Science.gov

Sample records for activities specific activities

  1. High specific activity silicon-32

    DOEpatents

    Phillips, Dennis R.; Brzezinski, Mark A.

    1996-01-01

    A process for preparation of silicon-32 is provided and includes contacting an irradiated potassium chloride target, including spallation products from a prior irradiation, with sufficient water, hydrochloric acid or potassium hydroxide to form a solution, filtering the solution, adjusting pH of the solution to from about 5.5 to about 7.5, admixing sufficient molybdate-reagent to the solution to adjust the pH of the solution to about 1.5 and to form a silicon-molybdate complex, contacting the solution including the silicon-molybdate complex with a dextran-based material, washing the dextran-based material to remove residual contaminants such as sodium-22, separating the silicon-molybdate complex from the dextran-based material as another solution, adding sufficient hydrochloric acid and hydrogen peroxide to the solution to prevent reformation of the silicon-molybdate complex and to yield an oxidization state of the molybdate adapted for subsequent separation by an anion exchange material, contacting the solution with an anion exchange material whereby the molybdate is retained by the anion exchange material and the silicon remains in solution, and optionally adding sufficient alkali metal hydroxide to adjust the pH of the solution to about 12 to 13. Additionally, a high specific activity silicon-32 product having a high purity is provided.

  2. High specific activity silicon-32

    DOEpatents

    Phillips, D.R.; Brzezinski, M.A.

    1996-06-11

    A process for preparation of silicon-32 is provided and includes contacting an irradiated potassium chloride target, including spallation products from a prior irradiation, with sufficient water, hydrochloric acid or potassium hydroxide to form a solution, filtering the solution, adjusting pH of the solution from about 5.5 to about 7.5, admixing sufficient molybdate-reagent to the solution to adjust the pH of the solution to about 1.5 and to form a silicon-molybdate complex, contacting the solution including the silicon-molybdate complex with a dextran-based material, washing the dextran-based material to remove residual contaminants such as sodium-22, separating the silicon-molybdate complex from the dextran-based material as another solution, adding sufficient hydrochloric acid and hydrogen peroxide to the solution to prevent reformation of the silicon-molybdate complex and to yield an oxidation state of the molybdate adapted for subsequent separation by an anion exchange material, contacting the solution with an anion exchange material whereby the molybdate is retained by the anion exchange material and the silicon remains in solution, and optionally adding sufficient alkali metal hydroxide to adjust the pH of the solution to about 12 to 13. Additionally, a high specific activity silicon-32 product having a high purity is provided.

  3. Arylesterase Phenotype-Specific Positive Association Between Arylesterase Activity and Cholinesterase Specific Activity in Human Serum

    PubMed Central

    Aoki, Yutaka; Helzlsouer, Kathy J.; Strickland, Paul T.

    2014-01-01

    Context: Cholinesterase (ChE) specific activity is the ratio of ChE activity to ChE mass and, as a biomarker of exposure to cholinesterase inhibitors, has a potential advantage over simple ChE activity. Objective: To examine the association of several potential correlates (serum arylesterase/paraoxonase activity, serum albumin, sex, age, month of blood collection, and smoking) with plasma ChE specific activity. Methods: We analyzed data from 195 cancer-free controls from a nested case-control study, accounting for potential confounding. Results: Arylesterase activity had an independent, statistically significant positive association with ChE specific activity, and its magnitude was the greatest for the arylesterase phenotype corresponding to the QQ PON1192 genotype followed by phenotypes corresponding to QR and RR genotypes. Serum albumin was positively associated with ChE specific activity. Conclusions: Plasma arylesterase activity was positively associated with plasma ChE specific activity. This observation is consistent with protection conferred by a metabolic phenotype resulting in reduced internal dose. PMID:24473115

  4. Production of high specific activity silicon-32

    SciTech Connect

    Phillips, D.R.; Brzezinski, M.A.

    1998-12-31

    This is the final report of a three-year, Laboratory Directed Research and Development Project (LDRD) at Los Alamos National Laboratory (LANL). There were two primary objectives for the work performed under this project. The first was to take advantage of capabilities and facilities at Los Alamos to produce the radionuclide {sup 32}Si in unusually high specific activity. The second was to combine the radioanalytical expertise at Los Alamos with the expertise at the University of California to develop methods for the application of {sup 32}Si in biological oceanographic research related to global climate modeling. The first objective was met by developing targetry for proton spallation production of {sup 32}Si in KCl targets and chemistry for its recovery in very high specific activity. The second objective was met by developing a validated field-useable, radioanalytical technique, based upon gas-flow proportional counting, to measure the dynamics of silicon uptake by naturally occurring diatoms.

  5. Chosen postures during specific sitting activities.

    PubMed

    Kamp, Irene; Kilincsoy, Umit; Vink, Peter

    2011-11-01

    This research study analysed the interaction between people's postures and activities while in semi-public/leisure situations and during transportation (journey by train). In addition, the use of small electronic devices received particular emphasis. Video recordings in German trains and photographs in Dutch semi-public spaces were analysed using a variation of Branton and Grayson's (An evaluation of train seats by observation of sitting behaviour. Ergonomics, 10 (1), (1967), 35-51) postural targeting forms and photos. The analysis suggests a significant relationship between most activities and the position of the head, trunk and arms during transportation situations. The relationship during public situations is less straightforward. Watching, talking/discussing and reading were the most observed activities for the transportation and leisure situations combined. Surprisingly, differences in head, trunk, arm and leg postures were not significant when using small electronic devices. Important issues not considered in this study include the duration of the activities, the gender and age of observed subjects and the influence of the time of day. These are interesting issues to consider and include for future research. STATEMENT OF RELEVANCE: This study shows what activities people choose to carry out and their related postures when not forced to a specific task (e.g. driving). The results of this study can be used for designing comfortable seating in the transportation industry (car passenger, train, bus and aircraft seats) and semi-public/leisure spaces.

  6. High specific activity platinum-195m

    SciTech Connect

    Mirzadeh, Saed; Du, Miting; Beets, Arnold L.; Knapp, Jr., Furn F.

    2004-10-12

    A new composition of matter includes .sup.195m Pt characterized by a specific activity of at least 30 mCi/mg Pt, generally made by method that includes the steps of: exposing .sup.193 Ir to a flux of neutrons sufficient to convert a portion of the .sup.193 Ir to .sup.195m Pt to form an irradiated material; dissolving the irradiated material to form an intermediate solution comprising Ir and Pt; and separating the Pt from the Ir by cation exchange chromatography to produce .sup.195m Pt.

