phases, such as schreibersite [(Fe,Ni)3P] within meteorites whose abundance on the early Earth would likely have been significant during a putative Hadean-Archean heavy bombardment. Here, we propose that the reduced oxidation state P-oxyacid, H-phosphite [HPO32−; Pi(III)] could have activated Pi(V) towards condensation via the intermediacy of the condensed oxyacid pyrophosphite [H2P2O52−; PPi(III)]. We provide geologically plausible provenance for PPi(III) along with evidence of its ability to activate Pi(V) towards PPi(V) formation under mild conditions (80 °C) in water. PMID:25369812

  10. Anti-biofilm activity of pseudoalteromonas haloplanktis tac125 against staphylococcus epidermidis biofilm: Evidence of a signal molecule involvement?

    PubMed

    Parrilli, E; Papa, R; Carillo, S; Tilotta, M; Casillo, A; Sannino, F; Cellini, A; Artini, M; Selan, L; Corsaro, M M; Tutino, M L

    2015-03-01

    Staphylococcus epidermidis is recognized as cause of biofilm-associated infections and interest in the development of new approaches for S. epidermidis biofilm treatment has increased. In a previous paper we reported that the supernatant of Antarctic bacterium Pseudoalteromonas haloplanktis TAC125 presents an anti-biofilm activity against S. epidermidis and preliminary physico-chemical characterization of the supernatant suggested that this activity is due to a polysaccharide. In this work we further investigated the chemical nature of the anti-biofilm P. haloplanktis TAC125 molecule. The production of the molecule was evaluated in different conditions, and reported data demonstrated that it is produced in all P. haloplanktis TAC125 biofilm growth stages, also in minimal medium and at different temperatures. By using a surface coating assay, the surfactant nature of the anti-biofilm compound was excluded. Moreover, a purification procedure was set up and the analysis of an enriched fraction demonstrated that the anti-biofilm activity is not due to a polysaccharide molecule but that it is due to small hydrophobic molecules that likely work as signal. The enriched fraction was also used to evaluate the effect on S. epidermidis biofilm formation in dynamic condition by BioFlux system. PMID:25816412

  11. Conformational, spectroscopic and nonlinear optical properties of biologically active N,N-dimethyltryptamine molecule: A theoretical study

    NASA Astrophysics Data System (ADS)

    Öner, Nazmiye; Tamer, Ömer; Avcı, Davut; Atalay, Yusuf

    2014-12-01

    The effective psychoactive properties of N,N-dimethyltryptamine (DMT) known as the near-death molecule have encouraged the imagination of many research disciplines for several decades. Although there is no theoretical study, a number of paper composed by experimental techniques have been reported for DMT molecule. In this study, the molecular modeling of DMT was carried out using B3LYP and HSEh1PBE levels of density functional theory (DFT). Our calculations showed that the energy gap between HOMO and LUMO is low, demonstrating that DMT is a biologically active molecule. Large hyperconjugation interaction energies imply that molecular charge transfer occurs in DMT. Moreover, NLO analysis indicates that DMT can be used an effective NLO material.

  12. Conformational, spectroscopic and nonlinear optical properties of biologically active N,N-dimethyltryptamine molecule: a theoretical study.

    PubMed

    Öner, Nazmiye; Tamer, Ömer; Avcı, Davut; Atalay, Yusuf

    2014-12-10

    The effective psychoactive properties of N,N-dimethyltryptamine (DMT) known as the near-death molecule have encouraged the imagination of many research disciplines for several decades. Although there is no theoretical study, a number of paper composed by experimental techniques have been reported for DMT molecule. In this study, the molecular modeling of DMT was carried out using B3LYP and HSEh1PBE levels of density functional theory (DFT). Our calculations showed that the energy gap between HOMO and LUMO is low, demonstrating that DMT is a biologically active molecule. Large hyperconjugation interaction energies imply that molecular charge transfer occurs in DMT. Moreover, NLO analysis indicates that DMT can be used an effective NLO material.

  13. Coupling factor 6 downregulates platelet endothelial cell adhesion molecule-1 via c-Src activation and acts as a proatherogenic molecule.

    PubMed

    Kumagai, Akiko; Osanai, Tomohiro; Katoh, Chisato; Tanaka, Makoto; Tomita, Hirofumi; Morimoto, Takeshi; Murakami, Reiichi; Magota, Koji; Okumura, Ken

    2008-09-01

    Coupling factor 6 (CF6), a component of ATP synthase, suppresses the generation of prostacyclin and nitric oxide (NO). Platelet endothelial cell adhesion molecule-1 (PECAM-1) is involved in shear-induced NO production. To investigate the linkage between the actions of CF6 and PECAM-1, we examined the effects of CF6 on PECAM-1 expression and shear-mediated NO release, comparatively with those of angiotensin II (AngII). Treatment of human umbilical vein endothelial cells (HUVEC) and aortic endothelial cells (HAEC) with CF6 at 10(-7)M or AngII at 10(-7)M for 24h suppressed PECAM-1 gene and protein expression. CF6 or AngII activated c-Src at 15 min in HUVEC, and blockade of c-Src with PP1, its specific inhibitor, restored them. Efrapeptin, an inhibitor of ATPase, attenuated CF6-induced suppression of PECAM-1 gene expression by blockade of acidification, whereas superoxide dismutase or apocinin, an inhibitor of NADPH oxidase, blocked AngII-induced suppression of PECAM-1. Exposure of the cells to shear stress at 25 dynes/cm(2) for 30 min enhanced phosphorylation of eNOS at Ser(1177) and NO release. Pretreatment with CF6 or AngII for 24h attenuated them in HUVEC and HAEC. These suggest that CF6 downregulates PECAM-1 expression via c-Src activation and attenuates shear-induced NO release presumably by suppressing eNOS phosphorylation. PMID:18243211

  14. Identification and biological activities of a new antiangiogenic small molecule that suppresses mitochondrial reactive oxygen species

    SciTech Connect

    Kim, Ki Hyun; Park, Ju Yeol; Jung, Hye Jin; Kwon, Ho Jeong

    2011-01-07

    Research highlights: {yields} YCG063 was screened as a new angiogenesis inhibitor which suppresses mitochondrial ROS generation in a phenotypic cell-based screening of a small molecule-focused library. {yields} The compound inhibited in vitro and in vivo angiogenesis in a dose-dependent manner. {yields} This new small molecule tool will provide a basis for a better understanding of angiogenesis driven under hypoxic conditions. -- Abstract: Mitochondrial reactive oxygen species (ROS) are associated with multiple cellular functions such as cell proliferation, differentiation, and apoptosis. In particular, high levels of mitochondrial ROS in hypoxic cells regulate many angiogenesis-related diseases, including cancer and ischemic disorders. Here we report a new angiogenesis inhibitor, YCG063, which suppressed mitochondrial ROS generation in a phenotypic cell-based screening of a small molecule-focused library with an ArrayScan HCS reader. YCG063 suppressed mitochondrial ROS generation under a hypoxic condition in a dose-dependent manner, leading to the inhibition of in vitro angiogenic tube formation and chemoinvasion as well as in vivo angiogenesis of the chorioallantoic membrane (CAM) at non-toxic doses. In addition, YCG063 decreased the expression levels of HIF-1{alpha} and its target gene, VEGF. Collectively, a new antiangiogenic small molecule that suppresses mitochondrial ROS was identified. This new small molecule tool will provide a basis for a better understanding of angiogenesis driven under hypoxic conditions.

  15. How water molecules affect the catalytic activity of hydrolases--a XANES study of the local structures of peptide deformylase.

    PubMed

    Cui, Peixin; Wang, Yu; Chu, Wangsheng; Guo, Xiaoyun; Yang, Feifei; Yu, Meijuan; Zhao, Haifeng; Dong, Yuhui; Xie, Yaning; Gong, Weimin; Wu, Ziyu

    2014-12-12

    Peptide deformylase (PDF) is a prokaryotic enzyme that catalyzes the deformylation of nascent peptides generated during protein synthesis and water molecules play a key role in these hydrolases. Using X-ray absorption near edge spectroscopy (XANES) and ab initio calculations we accurately probe the local atomic environment of the metal ion binding in the active site of PDF at different pH values and with different metal ions. This new approach is an effective way to monitor existing correlations among functions and structural changes. We show for the first time that the enzymatic activity depends on pH values and metal ions via the bond length of the nearest coordinating water (Wat1) to the metal ion. Combining experimental and theoretical data we may claim that PDF exhibits an enhanced enzymatic activity only when the distance of the Wat1 molecule with the metal ion falls in the limited range from 2.15 to 2.55 Å.

  16. Concerted regulation of inhibitory activity of alpha 1-antitrypsin by the native strain distributed throughout the molecule.

    PubMed

    Seo, Eun Joo; Lee, Cheolju; Yu, Myeong-Hee

    2002-04-19

    The native forms of common globular proteins are in their most stable state but the native forms of plasma serpins (serine protease inhibitors) show high energy state interactions. The high energy state strain of alpha(1)-antitrypsin, a prototype serpin, is distributed throughout the whole molecule, but the strain that regulates the function directly appears to be localized in the region where the reactive site loop is inserted during complex formation with a target protease. To examine the functional role of the strain at other regions of alpha(1)-antitrypsin, we increased the stability of the molecule greatly via combining various stabilizing single amino acid substitutions that did not affect the activity individually. The results showed that a substantial increase of stability, over 13 kcal mol(-1), affected the inhibitory activity with a correlation of 11% activity loss per kcal mol(-1). Addition of an activity affecting single residue substitution in the loop insertion region to these very stable substitutions caused a further activity decrease. The results suggest that the native strain of alpha(1)-antitrypsin distributed throughout the molecule regulates the inhibitory function in a concerted manner. PMID:11834734

  17. Biochemical and toxicological evaluation of nano-heparins in cell functional properties, proteasome activation and expression of key matrix molecules.

    PubMed

    Piperigkou, Zoi; Karamanou, Konstantina; Afratis, Nikolaos A; Bouris, Panagiotis; Gialeli, Chrysostomi; Belmiro, Celso L R; Pavão, Mauro S G; Vynios, Dimitrios H; Tsatsakis, Aristidis M

    2016-01-01

    The glycosaminoglycan heparin and its derivatives act strongly on blood coagulation, controlling the activity of serine protease inhibitors in plasma. Nonetheless, there is accumulating evidence highlighting different anticancer activities of these molecules in numerous types of cancer. Nano-heparins may have great biological significance since they can inhibit cell proliferation and invasion as well as inhibiting proteasome activation. Moreover, they can cause alterations in the expression of major modulators of the tumor microenvironment, regulating cancer cell behavior. In the present study, we evaluated the effects of two nano-heparin formulations: one isolated from porcine intestine and the other from the sea squirt Styela plicata, on a breast cancer cell model. We determined whether these nano-heparins are able to affect cell proliferation, apoptosis and invasion, as well as proteasome activity and the expression of extracellular matrix molecules. Specifically, we observed that nano-Styela compared to nano-Mammalian analogue has higher inhibitory role on cell proliferation, invasion and proteasome activity. Moreover, nano-Styela regulates cell apoptosis, expression of inflammatory molecules, such as IL-6 and IL-8 and reduces the expression levels of extracellular matrix macromolecules, such as the proteolytic enzymes MT1-MMP, uPA and the cell surface proteoglycans syndecan-1 and -2, but not on syndecan-4. The observations reported in the present article indicate that nano-heparins and especially ascidian heparin are effective agents for heparin-induced effects in critical cancer cell functions, providing an important possibility in pharmacological targeting.

  18. Shifting patterns of everyday activity in early dementia: experiences of men and their families.

    PubMed

    Phinney, Alison; Dahlke, Sherry; Purves, Barbara

    2013-08-01

    In this article we draw from a larger study to examine experiences of two men and their families as they negotiate changing patterns of everyday activity in the months after receiving a diagnosis of dementia. We conducted in-depth interpretive phenomenological analysis of interview and observational data that were gathered from the men and various members of their families (n = 7) over a period several months. Findings are presented as three themes: The best kind of man (highlighting participants' historical positioning); It's a little different now (recognizing challenges posed by the dementia); and You have to do something (showing how the men and their families responded to and accommodated these challenges). We discuss these findings in terms of how everyday activity is not only important for supporting personhood in dementia, but it also contributes to sustaining family identity, and does so in a way that is deeply influenced by gender and masculinity.

  19. The BCL-2 protein family: opposing activities that mediate cell death.

    PubMed

    Youle, Richard J; Strasser, Andreas

    2008-01-01

    BCL-2 family proteins, which have either pro- or anti-apoptotic activities, have been studied intensively for the past decade owing to their importance in the regulation of apoptosis, tumorigenesis and cellular responses to anti-cancer therapy. They control the point of no return for clonogenic cell survival and thereby affect tumorigenesis and host-pathogen interactions and regulate animal development. Recent structural, phylogenetic and biological analyses, however, suggest the need for some reconsideration of the accepted organizational principles of the family and how the family members interact with one another during programmed cell death. Although these insights into interactions among BCL-2 family proteins reveal how these proteins are regulated, a unifying hypothesis for the mechanisms they use to activate caspases remains elusive.

  20. Alkyne-tag Raman imaging of bio-active small molecules in live cells

    NASA Astrophysics Data System (ADS)

    Ando, Jun; Palonpon, Almar F.; Yamakoshi, Hiroyuki; Dodo, Kosuke; Kawata, Satoshi; Sodeoka, Mikiko; Fujita, Katsumasa

    2015-12-01

    Raman microscopy is useful for molecular imaging and analysis of biological specimens. Here, we used alkyne containing a carbon-carbon triple bond as a Raman tag for observing small molecules in live cells. Alkyne tags can maintain original properties of target molecules with providing high chemical specificity owing to its distinct peak in a Raman-silent window of biomolecules. For demonstrations, alkyne-tagged thymidine and coenzyme Q analogue in live cells were visualized with high-spatial resolution. We extended the application of alkyne-tag imaging to visualize cell organelles and specific lipid components in artificial monolayer membranes.

  1. Activation of Carbonyl-Containing Molecules with Solid Lewis Acids in Aqueous Media

    SciTech Connect

    Román-Leshkov, Yuriy; Davis, Mark E.

    2011-09-28

    Current interest in reacting carbonyl-containing molecules in aqueous media is primarily due to the growing emphasis on conversion of biomass to fuels and chemicals. Recently, solid Lewis acids have been shown to perform catalytic reactions with carbonyl-containing molecules such as sugars in aqueous media. Here, catalysis mediated by Lewis acids is briefly discussed, Lewis acid solids that perform catalysis in aqueous media are then described, and the review is concluded with a few comments on the outlook for the future.

  2. Activated leukocyte cell adhesion molecule regulates the interaction between pancreatic cancer cells and stellate cells

    PubMed Central

    Zhang, Wei-Wei; Zhan, Shu-Hui; Geng, Chang-Xin; Sun, Xin; Erkan, Mert; Kleeff, Jörg; Xie, Xiang-Jun

    2016-01-01

    Activated leukocyte cell adhesion molecule (ALCAM/CD166) is a transmembrane glycoprotein that is involved in tumor progression and metastasis. In the present study, the expression and functional role of ALCAM in pancreatic cancer cells and pancreatic stellate cells (PSCs) was investigated. Tissue specimens were obtained from patients with pancreatic ductal adenocarcinoma (n=56) or chronic pancreatitis (CP; n=10), who underwent pancreatic resection, and from normal pancreatic tissue samples (n=10). Immunohistochemistry was used to analyze the localization and expression of ALCAM in pancreatic tissues. Subsequently, reverse transcription-quantitative polymerase chain reaction and immunoblotting were applied to assess the expression of ALCAM in pancreatic cancer Panc-1 and T3M4 cells, as well as in PSCs. An enzyme-linked immunosorbent assay was used to measure ALCAM levels in cell culture medium stimulated by hypoxia, tumor necrosis factor (TNF)-α and transforming growth factor-β. Silencing of ALCAM was performed using ALCAM small interfering (si)RNA and immunocytochemistry was used to analyze the inhibition efficiency. An invasion assay and a cell interaction assay were performed to assess the invasive ability and co-cultured adhesive potential of Panc-1 and T3M4 cells, as well as PSCs. Histologically, ALCAM expression was generally weak or absent in pancreatic cancer cells, but was markedly upregulated in PSCs in pancreatic cancer tissues. ALCAM was highly expressed in PSCs from CP tissues and PSCs surrounding pancreatic intraepithelial neoplasias, as well as in pancreatic cancer cells. ALCAM mRNA was highly expressed in PSCs, with a low to moderate expression in T3M4 and Panc-1 cells. Similar to the mRNA expression, immunoblotting demonstrated that ALCAM protein levels were high in PSCs and T3M4 cells, but low in Panc-1 cells. The expression of TNF-α increased, while hypoxia decreased the secretion of ALCAM in pancreatic cancer Panc-1 and T3M4 cells, and also in

  3. Affinity of Drugs and Small Biologically Active Molecules to Carbon Nanotubes: A Pharmacodynamics and Nanotoxicity Factor?

    PubMed Central

    Liu, John; Yang, Liu; Hopfinger, Anton J.

    2009-01-01

    The MM-PBSA MD method was used to estimate the affinity, as represented by log kb, of each of a variety of biologically active molecules to a carbon nanotube in an aqueous environment. These ligand-receptor binding simulations were calibrated by first estimating the log kb values for eight ligands to human serum albumin, HSA, whose log kb values have been observed. A validation linear correlation equation was established [R2 = 0.888 Q2 = 0.603] between the observed and estimated log kb values to HSA. This correlation equation was then used to re-scale all MM-PBSA MD log kb values using a carbon nanotube as the receptor. The log kb of the eight HSA ligands, nine polar and/or rigid ligands and six nonpolar and/or flexible ligands to a carbon nanotube were estimated. The range in re-scaled log kb values across this set of 23 ligands is 0.25 to 7.14, essentially seven orders of magnitude. Some ligands, like PGI2, bind in the log kb = 7 range which corresponds to the lower limits of known drugs. Thus, such significant levels of binding of biologically relevant compounds to carbon nanotubes might lead to alterations in the normal pharmacodynamic profiles of these compounds and be a source of toxicity. Ligand binding potency to a carbon nanotube is largely controlled by the shape, polarity/nonpolarity distribution and flexibility of the ligand. HSA ligands exhibit the most limited binding to a carbon nanotube, and they are relatively rigid and of generally spherical shape. Polar and/or rigid ligands bind less strongly to the carbon nanotube, on average, than nonpolar and/or flexible ligands even though the chosen members of both classes of ligands in this study have chain-like shapes that facilitate binding. The introduction of only a few strategically spaced single bonds in the polar and/or rigid ligands markedly increases their binding to a carbon nanotube. PMID:19281188

  4. Value Questionnaires For Marriage and Family Living; A Creative Classroom Activity by Edu-Game.

    ERIC Educational Resources Information Center

    1975

    This booklet is a series of 10 unit-organized questionnaires to stimulate discussion on a variety of topics in the area of marriage and family life. These values clarification activities can help 11th and 12th grade students confirm or reevaluate personal attitudes. The units included in this activity are: Love and Marriage; Male and Female…

  5. Recipes for Fun: Play Activities and Games for Young Children with Disabilities and Their Families.

    ERIC Educational Resources Information Center

    Rappaport, Lisa

    Intended for friends and families of children with disabilities, the book contains information and suggestions on play activities, toys, and games for children (0-8) with mild and moderate disabilities. Activities are sequenced from simpler to more developmentally advanced within six major categories (1) exploring the senses (hid and find,…

  6. Physical Activity as a Dimension of Family Life for Lower Primary School Children

    ERIC Educational Resources Information Center

    Macdonald, Doune; Rodger, Sylvia; Ziviani, Jenny; Jenkins, David; Batch, Jenny; Jones, Judy

    2004-01-01

    While questions of children's engagement in physical activity are being widely debated, little is known about how physical activity is valued and managed within families. This paper reports on qualitative data from a multi-method study on lower primary aged children. The focus of the broader study was to determine the relationships between young…

  7. Low natural-killer-cell activity in familial melanoma patients and their relatives.

    PubMed

    Hersey, P; Edwards, A; Honeyman, M; McCarthy, W H

    1979-07-01

    Patients with melanoma who had one or more close relatives with melanoma were studied for their natural-killer-cell (NK) activity against cultured melanoma cells and Chang cells. A high proportion of the patients and their relatives were found to have low NK activity against these target cells. In most of the patients this could not be attributed to general depression of their immune function, since B- and T-cell numbers and the mitogenic response to PHA were within normal limits. The levels of NK activity of the patients and their relatives were found to be significantly correlated, suggesting that the NK activity in these families may have been genetically (or environmentally) determined. Several genetic markers were examined in the patients and their relatives for association with the disease state and NK activity. No association with HLA antigens or ABO blood groups was detected, but there was a low incidence of the Rhesus negative phenotype in the patients (the Rh phenotype had previously been associated with high NK activity). The present results indicate that NK activity has a familial association in families with a high incidence of melanoma, and raise the question whether low NK activity may be one of the predisposing factors in the development of familial melanoma.

  8. Modulation of Mitochondrial Complex I Activity Averts Cognitive Decline in Multiple Animal Models of Familial Alzheimer's Disease

    PubMed Central

    Zhang, Liang; Zhang, Song; Maezawa, Izumi; Trushin, Sergey; Minhas, Paras; Pinto, Matthew; Jin, Lee-Way; Prasain, Keshar; Nguyen, Thi D.T.; Yamazaki, Yu; Kanekiyo, Takahisa; Bu, Guojun; Gateno, Benjamin; Chang, Kyeong-Ok; Nath, Karl A.; Nemutlu, Emirhan; Dzeja, Petras; Pang, Yuan-Ping; Hua, Duy H.; Trushina, Eugenia

    2015-01-01

    Development of therapeutic strategies to prevent Alzheimer's disease (AD) is of great importance. We show that mild inhibition of mitochondrial complex I with small molecule CP2 reduces levels of amyloid beta and phospho-Tau and averts cognitive decline in three animal models of familial AD. Low-mass molecular dynamics simulations and biochemical studies confirmed that CP2 competes with flavin mononucleotide for binding to the redox center of complex I leading to elevated AMP/ATP ratio and activation of AMP-activated protein kinase in neurons and mouse brain without inducing oxidative damage or inflammation. Furthermore, modulation of complex I activity augmented mitochondrial bioenergetics increasing coupling efficiency of respiratory chain and neuronal resistance to stress. Concomitant reduction of glycogen synthase kinase 3β activity and restoration of axonal trafficking resulted in elevated levels of neurotrophic factors and synaptic proteins in adult AD mice. Our results suggest that metabolic reprogramming induced by modulation of mitochondrial complex I activity represents promising therapeutic strategy for AD. PMID:26086035

  9. The C1q domain containing proteins of the Mediterranean mussel Mytilus galloprovincialis: a widespread and diverse family of immune-related molecules.

    PubMed

    Gerdol, Marco; Manfrin, Chiara; De Moro, Gianluca; Figueras, Antonio; Novoa, Beatriz; Venier, Paola; Pallavicini, Alberto

    2011-06-01

    The key component of the classical complement pathway C1q is regarded as a major connecting link between innate and acquired immunity due to the highly adaptive binding properties of its trimeric globular domain gC1q. The gC1q domain also characterizes many non-complement proteins involved in a broad range of biological processes including apoptosis, inflammation, cell adhesion and cell differentiation. In molluscs and many other invertebrates lacking of adaptive immunity, C1q domain containing (C1qDC) proteins are abundant, they most probably emerged as lectins and subsequently evolved in a specialized class of pattern recognition molecules through the expanding interaction properties of gC1q. Here we report the identification of 168 C1qDC transcript sequences of Mytilus galloprovincialis. The remarkable abundance of C1qDC transcripts in the Mediterranean mussel suggests an evolutionary strategy of gene duplication, functional diversification and selection of many specific C1qDC variants. A comprehensive transcript sequence survey in Protostomia also revealed that the C1qDC family expansion observed in mussel could have occurred in some specific taxa independently from the events leading to the establishment of a large complement of C1qDC genes in the Chordates lineage. PMID:21295069

  10. Unequal Activities of Enantiomers via Biological Receptors: Examples of Chiral Drug, Pesticide, and Fragrance Molecules

    ERIC Educational Resources Information Center

    Mannschreck, Albrecht; Kiesswetter, Roland; von Angerer, Erwin

    2007-01-01

    A molecule coming from outside an organism can form a ligand-receptor complex. Upon its formation, a message is transmitted, for example, to certain cells. In this way, two enantiomers can emit messages that differ, either quantitatively or qualitatively. In the present article, these facts are taken as a common basis for the actions of chiral…

  11. Analyzing ligand and small molecule binding activity of solubilized GPCRs using biosensor technology.

    PubMed

    Navratilova, Iva; Dioszegi, Marianna; Myszka, David G

    2006-08-01

    We used Biacore technology to measure directly the binding of natural ligands and small molecules to the chemokine receptors CXCR4 and CCR5. Both G protein-coupled receptors were solubilized from whole cell pellets and captured on antibody surfaces for analysis. Our solubilization conditions maintained high-affinity binding of chemokines SDF-1alpha and RANTES to CXCR4 and CCR5, respectively. Surface density- and buffer-dependent binding responses, along with binding data for a selective ligand (RCP-168), further validated the biosensor assay. In addition, we showed that it is possible to collect high-quality binding responses for the archetypal small molecule inhibitors JM-2987 and TAK-779. Finally, using our biosensor-based method, we characterized the kinetics of 19 novel small molecule inhibitors of CCR5 and showed that their affinities correlated with values determined for the membrane-associated receptor. Together, the chemokine and small molecule binding data provide evidence that the solubilized receptors maintain native binding properties. These solubilized receptor preparations could be useful reagents for biophysical studies as well as for structural analysis.

  12. Cytotoxic activity of plants of family zygophyllaceae and euphorbiaceae.

    PubMed

    Dastagir, Ghulam; Hussain, Farrukh

    2014-07-01

    The methanolic and n-hexane extracts of studied plants showed significant toxicity to brine shrimps. The methanolic extract of Fagonia cretica had highest LD50 (117.72) value, while Peganum harmala showed low LD50 value (41.70) compared to n-hexane extract. The methanolic and n-hexane extracts of Tribulus terrestris showed similar LD50 values. The methanolic extract of Chrozophora tinctoria showed low LD50 value than the n-hexane extract. The methanolic extract of Ricinus communis showed highest LD50 value while the n-hexane extract showed lowest LD50 value. The LD50 value less than 100 was obtained for n-hexane extracts of Fagonia cretica, Peganum harmala and Ricinus communis. The n-hexane extracts of these plants also showed the highest toxicity as compare to methanolic extracts. The chemical constituents detected in the present investigation might be responsible for cytotoxic activity. PMID:25015443

  13. The ACTive Intervention in Hospice Interdisciplinary Team Meetings: Exploring family caregiver and hospice team communication

    PubMed Central

    Wittenberg-Lyles, Elaine; Parker Oliver, Debra; Demiris, George; Baldwin, Paula

    2010-01-01

    This paper presents the theoretical framework and rationale for the ACTive intervention which proposes the use of video technology to facilitate patient and family participation in hospice interdisciplinary team meetings where plans of care are determined. It is surmised that patient and family involvement will improve communication and compliance in hospice care. An analysis of data from a pilot project of the ACTive intervention was conducted to explore active participation among family caregivers and the hospice team. Through the use of videophone technology caregivers participated in video-recorded team meetings. The actual communication behaviors of caregivers and team members were analyzed for active participation. Findings revealed that team–prompted caregiver participation was most common, however, team use of supportive talk in this context was considerably less frequent. The study also found that the team’s use of active participation behaviors elicits caregiver active participation behaviors. The results of this study suggest the intervention was an effective way to involve family caregivers as active participants in the designing of care for their loved one. Findings also suggest that hospice staff would benefit from education and training on best practices for communicating with caregivers in the team meeting setting. PMID:20543889

  14. A bead-based activity screen for small-molecule inhibitors of signal transduction in chronic myelogenous leukemia cells

    PubMed Central

    Sylvester, Juliesta E.; Kron, Stephen J.

    2010-01-01

    Chronic myelogenous leukemia is characterized by the presence of the chimeric BCR-ABL gene, which is expressed as the constitutively active Bcr-Abl kinase. Although kinase activity is directly responsible for the clinical phenotype, current diagnostic and prognostic methods focus on a genetic classification system where molecularly distinct subcategories are used to predict patient responses to small-molecule inhibitors of the Bcr-Abl kinase. Point mutations in the kinase domain are a central factor regulating inhibitor resistance; however, compensatory signaling caused by the activation of unrelated kinases can influence inhibitor efficacy. Kinase activity profiling can be used as a complementary approach to genetic screening and allows direct screening of small-molecule inhibitors. We developed a quantitative assay to monitor tyrosine kinase activities and inhibitor sensitivities in a model of chronic myelogenous leukemia using peptide reporters covalently immobilized on Luminex beads. Kinase activity is quantified by non-linear regression from well-specific internal standard curves. Using optimized synthetic substrates and peptides derived from native substrates as probes, we measured kinase inhibition in cell lysates by the signal transduction inhibitors imatinib and dasatinib. Taking advantage of a convenient 96-well plate format, this assay also allows a straightforward and quantitative analysis of the differential effects of ATP and inhibitors on kinase activity. This method for analyzing a focused signaling network benefits from rigorous statistical analysis and short processing times, thereby offering a powerful tool for drug discovery and clinical testing. PMID:20423990

  15. Family Literacy Activities and Parental-Child Interactive Reading with Preschoolers from Asian Immigrant Families in the North Texas Area of the United States

    ERIC Educational Resources Information Center

    Suwannakhae, Yaowaluk

    2012-01-01

    There were two purposes of this descriptive research design. The first purpose of the study was to investigate family literacy activities with preschool-aged children and parental expectations of their children's development and future school success as reported by Asian immigrant families. The second purpose was to observe the interactive…

  16. SET7/9 Catalytic Mutants Reveal the Role of Active Site Water Molecules in Lysine Multiple Methylation*

    PubMed Central

    Del Rizzo, Paul A.; Couture, Jean-François; Dirk, Lynnette M. A.; Strunk, Bethany S.; Roiko, Marijo S.; Brunzelle, Joseph S.; Houtz, Robert L.; Trievel, Raymond C.

    2010-01-01

    SET domain lysine methyltransferases (KMTs) methylate specific lysine residues in histone and non-histone substrates. These enzymes also display product specificity by catalyzing distinct degrees of methylation of the lysine ϵ-amino group. To elucidate the molecular mechanism underlying this specificity, we have characterized the Y245A and Y305F mutants of the human KMT SET7/9 (also known as KMT7) that alter its product specificity from a monomethyltransferase to a di- and a trimethyltransferase, respectively. Crystal structures of these mutants in complex with peptides bearing unmodified, mono-, di-, and trimethylated lysines illustrate the roles of active site water molecules in aligning the lysine ϵ-amino group for methyl transfer with S-adenosylmethionine. Displacement or dissociation of these solvent molecules enlarges the diameter of the active site, accommodating the increasing size of the methylated ϵ-amino group during successive methyl transfer reactions. Together, these results furnish new insights into the roles of active site water molecules in modulating lysine multiple methylation by SET domain KMTs and provide the first molecular snapshots of the mono-, di-, and trimethyl transfer reactions catalyzed by these enzymes. PMID:20675860

  17. New Antibiotic Molecules: Bypassing the Membrane Barrier of Gram Negative Bacteria Increases the Activity of Peptide Deformylase Inhibitors

    PubMed Central

    Mamelli, Laurent; Petit, Sylvain; Chevalier, Jacqueline; Giglione, Carmela; Lieutaud, Aurélie; Meinnel, Thierry; Artaud, Isabelle; Pagès, Jean-Marie

    2009-01-01

    Background Multi-drug resistant (MDR) bacteria have become a major concern in hospitals worldwide and urgently require the development of new antibacterial molecules. Peptide deformylase is an intracellular target now well-recognized for the design of new antibiotics. The bacterial susceptibility to such a cytoplasmic target primarily depends on the capacity of the compound to reach and accumulate in the cytosol. Methodology/Principal Findings To determine the respective involvement of penetration (influx) and pumping out (efflux) mechanisms to peptide deformylase inhibitors (PDF-I) activity, the potency of various series was determined using various genetic contexts (efflux overproducers or efflux-deleted strains) and membrane permeabilizers. Depending on the structure of the tested molecules, two behaviors could be observed: (i) for actinonin the first PDF-I characterized, the AcrAB efflux system was the main parameter involved in the bacterial susceptibility, and (ii), for the lastest PDF-Is such as the derivatives of 2-(5-bromo-1H-indol-3-yl)-N-hydroxyacetamide, the penetration through the membrane was a important limiting step. Conclusions/Significance Our results clearly show that the bacterial membrane plays a key role in modulating the antibacterial activity of PDF-Is. The bacterial susceptibility for these new antibacterial molecules can be improved by two unrelated ways in MDR strains: by collapsing the Acr efflux activity or by increasing the uptake rate through the bacterial membrane. The efficiency of the second method is associated with the nature of the compound. PMID:19649280

  18. [Social support and physical activity in adolescents from public schools: the importance of family and friends].

    PubMed

    Prado, Crisley Vanessa; Lima, Alex Vieira; Fermino, Rogério César; Añez, Ciro Romelio Rodriguez; Reis, Rodrigo Siqueira

    2014-04-01

    The aim of this study was to verify the association between different types and sources of social support and physical activity among adolescents from Curitiba, Paraná State, Brazil. A school-based survey was conducted with a representative sample of adolescents from public schools (n = 1,469). Multiple regression models were used to test the association between weekly frequency and sources of social support from family and friends and weekly frequency of physical activity. Among boys, frequent company of family (PR: 2.88; 95%CI: 2.00-4.13) and friends (PR: 5.46; 95%CI: 2.33-12.78) and positive reinforcement from friends (PR: 1.81; 95%CI: 1.18-2.77) were positively associated with physical activity. Sporadic invitation by the family was negatively associated with physical activity (PR: 0.66; 95%CI: 0.46-1.14). For girls, frequent company of family (PR: 3.39; 95%CI: 1.49-7.69) and friends (PR: 4.06; 95%CI: 2.22-7.45) increased the likelihood of physical activity. Company of friends was the most important type of social support for physical activity among these adolescents.

  19. [Social support and physical activity in adolescents from public schools: the importance of family and friends].

    PubMed

    Prado, Crisley Vanessa; Lima, Alex Vieira; Fermino, Rogério César; Añez, Ciro Romelio Rodriguez; Reis, Rodrigo Siqueira

    2014-04-01

    The aim of this study was to verify the association between different types and sources of social support and physical activity among adolescents from Curitiba, Paraná State, Brazil. A school-based survey was conducted with a representative sample of adolescents from public schools (n = 1,469). Multiple regression models were used to test the association between weekly frequency and sources of social support from family and friends and weekly frequency of physical activity. Among boys, frequent company of family (PR: 2.88; 95%CI: 2.00-4.13) and friends (PR: 5.46; 95%CI: 2.33-12.78) and positive reinforcement from friends (PR: 1.81; 95%CI: 1.18-2.77) were positively associated with physical activity. Sporadic invitation by the family was negatively associated with physical activity (PR: 0.66; 95%CI: 0.46-1.14). For girls, frequent company of family (PR: 3.39; 95%CI: 1.49-7.69) and friends (PR: 4.06; 95%CI: 2.22-7.45) increased the likelihood of physical activity. Company of friends was the most important type of social support for physical activity among these adolescents. PMID:24896057

  20. On the determination of the diagonal components of the optical activity tensor in chiral molecules

    NASA Astrophysics Data System (ADS)

    Pelloni, Stefano; Lazzeretti, Paolo

    2014-02-01

    It is shown that the diagonal components of the mixed electric-magnetic dipole polarizability tensor, used to rationalize the optical rotatory power of chiral molecules, are origin independent, if they are referred to the coordinate system defined by the eigenvectors of the dynamic electric dipole polarizability, for a given value ω of the frequency of a monochromatic wave impinging on an ordered sample. Within this reference frame, the individual diagonal components of the mixed electric-magnetic dipole polarizability are separately measurable properties. The theoretical method is applied via a test calculation to the cyclic 1,2-M enantiomer of the dioxin molecule, using a large Gaussian basis set to estimate near Hartree-Fock values within a series of dipole length, velocity, and acceleration representations.

  1. Genetic regulation of inflammation-mediated activation of haemostasis: family-based approaches in population studies.

    PubMed

    Vohnout, B; Gianfagna, F; Lorenzet, R; Cerletti, C; de Gaetano, G; Donati, M B; Iacoviello, L

    2011-11-01

    Blood coagulation and inflammation play a key role in atherosclerosis and thrombosis. Candidate gene and genome wide association studies have identified potential specific genes that might have a causal role in these pathogenic processes. The analysis of quantitative traits is more powerful as they are closer to direct gene action than disease phenotypes. Thus linkage-based studies on extended families might be useful both to estimate the heritability and to map the genetic loci responsible for the regulation of the trait. Family-based studies may estimate high heritability for thrombosis and quantitative traits regarding both platelet aggregation and blood coagulation. Some specific loci relevant to thrombosis have been identified, with some of them showing a direct pleiotropic effect on the risk of thrombosis. Haemostasis factors can be activated by inflammatory stimuli. Fibrinogen level is genetically correlated with C-reactive protein levels with a link for both traits on chromosomes 12 and 21. Genes related to prostanoid biosynthesis, involved both in inflammation and thrombosis, show high heritability levels in both enzyme expression and prostanoid production. Considering that few large family-based linkage studies have as yet been performed on haemostasis and inflammation-related traits, additional studies are highly needed. We are performing a family-based linkage study on large pedigrees (750 subjects from 23 families with juvenile myocardial infarction and 31 control families), to identify genes responsible for quantitative traits involved in the pathway progressively going from inflammation to haemostasis, cell activation, thrombus formation and cardiovascular events.

  2. Structures of Clostridium Botulinum Neurotoxin Serotype A Light Chain Complexed with Small-Molecule Inhibitors Highlight Active-Site Flexibility

    SciTech Connect

    Silvaggi,N.; Boldt, G.; Hixon, M.; Kennedy, J.; Tzipori, S.; Janda, K.; Allen, K.

    2007-01-01

    The potential for the use of Clostridial neurotoxins as bioweapons makes the development of small-molecule inhibitors of these deadly toxins a top priority. Recently, screening of a random hydroxamate library identified a small-molecule inhibitor of C. botulinum Neurotoxin Serotype A Light Chain (BoNT/A-LC), 4-chlorocinnamic hydroxamate, a derivative of which has been shown to have in vivo efficacy in mice and no toxicity. We describe the X-ray crystal structures of BoNT/A-LC in complexes with two potent small-molecule inhibitors. The structures of the enzyme with 4-chlorocinnamic hydroxamate or 2,4-dichlorocinnamic hydroxamate bound are compared to the structure of the enzyme complexed with L-arginine hydroxamate, an inhibitor with modest affinity. Taken together, this suite of structures provides surprising insights into the BoNT/A-LC active site, including unexpected conformational flexibility at the S1' site that changes the electrostatic environment of the binding pocket. Information gained from these structures will inform the design and optimization of more effective small-molecule inhibitors of BoNT/A-LC.

  3. Using naturally occurring polysaccharides to align molecules with nonlinear optical activity

    NASA Technical Reports Server (NTRS)

    Prasthofer, Thomas

    1996-01-01

    The Biophysics and Advanced Materials Branch of the Microgravity Science and Applications Division at Marshall Space Flight Center has been investigating polymers with the potential for nonlinear optical (NLO) applications for a number of years. Some of the potential applications for NLO materials include optical communications, computing, and switching. To this point the branch's research has involved polydiacetylenes, phthalocyanins, and other synthetic polymers which have inherent NLO properties. The aim of the present research is to investigate the possibility of using naturally occurring polymers such as polysaccharides or proteins to trap and align small organic molecules with useful NLO properties. Ordering molecules with NLO properties enhances 3rd order nonlinear effects and is required for 2nd order nonlinear effects. Potential advantages of such a system are the flexibility to use different small molecules with varying chemical and optical properties, the stability and cost of the polymers, and the ability to form thin, optically transparent films. Since the quality of any polymer films depends on optimizing ordering and minimizing defects, this work is particularly well suited for microgravity experiments. Polysaccharide and protein polymers form microscopic crystallites which must align to form ordered arrays. The ordered association of crystallites is disrupted by gravity effects and NASA research on protein crystal growth has demonstrated that low gravity conditions can improve crystal quality.

  4. Staphylococcus-mediated T-cell activation and spontaneous natural killer cell activity in the absence of major histocompatibility complex class II molecules

    NASA Technical Reports Server (NTRS)

    Chapes, S. K.; Hoynowski, S. M.; Woods, K. M.; Armstrong, J. W.; Beharka, A. A.; Iandolo, J. J.; Spooner, B. S. (Principal Investigator)

    1993-01-01

    We used major histocompatibility complex class II antigen-deficient transgenic mice to show that in vitro natural killer cell cytotoxicity and T-cell activation by staphylococcal exotoxins (superantigens) are not dependent upon the presence of major histocompatibility complex class II molecules. T cells can be activated by exotoxins in the presence of exogenously added interleukin 1 or 2 or in the presence of specific antibody without exogenously added cytokines.

  5. Staphylococcus-mediated T-cell activation and spontaneous natural killer cell activity in the absence of major histocompatibility complex class II molecules.

    PubMed Central

    Chapes, S K; Hoynowski, S M; Woods, K M; Armstrong, J W; Beharka, A A; Iandolo, J J

    1993-01-01

    We used major histocompatibility complex class II antigen-deficient transgenic mice to show that in vitro natural killer cell cytotoxicity and T-cell activation by staphylococcal exotoxins (superantigens) are not dependent upon the presence of major histocompatibility complex class II molecules. T cells can be activated by exotoxins in the presence of exogenously added interleukin 1 or 2 or in the presence of specific antibody without exogenously added cytokines. PMID:8359928

  6. Activated peripheral lymphocytes with increased expression of cell adhesion molecules and cytotoxic markers are associated with dengue fever disease.

    PubMed

    Azeredo, Elzinandes L; Zagne, Sonia M O; Alvarenga, Allan R; Nogueira, Rita M R; Kubelka, Claire F; de Oliveira-Pinto, Luzia M

    2006-06-01

    The immune mechanisms involved in dengue fever and dengue hemorrhagic/dengue shock syndrome are not well understood. The ex vivo activation status of immune cells during the dengue disease in patients was examined. CD4 and CD8 T cells were reduced during the acute phase. Interestingly, CD8 T cells co-expressing activation marker HLA-DR, Q, P, and cytolytic granule protein-Tia-1 were significantly higher in dengue patients than in controls. Detection of adhesion molecules indicated that in dengue patients the majority of T cells (CD4 and CD8) express the activation/memory phenotype, characterized as CD44HIGH and lack the expression of the naïve cell marker, CD62L LOW. Also, the levels of T cells co-expressing ICAM-1 (CD54), VLA-4, and LFA-1 (CD11a) were significantly increased. CD8 T lymphocytes expressed predominantly low levels of anti-apoptotic molecule Bcl-2 in the acute phase, possibly leading to the exhibition of a phenotype of activated/effector cells. Circulating levels of IL-18, TGF-b1 and sICAM-1 were significantly elevated in dengue patients. Early activation events occur during acute dengue infection which might contribute to viral clearance. Differences in expression of adhesion molecules among CD4 and CD8 T cells might underlie the selective extravasation of these subsets from blood circulation into lymphoid organs and/or tissues. In addition, activated CD8 T cells would be more susceptible to apoptosis as shown by the alteration in Bcl-2 expression. Cytokines such as IL-18, TGF-b1, and sICAM-1 may be contributing by either stimulating or suppressing the adaptative immune response, during dengue infection, thereby perhaps establishing a relationship with disease severity.

  7. Near infrared emission from molecule-like silver clusters confined in zeolite A assisted by thermal activation

    SciTech Connect

    Lin, Hui Imakita, Kenji; Rong Gui, Sa Chu; Fujii, Minoru

    2014-07-07

    Strong and broad near infrared (NIR) emission peaked at ~855 nm upon optimal excitation at 342 nm has been observed from molecule-like silver clusters (MLSCs) confined in zeolite A assisted by thermal activation. To the best of our knowledge, this is the first observation of NIR emission peaked at longer than 800 nm from MLSCs confined in solid matrices. The decay time of the NIR emission is over 10 μs, which indicates that it is a spin-forbidden transition. The ~855 nm NIR emission shows strong dependence on the silver loading concentration and the thermal activation temperature.