  7. Production Of High Specific Activity Copper-67

    DOEpatents

    Jamriska, Sr., David J.; Taylor, Wayne A.; Ott, Martin A.; Fowler, Malcolm; Heaton, Richard C.

    2003-10-28

    A process for the selective production and isolation of high specific activity Cu.sup.67 from proton-irradiated enriched Zn.sup.70 target comprises target fabrication, target irradiation with low energy (<25 MeV) protons, chemical separation of the Cu.sup.67 product from the target material and radioactive impurities of gallium, cobalt, iron, and stable aluminum via electrochemical methods or ion exchange using both anion and cation organic ion exchangers, chemical recovery of the enriched Zn.sup.70 target material, and fabrication of new targets for re-irradiation is disclosed.

  8. Production Of High Specific Activity Copper-67

    DOEpatents

    Jamriska, Sr., David J.; Taylor, Wayne A.; Ott, Martin A.; Fowler, Malcolm; Heaton, Richard C.

    2002-12-03

    A process for the selective production and isolation of high specific activity cu.sup.67 from proton-irradiated enriched Zn.sup.70 target comprises target fabrication, target irradiation with low energy (<25 MeV) protons, chemical separation of the Cu.sup.67 product from the target material and radioactive impurities of gallium, cobalt, iron, and stable aluminum via electrochemical methods or ion exchange using both anion and cation organic ion exchangers, chemical recovery of the enriched Zn.sup.70 target material, and fabrication of new targets for re-irradiation is disclosed.

  9. Mechanisms of Specificity for Hox Factor Activity

    PubMed Central

    Zandvakili, Arya; Gebelein, Brian

    2016-01-01

    Metazoans encode clusters of paralogous Hox genes that are critical for proper development of the body plan. However, there are a number of unresolved issues regarding how paralogous Hox factors achieve specificity to control distinct cell fates. First, how do Hox paralogs, which have very similar DNA binding preferences in vitro, drive different transcriptional programs in vivo? Second, the number of potential Hox binding sites within the genome is vast compared to the number of sites bound. Hence, what determines where in the genome Hox factors bind? Third, what determines whether a Hox factor will activate or repress a specific target gene? Here, we review the current evidence that is beginning to shed light onto these questions. In particular, we highlight how cooperative interactions with other transcription factors (especially PBC and HMP proteins) and the sequences of cis-regulatory modules provide a basis for the mechanisms of Hox specificity. We conclude by integrating a number of the concepts described throughout the review in a case study of a highly interrogated Drosophila cis-regulatory module named “The Distal-less Conserved Regulatory Element” (DCRE). PMID:27583210

  10. Human task-specific somatosensory activation.

    PubMed

    Ginsberg, M D; Yoshii, F; Vibulsresth, S; Chang, J Y; Duara, R; Barker, W W; Boothe, T E

    1987-08-01

    We used positron emission tomography to study normal patterns of local cortical metabolic activation induced by somatosensory stimuli. Palpation and sorting of mah-jongg tiles by textured design increased local glucose metabolic rate (lCMRgl), by 18% on average, in contralateral somatosensory cortex. A graphesthesia task gave a similar result. In contrast, vigorous vibrotactile stimulation of fingers, face, or knee did not produce a consistent focus of activation. Our results indicate that lCMRgl activation is best achieved by somatosensory tasks requiring an active perceptual effort.

  11. Activity Specificity, Physical and Psychosocial Dimensions.

    ERIC Educational Resources Information Center

    Hatfield, Frederick C.

    The position is taken that the physical parameters of one's involvement in activity learning depend in large measure upon the objectives of the participant. General comments regarding the physical parameters of most activity classes are made. Underlying commonalities existing among these parameters are identified as: (1) freedom from disease; (2)…

  12. Production of high specific activity silicon-32

    DOEpatents

    Phillips, Dennis R.; Brzezinski, Mark A.

    1994-01-01

    A process for preparation of silicon-32 is provide and includes contacting an irradiated potassium chloride target, including spallation products from a prior irradiation, with sufficient water, hydrochloric acid or potassium hydroxide to form a solution, filtering the solution, adjusting pH of the solution to from about 5.5 to about 7.5, admixing sufficient molybdate-reagent to the solution to adjust the pH of the solution to about 1.5 and to form a silicon-molybdate complex, contacting the solution including the silicon-molybdate complex with a dextran-based material, washing the dextran-based material to remove residual contaminants such as sodium-22, separating the silicon-molybdate complex from the dextran-based material as another solution, adding sufficient hydrochloric acid and hydrogen peroxide to the solution to prevent reformation of the silicon-molybdate complex and to yield an oxidization state of the molybdate adapted for subsequent separation by an anion exchange material, contacting the solution with an anion exchange material whereby the molybdate is retained by the anion exchange material and the silicon remains in solution, and optionally adding sufficient alkali metal hydroxide to adjust the pH of the solution to about 12 to 13. Additionally, a high specific activity silicon-32 product having a high purity is provided.

  13. Activities.

    ERIC Educational Resources Information Center

    Kincaid, Charlene; And Others

    1993-01-01

    Presents an activity in which students collect and organize data from a real-world simulation of the scientific concept of half life. Students collect data using a marble sifter, analyze the data using a graphing calculator, and determine an appropriate mathematical model. Includes reproducible worksheets. (MDH)

  14. Activities.

    ERIC Educational Resources Information Center

    Mathematics Teacher, 1982

    1982-01-01

    The material presented is designed to help students explore geometric patterns involving Fibonnaci numbers and the golden ratio, and to aid in review of basic geometry skills. Worksheet masters intended for duplication are provided. Suggestions are made of possible classroom extensions to the initial activities. (MP)

  15. Suppression of Antigen-Specific Lymphocyte Activation in Simulated Microgravity

    NASA Technical Reports Server (NTRS)

    Cooper, David; Pride, Michael W.; Brown, Eric L.; Risin, Diana; Pellis, Neal R.

    1999-01-01

    Various parameters of immune suppression are observed in astronauts during and after spaceflight, and in isolated immune cells in true and simulated microgravity. Specifically, polyclonal activation of T cells is severely suppressed in true and simulated microgravity. These recent findings with various polyclonal activators suggests a suppression of oligoclonal lymphocyte activation in microgravity. We utilized rotating wall vessel (RWV) bioreactors that simulate aspects of microgravity for cell cultures to analyze three models of antigen-specific activation. A mixed-lymphocyte reaction (MLR), as a model for a primary immune response; a tetanus toxoid (TT) response and a B. burgdorferi (Bb) response, as models of a secondary immune response, were all suppressed in the RWV bioreactor. Our findings confirm that the suppression of activation observed with polyclonal models also encompasses oligoclonal antigen-specific activation.