  8. Activation of HER family members in gastric carcinoma cells mediates resistance to MET inhibition

    PubMed Central

    2010-01-01

    Background Gastric cancer is the second leading cause of cancer mortality in the world. The receptor tyrosine kinase MET is constitutively activated in many gastric cancers and its expression is strictly required for survival of some gastric cancer cells. Thus, MET is considered a good candidate for targeted therapeutic intervention in this type of tumor, and MET inhibitors recently entered clinical trials. One of the major problems of therapies targeting tyrosine kinases is that many tumors are not responsive to treatment or eventually develop resistance to the drugs. Perspective studies are thus mandatory to identify the molecular mechanisms that could cause resistance to these therapies. Results Our in vitro and in vivo results demonstrate that, in MET-addicted gastric cancer cells, the activation of HER (Human Epidermal Receptor) family members induces resistance to MET silencing or inhibition by PHA-665752 (a selective kinase inhibitor). We provide molecular evidences highlighting the role of EGFR, HER3, and downstream signaling pathways common to MET and HER family in resistance to MET inhibitors. Moreover, we show that an in vitro generated gastric cancer cell line resistant to MET-inhibition displays overexpression of HER family members, whose activation contributes to maintenance of resistance. Conclusions Our findings predict that gastric cancer tumors bearing constitutive activation of HER family members are poorly responsive to MET inhibition, even if this receptor is constitutively active. Moreover, the appearance of these alterations might also be responsible for the onset of resistance in initially responsive tumors. PMID:20500904

  9. Discovery of a small-molecule binder of the oncoprotein gankyrin that modulates gankyrin activity in the cell

    NASA Astrophysics Data System (ADS)

    Chattopadhyay, Anasuya; O’Connor, Cornelius J.; Zhang, Fengzhi; Galvagnion, Celine; Galloway, Warren R. J. D.; Tan, Yaw Sing; Stokes, Jamie E.; Rahman, Taufiq; Verma, Chandra; Spring, David R.; Itzhaki, Laura S.

    2016-04-01

    Gankyrin is an ankyrin-repeat oncoprotein whose overexpression has been implicated in the development of many cancer types. Elevated gankyrin levels are linked to aberrant cellular events including enhanced degradation of tumour suppressor protein p53, and inhibition of gankyrin activity has therefore been identified as an attractive anticancer strategy. Gankyrin interacts with several partner proteins, and a number of these protein-protein interactions (PPIs) are of relevance to cancer. Thus, molecules that bind the PPI interface of gankyrin and interrupt these interactions are of considerable interest. Herein, we report the discovery of a small molecule termed cjoc42 that is capable of binding to gankyrin. Cell-based experiments demonstrate that cjoc42 can inhibit gankyrin activity in a dose-dependent manner: cjoc42 prevents the decrease in p53 protein levels normally associated with high amounts of gankyrin, and it restores p53-dependent transcription and sensitivity to DNA damage. The results represent the first evidence that gankyrin is a “druggable” target with small molecules.

  10. Discovery of a small-molecule binder of the oncoprotein gankyrin that modulates gankyrin activity in the cell

    PubMed Central

    Chattopadhyay, Anasuya; O’Connor, Cornelius J.; Zhang, Fengzhi; Galvagnion, Celine; Galloway, Warren R. J. D.; Tan, Yaw Sing; Stokes, Jamie E.; Rahman, Taufiq; Verma, Chandra; Spring, David R.; Itzhaki, Laura S.

    2016-01-01

    Gankyrin is an ankyrin-repeat oncoprotein whose overexpression has been implicated in the development of many cancer types. Elevated gankyrin levels are linked to aberrant cellular events including enhanced degradation of tumour suppressor protein p53, and inhibition of gankyrin activity has therefore been identified as an attractive anticancer strategy. Gankyrin interacts with several partner proteins, and a number of these protein-protein interactions (PPIs) are of relevance to cancer. Thus, molecules that bind the PPI interface of gankyrin and interrupt these interactions are of considerable interest. Herein, we report the discovery of a small molecule termed cjoc42 that is capable of binding to gankyrin. Cell-based experiments demonstrate that cjoc42 can inhibit gankyrin activity in a dose-dependent manner: cjoc42 prevents the decrease in p53 protein levels normally associated with high amounts of gankyrin, and it restores p53-dependent transcription and sensitivity to DNA damage. The results represent the first evidence that gankyrin is a “druggable” target with small molecules. PMID:27046077

  11. Sub-lethal activity of small molecules from natural sources and their synthetic derivatives against biofilm forming nosocomial pathogens.

    PubMed

    Villa, Federica; Villa, Stefania; Gelain, Arianna; Cappitelli, Francesca

    2013-01-01

    Nowadays, the patient safety is seriously jeopardized by the emergence and spread of nosocomial pathogens in the form of biofilm that is resistant to traditional and affordable antimicrobials. Although advances in organic synthesis have extended the lifetime of classic antibiotics through synthetic modifications, the search of innovative antibiofilm compounds from natural sources can provide new templates, novel targets and unique mechanisms that should have advantages over known antimicrobial agents. Testing sub-lethal concentrations of crude extracts and/or isolated compounds from plants and microorganisms is critical to acting on mechanisms subtler than the killing activity, e.g. those influencing the multicellular behavior, offering an elegant way to develop novel antimicrobial-free antibiofilm strategies. Herein we discussed the search and biological activity of small molecules from natural sources and their synthetic derivatives able to modulate biofilm genesis of nosocomial pathogens through non-microbicidal mechanisms (sub-lethal concentrations). The present work offers an overview about the approaches applied to the discovery of lead small molecules including a) conventional drug design methods like screening of chemical compounds obtained from nature and b) computer- aided drug design approaches. Finally, a classification (not exhaustive but representative) based on the natural origin of small molecules and their synthetic derivatives was reported. The information presented in this review should be of interest to a broad range of disciplines and represents an effort to summarize experimental research and advances in this field. PMID:24200356

  12. A structure activity-relationship study of the bacterial signal molecule HHQ reveals swarming motility inhibition in Bacillus atrophaeus.

    PubMed

    Reen, F Jerry; Shanahan, Rachel; Cano, Rafael; O'Gara, Fergal; McGlacken, Gerard P

    2015-05-21

    The sharp rise in antimicrobial resistance has been matched by a decline in the identification and clinical introduction of new classes of drugs to target microbial infections. Thus new approaches are being sought to counter the pending threat of a post-antibiotic era. In that context, the use of non-growth limiting small molecules, that target virulence behaviour in pathogens, has emerged as a solution with real clinical potential. We have previously shown that two signal molecules (HHQ and PQS) from the nosocomial pathogen Pseudomonas aeruginosa have modulatory activity towards other microorganisms. This current study involves the synthesis and evaluation of analogues of HHQ towards swarming and biofilm virulence behaviour in Bacillus atrophaeus, a soil bacterium and co-inhibitor with P. aeruginosa. Compounds with altered C6-C8 positions on the anthranilate-derived ring of HHQ, display a surprising degree of biological specificity, with certain candidates displaying complete motility inhibition. In contrast, anti-biofilm activity of the parent molecule was completely lost upon alteration at any position indicating a remarkable degree of specificity and delineation of phenotype. PMID:25880413

  13. Lysine-Based Small Molecules That Disrupt Biofilms and Kill both Actively Growing Planktonic and Nondividing Stationary Phase Bacteria.

    PubMed

    Konai, Mohini M; Haldar, Jayanta

    2015-10-01

    The emergence of bacterial resistance is a major threat to global health. Alongside this issue, formation of bacterial biofilms is another cause of concern because most antibiotics are ineffective against these recalcitrant microbial communities. Ideal future antibacterial therapeutics should possess both antibacterial and anti-biofilm activities. In this study we engineered lysine-based small molecules, which showed not only commendable broad-spectrum antibacterial activity but also potent biofilm-disrupting properties. Synthesis of these lipophilic lysine-norspermidine conjugates was achieved in three simple reaction steps, and the resultant molecules displayed potent antibacterial activity against various Gram-positive (Staphylococcus aureus, Enterococcus faecium) and Gram-negative bacteria (Escherichia coli) including drug-resistant superbugs MRSA (methicillin-resistant S. aureus), VRE (vancomycin-resistant E. faecium), and β-lactam-resistant Klebsiella pneumoniae. An optimized compound in the series showed activity against planktonic bacteria in the concentration range of 3-10 μg/mL, and bactericidal activity against stationary phase S. aureus was observed within an hour. The compound also displayed about 120-fold selectivity toward both classes of bacteria (S. aureus and E. coli) over human erythrocytes. This rapidly bactericidal compound primarily acts on bacteria by causing significant membrane depolarization and K(+) leakage. Most importantly, the compound disrupted preformed biofilms of S. aureus and did not trigger bacterial resistance. Therefore, this class of compounds has high potential to be developed as future antibacterial drugs for treating infections caused by planktonic bacteria as well as bacterial biofilms. PMID:27623313

  14. Monocyte exosomes induce adhesion molecules and cytokines via activation of NF-κB in endothelial cells.

    PubMed

    Tang, Norina; Sun, Bing; Gupta, Archana; Rempel, Hans; Pulliam, Lynn

    2016-09-01

    HIV-infected individuals have activated monocytes with an IFNα phenotype and elevated levels of circulating LPS. These individuals also have a risk of premature cardiovascular disease. The effect of activated monocyte exosomes (Exos) on endothelial cells is unknown. To determine whether Exos from immune-activated monocytes could alter endothelial cell expression and contribute to monocyte/macrophage transmigration and adhesion, we isolated Exos from monocytes stimulated with IFNα, LPS, or both (I/L). We show that monocyte Exos contain different inflammatory microRNA cargo depending on stimulation. When LPS Exos or I/L Exos were added to HUVECs, we found a significant increase in adhesion molecule ICAM-1, chemokine ligand (CCL)-2, and cytokine IL-6 mRNAs and proteins compared with cells treated with IFNα Exos or Exos derived from unstimulated monocytes. Inhibition of transcription factor NF-κB, a common inflammatory cytokine pathway, prevented induction of CCL2, IL6, and ICAM1 Inhibition of TLR4 resulted in differential blockage of the targets. Our results demonstrate for the first time that primary human monocyte Exos enter endothelial cells and cause dysfunction via the TLR4 and NF-κB pathways, which may contribute to heart disease in HIV infection and other diseases involving chronic immune activation.-Tang, N., Sun, B., Gupta, A., Rempel, H., Pulliam, L. Monocyte exosomes induce adhesion molecules and cytokines via activation of NF-κB in endothelial cells. PMID:27226520

  15. X-ray Absorption and Emission Study of Dioxygen Activation by a Small-Molecule Manganese Complex

    PubMed Central

    Rees, Julian A.; Martin-Diaconescu, Vlad; Kovacs, Julie A.; DeBeer, Serena

    2015-01-01

    Manganese K-edge X-ray absorption (XAS) and Kβ emission (XES) spectroscopies were used to investigate the factors contributing to O–O bond activation in a small-molecule system. The recent structural characterization of a metastable peroxo-bridged dimeric Mn(III)2 complex derived from dioxygen has provided the first opportunity to obtain X-ray spectroscopic data on this type of species. Ground state and time-dependent density functional theory calculations have provided further insight into the nature of the transitions in XAS pre-edge and valence-to-core (VtC) XES spectral regions. An experimentally validated electronic structure description has also enabled the determination of structural and electronic factors that govern peroxo bond activation, and have allowed us to propose both a rationale for the metastability of this unique compound, as well as potential future ligand designs which may further promote or inhibit O–O bond scission. Finally, we have explored the potential of VtC XES as an element-selective probe of both the coordination mode and degree of activation of peroxomanganese adducts. The comparison of these results to a recent VtC XES study of iron-mediated dintrogen activation helps to illustrate the factors that may determine the success of this spectroscopic method for future studies of small-molecule activation at transition metal sites. PMID:26061165

  16. Lysine-Based Small Molecules That Disrupt Biofilms and Kill both Actively Growing Planktonic and Nondividing Stationary Phase Bacteria.

    PubMed

    Konai, Mohini M; Haldar, Jayanta

    2015-10-01

    The emergence of bacterial resistance is a major threat to global health. Alongside this issue, formation of bacterial biofilms is another cause of concern because most antibiotics are ineffective against these recalcitrant microbial communities. Ideal future antibacterial therapeutics should possess both antibacterial and anti-biofilm activities. In this study we engineered lysine-based small molecules, which showed not only commendable broad-spectrum antibacterial activity but also potent biofilm-disrupting properties. Synthesis of these lipophilic lysine-norspermidine conjugates was achieved in three simple reaction steps, and the resultant molecules displayed potent antibacterial activity against various Gram-positive (Staphylococcus aureus, Enterococcus faecium) and Gram-negative bacteria (Escherichia coli) including drug-resistant superbugs MRSA (methicillin-resistant S. aureus), VRE (vancomycin-resistant E. faecium), and β-lactam-resistant Klebsiella pneumoniae. An optimized compound in the series showed activity against planktonic bacteria in the concentration range of 3-10 μg/mL, and bactericidal activity against stationary phase S. aureus was observed within an hour. The compound also displayed about 120-fold selectivity toward both classes of bacteria (S. aureus and E. coli) over human erythrocytes. This rapidly bactericidal compound primarily acts on bacteria by causing significant membrane depolarization and K(+) leakage. Most importantly, the compound disrupted preformed biofilms of S. aureus and did not trigger bacterial resistance. Therefore, this class of compounds has high potential to be developed as future antibacterial drugs for treating infections caused by planktonic bacteria as well as bacterial biofilms.

  17. Chemical activation of molecules by metals: Experimental studies of electron distributions and bonding

    SciTech Connect

    Lichtenberger, D.L.

    1991-10-01

    The formal relationship between measured molecular ionization energies and thermodynamic bond dissociation energies has been developed into a single equation which unifies the treatment of covalent bonds, ionic bonds, and partially ionic bonds. This relationship has been used to clarify the fundamental thermodynamic information relating to metal-hydrogen, metal-alkyl, and metal-metal bond energies. We have been able to obtain a direct observation and measurement of the stabilization energy provided by the agostic interaction of the C-H bond with the metal. The ionization energies have also been used to correlate the rates of carbonyl substitution reactions of ({eta}{sup 5}-C{sub 5}H{sub 4}X)Rh(CO){sub 2} complexes, and to reveal the electronic factors that control the stability of the transition state. The extent that the electronic features of these bonding interactions transfer to other chemical systems is being investigated in terms of the principle of additivity of ligand electronic effects. Specific examples under study include metal- phosphines, metal-halides, and metallocenes. Especially interesting has been the recent application of these techniques to the characterization of the soccer-ball shaped C{sub 60} molecule, buckminsterfullerene, and its interaction with a metal surface. The high-resolution valence ionizations in the gas phase reveal the high symmetry of the molecule, and studies of thin films of C{sub 60} reveal weak intermolecular interactions. Scanning tunneling and atomic force microscopy reveal the arrangement of spherical molecules on gold substrates, with significant delocalization of charge from the metal surface. 21 refs.

  18. Recent advances in small organic molecules as DNA intercalating agents: synthesis, activity, and modeling.

    PubMed

    Rescifina, Antonio; Zagni, Chiara; Varrica, Maria Giulia; Pistarà, Venerando; Corsaro, Antonino

    2014-03-01

    The interaction of small molecules with DNA plays an essential role in many biological processes. As DNA is often the target for majority of anticancer and antibiotic drugs, study about the interaction of drug and DNA has a key role in pharmacology. Moreover, understanding the interactions of small molecules with DNA is of prime significance in the rational design of more powerful and selective anticancer agents. Two of the most important and promising targets in cancer chemotherapy include DNA alkylating agents and DNA intercalators. For these last the DNA recognition is a critical step in their anti-tumor action and the intercalation is not only one kind of the interactions in DNA recognition but also a pivotal step of several clinically used anti-tumor drugs such as anthracyclines, acridines and anthraquinones. To push clinical cancer therapy, the discovery of new DNA intercalators has been considered a practical approach and a number of intercalators have been recently reported. The intercalative binding properties of such molecules can also be harnessed as diagnostic probes for DNA structure in addition to DNA-directed therapeutics. Moreover, the problem of intercalation site formation in the undistorted B-DNA of different length and sequence is matter of tremendous importance in molecular modeling studies and, nowadays, three models of DNA intercalation targets have been proposed that account for the binding features of intercalators. Finally, despite DNA being an important target for several drugs, most of the docking programs are validated only for proteins and their ligands. Therefore, a default protocol to identify DNA binding modes which uses a modified canonical DNA as receptor is needed.

  19. Friction-induced enhancement in the optical activity of interacting chiral molecules

    NASA Astrophysics Data System (ADS)

    Bargueño, Pedro; Peñate-Rodríguez, Helen C.; Gonzalo, Isabel; Sols, Fernando; Miret-Artés, Salvador

    2011-11-01

    The stability of chiral molecules described by a non-linear two-state system which accounts for mean-field interactions between different isomers, including any external chiral influence (in particular, the parity violating energy difference) is investigated. By introducing the population and phase difference of the chiral states as a pair of canonical variables, driving an analogy to a bosonic Josephson junction, our study to include dissipative effects in condensed phase described by a Caldeira-Leggett like Hamiltonian is extended using the Langevin formalism. Dissipative effects produce an enhancement in the population difference, not leading to racemization.

  20. A new multiplexing single molecule technique for measuring restriction enzyme activity

    NASA Astrophysics Data System (ADS)

    Harbottle, Allison; Cavanaugh, Jillian; Gordon, Wendy; Loparo, Joseph; Price, Allen

    2012-02-01

    We present a new multiplexing single molecule method for observing the cleavage of DNAs by restriction enzymes. DNAs are attached to a surface at one end using a biotin-streptavidin link and to a micro bead at the other end via a digoxigenin-antidigoxigenin link. The DNAs are stretched by applying a flow. After introduction of the restriction enzyme, the exact time of cleavage of individual DNAs is recorded with video microscopy. We can image hundreds to thousands of DNAs in a single experiment. We are using our technique to search for the signature of facilitated diffusion in the measured rate dependence on ionic strength.

  1. Modification and structure-activity relationship of a small molecule HIV-1 inhibitor targeting the viral envelope glycoprotein gp120.

    PubMed

    Wang, Jingsong; Le, Nhut; Heredia, Alonso; Song, Haijing; Redfield, Robert; Wang, Lai-Xi

    2005-05-01

    This paper describes selected modification and structure-activity relationship of the small molecule HIV-1 inhibitor, 4-benzoyl-1-[(4-methoxy-1H-pyrrolo[2,3-b]pyridin-3-yl)oxoacetyl]-2-(R)-methylpiperazine (BMS-378806). The results revealed: i) that both the presence and configuration (R vs. S) of the 3-methyl group on the piperazine moiety are important for the antiviral activity, with the 3-(R)-methyl derivatives showing the highest activity; ii) that the electronegativity of the C-4 substituent on the indole or azaindole ring seems to be important for the activity, with a small, electron-donating group such as a fluoro or a methoxy group showing enhanced activity, while a nitro group diminishes the activity; iii) that the N-1 position of the indole ring is not eligible for modification without losing activity; and iv) that bulky groups around the C-4 position of the indole or azaindole ring diminish the activity, probably due to steric hindrance in the binding. We found that a synthetic bivalent compound with two BMS-378806 moieties being tethered by a spacer demonstrated about 5-fold enhanced activity in an nM range against HIV-1 infection than the corresponding monomeric inhibitor. But the polyacrylamide-based polyvalent compounds did not show inhibitory activity at up to 200 nM.

  2. Modification and structure-activity relationship of a small molecule HIV-1 inhibitor targeting the viral envelope glycoprotein gp120.

    PubMed

    Wang, Jingsong; Le, Nhut; Heredia, Alonso; Song, Haijing; Redfield, Robert; Wang, Lai-Xi

    2005-05-01

    This paper describes selected modification and structure-activity relationship of the small molecule HIV-1 inhibitor, 4-benzoyl-1-[(4-methoxy-1H-pyrrolo[2,3-b]pyridin-3-yl)oxoacetyl]-2-(R)-methylpiperazine (BMS-378806). The results revealed: i) that both the presence and configuration (R vs. S) of the 3-methyl group on the piperazine moiety are important for the antiviral activity, with the 3-(R)-methyl derivatives showing the highest activity; ii) that the electronegativity of the C-4 substituent on the indole or azaindole ring seems to be important for the activity, with a small, electron-donating group such as a fluoro or a methoxy group showing enhanced activity, while a nitro group diminishes the activity; iii) that the N-1 position of the indole ring is not eligible for modification without losing activity; and iv) that bulky groups around the C-4 position of the indole or azaindole ring diminish the activity, probably due to steric hindrance in the binding. We found that a synthetic bivalent compound with two BMS-378806 moieties being tethered by a spacer demonstrated about 5-fold enhanced activity in an nM range against HIV-1 infection than the corresponding monomeric inhibitor. But the polyacrylamide-based polyvalent compounds did not show inhibitory activity at up to 200 nM. PMID:15858664

  3. Differences in Home Food and Activity Environments between Obese and Healthy Weight Families of Preschool Children

    ERIC Educational Resources Information Center

    Boles, Richard E.; Scharf, Cynthia; Filigno, Stephanie S.; Saelens, Brian E.; Stark, Lori J.

    2013-01-01

    Objective: To develop and test a home food and activity instrument to discriminate between the home environments of obese and healthy weight preschool children. Design: A modified questionnaire about home environments was tested as an observation tool. Setting: Family homes. Participants: A total of 35 obese children with at least 1 obese…

  4. Sexually Active Adolescent Women: Assessing Family and Peer Relationships Using Event History Calendars

    ERIC Educational Resources Information Center

    Saftner, Melissa Ann; Martyn, Kristy Kiel; Lori, Jody Rae

    2011-01-01

    The purpose of this qualitative study is to explore family and peer relationships (including support and influence on risk behavior) among sexually active European American and African American adolescent girls in the context of risk behaviors documented on retrospective event history calendars (EHCs) and in interviews. The EHCs were completed by…

  5. Nutrition and Physical Activity Practices in Childcare Centers versus Family Childcare Homes

    ERIC Educational Resources Information Center

    Natale, Ruby; Page, Monica; Sanders, Lee

    2014-01-01

    Obesity rates among preschool-aged children have doubled in the past 10 years, and 60% of these children spend the majority of their day in childcare facilities. Few studies have examined the quality of nutrition and physical activity practices in childcare centers as compared to family childcare homes. The purpose of this study is to determine if…

  6. Excess screen time in US children: association with family rules and alternative activities.

    PubMed

    Gingold, Janet A; Simon, Alan E; Schoendorf, Kenneth C

    2014-01-01

    We describe the association of screen time in excess of American Academy of Pediatrics recommendations (≤2 h/d) with family television-use policies and regular nonscreen activities among US school-aged children. Data from the 2007 National Survey of Children's Health were used. The sum of minutes spent on television, videos, video games, and recreational computer use was calculated for children 6 to 17 years old. Bivariate and multivariate logistic regression models were used to calculate relative odds of exceeding American Academy of Pediatrics guidelines and of heavy screen use (>4 h/d) for varying family media-use policies and frequency of alternative activities (physical activity and family meals). In all, 49% of school-aged children had screen time >2 h/d and 16% had screen time >4 h/d. Lower frequency of family meals, presence of TV in the bedroom, absence of rules about TV viewing, and less physical activity were associated with both >2 and >4 hours per day of screen time. PMID:23922251

  7. Excess screen time in US children: association with family rules and alternative activities.

    PubMed

    Gingold, Janet A; Simon, Alan E; Schoendorf, Kenneth C

    2014-01-01

    We describe the association of screen time in excess of American Academy of Pediatrics recommendations (≤2 h/d) with family television-use policies and regular nonscreen activities among US school-aged children. Data from the 2007 National Survey of Children's Health were used. The sum of minutes spent on television, videos, video games, and recreational computer use was calculated for children 6 to 17 years old. Bivariate and multivariate logistic regression models were used to calculate relative odds of exceeding American Academy of Pediatrics guidelines and of heavy screen use (>4 h/d) for varying family media-use policies and frequency of alternative activities (physical activity and family meals). In all, 49% of school-aged children had screen time >2 h/d and 16% had screen time >4 h/d. Lower frequency of family meals, presence of TV in the bedroom, absence of rules about TV viewing, and less physical activity were associated with both >2 and >4 hours per day of screen time.

  8. The Role of Family Experiences for Adolescents' Readiness to Use and Participate in Illegal Political Activity

    ERIC Educational Resources Information Center

    Glatz, Terese; Dahl, Viktor

    2016-01-01

    This study used reactance theory as a starting point to explain what role a perceived undemocratic and controlling family has for adolescents' readiness to use illegal political activity. Additionally, we examined whether adolescents' readiness to use illegal political means was related to actual political behaviour, which has been lacking in…

  9. Children's Evaluations of Gender-Stereotypic Household Activities in the Family Context

    ERIC Educational Resources Information Center

    Schuette, Christine; Killen, Melanie

    2009-01-01

    Research Findings: The goal of this study was to analyze children's (N = 120) evaluations about parental decisions regarding gender-stereotypic household family activities. Children at 5, 8, and 10 years of age were individually interviewed. Contrary to prevailing findings, the majority of children used stereotypic expectations and…

  10. 24 CFR 1000.104 - What families are eligible for affordable housing activities?

    Code of Federal Regulations, 2013 CFR

    2013-04-01

    ... 24 Housing and Urban Development 4 2013-04-01 2013-04-01 false What families are eligible for affordable housing activities? 1000.104 Section 1000.104 Housing and Urban Development REGULATIONS RELATING TO HOUSING AND URBAN DEVELOPMENT (CONTINUED) OFFICE OF ASSISTANT SECRETARY FOR PUBLIC AND...

  11. 24 CFR 1000.104 - What families are eligible for affordable housing activities?

    Code of Federal Regulations, 2014 CFR

    2014-04-01

    ... 24 Housing and Urban Development 4 2014-04-01 2014-04-01 false What families are eligible for affordable housing activities? 1000.104 Section 1000.104 Housing and Urban Development REGULATIONS RELATING TO HOUSING AND URBAN DEVELOPMENT (CONTINUED) OFFICE OF ASSISTANT SECRETARY FOR PUBLIC AND...

  12. 77 FR 27542 - Agency Information Collection Activities (Bereaved Family Member Satisfaction Survey) Under OMB...

    Federal Register 2010, 2011, 2012, 2013, 2014

    2012-05-10

    ... AFFAIRS Agency Information Collection Activities (Bereaved Family Member Satisfaction Survey) Under OMB...: In compliance with the Paperwork Reduction Act (PRA) of 1995 (44 U.S.C. 3501-3521), this notice....Regulations.gov ; or to VA's OMB Desk Officer, OMB Human Resources and Housing Branch, New Executive...

  13. Activation of protein phosphatase 1 by a small molecule designed to bind to the enzyme's regulatory site.

    PubMed

    Tappan, Erin; Chamberlin, A Richard

    2008-02-01

    The activity of protein phosphatase 1 (PP1), a serine-threonine phosphatase that participates ubiquitously in cellular signaling, is controlled by a wide variety of regulatory proteins that interact with PP1 at an allosteric regulatory site that recognizes a "loose" consensus sequence (usually designated as RVXF) found in all such regulatory proteins. Peptides containing the regulatory consensus sequence have been found to recapitulate the binding and PP1 activity modulation of the regulatory proteins, suggesting that it might be possible to design small-molecule surrogates that activate PP1 rather than inhibiting it. This prospect constitutes a largely unexplored way of controlling signaling pathways that could be functionally complementary to the much more extensively explored stratagem of kinase inhibition. Based on these principles, we have designed a microcystin analog that activates PP1. PMID:18291321

  14. Histone deacetylase inhibitor givinostat: the small-molecule with promising activity against therapeutically challenging haematological malignancies.

    PubMed

    Ganai, Shabir Ahmad

    2016-08-01

    Histone acetyl transferases and histone deacetylases (HDACs) are counteracting epigenetic enzymes regulating the turnover of histone acetylation thereby regulating transcriptional events in a precise manner. Deregulation of histone acetylation caused by aberrant expression of HDACs plays a key role in tumour onset and progression making these enzymes as candidate targets for anticancer drugs and therapy. Small-molecules namely histone deacetylase inhibitors (HDACi) modulating the biological function of HDACs have shown multiple biological effects including differentiation, cell cycle arrest and apoptosis in tumour models. HDACi in general have been described in plethora of reviews with respect to various cancers. However, no review article is available describing thoroughly the role of inhibitor givinostat (ITF2357 or [6-(diethylaminomethyl) naphthalen-2-yl] methyl N-[4-(hydroxycarbamoyl) phenyl] carbamate) in haematological malignancies. Thus, the present review explores the intricate role of novel inhibitor givinostat in the defined malignancies including multiple myeloma, acute myelogenous leukaemia, Hodgkin's and non-Hodgkin's lymphoma apart from myeloproliferative neoplasms. The distinct molecular mechanisms triggered by this small-molecule inhibitor in these cancers to exert cytotoxic effect have also been dealt with. The article also highlights the combination strategy that can be used for enhancing the therapeutic efficiency of this inhibitor in the upcoming future. PMID:27121910

  15. Can Si-doped graphene activate or dissociate O2 molecule?

    PubMed

    Chen, Ying; Yang, Xiao-chun; Liu, Yue-jie; Zhao, Jing-xiang; Cai, Qing-hai; Wang, Xuan-zhang

    2013-02-01

    Recently, the adsorption and dissociation of oxygen molecule on a metal-free catalyst has attracted considerable attention due to the fundamental and industrial importance. In the present work, we have investigated the adsorption and dissociation of O(2) molecule on pristine and silicon-doped graphene, using density functional theory calculations. We found that O(2) is firstly adsorbed on Si-doped graphene by [2+1] or [2+2] cycloaddition, with adsorption energies of -1.439 and -0.856eV, respectively. Following this, the molecularly adsorbed O(2) can be dissociated in different pathways. In the most favorable reaction path, the dissociation barrier of adsorbed O(2) is significantly reduced from 3.180 to 0.206eV due to the doping of silicon into graphene. Our results may be useful to further develop effective metal-free catalysts for the oxygen reduction reactions (ORRs), thus greatly widening the potential applications of graphene. PMID:23261882

  16. Structural Insights into the Anti-HIV Activity of the Oscillatoria agardhii Agglutinin Homolog Lectin Family*

    PubMed Central

    Koharudin, Leonardus M. I.; Kollipara, Sireesha; Aiken, Christopher; Gronenborn, Angela M.

    2012-01-01

    Oscillatoria agardhii agglutinin homolog (OAAH) proteins belong to a recently discovered lectin family. All members contain a sequence repeat of ∼66 amino acids, with the number of repeats varying among different family members. Apart from data for the founding member OAA, neither three-dimensional structures, information about carbohydrate binding specificities, nor antiviral activity data have been available up to now for any other members of the OAAH family. To elucidate the structural basis for the antiviral mechanism of OAAHs, we determined the crystal structures of Pseudomonas fluorescens and Myxococcus xanthus lectins. Both proteins exhibit the same fold, resembling the founding family member, OAA, with minor differences in loop conformations. Carbohydrate binding studies by NMR and x-ray structures of glycan-lectin complexes reveal that the number of sugar binding sites corresponds to the number of sequence repeats in each protein. As for OAA, tight and specific binding to α3,α6-mannopentaose was observed. All the OAAH proteins described here exhibit potent anti-HIV activity at comparable levels. Altogether, our results provide structural details of the protein-carbohydrate interaction for this novel lectin family and insights into the molecular basis of their HIV inactivation properties. PMID:22865886

  17. Walking molecules.

    PubMed

    von Delius, Max; Leigh, David A

    2011-07-01

    Movement is intrinsic to life. Biologists have established that most forms of directed nanoscopic, microscopic and, ultimately, macroscopic movements are powered by molecular motors from the dynein, myosin and kinesin superfamilies. These motor proteins literally walk, step by step, along polymeric filaments, carrying out essential tasks such as organelle transport. In the last few years biological molecular walkers have inspired the development of artificial systems that mimic aspects of their dynamics. Several DNA-based molecular walkers have been synthesised and shown to walk directionally along a track upon sequential addition of appropriate chemical fuels. In other studies, autonomous operation--i.e. DNA-walker migration that continues as long as a complex DNA fuel is present--has been demonstrated and sophisticated tasks performed, such as moving gold nanoparticles from place-to-place and assistance in sequential chemical synthesis. Small-molecule systems, an order of magnitude smaller in each dimension and 1000× smaller in molecular weight than biological motor proteins or the walker systems constructed from DNA, have also been designed and operated such that molecular fragments can be progressively transported directionally along short molecular tracks. The small-molecule systems can be powered by light or chemical fuels. In this critical review the biological motor proteins from the kinesin, myosin and dynein families are analysed as systems from which the designers of synthetic systems can learn, ratchet concepts for transporting Brownian substrates are discussed as the mechanisms by which molecular motors need to operate, and the progress made with synthetic DNA and small-molecule walker systems reviewed (142 references). PMID:21416072

  18. Circadian activity rhythm in pre-pubertal and pubertal marmosets (Callithrix jacchus) living in family groups.

    PubMed

    Melo, Paula R; Gonçalves, Bruno S B; Menezes, Alexandre A L; Azevedo, Carolina V M

    2016-03-01

    In marmosets, a phase advance was observed in activity onset in pubertal animals living in captivity under semi-natural conditions which had stronger correlation with the times of sunrise over the course of the year than the age of the animal. In order to evaluate the effect of puberty on the circadian activity rhythm in male and female marmosets living in family groups in controlled lighting conditions, the activity of 5 dyads of twins (4 ♀/♂ and 1 ♂/♂) and their respective parents was continuously monitored by actiwatches between the 4th and 12th months of age. The families were kept under LD 12:12 h with constant humidity and temperature. The onset of puberty was identified by monitoring fecal steroids. Juveniles showed higher totals of daily activity and differences in the daily distribution of activity in relation to parents, in which the bimodal profile was characterized by higher levels in evening activity in relation to morning activity. Regarding the phase, the activity onset and offset, occurred later in relation to parents. After entering puberty, the activity onset and offset occurred later and there was an increase in total daily activity. On the other hand, when assessing the effect of sex, only females showed a delay in the activity offset and an increase in total daily activity. Therefore, the circadian activity rhythm in marmosets has peculiar characteristics in the juvenile stage in relation to the total of daily activity, the onset and offset of the active phase, and the distribution of activity during this phase. Besides, the entering puberty was associated with a phase delay and increase on total daily activity, with differences between sexes, possibly due to hormonal influences and/or social modulation on rhythm. PMID:26724713

  19. Circadian activity rhythm in pre-pubertal and pubertal marmosets (Callithrix jacchus) living in family groups.

    PubMed

    Melo, Paula R; Gonçalves, Bruno S B; Menezes, Alexandre A L; Azevedo, Carolina V M

    2016-03-01

    In marmosets, a phase advance was observed in activity onset in pubertal animals living in captivity under semi-natural conditions which had stronger correlation with the times of sunrise over the course of the year than the age of the animal. In order to evaluate the effect of puberty on the circadian activity rhythm in male and female marmosets living in family groups in controlled lighting conditions, the activity of 5 dyads of twins (4 ♀/♂ and 1 ♂/♂) and their respective parents was continuously monitored by actiwatches between the 4th and 12th months of age. The families were kept under LD 12:12 h with constant humidity and temperature. The onset of puberty was identified by monitoring fecal steroids. Juveniles showed higher totals of daily activity and differences in the daily distribution of activity in relation to parents, in which the bimodal profile was characterized by higher levels in evening activity in relation to morning activity. Regarding the phase, the activity onset and offset, occurred later in relation to parents. After entering puberty, the activity onset and offset occurred later and there was an increase in total daily activity. On the other hand, when assessing the effect of sex, only females showed a delay in the activity offset and an increase in total daily activity. Therefore, the circadian activity rhythm in marmosets has peculiar characteristics in the juvenile stage in relation to the total of daily activity, the onset and offset of the active phase, and the distribution of activity during this phase. Besides, the entering puberty was associated with a phase delay and increase on total daily activity, with differences between sexes, possibly due to hormonal influences and/or social modulation on rhythm.

  20. An uncharacterized member of the ribokinase family in Thermococcus kodakarensis exhibits myo-inositol kinase activity.

    PubMed

    Sato, Takaaki; Fujihashi, Masahiro; Miyamoto, Yukika; Kuwata, Keiko; Kusaka, Eriko; Fujita, Haruo; Miki, Kunio; Atomi, Haruyuki

    2013-07-19

    Here we performed structural and biochemical analyses on the TK2285 gene product, an uncharacterized protein annotated as a member of the ribokinase family, from the hyperthermophilic archaeon Thermococcus kodakarensis. The three-dimensional structure of the TK2285 protein resembled those of previously characterized members of the ribokinase family including ribokinase, adenosine kinase, and phosphofructokinase. Conserved residues characteristic of this protein family were located in a cleft of the TK2285 protein as in other members whose structures have been determined. We thus examined the kinase activity of the TK2285 protein toward various sugars recognized by well characterized ribokinase family members. Although activity with sugar phosphates and nucleosides was not detected, kinase activity was observed toward d-allose, d-lyxose, d-tagatose, d-talose, d-xylose, and d-xylulose. Kinetic analyses with the six sugar substrates revealed high Km values, suggesting that they were not the true physiological substrates. By examining activity toward amino sugars, sugar alcohols, and disaccharides, we found that the TK2285 protein exhibited prominent kinase activity toward myo-inositol. Kinetic analyses with myo-inositol revealed a greater kcat and much lower Km value than those obtained with the monosaccharides, resulting in over a 2,000-fold increase in kcat/Km values. TK2285 homologs are distributed among members of Thermococcales, and in most species, the gene is positioned close to a myo-inositol monophosphate synthase gene. Our results suggest the presence of a novel subfamily of the ribokinase family whose members are present in Archaea and recognize myo-inositol as a substrate.

  1. An Uncharacterized Member of the Ribokinase Family in Thermococcus kodakarensis Exhibits myo-Inositol Kinase Activity*

    PubMed Central

    Sato, Takaaki; Fujihashi, Masahiro; Miyamoto, Yukika; Kuwata, Keiko; Kusaka, Eriko; Fujita, Haruo; Miki, Kunio; Atomi, Haruyuki

    2013-01-01

    Here we performed structural and biochemical analyses on the TK2285 gene product, an uncharacterized protein annotated as a member of the ribokinase family, from the hyperthermophilic archaeon Thermococcus kodakarensis. The three-dimensional structure of the TK2285 protein resembled those of previously characterized members of the ribokinase family including ribokinase, adenosine kinase, and phosphofructokinase. Conserved residues characteristic of this protein family were located in a cleft of the TK2285 protein as in other members whose structures have been determined. We thus examined the kinase activity of the TK2285 protein toward various sugars recognized by well characterized ribokinase family members. Although activity with sugar phosphates and nucleosides was not detected, kinase activity was observed toward d-allose, d-lyxose, d-tagatose, d-talose, d-xylose, and d-xylulose. Kinetic analyses with the six sugar substrates revealed high Km values, suggesting that they were not the true physiological substrates. By examining activity toward amino sugars, sugar alcohols, and disaccharides, we found that the TK2285 protein exhibited prominent kinase activity toward myo-inositol. Kinetic analyses with myo-inositol revealed a greater kcat and much lower Km value than those obtained with the monosaccharides, resulting in over a 2,000-fold increase in kcat/Km values. TK2285 homologs are distributed among members of Thermococcales, and in most species, the gene is positioned close to a myo-inositol monophosphate synthase gene. Our results suggest the presence of a novel subfamily of the ribokinase family whose members are present in Archaea and recognize myo-inositol as a substrate. PMID:23737529

  2. Identification of small molecule activators of the janus kinase/signal transducer and activator of transcription pathway using a cell-based screen.

    PubMed

    Tai, Zheng Fu; Zhang, Guo Lin; Wang, Fei

    2012-01-01

    Type I interferons (IFN-α/β) have been widely used in the treatment of many viral and malignant diseases by activation of the Janus kinase/signal transducer and activator of transcription (JAK/STAT) signaling pathway, but the side effects of protein-based IFN therapy severely limit their clinical use. Discovering small molecules to activate the JAK/STAT pathway will greatly facilitate the development of new drugs which have similar pharmacological function to IFNs but with fewer side effects. To screen a natural products-based library, we established a cell-based screening assay using human hepatoma HepG2 cells stably transfected with a plasmid where the luciferase reporter activity is driven by interferon α-stimulated response element (ISRE), the motif specifically recognized by type I IFN-induced activation of JAK/STAT pathway. Among 1,431 natural product compounds screened, four compounds (emodin, quercetin, apigenin and luteolin) were identified as activators of the JAK/STAT pathway. Further studies demonstrated that these four compounds could increase the endogenous antiviral gene expression regulated by the IFN-activated JAK/STAT pathway. The identified small molecule activators are valuable for structural modification and warrant further investigation for use in new antiviral drugs as IFN mimics or adjuvants.

  3. Identification of Novel Small Molecule Activators of Nuclear Factor-κB With Neuroprotective Action Via High-Throughput Screening

    PubMed Central

    Manuvakhova, Marina S.; Johnson, Guyla G.; White, Misti C.; Ananthan, Subramaniam; Sosa, Melinda; Maddox, Clinton; McKellip, Sara; Rasmussen, Lynn; Wennerberg, Krister; Hobrath, Judith V.; White, E. Lucile; Maddry, Joseph A.; Grimaldi, Maurizio

    2012-01-01

    Neuronal noncytokine-dependent p50/p65 nuclear factor-κB (the primary NF-κB complex in the brain) activation has been shown to exert neuroprotective actions. Thus neuronal activation of NF-κB could represent a viable neuroprotective target. We have developed a cell-based assay able to detect NF-κB expression enhancement, and through its use we have identified small molecules able to up-regulate NF-κB expression and hence trigger its activation in neurons. We have successfully screened approximately 300,000 compounds and identified 1,647 active compounds. Cluster analysis of the structures within the hit population yielded 14 enriched chemical scaffolds. One high-potency and chemically attractive representative of each of these 14 scaffolds and four singleton structures were selected for follow-up. The experiments described here highlighted that seven compounds caused noncanonical long-lasting NF-κB activation in primary astrocytes. Molecular NF-κB docking experiments indicate that compounds could be modulating NF-κB-induced NF-κB expression via enhancement of NF-κB binding to its own promoter. Prototype compounds increased p65 expression in neurons and caused its nuclear translocation without affecting the inhibitor of NF-κB (I-κB). One of the prototypical compounds caused a large reduction of glutamate-induced neuronal death. In conclusion, we have provided evidence that we can use small molecules to activate p65 NF-κB expression in neurons in a cytokine receptor-independent manner, which results in both long-lasting p65 NF-κB translocation/activation and decreased glutamate neurotoxicity. PMID:21046675

  4. A novel AKT inhibitor, AZD5363, inhibits phosphorylation of AKT downstream molecules, and activates phosphorylation of mTOR and SMG-1 dependent on the liver cancer cell type

    PubMed Central

    ZHANG, YUNCHENG; ZHENG, YUANWEN; FAHEEM, ALI; SUN, TIANTONG; LI, CHUNYOU; LI, ZHE; ZHAO, DIANTANG; WU, CHAO; LIU, JUN

    2016-01-01

    Due to frequent phosphoinositide 3-kinase (PI3K)/AKT/mammalian target of rapamycin (mTOR) signaling pathway dysregulation, AKT is typically accepted as a promising anticancer therapeutic target. mTOR, in particular, represents a suitable therapeutic target for hepatocellular carcinoma, whilst suppressor with morphogenetic effect on genitalia family member-1 (SMG-1) is believed to serve a potential tumor suppressor role in human cancer. Despite SMG-1 and mTOR belonging to the same PI3K-related kinase family, the interactions between them are not yet fully understood. In the present study, a novel pyrrolopyrimidine-derived compound, AZD5363, was observed to suppress proliferation in liver cancer Hep-G2 and Huh-7 cells by inhibiting the phosphorylation of downstream molecules in the AKT signal pathway, in a dose- and time-dependent manner. AZD5363 activated the phosphorylation of mTOR, dependent on the liver cancer cell type, as it may have differing effects in various liver cancer cell lines. Additionally, AZD5363 also activated SMG-1 within the same liver cancer cells types, which subsequently activated the phosphorylation of mTOR. In conclusion, the present study indicates that AZD5363 inhibited phosphorylation of AKT downstream molecules, and activated phosphorylation of mTOR and SMG-1, dependent on the liver cancer type. PMID:26998062

  5. The time-dependent generalized active space configuration interaction approach to correlated ionization dynamics of diatomic molecules

    NASA Astrophysics Data System (ADS)

    Bauch, S.; Larsson, H. R.; Hinz, C.; Bonitz, M.

    2016-03-01

    In this contribution, we review the time-dependent generalized-active-space configuration interaction (TD-GAS-CI) approach to the photoionization dynamics of atoms and molecules including electron correlation effects. It is based on the configuration interaction (CI) expansion of the many-body wave function and the restriction of the determinantal space to a reduced subspace. For its numerically efficient application to photoionization, a partially-rotated basis set is used which adopts features of a localized basis with a good reference description and a grid representation for escaping wave packets. After reviewing earlier applications of the theory, we address the strong-field ionization of a one-dimensional model of the four-electron LiH molecule using TD-GAS-CI and demonstrate the importance of electron-electron correlations in the ionization yield for different orientations of the molecule w.r.t the peak of the linearly polarized laser field. A pronounced orientation-dependent variation of the yield with the pulse duration and the level of considered electron-electron correlations is observed.