  16. Calf thymus ribonuclease H IIa activity lacks ribonuclease H specificity.

    PubMed

    Vonwirth, H; Frank, P; Büsen, W

    1990-03-15

    Less purified fractions of ribonuclease H IIa activity of calf thymus display divalent cation-dependent ribonuclease H activity and divalent cation-independent ribonuclease activity. Because the ratio of the two enzyme activities does not change during successive chromatographic procedures, we suggest that ribonuclease H IIa activity is indeed able to degrade both ssRNA and the RNA moiety of RNA.DNA-hybrids. Ribonuclease H IIa activity can therefore be differentiated from calf thymus ribonuclease H I and H IIb by its lack of ribonuclease H specificity. The native molecular mass of ribonuclease H IIa activity is between 23 and 28 kDa. Under denaturing conditions a 23 kDa-protein band copurifies with the enzyme activity suggesting that this enzyme is monomeric.

  17. Mechanism of dual specificity kinase activity of DYRK1A.

    PubMed

    Walte, Agnes; Rüben, Katharina; Birner-Gruenberger, Ruth; Preisinger, Christian; Bamberg-Lemper, Simone; Hilz, Nikolaus; Bracher, Franz; Becker, Walter

    2013-09-01

    The function of many protein kinases is controlled by the phosphorylation of a critical tyrosine residue in the activation loop. Dual specificity tyrosine-phosphorylation-regulated kinases (DYRKs) autophosphorylate on this tyrosine residue but phosphorylate substrates on aliphatic amino acids. This study addresses the mechanism of dual specificity kinase activity in DYRK1A and related kinases. Tyrosine autophosphorylation of DYRK1A occurred rapidly during in vitro translation and did not depend on the non-catalytic domains or other proteins. Expression in bacteria as well as in mammalian cells revealed that tyrosine kinase activity of DYRK1A is not restricted to the co-translational autophosphorylation in the activation loop. Moreover, mature DYRK1A was still capable of tyrosine autophosphorylation. Point mutants of DYRK1A and DYRK2 lacking the activation loop tyrosine showed enhanced tyrosine kinase activity. A series of structurally diverse DYRK1A inhibitors was used to pharmacologically distinguish different conformational states of the catalytic domain that are hypothesized to account for the dual specificity kinase activity. All tested compounds inhibited substrate phosphorylation with higher potency than autophosphorylation but none of the tested inhibitors differentially inhibited threonine and tyrosine kinase activity. Finally, the related cyclin-dependent kinase-like kinases (CLKs), which lack the activation loop tyrosine, autophosphorylated on tyrosine both in vitro and in living cells. We propose a model of DYRK autoactivation in which tyrosine autophosphorylation in the activation loop stabilizes a conformation of the catalytic domain with enhanced serine/threonine kinase activity without disabling tyrosine phosphorylation. The mechanism of dual specificity kinase activity probably applies to related serine/threonine kinases that depend on tyrosine autophosphorylation for maturation. PMID:23809146

  18. Flexible task-specific control using active vision

    NASA Astrophysics Data System (ADS)

    Firby, Robert J.; Swain, Michael J.

    1992-04-01

    This paper is about the interface between continuous and discrete robot control. We advocate encapsulating continuous actions and their related sensing strategies into behaviors called situation specific activities, which can be constructed by a symbolic reactive planner. Task- specific, real-time perception is a fundamental part of these activities. While researchers have successfully used primitive touch and sonar sensors in such situations, it is more problematic to achieve reasonable performance with complex signals such as those from a video camera. Active vision routines are suggested as a means of incorporating visual data into real time control and as one mechanism for designating aspects of the world in an indexical-functional manner. Active vision routines are a particularly flexible sensing methodology because different routines extract different functional attributes from the world using the same sensor. In fact, there will often be different active vision routines for extracting the same functional attribute using different processing techniques. This allows an agent substantial leeway to instantiate its activities in different ways under different circumstances using different active vision routines. We demonstrate the utility of this architecture with an object tracking example. A control system is presented that can be reconfigured by a reactive planner to achieve different tasks. We show how this system allows us to build interchangeable tracking activities that use either color histogram or motion based active vision routines.

  19. UML activity diagrams in requirements specification of logic controllers

    NASA Astrophysics Data System (ADS)

    Grobelna, Iwona; Grobelny, Michał

    2015-12-01

    Logic controller specification can be prepared using various techniques. One of them is the wide understandable and user-friendly UML language and its activity diagrams. Using formal methods during the design phase increases the assurance that implemented system meets the project requirements. In the approach we use the model checking technique to formally verify a specification against user-defined behavioral requirements. The properties are usually defined as temporal logic formulas. In the paper we propose to use UML activity diagrams in requirements definition and then to formalize them as temporal logic formulas. As a result, UML activity diagrams can be used both for logic controller specification and for requirements definition, what simplifies the specification and verification process.

  20. Stage-specific fucosyltransferase activity during mouse spermatogenesis

    SciTech Connect

    Cardullo, R.A.; Armant, D.R.; Millette, C.F.

    1986-05-01

    This laboratory is involved in the biochemical characterization of developing spermatogenic cells. The authors have measured the in vitro activity of fucosyltransferase (FT) in germ cells. FT activity was assayed with a procedure modified from Letts et al. using GDP-(/sup 14/C)-fucose and asialofetuin as substrates. After incubation for 15 minutes at 33/sup 0/C, the reaction was stopped by adding cold 500 mM EDTA. Radiolabeled asialofetuin was isolated using Bio-Gel P-10 chromatography. The FT activity of germ cells purified from seminiferous tubules was 18.5 +/- 1.7 pmol/mg protein-min. To see if this activity varied at different stages of development, germ cells were further separated in a STAPUT chamber using a 2-4% BSA gradient. Pachytene spermatocytes or round spermatids were purified to at least 87%. The FT activity in isolated pachytene spermatocytes was 24.4 +/- 1.2 pmol/mg protein-min while the activity in isolated round spermatids was 49.0 +/- 7.2 pmol/mg protein-min. These results suggest that the highest FT activity is in developing spermatogenic cells with round spermatids having nearly twice the FT activity as pachytene spermatocytes. This increase in FT activity may be biologically significant since it occurs at a time when the Golgi apparatus is undergoing differentiation and when stage-specific fucosylated proteins appear.

  1. High efficiency cell-specific targeting of cytokine activity

    NASA Astrophysics Data System (ADS)

    Garcin, Geneviève; Paul, Franciane; Staufenbiel, Markus; Bordat, Yann; van der Heyden, José; Wilmes, Stephan; Cartron, Guillaume; Apparailly, Florence; de Koker, Stefaan; Piehler, Jacob; Tavernier, Jan; Uzé, Gilles

    2014-01-01

    Systemic toxicity currently prevents exploiting the huge potential of many cytokines for medical applications. Here we present a novel strategy to engineer immunocytokines with very high targeting efficacies. The method lies in the use of mutants of toxic cytokines that markedly reduce their receptor-binding affinities, and that are thus rendered essentially inactive. Upon fusion to nanobodies specifically binding to marker proteins, activity of these cytokines is selectively restored for cell populations expressing this marker. This ‘activity-by-targeting’ concept was validated for type I interferons and leptin. In the case of interferon, activity can be directed to target cells in vitro and to selected cell populations in mice, with up to 1,000-fold increased specific activity. This targeting strategy holds promise to revitalize the clinical potential of many cytokines.

  2. Thyroid-specific transcription factors control Hex promoter activity

    PubMed Central

    Puppin, Cinzia; D'Elia, Angela V.; Pellizzari, Lucia; Russo, Diego; Arturi, Franco; Presta, Ivan; Filetti, Sebastiano; Bogue, Clifford W.; Denson, Lee A.; Damante, Giuseppe

    2003-01-01

    The homeobox-containing gene Hex is expressed in several cell types, including thyroid follicular cells, in which it regulates the transcription of tissue- specific genes. In this study the regulation of Hex promoter activity was investigated. Using co- transfection experiments, we demonstrated that the transcriptional activity of the Hex gene promoter in rat thyroid FRTL-5 cells is ∼10-fold greater than that observed in HeLa and NIH 3T3 cell lines (which do not normally express the Hex gene). To identify the molecular mechanisms underlying these differences, we evaluated the effect of the thyroid- specific transcription factor TTF-1 on the Hex promoter activity. TTF-1 produced 3–4-fold increases in the Hex promoter activity. Gel- retardation assays and mutagenesis experiments revealed the presence of functionally relevant TTF-1 binding sites in the Hex promoter region. These in vitro data may also have functional relevance in vivo, since a positive correlation between TTF-1 and Hex mRNAs was demonstrated in human thyroid tissues by means of RT–PCR analysis. The TTF-1 effect, however, is not sufficient to explain the difference in Hex promoter activity between FRTL-5 and cells that do not express the Hex gene. For this reason, we tested whether Hex protein is able to activate the Hex promoter. Indeed, co-transfection experiments indicate that Hex protein is able to increase the activity of its own promoter in HeLa cells ∼4-fold. TTF-1 and Hex effects are additive: when transfected together in HeLa cells, the Hex promoter activity is increased 6–7-fold. Thus, the contemporary presence of both TTF-1 and Hex could be sufficient to explain the higher transcriptional activity of the Hex promoter in thyroid cells with respect to cell lines that do not express the Hex gene. These findings demonstrate the existence of direct cross-regulation between thyroid-specific transcription factors. PMID:12655000

  3. Photodynamic activity of thiophene-derived lysosome-specific dyes.

    PubMed

    Baldassarre, Francesca; Foglietta, Federica; Vergaro, Viviana; Barbero, Nadia; Capodilupo, Agostina L; Serpe, Loredana; Visentin, Sonja; Tepore, Antonio; Ciccarella, Giuseppe

    2016-05-01

    The photodynamic activity occurring through the lysosome photo-damage is effective in terms of triggered synergic effects which can avoid chemo-resistance pathways. The potential photodynamic activity of two fluorescent lysosome-specific probes was studied providing their interaction with human serum albumin, demonstrating their in vitro generation of singlet oxygen and investigating the resulted photo-toxic effect in human cancer cells. PMID:26930158

  4. Stage-specific effects of Notch activation during skeletal myogenesis

    PubMed Central

    Bi, Pengpeng; Yue, Feng; Sato, Yusuke; Wirbisky, Sara; Liu, Weiyi; Shan, Tizhong; Wen, Yefei; Zhou, Daoguo; Freeman, Jennifer; Kuang, Shihuan

    2016-01-01

    Skeletal myogenesis involves sequential activation, proliferation, self-renewal/differentiation and fusion of myogenic stem cells (satellite cells). Notch signaling is known to be essential for the maintenance of satellite cells, but its function in late-stage myogenesis, i.e. post-differentiation myocytes and post-fusion myotubes, is unknown. Using stage-specific Cre alleles, we uncovered distinct roles of Notch1 in mononucleated myocytes and multinucleated myotubes. Specifically, constitutive Notch1 activation dedifferentiates myocytes into Pax7 quiescent satellite cells, leading to severe defects in muscle growth and regeneration, and postnatal lethality. By contrast, myotube-specific Notch1 activation improves the regeneration and exercise performance of aged and dystrophic muscles. Mechanistically, Notch1 activation in myotubes upregulates the expression of Notch ligands, which modulate Notch signaling in the adjacent satellite cells to enhance their regenerative capacity. These results highlight context-dependent effects of Notch activation during myogenesis, and demonstrate that Notch1 activity improves myotube’s function as a stem cell niche. DOI: http://dx.doi.org/10.7554/eLife.17355.001 PMID:27644105

  5. Specific activity effect in the metabolism of Tc

    SciTech Connect

    Ennis, M.E. Jr.; Johnson, J.E.; Ward, G.M.; Voigt, G.M.

    1988-02-01

    A study of transfer to milk of three isotopes of Tc indicated different results for /sup 99m/TcO/sub 4/-, /sup 95m/TcO/sub 4/-, and /sup 99/TcO/sub 4/-. When the three isotopes were given orally to lactating goats in separate experiments, the milk transfer coefficients increased with decreasing specific activity. Gut absorption of Tc administered as pertechnetate (TcO/sub 4/-) was decreased by reduction of TcO/sub 4/- in the rumen. The rate of reduction of TcO/sub 4/- in the rumen was greater for higher specific activity doses of Tc. These results suggest that the specific activity of a radioisotope may significantly influence the estimated dose to humans from contaminated milk and, probably, other food products.