  6. Xalpha-DVM investigation of double water molecule interactions with active sites of alpha- and beta-subunits of hemoglobin

    NASA Astrophysics Data System (ADS)

    Yuryeva, Elmira I.

    In this work, the results of Xalpha-discrete variation method calculations of the electronic structure and interatomic parameters of chemical bonding between iron (II) and oxygen molecule with and without extra electrons and protons in active site (AS) of alpha- and beta-subunits of oxyhemoglobin are presented. The Skulachev model of O2 molecule existing in respiration medium in the 2H2O form was used. The introduction of extra electrons does not change considerably the interaction of the iron atom with the O2 oxygen molecule, but strengthens the repulsion in the Fe bond N bonds. In this case, the estimated effective charge of the iron atom is +1.8/1.5e for AS of alpha-/beta-subunits of oxyhemoglobin, and the magnetic moment of iron atoms becomes zero. The deoxygenation effect of the AS of the alpha- and beta-subunits of oxyhemoglobin is due to the ability of extra protons to break down covalent attraction between the iron atom and the nearest oxygen atom and also to weakening of the repulsive component of the covalent Fe bond N interactions.

  7. Surface active molecules: preparation and properties of long chain n-acyl-l-alpha-amino-omega-guanidine alkyl acid derivatives.

    PubMed

    Infante, R; Dominguez, J G; Erra, P; Julia, R; Prats, M

    1984-12-01

    Synopsis A new route for the synthesis of long chain N(alpha)-acyl-l-alpha-amino-omega-guamdine alkyl acid derivatives, with cationic or amphoteric character has been established. The general formula of these compounds is shown below. A physico-chemical and antimicrobial study of these products as a function of the alkyl ester or sodium salt (R), the straight chain length of the fatty acid residue (x) and the number of carbons between the omega-guanidine and omega-carboxyl group (n) has been investigated. The water solubility, surface tension, critical micelle concentration (c.m.c.) and minimum inhibitory concentration (MIC) against Gram-positive and Gram-negative bacteria (including Pseudomonas) has been determined. Dicyclohexylcarbodiimide has been used to condense fatty acids and alpha-amino-omega-guanidine alkyl acids. In these conditions protection of the omega-guanidine group is not necessary. The main characteristic of this synthetic procedure is the use of very mild experimental conditions (temperature, pH) to form the amide linkage which leads to pure optical compounds in high yield in the absence of electrolytes. The results show that some structural modifications, particularly the protection of the carboxyl group, promote variations of the surfactant and antimicrobial properties. Only those molecules with the blocked carboxyl group (cationic molecules, where R = Me, Et or Pr) showed a good surfactant and antimicrobial activity. When the carboxyl group was unprotected (amphoteric molecules, where R = Na(+)) the resulting compounds were inactive.

  8. Understanding the adsorption behavior of surface active molecules on ZnO nanostructures by experimental and first-principles calculations.

    PubMed

    Singh, Baljinder; Singh, Satvinder; Singh, Janpreet; Saini, G S S; Mehta, D S; Singh, Gurinder; Tripathi, S K; Kaura, Aman

    2015-11-11

    Zinc oxide (ZnO) nanostructures with different morphologies are prepared in the presence of surface active molecules such as sodium dodecyl sulphate (SDS), Tween 80 and Triton X-100 by a chemical method. The experimental and first principles methods are employed to understand the microscopic origin of the asymmetric growth mechanism of ZnO in the presence of various surface active molecules. Effect of increase in the amount of surface active molecules and temperature is studied on the growth morphology of ZnO. An innovative method is developed to synthesize ZnO nanowires (NWs) in the presence of SDS. Spherical nanoparticles (NPs) to spherical clusters are obtained in the presence of Triton X-100 and Tween 80. These results are then supported by first principles calculations. The adsorption of the -OH functional group on both polar and nonpolar surfaces of ZnO is modelled by using density functional theory (DFT). The calculated binding energy (BE) is almost equivalent on both the surfaces with no preference on any particular surface. The calculated value of BE shows that the -OH group is physio-adsorbed on both the surfaces. This results in the spherical morphology of nanoparticles prepared in the presence of Tween 80. Bader charge analysis shows that the charge transfer mainly takes place on top two layers of the ZnO(101[combining macron]0) surface. The absence of high values of electron localization function (ELF) reflects the lack of covalent bonding between the -OH group and the ZnO(101[combining macron]0) surface. PMID:26510134

  9. Activity-dependent mobilization of the adhesion molecule polysialic NCAM to the cell surface of neurons and endocrine cells.

    PubMed Central

    Kiss, J Z; Wang, C; Olive, S; Rougon, G; Lang, J; Baetens, D; Harry, D; Pralong, W F

    1994-01-01

    The alpha-2,8-linked sialic acid polymer (PSA) on the neural cell adhesion molecule (NCAM) is an important regulator of cell surface interactions. We have examined the translocation of PSA-NCAM to the surface of cultured cortical neurons and insulin secreting beta cells under different conditions of cell activity. Endoneuraminidase N, an enzyme that specifically cleaves PSA chains, was used to remove pre-existing PSA from the plasma membrane and the re-expression of the molecule was monitored by immunocytochemistry. Punctate PSA immunostaining was restored on the surface of 68% of neurons within 1 h. This recovery was almost completely prevented by tetrodotoxin, suggesting that spontaneous electrical activity is required. K+ depolarization (50 mM) allowed recovery of PSA surface staining in the presence of tetrodotoxin and this effect required the presence of extracellular Ca2+. Rapid redistribution of PSA-NCAM to the surface of beta cells was observed under conditions that stimulate insulin secretion. Ca2+ channel inhibition decreased both PSA-NCAM expression and insulin secretion to control, non-stimulated levels. Finally, subcellular fractionation of an insulin-secreting cell line showed that the secretory vesicle fraction is highly enriched in PSA-NCAM. These results suggest that PSA-NCAM can be translocated to the cell surface via regulated exocytosis. Taken together, our results provide unprecedented evidence linking cell activity and PSA-NCAM expression, and suggest a mechanism for rapid modulation of cell surface interactions. Images PMID:7957094

  10. A small molecule inhibitor for ATPase activity of Hsp70 and Hsc70 enhances the immune response to protein antigens

    NASA Astrophysics Data System (ADS)

    Baek, Kyung-Hwa; Zhang, Haiying; Lee, Bo Ryeong; Kwon, Young-Guen; Ha, Sang-Jun; Shin, Injae

    2015-12-01

    The ATPase activities of Hsp70 and Hsc70 are known to be responsible for regulation of various biological processes. However, little is known about the roles of Hsp70 and Hsc70 in modulation of immune responses to antigens. In the present study, we investigated the effect of apoptozole (Az), a small molecule inhibitor of Hsp70 and Hsc70, on immune responses to protein antigens. The results show that mice administered with both protein antigen and Az produce more antibodies than those treated with antigen alone, showing that Az enhances immune responses to administered antigens. Treatment of mice with Az elicits production of antibodies with a high IgG2c/IgG1 ratio and stimulates the release of Th1 and Th2-type cytokines, suggesting that Az activates the Th1 and Th2 immune responses. The observations made in the present study suggest that inhibition of Hsp70 and Hsc70 activities could be a novel strategy designing small molecule-based adjuvants in protein vaccines.

  11. Evolution of the ONSEN retrotransposon family activated upon heat stress in Brassicaceae.

    PubMed

    Ito, Hidetaka; Yoshida, Takanori; Tsukahara, Sayuri; Kawabe, Akira

    2013-04-15

    A Ty1/Copia-like retrotransposon, ONSEN, is activated by heat stress in Arabidopsis thaliana, and its de novo integrations that were observed preferentially within genes implies its regulation of neighboring genes. Here we show that ONSEN related copies were found in most species of Brassicaceae, forming a cluster with each species in phylogenetic tree. Most copies were localized close to genes in Arabidopsis lyrata and Brassica rapa, suggesting conserved integration specificity of ONSEN family into genic or open chromatin. In addition, we found heat-induced transcriptional activation of ONSEN family in several species of Brassicaceae. These results suggest that ONSEN has conserved transcriptional activation promoted by environmental heat stress in some Brassicaceae species.

  12. Broad Spectrum Activity of a Lectin-Like Bacterial Serine Protease Family on Human Leukocytes

    PubMed Central

    Ayala-Lujan, Jorge Luis; Vijayakumar, Vidhya; Gong, Mei; Smith, Rachel; Santiago, Araceli E.; Ruiz-Perez, Fernando

    2014-01-01

    The serine protease autotransporter from Enterobacteriaceae (SPATE) family, which number more than 25 proteases with apparent diverse functions, have been phylogenetically divided into two distinct classes, designated 1 and 2. We recently demonstrated that Pic and Tsh, two members of the class-2 SPATE family produced by intestinal and extraintestinal pathogenic E. coli, were able to cleave a number of O-glycosylated proteins on neutrophils and lymphocytes resulting in impaired leukocyte functions. Here we show that most members of the class-2 SPATE family have lectin-like properties and exhibit differential protease activity reliant on glycoprotein type and cell lineage. Protease activity was seen in virtually all tested O-glycosylated proteins including CD34, CD55, CD164, TIM1, TIM3, TIM4 and C1-INH. We also show that although SPATE proteins bound and cleaved glycoproteins more efficiently on granulocytes and monocytes, they also targeted glycoproteins on B, T and natural killer lymphocytes. Finally, we found that the characteristic domain-2 of class-2 SPATEs is not required for glycoprotease activity, but single amino acid mutations in Pic domain-1 to those residues naturally occurring in domain-1 of SepA, were sufficient to hamper Pic glycoprotease activity. This study shows that most class-2 SPATEs have redundant activities and suggest that they may function as immunomodulators at several levels of the immune system. PMID:25251283

  13. Chlorogenic acid protects MSCs against oxidative stress by altering FOXO family genes and activating intrinsic pathway.

    PubMed

    Li, Shiyong; Bian, Hetao; Liu, Zhe; Wang, Ye; Dai, Jianghua; He, Wenfeng; Liao, Xingen; Liu, Rongrong; Luo, Jun

    2012-01-15

    Chlorogenic acid as an antioxidant exists widely in edible and medicinal plants, and can protect cell against apoptosis induced by oxidative stress. However, its molecular mechanisms remain largely unknown. Here, we showed that Chlorogenic acid suppressed reactive oxygen species increase by activation of Akt phosphorylation,and increased FOXO family genes and anti-apoptotic protein Bcl-2 expression in MSCs culturing under oxidative stress. In addition, PI-3Kinase Inhibitor (2-(4-Morpholinyl)-8-phenyl-4H-1-benzopyran-4-one, LY294002) could suppress the Chlorogenic acid-induced: (1) the cellular protective role, (2) the increase of the FOXO family genes expression, (3) increased expression of Bcl-2. These results suggested that Chlorogenic acid protected MSCs against apoptosis via PI3K/AKT signal and FOXO family genes.

  14. Investigating diet and physical activity in Malaysia: education and family history of diabetes relate to lower levels of physical activity

    PubMed Central

    Tam, Cai Lian; Bonn, Gregory; Yeoh, Si Han; Wong, Chee Piau

    2014-01-01

    The National Health and Morbidity Survey (NHMS, 2011), estimates that the number of Malaysian adults suffering from type 2 diabetes has increased from 8.3 to 31.2% since 1996. This study is a preliminary investigation of possible factors contributing to this epidemic. Knowledge of diabetes, health locus of control, diet and exercise habits, as well as family history, education level and other demographic factors to better understand the correlates of risky and healthy behaviors. This was done as part of a larger initiative to improve prevention efforts. Questionnaires were completed by 770 individuals from three Malaysian states: Selangor, Penang, and Terengganu. Findings showed that people with better health knowledge and those who have a family history of type 2 diabetes were more likely to have healthy diets. Also, health knowledge related to lower alcohol consumption. Participants with diabetic family members, however, also reported higher levels of stress. Counterintuitively, higher educational levels, higher internal locus of control, better health knowledge, as well as a family history of diabetes all correlated with lower levels of physical activity. Thus, it is suggested that, while increasing health knowledge will be important in addressing the type 2 diabetes epidemic in Malaysia, especially in relation to diet, other cultural factors, specifically norms related to exercise and physical activity, also need to be addressed if the spread of type 2 diabetes is to be addressed over the long term. PMID:25520676

  15. Investigating diet and physical activity in Malaysia: education and family history of diabetes relate to lower levels of physical activity.

    PubMed

    Tam, Cai Lian; Bonn, Gregory; Yeoh, Si Han; Wong, Chee Piau

    2014-01-01

    The National Health and Morbidity Survey (NHMS, 2011), estimates that the number of Malaysian adults suffering from type 2 diabetes has increased from 8.3 to 31.2% since 1996. This study is a preliminary investigation of possible factors contributing to this epidemic. Knowledge of diabetes, health locus of control, diet and exercise habits, as well as family history, education level and other demographic factors to better understand the correlates of risky and healthy behaviors. This was done as part of a larger initiative to improve prevention efforts. Questionnaires were completed by 770 individuals from three Malaysian states: Selangor, Penang, and Terengganu. Findings showed that people with better health knowledge and those who have a family history of type 2 diabetes were more likely to have healthy diets. Also, health knowledge related to lower alcohol consumption. Participants with diabetic family members, however, also reported higher levels of stress. Counterintuitively, higher educational levels, higher internal locus of control, better health knowledge, as well as a family history of diabetes all correlated with lower levels of physical activity. Thus, it is suggested that, while increasing health knowledge will be important in addressing the type 2 diabetes epidemic in Malaysia, especially in relation to diet, other cultural factors, specifically norms related to exercise and physical activity, also need to be addressed if the spread of type 2 diabetes is to be addressed over the long term.

  16. Identification of novel orosensory active molecules in cured vanilla Beans (Vanilla planifolia).

    PubMed

    Schwarz, Bernd; Hofmann, Thomas

    2009-05-13

    Sequential application of solvent extraction, gel permeation chromatography, and HPLC in combination with taste dilution analyses, followed by LC-MS and 1D/2D NMR experiments, led to the discovery of seven velvety mouth-coating molecules in cured beans of Vanilla planifolia . Among these, 5-(4-hydroxybenzyl)vanillin, 4-(4-hydroxybenzyl)-2-methoxyphenol, 4-hydroxy-3-(4-hydroxy-3-methoxybenzyl)-5-methoxybenzaldehyde, (1-O-vanilloyl)-(6-O-feruloyl)-beta-d-glucopyranoside, americanin A, and 4',6'-dihydroxy-3',5-dimethoxy-[1,1'-biphenyl]-3-carboxaldehyde were previously not reported in vanilla beans. Sensory studies revealed human recognition thresholds for the velvety mouth-coating sensation between 1.0 and 5.0 mumol/kg (water). Interestingly, the biphenyl derivatives were found to enhance the perception of creaminess and fatty body of sweetened skim milk, among which 4',6'-dihydroxy-3',5-dimethoxy-[1,1'-biphenyl]-3-carboxaldehyde showed the lowest threshold level of 5 mumol/kg. Quantitative analysis of these compounds in cured vanilla beans from different origins as well as in noncured beans revealed that, with the exception of americanin A, all of the other taste compounds are not present in the green vanilla beans and are formed during the bean curing process.

  17. Profiling T cell activation using single-molecule fluorescence in situ hybridization and flow cytometry.

    PubMed

    Bushkin, Yuri; Radford, Felix; Pine, Richard; Lardizabal, Alfred; Mangura, Bonita T; Gennaro, Maria Laura; Tyagi, Sanjay

    2015-01-15

    Flow cytometric characterization of Ag-specific T cells typically relies on detection of protein analytes. Shifting the analysis to detection of RNA would provide several significant advantages, which we illustrate by developing a new host immunity-based platform for detection of infections. Cytokine mRNAs synthesized in response to ex vivo stimulation with pathogen-specific Ags are detected in T cells with single-molecule fluorescence in situ hybridization followed by flow cytometry. Background from pre-existing in vivo analytes is lower for RNAs than for proteins, allowing greater sensitivity for detection of low-frequency cells. Moreover, mRNA analysis reveals kinetic differences in cytokine expression that are not apparent at the protein level but provide novel insights into gene expression programs expected to define different T cell subsets. The utility of probing immunological memory of infections is demonstrated by detecting T cells that recognize mycobacterial and viral Ags in donors exposed to the respective pathogens. PMID:25505292

  18. Activation of family C G-protein-coupled receptors by the tripeptide glutathione.

    PubMed

    Wang, Minghua; Yao, Yi; Kuang, Donghui; Hampson, David R

    2006-03-31

    The Family C G-protein-coupled receptors include the metabotropic glutamate receptors, the gamma-aminobutyric acid, type B (GABAB) receptor, the calcium-sensing receptor (CaSR), which participates in the regulation of calcium homeostasis in the body, and a diverse group of sensory receptors that encompass the amino acid-activated fish 5.24 chemosensory receptor, the mammalian T1R taste receptors, and the V2R pheromone receptors. A common feature of Family C receptors is the presence of an amino acid binding site. In this study, a preliminary in silico analysis of the size and shape of the amino acid binding pocket in selected Family C receptors suggested that some members of this family could accommodate larger ligands such as peptides. Subsequent screening and docking experiments identified GSH as a potential ligand or co-ligand at the fish 5.24 receptor and the rat CaSR. These in silico predictions were confirmed using an [3H]GSH radioligand binding assay and a fluorescence-based functional assay performed on wild-type and chimeric receptors. Glutathione was shown to act as an orthosteric agonist at the 5.24 receptor and as a potent enhancer of calcium-induced activation of the CaSR. Within the mammalian receptors, this effect was specific to the CaSR because GSH neither directly activated nor potentiated other Family C receptors including GPRC6A (the putative mammalian homolog of the fish 5.24 receptor), the metabotropic glutamate receptors, or the GABAB receptor. Our findings reveal a potential new role for GSH and suggest that this peptide may act as an endogenous modulator of the CaSR in the parathyroid gland where this receptor is known to control the release of parathyroid hormone, and in other tissues such as the brain and gastrointestinal tract where the role of the calcium receptor appears to subserve other, as yet unknown, physiological functions. PMID:16455645

  19. CD26 surface molecule involvement in T cell activation and lymphokine synthesis in rheumatoid and other inflammatory synovitis.

    PubMed

    Gerli, R; Muscat, C; Bertotto, A; Bistoni, O; Agea, E; Tognellini, R; Fiorucci, G; Cesarotti, M; Bombardieri, S

    1996-07-01

    T cell surface expression and the functional role of CD26 antigen (Ag), a surface ectoenzyme involved in T cell activation and migration across the extracellular matrix, were analyzed in the peripheral blood (PB) and synovial fluid (SF) from patients with inflammatory arthritides. CD26 membrane expression on T cells was detected by cytofluorometry using two different monoclonal antibodies, anti-Ta1 and anti-1F7, while cell proliferation and both IL-2 and IFN-gamma production were evaluated in anti-CD3- or anti-CD2-stimulated cell cultures after Ag surface modulation with anti-1F7. The results showed that Ta1 and 1F7 Ag expression were increased on T cells from PB of patients with active, but not inactive, rheumatoid arthritis (RA). Most SF T cells from RA or other inflammatory arthritides displayed the memory marker CD45R0 and the Ta1 Ag, but lacked the 1F7 molecule. In addition, in vitro 1F7 modulation, which enhanced RA PB T cell proliferation and both IL-2 and IFN-gamma synthesis, did not synergize with anti-CD3 or anti-CD2 in inducing IL-2-dependent activation of SF T cells, but reduced IFN-gamma production. A spontaneous reappearance of 1F7 Ag on the SF T cell surface was seen after 2-5 days in culture. Phorbol myristate acetate, able to accelerate its reexpression, also restored a normal response of SF T cells to anti-1F7 comitogenic effects. These data confirm a role of the CD26 surface molecule in regulating T cell activation and lymphokine synthesis. This observation may have important implications in the regulation of T cell activity at the joint level during chronic inflammatory processes. PMID:8674237

  20. Activation of macrophages for destruction of Francisella tularensis: identification of cytokines, effector cells, and effector molecules.

    PubMed Central

    Fortier, A H; Polsinelli, T; Green, S J; Nacy, C A

    1992-01-01

    Francisella tularensis live vaccine strain (LVS) was grown in culture with nonadherent resident, starch-elicited, or Proteose Peptone-elicited peritoneal cells. Numbers of bacteria increased 4 logs over the input inoculum in 48 to 72 h. Growth rates were faster in inflammatory cells than in resident cells: generation times for the bacterium were 3 h in inflammatory cells and 6 h in resident macrophages. LVS-infected macrophage cultures treated with lymphokines did not support growth of the bacterium, although lymphokines alone had no inhibitory effects on replication of LVS in culture medium devoid of cells. Removal of gamma interferon (IFN-gamma) by immunoaffinity precipitation rendered lymphokines ineffective for induction of macrophage anti-LVS activity, and recombinant IFN-gamma stimulated both resident and inflammatory macrophage populations to inhibit LVS growth in vitro. Inflammatory macrophages were more sensitive to effects of IFN-gamma: half-maximal activity was achieved at 5 U/ml for inflammatory macrophages and 20 U/ml for resident macrophages. IFN-gamma-induced anti-LVS activity correlated with the production of nitrite (NO2-), an oxidative end product of L-arginine-derived nitric oxide (NO). Anti-LVS activity and nitrite production were both completely inhibited by the addition of either the L-arginine analog NG-monomethyl-L-arginine or anti-tumor necrosis factor antibodies to activated macrophage cultures. Thus, macrophages can be activated by IFN-gamma to suppress the growth of F. tularensis by generation of toxic levels of NO, and inflammatory macrophages are substantially more sensitive to activation activities of IFN-gamma for this effector reaction than are more differentiated resident cells. PMID:1541555

  1. Differences in home food and activity environments between obese and healthy weight families of preschool children

    PubMed Central

    Boles, Richard E.; Scharf, Cynthia; Filigno, Stephanie S.; Saelens, Brian E.; Stark, Lori J.

    2013-01-01

    Objective To develop and test a home food and activity instrument to discriminate between the home environments of obese and healthy weight preschool children. Design A modified questionnaire about home environments was tested as an observation tool. Setting Family homes. Participants Thirty-five obese children with at least one obese caregiver were compared to forty-seven healthy weight children with no obese caregivers. Main Outcome Measures Home observation assessments were conducted to evaluate the availability of devices supporting activity behaviors and foods based on availability, accessibility, and readiness to be eaten. Analysis Agreement statistics were conducted to analyze psychometrics and MANOVAs were conducted to assess group differences, significance, P < .05. Results Home observations showed acceptable agreement statistics between independent coders across food and activity items. Families of obese preschoolers were significantly less likely to have fresh vegetables available or accessible in the home, were more likely to have a TV in the obese child’s bedroom and had fewer physical activity devices compared to healthy weight preschoolers. Conclusions and Implications Families of young children live in home environments that were discriminatively characterized based on home observations. Future tool refinement will further clarify the impact of the home environment on early growth. PMID:23380192

  2. Isolation and Molecular Characterization of Biofouling Bacteria and Profiling of Quorum Sensing Signal Molecules from Membrane Bioreactor Activated Sludge

    PubMed Central

    Lade, Harshad; Paul, Diby; Kweon, Ji Hyang

    2014-01-01

    The formation of biofilm in a membrane bioreactor depends on the production of various signaling molecules like N-acyl homoserine lactones (AHLs). In the present study, a total of 200 bacterial strains were isolated from membrane bioreactor activated sludge and screened for AHLs production using two biosensor systems, Chromobacterium violaceum CV026 and Agrobacterium tumefaciens A136. A correlation between AHLs production and biofilm formation has been made among screened AHLs producing strains. The 16S rRNA gene sequence analysis revealed the dominance of Aeromonas and Enterobacter sp. in AHLs production; however few a species of Serratia, Leclercia, Pseudomonas, Klebsiella, Raoultella and Citrobacter were also identified. The chromatographic characterization of sludge extract showed the presence of a broad range of quorum sensing signal molecules. Further identification of sludge AHLs by thin layer chromatography bioassay and high performance liquid chromatography confirms the presence of C4-HSL, C6-HSL, C8-HSL, 3-oxo-C8-HSL, C10-HSL, C12-HSL, 3-oxo-C12-HSL and C14-HSL. The occurrence of AHLs in sludge extract and dominance of Aeromonas and Enterobacter sp. in activated sludge suggests the key role of these bacterial strains in AHLs production and thereby membrane fouling. PMID:24499972

  3. Small molecule inhibitor screen identifies synergistic activity of the bromodomain inhibitor CPI203 and bortezomib in drug resistant myeloma

    PubMed Central

    Siegel, Matthew B.; Liu, Selina Qiuying; Davare, Monika A.; Spurgeon, Stephen E.; Loriaux, Marc M.; Druker, Brian J.

    2015-01-01

    Purpose Despite significant therapeutic progress in multiple myeloma, drug resistance is uniformly inevitable and new treatments are needed. Our aim was to identify novel, efficacious small-molecule combinations for use in drug resistant multiple myeloma. Experimental Design A panel of 116 small molecule inhibitors was used to screen resistant myeloma cell lines for potential therapeutic targets. Agents found to have enhanced activity in the bortezomib or melphalan resistant myeloma cell lines were investigated further in combination. Synergistic combinations of interest were evaluated in primary patient cells. Results The overall single-agent drug sensitivity profiles were dramatically different between melphalan and bortezomib resistant cells, however, the bromodomain inhibitor, CPI203, was observed to have enhanced activity in both the bortezomib and melphalan resistant lines compared to their wild-type counterparts. The combination of bortezomib and CPI203 was found to be synergistic in both the bortezomib and melphalan resistant cell lines as well as in a primary multiple myeloma sample from a patient refractory to recent proteasome inhibitor treatment. The CPI203-bortezomib combination led to enhanced apoptosis and anti-proliferative effects. Finally, in contrast to prior reports of synergy between bortezomib and other epigenetic modifying agents, which implicated MYC downregulation or NOXA induction, our analyses suggest that CPI203-bortezomib synergy is independent of these events. Conclusion Our preclinical data supports a role for the clinical investigation of the bromodomain inhibitor CPI203 combined with bortezomib or alkylating agents in resistant multiple myeloma. PMID:26254279

  4. Stimulation of mammalian translation initiation factor eIF4A activity by a small molecule inhibitor of eukaryotic translation

    PubMed Central

    Bordeleau, Marie-Eve; Matthews, James; Wojnar, Joanna M.; Lindqvist, Lisa; Novac, Olivia; Jankowsky, Eckhard; Sonenberg, Nahum; Northcote, Peter; Teesdale-Spittle, Paul; Pelletier, Jerry

    2005-01-01

    RNA helicases are the largest group of enzymes in eukaryotic RNA metabolism. The DEXD/H-box putative RNA helicases form the helicase superfamily II, whose members are defined by seven highly conserved amino acid motifs, making specific targeting of selected members a challenging pharmacological problem. The translation initiation factor eIF4A is the prototypical DEAD-box RNA helicase that works in conjunction with eIF4B and eIF4H and as a subunit of eIF4F to prepare the mRNA template for ribosome binding, possibly by unwinding the secondary structure proximal to the 5′ m7GpppN cap structure. We report the identification and characterization of a small molecule inhibitor of eukaryotic translation initiation that acts in an unusual manner by stimulating eIF4A-associated activities. Our results suggest that proper control of eIF4A helicase activity is necessary for efficient ribosome binding and demonstrate the feasibility of selectively targeting DEAD-box RNA helicases with small molecules. PMID:16030146

  5. Isolation and molecular characterization of biofouling bacteria and profiling of quorum sensing signal molecules from membrane bioreactor activated sludge.

    PubMed

    Lade, Harshad; Paul, Diby; Kweon, Ji Hyang

    2014-02-04

    The formation of biofilm in a membrane bioreactor depends on the production of various signaling molecules like N-acyl homoserine lactones (AHLs). In the present study, a total of 200 bacterial strains were isolated from membrane bioreactor activated sludge and screened for AHLs production using two biosensor systems, Chromobacterium violaceum CV026 and Agrobacterium tumefaciens A136. A correlation between AHLs production and biofilm formation has been made among screened AHLs producing strains. The 16S rRNA gene sequence analysis revealed the dominance of Aeromonas and Enterobacter sp. in AHLs production; however few a species of Serratia, Leclercia, Pseudomonas, Klebsiella, Raoultella and Citrobacter were also identified. The chromatographic characterization of sludge extract showed the presence of a broad range of quorum sensing signal molecules. Further identification of sludge AHLs by thin layer chromatography bioassay and high performance liquid chromatography confirms the presence of C4-HSL, C6-HSL, C8-HSL, 3-oxo-C8-HSL, C10-HSL, C12-HSL, 3-oxo-C12-HSL and C14-HSL. The occurrence of AHLs in sludge extract and dominance of Aeromonas and Enterobacter sp. in activated sludge suggests the key role of these bacterial strains in AHLs production and thereby membrane fouling.

  6. The B7-2 (B70) costimulatory molecule expressed by monocytes and activated B lymphocytes is the CD86 differentiation antigen.

    PubMed

    Engel, P; Gribben, J G; Freeman, G J; Zhou, L J; Nozawa, Y; Abe, M; Nadler, L M; Wakasa, H; Tedder, T F

    1994-09-01

    T-cell activation is initiated after T-cell receptor binding to antigen, but also requires interactions between costimulatory molecules expressed on antigen-presenting cells. An important costimulatory molecule expressed by monocytes and activated B lymphocytes has been recently identified and termed B7-2 or B70. Independently, a new Cluster of Differentiation was defined in the Fifth International Leukocyte Differentiation Antigen Workshop as CD86, a molecule predominantly expressed by monocytes and activated B lymphocytes. In this study, the two monoclonal antibodies that defined CD86, FUN-1 and BU-63, were shown to bind to cDNA transfected cells expressing B7-2/B70. The FUN-1 monoclonal antibody also completely blocked the costimulatory activity of B7-2/B70 in functional assays. Therefore, the serologically defined CD86 differentiation antigen is the B7-2/B70 molecule.

  7. Functionally stable plasminogen activator inhibitor-1 in a family with cardiovascular disease and vitiligo.

    PubMed

    Agirbasli, Mehmet; Eren, Mesut; Yasar, Songul; Delil, Kenan; Goktay, Fatih; Oner, Ebru Toksoy; Vaughan, Douglas E

    2014-07-01

    Vitiligo is a common skin condition with a complex pathophysiology characterized by the lack of pigmentation due to melanocyte degeneration. In this study, we investigated PAI-1 antigen (Ag) and activity levels in a 34 year old male with extensive vascular disease, alopecia areata and vitiligo. Fasting PAI-1 Ag and activity levels were measured at 9 a.m. in the subject and family members. Both PAI-1 Ag (67 ± 38 vs. 18.6 ± 6.5 ng/ml, P < 0.001) and specific activity (15.8 ± 10.0 vs. 7.6 ± 6.0 IU/pmol, P < 0.04) levels of PAI-1 were moderately elevated in subjects compared to the controls. PAI-1 kinetic studies demonstrated a markedly enhanced stability of plasma PAI-1 activity in the family members. Specific activity at 16 h was significantly higher than expected activity levels (0.078 ± 0.072 vs. 0.001 ± 0.001 IU/ng/ml, P < 0.001). While the exact mechanism of increased stability of PAI-1 activity in vitiligo is not known, it is likely due to post-translational modifications or increased binding affinity for a stabilizing cofactor. In conclusion, enhanced stability of PAI-1 may contribute to the pathophysiology of vascular disease and associated melanocyte degeneration. Systemic or local treatment with PAI-1 inhibitors may offer a potential treatment alternative to the near orphan status for vitiligo drug development. PMID:24197654

  8. Neutrophil-Derived Proteases Escalate Inflammation through Activation of IL-36 Family Cytokines.

    PubMed

    Henry, Conor M; Sullivan, Graeme P; Clancy, Danielle M; Afonina, Inna S; Kulms, Dagmar; Martin, Seamus J

    2016-02-01

    Recent evidence has strongly implicated the IL-1 family cytokines IL-36α, IL-36β, and IL-36γ as key initiators of skin inflammation. Similar to the other members of the IL-1 family, IL-36 cytokines are expressed as inactive precursors and require proteolytic processing for activation; however, the responsible proteases are unknown. Here, we show that IL-36α, IL-36β, and IL-36γ are activated differentially by the neutrophil granule-derived proteases cathepsin G, elastase, and proteinase-3, increasing their biological activity ~500-fold. Active IL-36 promoted a strong pro-inflammatory signature in primary keratinocytes and was sufficient to perturb skin differentiation in a reconstituted 3D human skin model, producing features resembling psoriasis. Furthermore, skin eluates from psoriasis patients displayed significantly elevated cathepsin G-like activity that was sufficient to activate IL-36β. These data identify neutrophil granule proteases as potent IL-36-activating enzymes, adding to our understanding of how neutrophils escalate inflammatory reactions. Inhibition of neutrophil-derived proteases may therefore have therapeutic benefits in psoriasis. PMID:26776523

  9. Modulation of Pantothenate Kinase 3 Activity by Small Molecules that Interact with the Substrate/Allosteric Regulatory Domain

    SciTech Connect

    Leonardi, Roberta; Zhang, Yong-Mei; Yun, Mi-Kyung; Zhou, Ruobing; Zeng, Fu-Yue; Lin, Wenwei; Cui, Jimmy; Chen, Taosheng; Rock, Charles O.; White, Stephen W.; Jackowski, Suzanne

    2010-09-27

    Pantothenate kinase (PanK) catalyzes the rate-controlling step in coenzyme A (CoA) biosynthesis. PanK3 is stringently regulated by acetyl-CoA and uses an ordered kinetic mechanism with ATP as the leading substrate. Biochemical analysis of site-directed mutants indicates that pantothenate binds in a tunnel adjacent to the active site that is occupied by the pantothenate moiety of the acetyl-CoA regulator in the PanK3 acetyl-CoA binary complex. A high-throughput screen for PanK3 inhibitors and activators was applied to a bioactive compound library. Thiazolidinediones, sulfonylureas and steroids were inhibitors, and fatty acyl-amides and tamoxifen were activators. The PanK3 activators and inhibitors either stimulated or repressed CoA biosynthesis in HepG2/C3A cells. The flexible allosteric acetyl-CoA regulatory domain of PanK3 also binds the substrates, pantothenate and pantetheine, and small molecule inhibitors and activators to modulate PanK3 activity.

  10. Nrf2 and HSF-1 Pathway Activation via Hydroquinone-Based Proelectrophilic Small Molecules Is Regulated by Electrochemical Oxidation Potential

    PubMed Central

    Stalder, Romain; McKercher, Scott R.; Williamson, Robert E.; Roth, Gregory P.; Lipton, Stuart A.

    2015-01-01

    Activation of the Kelch-like ECH-associated protein 1/nuclear factor (erythroid-derived 2)-like 2 and heat-shock protein 90/heat-shock factor-1 signal-transduction pathways plays a central role in combatting cellular oxidative damage and related endoplasmic reticulum stress. Electrophilic compounds have been shown to be activators of these transcription-mediated responses through S-alkylation of specific regulatory proteins. Previously, we reported that a prototype compound (D1, a small molecule representing a proelectrophilic, para-hydroquinone species) exhibited neuroprotective action by activating both of these pathways. We hypothesized that the para-hydroquinone moiety was critical for this activation because it enhanced transcription of these neuroprotective pathways to a greater degree than that of the corresponding ortho-hydroquinone isomer. This notion was based on the differential oxidation potentials of the isomers for the transformation of the hydroquinone to the active, electrophilic quinone species. Here, to further test this hypothesis, we synthesized a pair of para- and ortho-hydroquinone-based proelectrophilic compounds and measured their redox potentials using analytical cyclic voltammetry. The redox potential was then compared with functional biological activity, and the para-hydroquinones demonstrated a superior neuroprotective profile. PMID:26243592

  11. Small Molecule APY606 Displays Extensive Antitumor Activity in Pancreatic Cancer via Impairing Ras-MAPK Signaling

    PubMed Central

    Guo, Na; Liu, Zuojia; Zhao, Wenjing; Wang, Erkang; Wang, Jin

    2016-01-01

    Pancreatic cancer has been found with abnormal expression or mutation in Ras proteins. Oncogenic Ras activation exploits their extensive signaling reach to affect multiple cellular processes, in which the mitogen-activated protein kinase (MAPK) signaling exerts important roles in tumorigenesis. Therapies targeted Ras are thus of major benefit for pancreatic cancer. Although small molecule APY606 has been successfully picked out by virtual drug screening based on Ras target receptor, its in-depth mechanism remains to be elucidated. We herein assessed the antitumor activity of APY606 against human pancreatic cancer Capan-1 and SW1990 cell lines and explored the effect of Ras-MAPK and apoptosis-related signaling pathway on the activity of APY606. APY606 treatment resulted in a dose- and time-dependent inhibition of cancer cell viability. Additionally, APY606 exhibited strong antitumor activity, as evidenced not only by reduction in tumor cell invasion, migration and mitochondrial membrane potential but also by alteration in several apoptotic indexes. Furthermore, APY606 treatment directly inhibited Ras-GTP and the downstream activation of MAPK, which resulted in the down-regulation of anti-apoptotic protein Bcl-2, leading to the up-regulation of mitochondrial apoptosis pathway-related proteins (Bax, cytosolic Cytochrome c and Caspase 3) and of cyclin-dependent kinase 2 and Cyclin A, E. These data suggest that impairing Ras-MAPK signaling is a novel mechanism of action for APY606 during therapeutic intervention in pancreatic cancer. PMID:27223122

  12. Cyclodextrin-derived host molecules as reversal agents for the neuromuscular blocker rocuronium bromide: synthesis and structure-activity relationships.

    PubMed

    Adam, Julia M; Bennett, D Jonathan; Bom, Anton; Clark, John K; Feilden, Helen; Hutchinson, Edward J; Palin, Ronald; Prosser, Alan; Rees, David C; Rosair, Georgina M; Stevenson, Donald; Tarver, Gary J; Zhang, Ming-Qiang

    2002-04-25

    A series of mono- and per-6-substituted cyclodextrin derivatives were synthesized as synthetic receptors (or host molecules) of rocuronium bromide, the most widely used neuromuscular blocker in anaesthesia. By forming host-guest complexes with rocuronium, these cyclodextrin derivatives reverse the muscle relaxation induced by rocuronium in vitro and in vivo and therefore can be used as reversal agents of the neuromuscular blocker to assist rapid recovery of patients after surgery. Because this supramolecular mechanism of action does not involve direct interaction with the cholinergic system, the reversal by these compounds, e.g., compound 14 (Org 25969), is not accompanied by cardiovascular side effects usually attendant with acetylcholinesterase inhibitors such as neostigmine. The structure-activity relationships are consistent with this supramolecular mechanism of action and are discussed herein. These include the effects of binding cavity size and hydrophobic and electrostatic interaction on the reversal activities of these compounds.

  13. Spatiotemporal regulation of chemical reaction kinetics of cell surface molecules by active remodeling of cortical actin

    NASA Astrophysics Data System (ADS)

    Bhattacharyya, Bhaswati; Chaudhuri, Abhishek; Gowrishankar, Kripa; Mayor, Satyajit; Rao, Madan

    2010-03-01

    Cell surface proteins such as lipid tethered GPI-anchored proteins and Ras-proteins are distributed as monomers and nanoclusters on the surface of living cells. Recent work from our laboratory suggests that the spatial distribution and dynamics of formation and breakup of these nanoclusters is controlled by the active remodeling dynamics of the underlying cortical actin. To explain these observations, we propose a novel mechanism of nanoclustering, involving the transient binding to and advection along constitutively occuring ``asters'' of cortical actin. Here we study the consequences of such active actin based clustering, in the context of chemical reactions involving conformational changes of cell surface proteins. We find that active remodeling of cortical actin, can give rise to a dramatic increase in the reaction efficiency and output levels. In general, such actin driven clustering of membrane proteins could be a cellular mechanism to spatiotemporally regulate and amplify local chemical reaction rates, in the context of signalling and endocytosis.

  14. The Influence of AD Family History and APOE4 on Mesial Temporal Lobe Activation

    PubMed Central

    Johnson, Sterling C.; Schmitz, Taylor W.; Trivedi, Mehul A.; Ries, Michele L.; Torgerson, Britta M.; Carlsson, Cynthia; Asthana, Sanjay; Hermann, Bruce P.; Sager, Mark A.

    2009-01-01

    First-degree family history of sporadic Alzheimer Disease (AD) and the apolipoprotein E ε4 (APOE4) are risk factors for developing AD. Although the role of APOE4 in AD pathogenesis has been well studied, family history remains a rarely studied and poorly understood risk factor. Both putatively cause early brain changes prior to symptomatic disease, but the relative contribution of each on brain function is unknown. We examined 68 middle-aged participants with a parent diagnosed with AD (+FH) and 64 age- and education-matched controls without a first-degree family history of any dementia (−FH). All underwent cognitive testing, APOE genotyping and a functional MRI (fMRI) encoding task that required discrimination of novel items from previously learned items. A 2 × 2 factorial ANOVA (presence/absence of parental family history and presence/absence of the APOE4) was used to detect group effects. A greater response to novel items was detected in the mesial temporal lobe and fusiform gyrus bilaterally among persons without a first-degree family history of AD. In hippocampal areas, the −FH +ε4 group exhibited the greatest signal change, and the +FH +ε4 group the least. These findings indicate that FH of AD is an important predictor of hippocampal activation during encoding and that FH may modulate the effect of APOE4 in these middle-aged adults, suggesting that an as yet unspecified factor embodied in first-degree family history of AD is influencing the expression of APOE4 on brain function. PMID:16738250

  15. Integrin Activation Through the Hematopoietic Adapter Molecule ADAP Regulates Dendritic Development of Hippocampal Neurons

    PubMed Central

    Thiere, Marlen; Kliche, Stefanie; Müller, Bettina; Teuber, Jan; Nold, Isabell; Stork, Oliver

    2016-01-01

    Integrin-mediated cell adhesion and signaling is of critical importance for neuronal differentiation. Recent evidence suggests that an “inside-out” activation of β1-integrin, similar to that observed in hematopoietic cells, contributes to the growth and branching of dendrites. In this study, we investigated the role of the hematopoietic adaptor protein adhesion and degranulation promoting adapter protein (ADAP) in these processes. We demonstrate the expression of ADAP in the developing and adult nervous hippocampus, and in outgrowing dendrites of primary hippocampal neurons. We further show that ADAP occurs in a complex with another adaptor protein signal-transducing kinase-associated phosphoprotein-homolog (SKAP-HOM), with the Rap1 effector protein RAPL and the Hippo kinase macrophage-stimulating 1 (MST1), resembling an ADAP/SKAP module that has been previously described in T-cells and is critically involved in “inside-out” activation of integrins. Knock down of ADAP resulted in reduced expression of activated β1-integrin on dendrites. It furthermore reduced the differentiation of developing neurons, as indicated by reduced dendrite growth and decreased expression of the dendritic marker microtubule-associated protein 2 (MAP2). Our data suggest that an ADAP-dependent integrin-activation similar to that described in hematopoietic cells contributes to the differentiation of neuronal cells. PMID:27746719

  16. Screening a Commercial Library of Pharmacologically Active Small Molecules against Staphylococcus aureus Biofilms.

    PubMed

    Torres, Nelson S; Abercrombie, Johnathan J; Srinivasan, Anand; Lopez-Ribot, Jose L; Ramasubramanian, Anand K; Leung, Kai P

    2016-10-01

    It is now well established that bacterial infections are often associated with biofilm phenotypes that demonstrate increased resistance to common antimicrobials. Further, due to the collective attrition of new antibiotic development programs by the pharmaceutical industries, drug repurposing is an attractive alternative. In this work, we screened 1,280 existing commercially available drugs in the Prestwick Chemical Library, some with previously unknown antimicrobial activity, against Staphylococcus aureus, one of the commonly encountered causative pathogens of burn and wound infections. From the primary screen of the entire Prestwick Chemical Library at a fixed concentration of 10 μM, 104 drugs were found to be effective against planktonic S. aureus strains, and not surprisingly, these were mostly antimicrobials and antiseptics. The activity of 18 selected repurposing candidates, that is, drugs that show antimicrobial activity that are not already considered antimicrobials, observed in the primary screen was confirmed in dose-response experiments. Finally, a subset of nine of these drug candidates was tested against preformed biofilms of S. aureus We found that three of these drugs, niclosamide, carmofur, and auranofin, possessed antimicrobial activity against preformed biofilms, making them attractive candidates for repurposing as novel antibiofilm therapies.