  6. Suppression of antigen-specific lymphocyte activation in modeled microgravity

    NASA Technical Reports Server (NTRS)

    Cooper, D.; Pride, M. W.; Brown, E. L.; Risin, D.; Pellis, N. R.; McIntire, L. V. (Principal Investigator)

    2001-01-01

    Various parameters of immune suppression are observed in lymphocytes from astronauts during and after a space flight. It is difficult to ascribe this suppression to microgravity effects on immune cells in crew specimens, due to the complex physiological response to space flight and the resultant effect on in vitro immune performance. Use of isolated immune cells in true and modeled microgravity in immune performance tests, suggests a direct effect of microgravity on in vitro cellular function. Specifically, polyclonal activation of T-cells is severely suppressed in true and modeled microgravity. These recent findings suggest a potential suppression of oligoclonal antigen-specific lymphocyte activation in microgravity. We utilized rotating wall vessel (RWV) bioreactors as an analog of microgravity for cell cultures to analyze three models of antigen-specific activation. A mixed-lymphocyte reaction, as a model for a primary immune response, a tetanus toxoid response and a Borrelia burgdorferi response, as models of a secondary immune response, were all suppressed in the RWV bioreactor. Our findings confirm that the suppression of activation observed with polyclonal models also encompasses oligoclonal antigen-specific activation.

  7. Stress causes tissue-specific changes in the sialyltransferase activity.

    PubMed

    Dabelic, Sanja; Flögel, Mirna; Maravić, Gordana; Lauc, Gordan

    2004-01-01

    Numerous pathological conditions are associated with specific changes in glycosylation. Recent studies clearly demonstrated a link between stress and the development and course of many diseases. Biochemical mechanisms that link stress and diseases are still not fully understood, but there are some indications that changes in glycosylation are involved in this process. Influence of acute and chronic psychological stress on protein sialylation as well as the activity of sialyltransferases, enzymes that synthesize sialoglycoproteins, has been studied on Fischer rats. Liver, spleen, kidney, skeletal muscle, heart, adrenal gland, serum, cerebellum, hippocampus, medulla oblongata and cortex have been analyzed. Statistically significant tissue- and type of stress-specific changes in total sialyltransferase (ST) activity were observed. Acute stress resulted in 39% increase of ST activity in liver and spleen, while at the same time there was 43% decrease in ST activity in cerebellum. In chronic stress, ST activity increased in spleen (93%) and decreased in liver (17%), cerebellum (38%) and hippocampus (64%). Western-blot analysis using Maackia amurensis and Sambucus nigra lectins did not reveal any difference in protein sialylation. The results of serum corticosterone analysis indicate that showed increase in acute stress and decrease in chronic stress are in good accordance with the hypothesis that corticosterone has a role in the regulation of liver ST activity. PMID:15241940

  8. Stress causes tissue-specific changes in the sialyltransferase activity.

    PubMed

    Dabelic, Sanja; Flögel, Mirna; Maravić, Gordana; Lauc, Gordan

    2004-01-01

    Numerous pathological conditions are associated with specific changes in glycosylation. Recent studies clearly demonstrated a link between stress and the development and course of many diseases. Biochemical mechanisms that link stress and diseases are still not fully understood, but there are some indications that changes in glycosylation are involved in this process. Influence of acute and chronic psychological stress on protein sialylation as well as the activity of sialyltransferases, enzymes that synthesize sialoglycoproteins, has been studied on Fischer rats. Liver, spleen, kidney, skeletal muscle, heart, adrenal gland, serum, cerebellum, hippocampus, medulla oblongata and cortex have been analyzed. Statistically significant tissue- and type of stress-specific changes in total sialyltransferase (ST) activity were observed. Acute stress resulted in 39% increase of ST activity in liver and spleen, while at the same time there was 43% decrease in ST activity in cerebellum. In chronic stress, ST activity increased in spleen (93%) and decreased in liver (17%), cerebellum (38%) and hippocampus (64%). Western-blot analysis using Maackia amurensis and Sambucus nigra lectins did not reveal any difference in protein sialylation. The results of serum corticosterone analysis indicate that showed increase in acute stress and decrease in chronic stress are in good accordance with the hypothesis that corticosterone has a role in the regulation of liver ST activity.

  9. Spontaneous and specific activation of chemical bonds in macromolecular fluids.

    PubMed

    Park, Insun; Shirvanyants, David; Nese, Alper; Matyjaszewski, Krzysztof; Rubinstein, Michael; Sheiko, Sergei S

    2010-09-01

    Mechanical activation of chemical bonds typically involves the application of external forces, which implies a broad distribution of bond tensions. We demonstrate that controlling the flow profile of a macromolecular fluid generates and delineates mechanical force concentration, enabling a hierarchical activation of chemical bonds on different length scales from the macroscopic to the molecular. Bond tension is spontaneously generated within brushlike macromolecules as they spread on a solid substrate. The molecular architecture creates an uneven distribution of tension in the covalent bonds, leading to spatially controlled bond scission. By controlling the flow rate and the gradient of the film pressure, one can sever the flowing macromolecules with high precision. Specific chemical bonds are activated within distinct macromolecules located in a defined area of a thin film. Furthermore, the flow-controlled loading rate enables quantitative analysis of the bond activation parameters.

  10. 50 CFR 654.27 - Specifically authorized activities.

    Code of Federal Regulations, 2011 CFR

    2011-10-01

    ... 50 Wildlife and Fisheries 10 2011-10-01 2011-10-01 false Specifically authorized activities. 654.27 Section 654.27 Wildlife and Fisheries FISHERY CONSERVATION AND MANAGEMENT, NATIONAL OCEANIC AND ATMOSPHERIC ADMINISTRATION, DEPARTMENT OF COMMERCE STONE CRAB FISHERY OF THE GULF OF MEXICO...

  11. 50 CFR 654.27 - Specifically authorized activities.

    Code of Federal Regulations, 2010 CFR

    2010-10-01

    ... 50 Wildlife and Fisheries 8 2010-10-01 2010-10-01 false Specifically authorized activities. 654.27 Section 654.27 Wildlife and Fisheries FISHERY CONSERVATION AND MANAGEMENT, NATIONAL OCEANIC AND ATMOSPHERIC ADMINISTRATION, DEPARTMENT OF COMMERCE STONE CRAB FISHERY OF THE GULF OF MEXICO...