  17. Screening a Commercial Library of Pharmacologically Active Small Molecules against Staphylococcus aureus Biofilms.

    PubMed

    Torres, Nelson S; Abercrombie, Johnathan J; Srinivasan, Anand; Lopez-Ribot, Jose L; Ramasubramanian, Anand K; Leung, Kai P

    2016-10-01

    It is now well established that bacterial infections are often associated with biofilm phenotypes that demonstrate increased resistance to common antimicrobials. Further, due to the collective attrition of new antibiotic development programs by the pharmaceutical industries, drug repurposing is an attractive alternative. In this work, we screened 1,280 existing commercially available drugs in the Prestwick Chemical Library, some with previously unknown antimicrobial activity, against Staphylococcus aureus, one of the commonly encountered causative pathogens of burn and wound infections. From the primary screen of the entire Prestwick Chemical Library at a fixed concentration of 10 μM, 104 drugs were found to be effective against planktonic S. aureus strains, and not surprisingly, these were mostly antimicrobials and antiseptics. The activity of 18 selected repurposing candidates, that is, drugs that show antimicrobial activity that are not already considered antimicrobials, observed in the primary screen was confirmed in dose-response experiments. Finally, a subset of nine of these drug candidates was tested against preformed biofilms of S. aureus We found that three of these drugs, niclosamide, carmofur, and auranofin, possessed antimicrobial activity against preformed biofilms, making them attractive candidates for repurposing as novel antibiofilm therapies. PMID:27401577

  18. Single Molecule Behavior of Inhibited and Active States of Escherichia coli ATP Synthase F1 Rotation*

    PubMed Central

    Sekiya, Mizuki; Hosokawa, Hiroyuki; Nakanishi-Matsui, Mayumi; Al-Shawi, Marwan K.; Nakamoto, Robert K.; Futai, Masamitsu

    2010-01-01

    ATP hydrolysis-dependent rotation of the F1 sector of the ATP synthase is a successive cycle of catalytic dwells (∼0.2 ms at 24 °C) and 120° rotation steps (∼0.6 ms) when observed under Vmax conditions using a low viscous drag 60-nm bead attached to the γ subunit (Sekiya, M., Nakamoto, R. K., Al-Shawi, M. K., Nakanishi-Matsui, M., and Futai, M. (2009) J. Biol. Chem. 284, 22401–22410). During the normal course of observation, the γ subunit pauses in a stochastic manner to a catalytically inhibited state that averages ∼1 s in duration. The rotation behavior with adenosine 5′-O-(3-thiotriphosphate) as the substrate or at a low ATP concentration (4 μm) indicates that the rotation is inhibited at the catalytic dwell when the bound ATP undergoes reversible hydrolysis/synthesis. The temperature dependence of rotation shows that F1 requires ∼2-fold higher activation energy for the transition from the active to the inhibited state compared with that for normal steady-state rotation during the active state. Addition of superstoichiometric ϵ subunit, the inhibitor of F1-ATPase, decreases the rotation rate and at the same time increases the duration time of the inhibited state. Arrhenius analysis shows that the ϵ subunit has little effect on the transition between active and inhibited states. Rather, the ϵ subunit confers lower activation energy of steady-state rotation. These results suggest that the ϵ subunit plays a role in guiding the enzyme through the proper and efficient catalytic and transport rotational pathway but does not influence the transition to the inhibited state. PMID:20974856

  19. The structure and activation of substrate water molecules in Sr(2+)-substituted photosystem II.

    PubMed

    Chatterjee, Ruchira; Milikisiyants, Sergey; Coates, Christopher S; Koua, Faisal H M; Shen, Jian-Ren; Lakshmi, K V

    2014-10-14

    The mechanism of solar water oxidation by photosystem II (PSII) is of fundamental interest and it is the object of extensive studies both in the past and present. The solar water oxidation reaction of PSII occurs in the oxygen-evolving complex (OEC). The OEC consists of a tetranuclear manganese calcium-oxo (Mn4Ca-oxo) cluster that is surrounded by amino acid residues and inorganic cofactors. The role of the Ca(2+) ion in the water oxidation reaction is one of the most interesting questions that is yet to be answered. In this study, we probe the structural and functional differences induced by metal ion substitution in the Mn4Ca-oxo cluster by substituting the Ca(2+) ion in the OEC by a Sr(2+) ion. We apply two-dimensional (2D) hyperfine sublevel correlation (HYSCORE) spectroscopy to detect weak magnetic interactions between the paramagnetic Mn4Sr-oxo cluster and the surrounding protons in the S2 state of the OEC of Sr(2+)-substituted PSII. We identify three groups of protons that are magnetically interacting with the Mn4Sr-oxo cluster. Using the recently reported 1.9 Å resolution X-ray structure of the OEC in the S1 state [Umena et al.] and the high-resolution 2D HYSCORE spectroscopy studies of the S2 state of the OEC of Ca(2+)-containing PSII [Milikisiyants et al., Energy Environ. Sci., 2012, 5, 7747], we discuss the assignments of the three groups of protons that are magnetically coupled to the Mn4Sr-oxo cluster. Since hyperfine interactions are highly sensitive to small perturbations in the electronic and geometric structure of paramagnetic centers, a comparison of the 2D HYSCORE spectra of Sr(2+)-substituted and Ca(2+)-containing PSII allows us to draw important conclusions with respect to the structure of the substrate water molecules in the OEC and the role of the Ca(2+) ion in the water oxidation reaction. In addition, for the first time, we determine the experimental value of the spin projection factor for the Mn(III) ion of the Mn4Ca-oxo cluster as ρ1

  20. Plant signal molecules activate the syrB gene, which is required for syringomycin production by Pseudomonas syringae pv. syringae.

    PubMed Central

    Mo, Y Y; Gross, D C

    1991-01-01

    The syrB gene is required for syringomycin production by Pseudomonas syringae pv. syringae and full virulence during plant pathogenesis. Strain B3AR132 containing a syrB::lacZ fusion was used to detect transcriptional activation of the syrB gene in syringomycin minimal medium by plant metabolites with signal activity. Among 34 plant phenolic compounds tested, arbutin, phenyl-beta-D-glucopyranoside, and salicin were shown to be strong inducers of syrB, giving rise to approximately 1,200 U of beta-galactosidase activity at 100 microM; esculin and helicin were moderate inducers, with about 250 to 400 U of beta-galactosidase activity at 100 microM. Acetosyringone and flavonoids that serve as signal molecules in Agrobacterium and Rhizobium species, respectively, did not induce the syrB::lacZ fusion. All syrB inducers were phenolic glucosides and none of the aglucone derivatives were active, suggesting that the beta-glycosidic linkage was necessary for signal activity. Phenyl-beta-D-galactopyranoside containing galactose substituted for glucose in the beta-glycosidic linkage also lacked inducer activity. Phenolic signal activity was enhanced two- to fivefold by specific sugars common to plant tissues, including D-fructose, D-mannose, and sucrose. The effect of sugars on syrB induction was most noticeable at low concentrations of phenolic glucoside (i.e., 1 to 10 microM), indicating that sugars such as D-fructose increase the sensitivity of P. syringae pv. syringae to the phenolic plant signal. Besides induction of syrB, syringomycin biosynthesis by parental strain B3A-R was induced to yield over 250 U of toxin by the additions of arbutin and D-fructose to syringomycin minimal medium. These data indicate that syringomycin production by most strains of P. syringae pv. syringae is modulated by the perception of two classes of plant signal molecules and transduced to the transcriptional apparatus of syringomycin (syr) genes such as syrB. PMID:1885550

  1. Time-dependent generalized-active-space configuration-interaction approach to photoionization dynamics of atoms and molecules

    NASA Astrophysics Data System (ADS)

    Bauch, S.; Sørensen, L. K.; Madsen, L. B.

    2014-12-01

    We present a wave-function-based method to solve the time-dependent many-electron Schrödinger equation with special emphasis on strong-field ionization phenomena. The theory builds on the configuration-interaction (CI) approach supplemented by the generalized-active-space concept from quantum chemistry. The latter allows for a controllable reduction in the number of configurations in the CI expansion by imposing restrictions on the active orbital space. The method is similar to the recently formulated time-dependent restricted-active-space CI method [D. Hochstuhl and M. Bonitz, Phys. Rev. A 86, 053424 (2012), 10.1103/PhysRevA.86.053424]. We present details of our implementation and address convergence properties with respect to the active spaces and the associated account of electron correlation in both ground-state and excitation scenarios. We apply the time-dependent generalized-active-space CI theory to strong-field ionization of polar diatomic molecules and illustrate how the method allows us to uncover a strong correlation-induced shift of the preferred direction of emission of photoelectrons.

  2. Interleukin 3 stimulates proliferation and triggers endothelial-leukocyte adhesion molecule 1 gene activation of human endothelial cells.

    PubMed

    Brizzi, M F; Garbarino, G; Rossi, P R; Pagliardi, G L; Arduino, C; Avanzi, G C; Pegoraro, L

    1993-06-01

    Proliferation and functional activation of endothelial cells within a tissue site of inflammation are regulated by humoral factors released by cells, such as T lymphocytes and monocytes, infiltrating the perivascular space. In the present study we investigated the effects of interleukin 3 (IL-3), an activated T lymphocyte-derived cytokine, on cultured human umbilical vein endothelial cells (HUVEC). Proliferative activity, evaluated both by estimation of the fraction of cells in the S phase and by direct cell count demonstrated that IL-3, at the dose of 25 ng/ml, enhances more than threefold both DNA synthesis and cell proliferation above baseline control conditions. Binding studies with radioiodinated ligand demonstrated that HUVEC constitutively express a smaller number of IL-3 binding sites (approximately 99 binding sites per cell, with an apparent Kd of 149 pM). Accordingly, molecular analysis showed the presence of transcripts for both alpha and beta subunits of the IL-3 receptor. Functional activation of endothelial cells was evaluated by the expression of the endothelial-leukocyte adhesion molecule 1 (ELAM-1) transcript and by leukocyte adhesion. The ELAM-1 gene transcript was clearly detectable 4 h after IL-3 addition and started to decrease after 12 h. Moreover, IL-3-induced ELAM-1 transcription was followed by enhanced adhesion of neutrophils and CD4+ T cells to HUVEC. The findings that IL-3 can stimulate both proliferation and functional activation of endothelial cells suggest that this cytokine can be involved in sustaining the process of chronic inflammation.

  3. Detection of a water molecule in the active-site of bacteriorhodopsin: hydrogen bonding changes during the primary photoreaction.

    PubMed

    Fischer, W B; Sonar, S; Marti, T; Khorana, H G; Rothschild, K J

    1994-11-01

    FTIR-difference spectroscopy in combination with site-directed mutagenesis has been used to investigate the role of water during the photocycle of bacteriorhodopsin. At least one water molecule is detected which undergoes an increase in H-bonding during the primary bR-->K phototransition. Bands due to water appear in the OH stretch region of the bR-->K FTIR-difference spectrum which downshift by approximately 12 cm-1 when the sample is hydrated with H2(18)O. In contrast to 2H2O, the H2(18)O-induced shift is not complete, even after 24 h of hydration. This indicates that even though water is still able to exchange protons with the outside medium, it is partially trapped in the interior of the protein. In the mutant Y57D, these bands are absent while a new set of bands appear at much lower frequencies which undergo H2(18)O-induced shifts. It is concluded that the water molecule we detect is located inside the bR active-site and may interact with Tyr-57. The change in its hydrogen-bonding strength is most likely due to the photoinduced all-trans-->13-cis isomerization of the retinal chromophore and the associated movement of the positively charged Schiff base during the bR-->K transition. In contrast, a second water molecule, whose infrared difference bands are not affected by the Y57D mutation, appears to undergo a decrease in hydrogen bonding during the K-->L and L-->M transitions.

  4. Cellular Activity of New Small Molecule Protein Arginine Deiminase 3 (PAD3) Inhibitors.

    PubMed

    Jamali, Haya; Khan, Hasan A; Tjin, Caroline C; Ellman, Jonathan A

    2016-09-01

    The protein arginine deiminases (PADs) catalyze the post-translational deimination of arginine side chains. Multiple PAD isozymes have been characterized, and abnormal PAD activity has been associated with several human disease states. PAD3 has been characterized as a modulator of cell growth via apoptosis inducing factor and has been implicated in the neurodegenerative response to spinal cord injury. Here, we describe the design, synthesis, and evaluation of conformationally constrained versions of the potent and selective PAD3 inhibitor 2. The cell activity of representative inhibitors in this series was also demonstrated for the first time by rescue of thapsigargin-induced cell death in PAD3-expressing HEK293T cells. PMID:27660689

  5. Small-molecule FRET probes for protein kinase activity monitoring in living cells

    SciTech Connect

    Vaasa, Angela; Lust, Marje; Terrin, Anna; Uri, Asko; Zaccolo, Manuela

    2010-07-09

    In this study, the applicability of fluorescently labeled adenosine analogue-oligoarginine conjugates (ARC-Photo probes) for monitoring of protein kinase A (PKA) activity in living cells was demonstrated. ARC-Photo probes possessing subnanomolar affinity towards the catalytic subunit of PKA (PKAc) and competitive with the regulatory subunit (PKAr), penetrate cell plasma membrane and associate with PKAc fused with yellow fluorescent protein (PKAc-YFP). Detection of inter-molecular Foerster resonance energy transfer (FRET) efficiency between the fluorophores of the fusion protein and ARC-Photo probe can be used for both the evaluation of non-labeled inhibitors of PKAc and for monitoring of cAMP signaling via detection of changes in the activity of PKA as a cAMP downstream effector.

  6. Discovery of Structurally Diverse Small-Molecule Compounds with Broad Antiviral Activity against Enteroviruses

    PubMed Central

    Zuo, Jun; Kye, Steve; Quinn, Kevin K.; Cooper, Paige; Damoiseaux, Robert

    2015-01-01

    Antiviral drugs do not currently exist for the treatment of enterovirus infections, which are often severe and potentially life-threatening. We conducted high-throughput molecular screening and identified a structurally diverse set of compounds that inhibit the replication of coxsackievirus B3, a commonly encountered enterovirus. These compounds did not interfere with the function of the viral internal ribosome entry site or with the activity of the viral proteases, but they did drastically reduce the synthesis of viral RNA and viral proteins in infected cells. Sequence analysis of compound-resistant mutants suggests that the viral 2C protein is targeted by most of these compounds. These compounds demonstrated antiviral activity against a panel of the most commonly encountered enteroviruses and thus represent potential leads for the development of broad-spectrum anti-enteroviral drugs. PMID:26711750

  7. Surface enhanced Raman optical activity of molecules on orientationally averaged substrates: theory of electromagnetic effects.

    PubMed

    Janesko, Benjamin G; Scuseria, Gustavo E

    2006-09-28

    We present a model for electromagnetic enhancements in surface enhanced Raman optical activity (SEROA) spectroscopy. The model extends previous treatments of SEROA to substrates, such as metal nanoparticles in solution, that are orientationally averaged with respect to the laboratory frame. Our theoretical treatment combines analytical expressions for unenhanced Raman optical activity with molecular polarizability tensors that are dressed by the substrate's electromagnetic enhancements. We evaluate enhancements from model substrates to determine preliminary scaling laws and selection rules for SEROA. We find that dipolar substrates enhance Raman optical activity (ROA) scattering less than Raman scattering. Evanescent gradient contributions to orientationally averaged ROA scale to first or higher orders in the gradient of the incident plane-wave field. These evanescent gradient contributions may be large for substrates with quadrupolar responses to the plane-wave field gradient. Some substrates may also show a ROA contribution that depends only on the molecular electric dipole-electric dipole polarizability. These conclusions are illustrated via numerical calculations of surface enhanced Raman and ROA spectra from (R)-(-)-bromochlorofluoromethane on various model substrates.

  8. Dissecting Dynamic Allosteric Pathways Using Chemically Related Small-Molecule Activators.

    PubMed

    Lisi, George P; Manley, Gregory A; Hendrickson, Heidi; Rivalta, Ivan; Batista, Victor S; Loria, J Patrick

    2016-07-01

    The allosteric mechanism of the heterodimeric enzyme imidazole glycerol phosphate synthase was studied in detail with solution nuclear magnetic resonance spectroscopy and molecular dynamics simulations. We studied IGPS in complex with a series of allosteric activators corresponding to a large range of catalytic rate enhancements (26- to 4,900-fold), in which ligand binding is entropically driven. Conformational flexibility on the millisecond timescale plays a crucial role in intersubunit communication. Carr-Purcell-Meiboom-Gill relaxation dispersion experiments probing Ile, Leu, and Val methyl groups reveal that the apo- and glutamine-mimicked complexes are static on the millisecond timescale. Domain-wide motions are stimulated in the presence of the allosteric activators. These studies, in conjunction with ligand titrations, demonstrate that the allosteric network is widely dispersed and varies with the identity of the effector. Furthermore, we find that stronger allosteric ligands create more conformational flexibility on the millisecond timescale throughout HisF. This domain-wide loosening leads to maximum catalytic activity. PMID:27238967

  9. Proapoptotic and antiinvasive activity of Rac1 small molecule inhibitors on malignant glioma cells

    PubMed Central

    Cardama, Georgina A; Gonzalez, Nazareno; Ciarlantini, Matias; Gandolfi Donadío, Lucia; Comin, María Julieta; Alonso, Daniel F; Menna, Pablo Lorenzano; Gomez, Daniel E

    2014-01-01

    Malignant gliomas are characterized by an intrinsic ability to invade diffusely throughout the normal brain tissue. This feature contributes mainly to the failure of existing therapies. Deregulation of small GTPases signaling, in particular Rac1 activity, plays a key role in the invasive phenotype of gliomas. Here we report the effect of ZINC69391, a specific Rac1 inhibitor developed by our group, on human glioma cell lines LN229 and U-87 MG. ZINC69391 is able to interfere with the interaction of Rac1 with Dock180, a relevant Rac1 activator in glioma invasion, and to reduce Rac1-GTP levels. The kinase Pak1, a downstream effector of Dock180–Rac1 signaling, was also downregulated upon ZINC69391 treatment. ZINC69391 reduced cell proliferation, arrested cells in G1 phase, and triggered apoptosis in glioma cells. Importantly, ZINC69391 dramatically affected cell migration and invasion in vitro, interfering with actin cytoskeleton dynamics. We also evaluated the effect of analog 1A-116, a compound derived from ZINC69391 structure. 1A-116 showed an improved antiproliferative and antiinvasive activity on glioma cells. These findings encourage further preclinical testing in clinically relevant animal models. PMID:25378937

  10. Accommodation of two diatomic molecules in cytochrome bo3: insights into NO reductase activity in terminal oxidases†

    PubMed Central

    Hayashi, Takahiro; Lin, Myat T.; Ganesan, Krithika; Chen, Ying; Fee, James A.; Gennis, Robert B.; Moënne-Loccoz, Pierre

    2009-01-01

    Bacterial heme-copper terminal oxidases react quickly with NO to form a heme-nitrosyl complex, which, in some of these enzymes, can further react with a second NO molecule to produce N2O. Previously, we characterized the heme a3-NO complex formed in cytochrome ba3 from Thermus thermophilus and the product of its low-temperature illumination. We showed that the photolyzed NO group binds to CuB(I) to form an end-on NO-CuB or side-on copper-nitrosyl complex which is likely to represent the binding characteristics of the second NO molecule at the heme-copper active site. Here we present a comparative study with cytochrome bo3 from Escherichia coli. Both terminal oxidases are shown to catalyze the same two-electron reduction of NO to N2O. The EPR and resonance Raman signatures of the heme o3-NO complex are comparable to those of the a3-NO complex. However, low-temperature FTIR experiments reveal that photolysis of the heme o3-NO complex does not produce a CuB-nitrosyl complex, but that instead, the NO remains unbound in the active-site cavity. Additional FTIR photolysis experiments on the heme-nitrosyl complexes of these terminal oxidases, in the presence of CO demonstrate that an [o3–NO • OC–CuB] tertiary complex can form in bo3 but not in ba3. We assign these differences to a greater iron-copper distance in the reduced form of bo3 compared to that of ba3. Because this difference in metal-metal distance does not appear to affect the NO reductase activity, our results suggest that the coordination of the second NO to CuB is not an essential step of the reaction mechanism. PMID:19187032

  11. Evolutionary origins of retinoid active short-chain dehydrogenases/reductases of SDR16C family

    PubMed Central

    Belyaeva, Olga V.; Chang, Chenbei; Berlett, Michael C; Kedishvili, Natalia Y.

    2014-01-01

    Vertebrate enzymes that belong to the 16C family of short-chain dehydrogenases/reductases (SDR16C) were shown to play an essential role in the control of retinoic acid (RA) levels during development. To trace the evolution of enzymatic function of SDR16C family, and to examine the origins of the pathway for RA biosynthesis from vitamin A, we identified putative SDR16C enzymes through the extensive search of available genome sequencing data in a subset of species representing major metazoan phyla. The phylogenetic analysis revealed that enzymes from protostome, non-chordate deuterostome and invertebrate chordate species are found in three clades of SDR16C family containing retinoid active enzymes, which are retinol dehydrogenase 10 (RDH10), retinol dehydrogenases E2 (RDHE2) and RDHE2-similar, and dehydrogenase reductase (SDR family) member 3 (DHRS3). For the initial functional analysis, we cloned RDH10- and RDHE2-related enzymes from the early developmental stages of a non-chordate deuterostome, green sea urchin Lytechinus variegatus, and an invertebrate chordate, sea squirt Ciona intestinalis. In situ hybridization revealed that these proteins are expressed in a pattern relevant to development, while assays performed on proteins expressed in mammalian cell culture showed that they possess retinol-oxidizing activity as their vertebrate homologs. The existence of invertebrate homologs of DHRS3 was inferred from the analysis of phylogeny and cofactor-binding residues characteristic of preference for NADP(H). The presence of invertebrate homologs in the DHRS3 group of SDR16C is interesting in light of the complex mutually activating interaction, which we have recently described for human RDH10 and DHRS3 enzymes. Further functional analysis of these homologs will establish whether this interaction evolved to control retinoid homeostasis only in vertebrates, or is also conserved in pre-vertebrates. PMID:25451586

  12. The functional importance of blood group-active molecules in human red blood cells.

    PubMed

    Anstee, D J

    2011-01-01

    Antigens of 23 of the 30 human blood group systems are defined by the amino acid sequence of red cell membrane proteins. The antigens of DI, RH, RHAG, MNS, GE and CO systems are carried on blood group-active proteins (Band 3, D and CE polypeptides, RhAG, Glycophorins A and B, Glycophorins C and D and Aquaporin 1, respectively) which are expressed at high levels (>200,000 copies/red cell). These major proteins contribute to essential red cell functions either directly as membrane transporters and by providing linkage to the underlying red cell skeleton or by facilitating the membrane assembly of the protein complexes involved in these processes. The proteins expressing antigens of the remaining 17 blood group systems are much less abundant (<20,000 copies/red cell) and their functional importance for the circulating red cell is largely unknown. Human gene knock-outs (null phenotypes) have been described for many of these minor blood group-active proteins, but only absence of Kx glycoprotein has been clearly linked with pathology directly related to the function of circulating red cells. Recent evidence suggesting the normal quality control system for glycoprotein synthesis is altered during the latter stages of red cell production raises the possibility that many of these low abundance blood group-active proteins are vestigial. In sickle cell disease and polycythaemia vera, elevated Lutheran glycoprotein expression may contribute to pathology. Dyserythropoiesis with reduced antigen expression can result from mutations in the erythroid transcription factors GATA-1 and EKLF.