  12. Unconscious Semantic Activation Depends on Feature-Specific Attention Allocation

    ERIC Educational Resources Information Center

    Spruyt, Adriaan; De Houwer, Jan; Everaert, Tom; Hermans, Dirk

    2012-01-01

    We examined whether semantic activation by subliminally presented stimuli is dependent upon the extent to which participants assign attention to specific semantic stimulus features and stimulus dimensions. Participants pronounced visible target words that were preceded by briefly presented, masked prime words. Both affective and non-affective…

  13. Essentials of specifications for activated alumina in defluoridation technology.

    PubMed

    Misra, S K

    2006-10-01

    Worldwide, fluoride occurs naturally in some aquifers at concentrations above the WHO guideline values or Bureau of Indian Standards or CPHEEO - MUD - GOI parametric values. Fluoride in excess of the permissible limits in drinking water causes a number of endemic conditions referred to collectively as "fluorosis". Endemic fluorosis remains a challenging national health problem in India and Rajasthan is one of the worst affected states in India though a wide range of chemical and physical defluoridation systems were evolved and tried. Still activated alumina is one of the most widely used and liked defluoridation material currently available. Boom in the advanced and versatile alumina technology has opened new vistas to avail the strong potential of selective alumina adsorbents which are application-specific. Non-regenerable and specialty alumina offer tremendous scope to defluoridate drinking water. Indian industries are manufacturing regenerable activated alumina for defluoridation of drinking water. In order to ensure application of an adsorbent, which caters the desired results with minimum interferences, health risks and long service life span, it is inevitable to draw out dimensions which define precisely the attributes of activated alumina. Specifications for activated alumina intended for defluoridation of drinking water, specific operating and performance requirements, and limitations expressed by critical analysis of cardinal characteristics pave way for adoption of acceptable specifications and code of practice at national level.

  14. [Specific risks of physical activity in the elderly].

    PubMed

    Paillard, T

    2015-01-01

    The aim of this paper is to present the specific risks of physical activity in elderly subjects. These risks mainly consist of the loss of physical integrity and the weakening of the capabilities of metabolic regulation. The risk of impairment of physical integrity (e.g. injury) related to regular physical activity is not overall greater in elderly subjects than in young subjects. The choice of a physical activity that is suited to the elderly subject's physical and cognitive abilities largely limits these risks. When physical activity is adapted to suit elderly subjects, the number of accidents in relation to the number of participants is actually very low. In fact, participation in a program of education for prevention related to physical activity reduces the risk of accidents and injuries (and, thus, falls) occurring thereafter. In the case of metabolic risks, isometric muscular contractions carried out under certain conditions (duration: > 6 seconds; intensity: > 50% of maximal voluntary contraction) are inappropriate. Physical activity carried out in extreme thermal atmospheres (0-5° < and > 25-30°) should be avoided. Hydration is very important and liquids should be drunk well before any thirst sensation occurs.

  15. Functional Specificity of the Visual Word Form Area: General Activation for Words and Symbols but Specific Network Activation for Words

    ERIC Educational Resources Information Center

    Reinke, Karen; Fernandes, Myra; Schwindt, Graeme; O'Craven, Kathleen; Grady, Cheryl L.

    2008-01-01

    The functional specificity of the brain region known as the Visual Word Form Area (VWFA) was examined using fMRI. We explored whether this area serves a general role in processing symbolic stimuli, rather than being selective for the processing of words. Brain activity was measured during a visual 1-back task to English words, meaningful symbols…

  16. Specific activation of the paralemniscal pathway during nociception.

    PubMed

    Frangeul, Laura; Porrero, Cesar; Garcia-Amado, Maria; Maimone, Benedetta; Maniglier, Madlyne; Clascá, Francisco; Jabaudon, Denis

    2014-05-01

    Two main neuronal pathways connect facial whiskers to the somatosensory cortex in rodents: (i) the lemniscal pathway, which originates in the brainstem principal trigeminal nucleus and is relayed in the ventroposterior thalamic nucleus and (ii) the paralemniscal pathway, originating in the spinal trigeminal nucleus and relayed in the posterior thalamic nucleus. While lemniscal neurons are readily activated by whisker contacts, the contribution of paralemniscal neurons to perception is less clear. Here, we functionally investigated these pathways by manipulating input from the whisker pad in freely moving mice. We report that while lemniscal neurons readily respond to neonatal infraorbital nerve sectioning or whisker contacts in vivo, paralemniscal neurons do not detectably respond to these environmental changes. However, the paralemniscal pathway is specifically activated upon noxious stimulation of the whisker pad. These findings reveal a nociceptive function for paralemniscal neurons in vivo that may critically inform context-specific behaviour during environmental exploration.

  17. Novel strategies for ultrahigh specific activity targeted nanoparticles

    SciTech Connect

    Zhou, Dong

    2012-12-13

    We have developed novel strategies optimized for preparing high specific activity radiolabeled nanoparticles, targeting nuclear imaging of low abundance biomarkers. Several compounds have been labeled with F-18 and Cu-64 for radiolabeling of SCK-nanoparticles via Copper(I) catalyzed or copper-free alkyne-azide cyclolization. Novel strategies have been developed to achieve ultrahigh specific activity with administrable amount of dose for human study using copper-free chemistry. Ligands for carbonic anhydrase 12 (CA12), a low abundance extracellular biomarker for the responsiveness of breast cancer to endocrine therapie, have been labeled with F-18 and Cu-64, and one of them has been evaluated in animal models. The results of this project will lead to major improvements in the use of nanoparticles in nuclear imaging and will significantly advance their potential for detecting low abundance biomarkers of medical importance.

  18. Method of preparing high specific activity platinum-195m

    SciTech Connect

    Mirzadeh, Saed; Du, Miting; Beets, Arnold L.; Knapp, Jr., Furn F.

    2004-06-15

    A method of preparing high-specific-activity .sup.195m Pt includes the steps of: exposing .sup.193 Ir to a flux of neutrons sufficient to convert a portion of the .sup.193 Ir to .sup.195m Pt to form an irradiated material; dissolving the irradiated material to form an intermediate solution comprising Ir and Pt; and separating the Pt from the Ir by cation exchange chromatography to produce .sup.195m Pt.

  19. Method for preparing high specific activity 177Lu

    SciTech Connect

    Mirzadeh, Saed; Du, Miting; Beets, Arnold L.; Knapp, Jr., Furn F.

    2004-04-06

    A method of separating lutetium from a solution containing Lu and Yb, particularly reactor-produced .sup.177 Lu and .sup.177 Yb, includes the steps of: providing a chromatographic separation apparatus containing LN resin; loading the apparatus with a solution containing Lu and Yb; and eluting the apparatus to chromatographically separate the Lu and the Yb in order to produce high-specific-activity .sup.177 Yb.

  20. Regulation of platelet activating factor receptor coupled phosphoinositide-specific phospholipase C activity

    SciTech Connect

    Morrison, W.J.

    1988-01-01

    The major objectives of this study were two-fold. The first was to establish whether binding of platelet activating factor (PAF) to its receptor was integral to the stimulation of polyphosphoinositide-specific phospholipase C (PLC) in rabbit platelets. The second was to determine regulatory features of this receptor-coupled mechanism. ({sup 3}H)PAF binding demonstrated two binding sites, a high affinity site with a inhibitory constant (Ki) of 2.65 nM and a low affinity site with a Ki of 0.80 {mu}M. PAF receptor coupled activation of phosphoinositide-specific PLC was studied in platelets which were made refractory, by short term pretreatments, to either PAF or thrombin. Saponin-permeabilized rabbit platelets continue to regulate the mechanism(s) coupling PAF receptors to PLC stimulation. However, TRP{gamma}S and GDP{beta}S, which affect guanine nucleotide regulatory protein functions, were unable to modulate the PLC activity to any appreciable extent as compared to PAF. The possible involvement of protein kinase C (PKC) activation in regulating PAF-stimulated PLC activity was studied in rabbit platelets pretreated with staurosporine followed by pretreatments with PAF or phorbol 12-myristate 13-acetate (PMA).

  1. GTP-specific fab fragment-based GTPase activity assay.

    PubMed

    Kopra, Kari; Rozwandowicz-Jansen, Anita; Syrjänpää, Markku; Blaževitš, Olga; Ligabue, Alessio; Veltel, Stefan; Lamminmäki, Urpo; Abankwa, Daniel; Härmä, Harri

    2015-03-17

    GTPases are central cellular signaling proteins, which cycle between a GDP-bound inactive and a GTP-bound active conformation in a controlled manner. Ras GTPases are frequently mutated in cancer and so far only few experimental inhibitors exist. The most common methods for monitoring GTP hydrolysis rely on luminescent GDP- or GTP-analogs. In this study, the first GTP-specific Fab fragment and its application are described. We selected Fab fragments using the phage display technology. Six Fab fragments were found against 2'/3'-GTP-biotin and 8-GTP-biotin. Selected antibody fragments allowed specific detection of endogenous, free GTP. The most potent Fab fragment (2A4(GTP)) showed over 100-fold GTP-specificity over GDP, ATP, or CTP and was used to develop a heterogeneous time-resolved luminescence based assay for the monitoring of GTP concentration. The method allows studying the GEF dependent H-Ras activation (GTP binding) and GAP-catalyzed H-Ras deactivation (GTP hydrolysis) at nanomolar protein concentrations.

  2. In Vitro Antibody-Enzyme Conjugates with Specific Bactericidal Activity

    PubMed Central

    Knowles, Daniel M.; Sullivan, Timothy J.; Parker, Charles W.; Williams, Ralph C.

    1973-01-01

    IgG with antibacterial antibody opsonic activity was isolated from rabbit antisera produced by intravenous hyperimmunization with several test strains of pneumococci, Group A β-hemolytic streptococci, Staphylococcus aureus, Proteus mirabilis, Pseudomonas aeruginosa, and Escherichia coli. Antibody-enzyme conjugates were prepared, using diethylmalonimidate to couple glucose oxidase to IgG antibacterial antibody preparations. Opsonic human IgG obtained from serum of patients with subacute bacterial endocarditis was also conjugated to glucose oxidase. Antibody-enzyme conjugates retained combining specificity for test bacteria as demonstrated by indirect immunofluorescence. In vitro test for bactericidal activity of antibody-enzyme conjugates utilized potassium iodide, lactoperoxidase, and glucose as cofactors. Under these conditions glucose oxidase conjugated to antibody generates hydrogen peroxide, and lactoperoxidase enzyme catalyzes the reduction of hydrogen peroxide with simultaneous oxidation of I- and halogenation and killing of test bacteria. Potent in vitro bactericidal activity of this system was repeatedly demonstrated for antibody-enzyme conjugates against pneumococci, streptococci, S. aureus, P. mirabilis, and E. coli. However, no bactericidal effect was demonstrable with antibody-enzyme conjugates and two test strains of P. aeruginosa. Bactericidal activity of antibody-enzyme conjugates appeared to parallel original opsonic potency of unconjugated IgG preparations. Antibody-enzyme conjugates at concentrations as low as 0.01 mg/ml were capable of intense bactericidal activity producing substantial drops in surviving bacterial counts within 30-60 min after initiation of assay. These in vitro bactericidal systems indicate that the concept of antibacterial antibody-enzyme conjugates may possibly be adaptable as a mechanism for treatment of patients with leukocyte dysfunction or fulminant bacteremia. PMID:4145026

  3. Low Specific Activity materials concepts are being reevaluated

    SciTech Connect

    Rawl, R.R.

    1993-10-01

    Many types of radioactive low-level waste are classified, packaged, and transported as Low-Specific Activity (LSA) material. The transportation regulations allow LSA materials to be shipped in economical packagings and, under certain conditions, waives compliance with other detailed requirements such as labeling. The fundamental concepts which support the LSA category are being thoroughly reevaluated to determine the defensibility of the provisions. A series of national and international events are leading to the development of new dose models which are likely to fundamentally change the ways these materials are defined. Similar basis changes are likely for the packaging requirements applicable to these materials.

  4. The Activation of Modality-Specific Representations During Discourse Processing

    PubMed Central

    Kurby, Christopher A.; Zacks, Jeffrey M.

    2013-01-01

    Previous research has shown that readers generate mental images of events. Most studies have investigated imagery during the reading of short texts, which also included explicit judgment tasks. In two fMRI studies, we assessed whether modality-specific imagery occurs during naturalistic, discourse comprehension. We identified clauses in the texts that elicited auditory, motor, or visual imagery. In both studies, reading motor imagery clauses was associated with increases in activity in left postcentral and precentral sulci, and reading auditory imagery clauses was associated with increases in left superior temporal gyrus and perisylvian language-related regions. Study 2 compared presentation of connected discourse to a condition in which unconnected sentences were presented, preventing the establishment of global coherence. Sensorimotor imagery was strongest when readers were able to generate a globally coherent discourse representation. Overall, these results suggest that modality-specific imagery occurs during discourse comprehension and it is dependent on the development of discourse-level representations. PMID:23933473

  5. Specific RNA-cleaving activities from HeLa cells.

    PubMed Central

    Ferrari, S; Yehle, C O; Robertson, H D; Dickson, E

    1980-01-01

    Subcellular fractionation of HeLa cells was carried out under gentle conditions to isolate enzymes that cleave RNA precursors in a specific manner. Four separate activities--cleavage of HeLa cell heterogeneous nuclear RNA, the HeLa cell 45S rRNA precursor, RNA . DNA hybrids (RNase H), and the Escherichia coli tRNATyr precursor (RNase P)--were revealed by these studies. The specificity and limited nature of these cleavages suggest that they are due to eukaryotic RNA-processing enzymes. The virtual absence of random nucleases from these enzymes was demonstrated by their inability to cleave the 8000-base early mRNA precursor of bacteriophage T7, E. coli 30S rRNA precursor, or HeLa cytoplasmic poly(A)-containing RNA. Images PMID:6930639