  13. Modelling active sites for the Beckmann rearrangement reaction in boron-containing zeolites and their interaction with probe molecules.

    PubMed

    Lezcano-González, Inés; Vidal-Moya, Alejandro; Boronat, Mercedes; Blasco, Teresa; Corma, Avelino

    2010-06-28

    Theoretical calculations and in situ solid state NMR spectroscopy have been combined to get insight on the nature of the active sites for the Beckmann rearrangement reaction in borosilicate zeolites. The interaction of a B site in zeolite Beta with a series of probe molecules (ammonia, pyridine, acetone and water) has been modelled and the (15)N and (11)B NMR isotropic chemical shift of the resulting complexes calculated and compared with experimental in situ NMR results. This approach has allowed validation of the methodology to model the adsorption on a zeolite boron site of molecules of varying basicity which are either protonated or non-protonated. The limitation is that theoretical calculations overestimate the effect of molecular adsorption through hydrogen bonds on the calculated isotropic (11)B NMR chemical shift.Theoretical and experimental results on the adsorption of acetophenone and cyclohexanone oximes on zeolite B-Beta indicate that Brønsted acid sites protonate the oximes, changing the boron coordination from trigonal to tetrahedral. Comparison of theoretical and experimental (15)N NMR chemical shifts of the adsorbed amides (acetanilide and epsilon-caprolactam) indicates that they are non-protonated, and the (11)B NMR spectra show that, as expected, boron remains in trigonal coordination with an isotropic delta(11)B(exp) which differs from the calculated value delta(11)B(calc).

  14. Modelling active sites for the Beckmann rearrangement reaction in boron-containing zeolites and their interaction with probe molecules.

    PubMed

    Lezcano-González, Inés; Vidal-Moya, Alejandro; Boronat, Mercedes; Blasco, Teresa; Corma, Avelino

    2010-06-28

    Theoretical calculations and in situ solid state NMR spectroscopy have been combined to get insight on the nature of the active sites for the Beckmann rearrangement reaction in borosilicate zeolites. The interaction of a B site in zeolite Beta with a series of probe molecules (ammonia, pyridine, acetone and water) has been modelled and the (15)N and (11)B NMR isotropic chemical shift of the resulting complexes calculated and compared with experimental in situ NMR results. This approach has allowed validation of the methodology to model the adsorption on a zeolite boron site of molecules of varying basicity which are either protonated or non-protonated. The limitation is that theoretical calculations overestimate the effect of molecular adsorption through hydrogen bonds on the calculated isotropic (11)B NMR chemical shift.Theoretical and experimental results on the adsorption of acetophenone and cyclohexanone oximes on zeolite B-Beta indicate that Brønsted acid sites protonate the oximes, changing the boron coordination from trigonal to tetrahedral. Comparison of theoretical and experimental (15)N NMR chemical shifts of the adsorbed amides (acetanilide and epsilon-caprolactam) indicates that they are non-protonated, and the (11)B NMR spectra show that, as expected, boron remains in trigonal coordination with an isotropic delta(11)B(exp) which differs from the calculated value delta(11)B(calc). PMID:20454729

  15. Human L-ficolin, a recognition molecule of the lectin activation pathway of complement, activates complement by binding to pneumolysin, the major toxin of Streptococcus pneumoniae.

    PubMed

    Ali, Youssif M; Kenawy, Hany I; Muhammad, Adnan; Sim, Robert B; Andrew, Peter W; Schwaeble, Wilhelm J

    2013-01-01

    The complement system is an essential component of the immune response, providing a critical line of defense against different pathogens including S. pneumoniae. Complement is activated via three distinct pathways: the classical (CP), the alternative (AP) and the lectin pathway (LP). The role of Pneumolysin (PLY), a bacterial toxin released by S. pneumoniae, in triggering complement activation has been studied in vitro. Our results demonstrate that in both human and mouse sera complement was activated via the CP, initiated by direct binding of even non-specific IgM and IgG3 to PLY. Absence of CP activity in C1q(-/-) mouse serum completely abolished any C3 deposition. However, C1q depleted human serum strongly opsonized PLY through abundant deposition of C3 activation products, indicating that the LP may have a vital role in activating the human complement system on PLY. We identified that human L-ficolin is the critical LP recognition molecule that drives LP activation on PLY, while all of the murine LP recognition components fail to bind and activate complement on PLY. This work elucidates the detailed interactions between PLY and complement and shows for the first time a specific role of the LP in PLY-mediated complement activation in human serum.

  16. A conserved activation cluster is required for allosteric communication in HtrA-family proteases

    PubMed Central

    de Regt, Anna; Kim, Seokhee; Sohn, Jungsan; Grant, Robert A.; Baker, Tania A.; Sauer, Robert T.

    2015-01-01

    Summary In E. coli, outer-membrane stress causes a transcriptional response through a signaling cascade initiated by DegS cleavage of a transmembrane anti-sigma factor. Each subunit of DegS, an HtrA-family protease, contains a protease domain and a PDZ domain. The trimeric protease domain is autoinhibited by the unliganded PDZ domains. Allosteric activation requires binding of unassembled outer-membrane proteins (OMPs) to the PDZ domains and protein-substrate binding. Here, we identify a set of DegS residues that cluster together at subunit-subunit interfaces in the trimer, link the active sites and substrate-binding sites, and are crucial for stabilizing the active enzyme conformation in response to OMP signaling. These residues are conserved across the HtrA-protease family, including orthologs linked to human disease, supporting a common mechanism of allosteric activation. Indeed, mutation of residues at homologous positions in the DegP quality-control protease also eliminates allosteric activation. PMID:25703375

  17. Macin Family of Antimicrobial Proteins Combines Antimicrobial and Nerve Repair Activities*

    PubMed Central

    Jung, Sascha; Sönnichsen, Frank D.; Hung, Chien-Wen; Tholey, Andreas; Boidin-Wichlacz, Céline; Haeusgen, Wiebke; Gelhaus, Christoph; Desel, Christine; Podschun, Rainer; Waetzig, Vicki; Tasiemski, Aurélie; Leippe, Matthias; Grötzinger, Joachim

    2012-01-01

    The tertiary structures of theromacin and neuromacin confirmed the macin protein family as a self-contained family of antimicrobial proteins within the superfamily of scorpion toxin-like proteins. The macins, which also comprise hydramacin-1, are antimicrobially active against Gram-positive and Gram-negative bacteria. Despite high sequence identity, the three proteins showed distinct differences with respect to their biological activity. Neuromacin exhibited a significantly stronger capacity to permeabilize the cytoplasmic membrane of Bacillus megaterium than theromacin and hydramacin-1. Accordingly, it is the only macin that displays pore-forming activity and that was potently active against Staphylococcus aureus. Moreover, neuromacin and hydramacin-1 led to an aggregation of bacterial cells that was not observed with theromacin. Analysis of the molecular surface properties of macins allowed confirmation of the barnacle model as the mechanistic model for the aggregation effect. Besides being antimicrobially active, neuromacin and theromacin, in contrast to hydramacin-1, were able to enhance the repair of leech nerves ex vivo. Notably, all three macins enhanced the viability of murine neuroblastoma cells, extending their functional characteristics. As neuromacin appears to be both a functional and structural chimera of hydramacin-1 and theromacin, the putative structural correlate responsible for the nerve repair capacity in leech was located to a cluster of six amino acid residues using the sequence similarity of surface-exposed regions. PMID:22396551

  18. Elucidating the Activation Mechanism of the Insulin-Family Proteins with Molecular Dynamics Simulations

    PubMed Central

    Papaioannou, Anastasios; Kuyucak, Serdar; Kuncic, Zdenka

    2016-01-01

    The insulin-family proteins bind to their own receptors, but insulin-like growth factor II (IGF-II) can also bind to the A isoform of the insulin receptor (IR-A), activating unique and alternative signaling pathways from those of insulin. Although extensive studies of insulin have revealed that its activation is associated with the opening of the B chain-C terminal (BC-CT), the activation mechanism of the insulin-like growth factors (IGFs) still remains unknown. Here, we present the first comprehensive study of the insulin-family proteins comparing their activation process and mechanism using molecular dynamics simulations to reveal new insights into their specificity to the insulin receptor. We have found that all the proteins appear to exhibit similar stochastic dynamics in their conformational change to an active state. For the IGFs, our simulations show that activation involves two opening locations: the opening of the BC-CT section away from the core, similar to insulin; and the additional opening of the BC-CT section away from the C domain. Furthermore, we have found that these two openings occur simultaneously in IGF-I, but not in IGF-II, where they can occur independently. This suggests that the BC-CT section and the C domain behave as a unified domain in IGF-I, but as two independent domains in IGF-II during the activation process, implying that the IGFs undergo different activation mechanisms for receptor binding. The probabilities of the active and inactive states of the proteins suggest that IGF-II is hyperactive compared to IGF-I. The hinge residue and the hydrophobic interactions in the core are found to play a critical role in the stability and activity of IGFs. Overall, our simulations have elucidated the crucial differences and similarities in the activation mechanisms of the insulin-family proteins, providing new insights into the molecular mechanisms responsible for the observed differences between IGF-I and IGF-II in receptor binding. PMID

  19. Activation of cell signaling via optical manipulation of gold-coated liposomes encapsulating signaling molecules

    NASA Astrophysics Data System (ADS)

    Orsinger, Gabriel V.; Leung, Sarah J.; Romanowski, Marek

    2013-02-01

    Many diseases involve changes in cell signaling cascades, as seen commonly in drug resistant cancers. To better understand these intricate signaling events in diseased cells and tissues, experimental methods of probing cellular communication at a single to multi-cell level are required. We recently introduced a general platform for activation of selected signaling pathways by optically controlled delivery and release of water soluble factors using gold-coated liposomes. In the example presented here, we encapsulated inositol trisphosphate (IP3), a ubiquitous intracellular secondary messenger involved in GPCR and Akt signaling cascades, within 100 nm gold-coated liposomes. The high polarizability of the liposome's unique gold pseudo-shell allows stable optical trapping for subcellular manipulation in the presence of cells. We take this optical manipulation further by optically injecting IP3-containing liposomes into the cytosol of a single cell to initiate localized cell signaling. Upon optical injection of liposomal IP3 into a single ovarian carcinoma cell, we observed localized activation as reported by changes in Indo-1 fluorescence intensity. With established gap junctions between the injected cell and neighboring cells, we monitored propagation of this signaling to and through nearby cells.

  20. Anticancer Activity of Small Molecule and Nanoparticulate Arsenic(III) Complexes

    PubMed Central

    Swindell, Elden P.; Hankins, Patrick L.; Chen, Haimei; Miodragović, Ðenana U.; O'Halloran, Thomas V.

    2014-01-01

    Starting in ancient China and Greece, arsenic-containing compounds have been used in the treatment of disease for over 3000 years. They were used for a variety of diseases in the 20th century, including parasitic and sexually transmitted illnesses. A resurgence of interest in the therapeutic application of arsenicals has been driven by the discovery that low doses of a 1% aqueous solution of arsenic trioxide (i.e. arsenous acid) leads to complete remission of certain types of leukemia. Since FDA approval of arsenic trioxide (As2O3) for treatment of acute promyelocytic leukemia (APL) in 2000, it has become a front line therapy in this indication. There are currently over 100 active clinical trials involving inorganic arsenic or organoarsenic compounds registered with the FDA for the treatment of cancers. New generations of inorganic and organometallic arsenic compounds with enhanced activity or targeted cytotoxicity are being developed to overcome some of the shortcomings of arsenic therapeutics, namely short plasma half-lives and narrow therapeutic window. PMID:24147771

  1. Activation of CD4+ T lymphocytes form interleukin 2-deficient mice by costimulatory B7 molecules.

    PubMed Central

    Razi-Wolf, Z; Höllander, G A; Reiser, H

    1996-01-01

    Interleukin 2 (IL-2)-deficient (IL-2-/-) mice develop hemolytic anemia and chronic inflammatory bowel disease. Importantly, the induction of disease in IL-2-deficient mice is critically dependent on CD4+ T cells. We have studied the requirements of T cells from IL-2-deficient mice for costimulation with B7 antigens. Stable B7-1 or B7-2 chinese hamster ovary (CHO) cell transfectants could synergize with anti-CD3 monoclonal antibody (mAb) to induce the proliferation of CD4+ T cells from IL-2-/- mutant mice. Further mechanistic studies established that B7-induced activation resulted in surface expression of the alpha chain of the IL-2 receptor. B7-induced proliferation occurred independently of IL-4 and was largely independent of the common gamma chain of the IL-2, IL-4, IL-7, IL-9, and IL-15 receptors. Finally, anti-B7-2 but not anti-B7-1 mAb was able to inhibit the activation of IL-2-/- T cells induced by anti-CD3 mAb in the presence of syngeneic antigen-presenting cells. The results of our experiments indicate that IL-2-/- CD4+ T cells remain responsive to B7 stimulation and raise the possibility that B7 antagonists have a role in the prevention/treatment of inflammatory bowel disease. Images Fig. 3 PMID:8610140

  2. Antibody nanoparticle dispersions formed with mixtures of crowding molecules retain activity and in vivo bioavailability

    PubMed Central

    Miller, Maria A.; Khan, Tarik A.; Kaczorowski, Kevin J.; Wilson, Brian K.; Dinin, Aileen K.; Borwankar, Ameya U.; Rodrigues, Miguel A.; Truskett, Thomas M.; Johnston, Keith P.; Maynard, Jennifer A.

    2013-01-01

    Monoclonal antibodies continue to command a large market for treatment of a variety of diseases. In many cases, the doses required for therapeutic efficacy are large, limiting options for antibody delivery and administration. We report a novel formulation strategy based on dispersions of antibody nanoclusters that allows for subcutaneous injection of highly concentrated antibody (~190 mg/ml). A solution of monoclonal antibody 1B7 was rapidly frozen and lyophilized using a novel spiral-wound in situ freezing technology (SWIFT) to generate amorphous particles. Upon gentle stirring, a translucent dispersion of ~430 nm protein clusters low apparent viscosity (~24 cp) formed rapidly in buffer containing the pharmaceutically acceptable crowding agents, trehalose, polyethylene glycol and n-methyl-2-pyrrolidone. Upon in vitro dilution of the dispersion, the nanoclusters rapidly reverted to monomeric protein with full activity, as monitored by dynamic light scattering and antigen binding. When administered to mice as an intravenous solution, subcutaneous solution or subcutaneous dispersion at similar (4.6-7.3 mg/kg) or ultra-high dosages (51.6 mg/kg), the distribution and elimination kinetics were within error and the protein retained full activity. Overall, this method of generating high-concentration, low-viscosity dispersions of antibody nanoclusters could lead to improved administration and patient compliance, providing new opportunities for the biotechnology industry. PMID:22777686

  3. Antibody nanoparticle dispersions formed with mixtures of crowding molecules retain activity and in vivo bioavailability.

    PubMed

    Miller, Maria A; Khan, Tarik A; Kaczorowski, Kevin J; Wilson, Brian K; Dinin, Aileen K; Borwankar, Ameya U; Rodrigues, Miguel A; Truskett, Thomas M; Johnston, Keith P; Maynard, Jennifer A

    2012-10-01

    Monoclonal antibodies continue to command a large market for treatment of a variety of diseases. In many cases, the doses required for therapeutic efficacy are large, limiting options for antibody delivery and administration. We report a novel formulation strategy based on dispersions of antibody nanoclusters that allows for subcutaneous injection of highly concentrated antibody (≈ 190 mg/mL). A solution of monoclonal antibody 1B7 was rapidly frozen and lyophilized using a novel spiral-wound in-situ freezing technology to generate amorphous particles. Upon gentle stirring, a translucent dispersion of approximately 430 nm protein clusters with low apparent viscosity (≈ 24 cp) formed rapidly in buffer containing the pharmaceutically acceptable crowding agents such as trehalose, polyethylene glycol, and n-methyl-2-pyrrolidone. Upon in vitro dilution of the dispersion, the nanoclusters rapidly reverted to monomeric protein with full activity, as monitored by dynamic light scattering and antigen binding. When administered to mice as an intravenous solution, subcutaneous solution, or subcutaneous dispersion at similar (4.6-7.3 mg/kg) or ultra-high dosages (51.6 mg/kg), the distribution and elimination kinetics were within error and the protein retained full activity. Overall, this method of generating high-concentration, low-viscosity dispersions of antibody nanoclusters could lead to improved administration and patient compliance, providing new opportunities for the biotechnology industry.

  4. Evaluation of Endothelial Cell Adhesion Molecules and Anti-C1q Antibody in Discriminating between Active and Non-Active Systemic Lupus Erythematosus

    PubMed Central

    Mahayidin, Hasni; Yahya, Nurul Khaiza; Wan Ghazali, Wan Syamimee; Mohd Ismail, Asmahan; Wan Ab Hamid, Wan Zuraida

    2016-01-01

    Background Detecting the active state of systemic lupus erythematosus (SLE) is important but challenging. This study aimed to determine the diagnostic accuracy of serum endothelial cell adhesion molecules (ICAM-1 and VCAM-1) and anti-C1q antibody in discriminating between active and non-active SLE. Methods Using SELENA-SLE disease activity index (SLEDAI), 95 SLE patients (45 active and 50 non-active) were assessed. A score above five was considered indicative of active SLE. The blood samples were tested for serum ICAM-1, VCAM-1 and anti-C1q antibody using enzyme-linked immunosorbent assay (ELISA). Results The levels of serum VCAM-1 and anti-C1q antibody were significantly higher in active SLE patients. Both VCAM-1 and anti-C1q were able to discriminate between active and non-active SLE (p-value < 0.001 and 0.005, respectively). From the receiver operating characteristic curves (ROCs) constructed, the optimal cut-off values for VCAM-1 and anti-C1q antibody in discriminating between active and non-active SLE were 30.5 ng/mL (69.0% sensitivity, 60.0% specificity, PPV 58.5%, NPV 66.7%) and 7.86 U/mL (75.6% sensitivity, 80% specificity, PPV 77.3%, NPV 78.4%), respectively. However, serum ICAM-1 level was unable to discriminate between the two groups (p-value = 0.193). Conclusion Anti-C1q antibody demonstrated the best diagnostic accuracy in discriminating between active and non-active SLE patients. PMID:27418866

  5. Identification of Quorum-Sensing Signal Molecules and a Biosynthetic Gene in Alicycliphilus sp. Isolated from Activated Sludge.

    PubMed

    Morohoshi, Tomohiro; Okutsu, Noriya; Xie, Xiaonan; Ikeda, Tsukasa

    2016-01-01

    Activated sludge is a complicated mixture of various microorganisms that is used to treat sewage and industrial wastewater. Many bacteria produce N-acylhomoserine lactone (AHL) as a quorum-sensing signal molecule to regulate the expression of the exoenzymes used for wastewater treatment. Here, we isolated an AHL-producing bacteria from an activated sludge sample collected from an electronic component factory, which we named Alicycliphilus sp. B1. Clone library analysis revealed that Alicycliphilus was a subdominant genus in this sample. When we screened the activated sludge sample for AHL-producing strains, 12 of 14 the AHL-producing isolates were assigned to the genus Alicycliphilus. A putative AHL-synthase gene, ALISP_0667, was cloned from the genome of B1 and transformed into Escherichia coli DH5α. The AHLs were extracted from the culture supernatants of the B1 strain and E. coli DH5α cells harboring the ALISP_0667 gene and were identified by liquid chromatography-mass spectrometry as N-(3-hydroxydecanoyl)-l-homoserine lactone and N-(3-hydroxydodecanoyl)-l-homoserine lactone. The results of comparative genomic analysis suggested that the quorum-sensing genes in the B1 strain might have been acquired by horizontal gene transfer within activated sludge. PMID:27490553

  6. Identification of Quorum-Sensing Signal Molecules and a Biosynthetic Gene in Alicycliphilus sp. Isolated from Activated Sludge

    PubMed Central

    Morohoshi, Tomohiro; Okutsu, Noriya; Xie, Xiaonan; Ikeda, Tsukasa

    2016-01-01

    Activated sludge is a complicated mixture of various microorganisms that is used to treat sewage and industrial wastewater. Many bacteria produce N-acylhomoserine lactone (AHL) as a quorum-sensing signal molecule to regulate the expression of the exoenzymes used for wastewater treatment. Here, we isolated an AHL-producing bacteria from an activated sludge sample collected from an electronic component factory, which we named Alicycliphilus sp. B1. Clone library analysis revealed that Alicycliphilus was a subdominant genus in this sample. When we screened the activated sludge sample for AHL-producing strains, 12 of 14 the AHL-producing isolates were assigned to the genus Alicycliphilus. A putative AHL-synthase gene, ALISP_0667, was cloned from the genome of B1 and transformed into Escherichia coli DH5α. The AHLs were extracted from the culture supernatants of the B1 strain and E. coli DH5α cells harboring the ALISP_0667 gene and were identified by liquid chromatography-mass spectrometry as N-(3-hydroxydecanoyl)-l-homoserine lactone and N-(3-hydroxydodecanoyl)-l-homoserine lactone. The results of comparative genomic analysis suggested that the quorum-sensing genes in the B1 strain might have been acquired by horizontal gene transfer within activated sludge. PMID:27490553

  7. Identification of Quorum-Sensing Signal Molecules and a Biosynthetic Gene in Alicycliphilus sp. Isolated from Activated Sludge.

    PubMed

    Morohoshi, Tomohiro; Okutsu, Noriya; Xie, Xiaonan; Ikeda, Tsukasa

    2016-08-02

    Activated sludge is a complicated mixture of various microorganisms that is used to treat sewage and industrial wastewater. Many bacteria produce N-acylhomoserine lactone (AHL) as a quorum-sensing signal molecule to regulate the expression of the exoenzymes used for wastewater treatment. Here, we isolated an AHL-producing bacteria from an activated sludge sample collected from an electronic component factory, which we named Alicycliphilus sp. B1. Clone library analysis revealed that Alicycliphilus was a subdominant genus in this sample. When we screened the activated sludge sample for AHL-producing strains, 12 of 14 the AHL-producing isolates were assigned to the genus Alicycliphilus. A putative AHL-synthase gene, ALISP_0667, was cloned from the genome of B1 and transformed into Esche