  6. The activation of modality-specific representations during discourse processing.

    PubMed

    Kurby, Christopher A; Zacks, Jeffrey M

    2013-09-01

    Previous research has shown that readers generate mental images of events. Most studies have investigated imagery during the reading of short texts, which also included explicit judgment tasks. In two fMRI studies, we assessed whether modality-specific imagery occurs during naturalistic, discourse comprehension. We identified clauses in the texts that elicited auditory, motor, or visual imagery. In both studies, reading motor imagery clauses was associated with increases in activity in left postcentral and precentral sulci, and reading auditory imagery clauses was associated with increases in left superior temporal gyrus and perisylvian language-related regions. Study 2 compared presentation of connected discourse to a condition in which unconnected sentences were presented, preventing the establishment of global coherence. Sensorimotor imagery was strongest when readers were able to generate a globally coherent discourse representation. Overall, these results suggest that modality-specific imagery occurs during discourse comprehension and it is dependent on the development of discourse-level representations.

  7. Prostaglandin endoperoxide H synthases: peroxidase hydroperoxide specificity and cyclooxygenase activation.

    PubMed

    Liu, Jiayan; Seibold, Steve A; Rieke, Caroline J; Song, Inseok; Cukier, Robert I; Smith, William L

    2007-06-22

    The cyclooxygenase (COX) activity of prostaglandin endoperoxide H synthases (PGHSs) converts arachidonic acid and O2 to prostaglandin G2 (PGG2). PGHS peroxidase (POX) activity reduces PGG2 to PGH2. The first step in POX catalysis is formation of an oxyferryl heme radical cation (Compound I), which undergoes intramolecular electron transfer forming Intermediate II having an oxyferryl heme and a Tyr-385 radical required for COX catalysis. PGHS POX catalyzes heterolytic cleavage of primary and secondary hydroperoxides much more readily than H2O2, but the basis for this specificity has been unresolved. Several large amino acids form a hydrophobic "dome" over part of the heme, but when these residues were mutated to alanines there was little effect on Compound I formation from H2O2 or 15-hydroperoxyeicosatetraenoic acid, a surrogate substrate for PGG2. Ab initio calculations of heterolytic bond dissociation energies of the peroxyl groups of small peroxides indicated that they are almost the same. Molecular Dynamics simulations suggest that PGG2 binds the POX site through a peroxyl-iron bond, a hydrogen bond with His-207 and van der Waals interactions involving methylene groups adjoining the carbon bearing the peroxyl group and the protoporphyrin IX. We speculate that these latter interactions, which are not possible with H2O2, are major contributors to PGHS POX specificity. The distal Gln-203 four residues removed from His-207 have been thought to be essential for Compound I formation. However, Q203V PGHS-1 and PGHS-2 mutants catalyzed heterolytic cleavage of peroxides and exhibited native COX activity. PGHSs are homodimers with each monomer having a POX site and COX site. Cross-talk occurs between the COX sites of adjoining monomers. However, no cross-talk between the POX and COX sites of monomers was detected in a PGHS-2 heterodimer comprised of a Q203R monomer having an inactive POX site and a G533A monomer with an inactive COX site.

  8. DNA methylation status predicts cell type-specific enhancer activity

    PubMed Central

    Wiench, Malgorzata; John, Sam; Baek, Songjoon; Johnson, Thomas A; Sung, Myong-Hee; Escobar, Thelma; Simmons, Catherine A; Pearce, Kenneth H; Biddie, Simon C; Sabo, Pete J; Thurman, Robert E; Stamatoyannopoulos, John A; Hager, Gordon L

    2011-01-01

    Cell-selective glucocorticoid receptor (GR) binding to distal regulatory elements is associated with cell type-specific regions of locally accessible chromatin. These regions can either pre-exist in chromatin (pre-programmed) or be induced by the receptor (de novo). Mechanisms that create and maintain these sites are not well understood. We observe a global enrichment of CpG density for pre-programmed elements, and implicate their demethylated state in the maintenance of open chromatin in a tissue-specific manner. In contrast, sites that are actively opened by GR (de novo) are characterized by low CpG density, and form a unique class of enhancers devoid of suppressive effect of agglomerated methyl-cytosines. Furthermore, treatment with glucocorticoids induces rapid changes in methylation levels at selected CpGs within de novo sites. Finally, we identify GR-binding elements with CpGs at critical positions, and show that methylation can affect GR–DNA interactions in vitro. The findings present a unique link between tissue-specific chromatin accessibility, DNA methylation and transcription factor binding and show that DNA methylation can be an integral component of gene regulation by nuclear receptors. PMID:21701563

  9. Site-specific PEGylation of lidamycin and its antitumor activity.

    PubMed

    Li, Liang; Shang, Boyang; Hu, Lei; Shao, Rongguang; Zhen, Yongsu

    2015-05-01

    In this study, N-terminal site-specific mono-PEGylation of the recombinant lidamycin apoprotein (rLDP) of lidamycin (LDM) was prepared using a polyethyleneglycol (PEG) derivative (M w 20 kDa) through a reactive terminal aldehyde group under weak acidic conditions (pH 5.5). The biochemical properties of mPEG-rLDP-AE, an enediyne-integrated conjugate, were analyzed by SDS-PAGE, RP-HPLC, SEC-HPLC and MALDI-TOF. Meanwhile, in vitro and in vivo antitumor activity of mPEG-rLDP-AE was evaluated by MTT assays and in xenograft model. The results indicated that mPEG-rLDP-AE showed significant antitumor activity both in vitro and in vivo. After PEGylation, mPEG-rLDP still retained the binding capability to the enediyne AE and presented the physicochemical characteristics similar to that of native LDP. It is of interest that the PEGylation did not diminish the antitumor efficacy of LDM, implying the possibility that this derivative may function as a payload to deliver novel tumor-targeted drugs. PMID:26579455

  10. Allele-specific DNA methylation reinforces PEAR1 enhancer activity.

    PubMed

    Izzi, Benedetta; Pistoni, Mariaelena; Cludts, Katrien; Akkor, Pinar; Lambrechts, Diether; Verfaillie, Catherine; Verhamme, Peter; Freson, Kathleen; Hoylaerts, Marc F

    2016-08-18

    Genetic variation in the PEAR1 locus is linked to platelet reactivity and cardiovascular disease. The major G allele of rs12041331, an intronic cytosine guanine dinucleotide-single-nucleotide polymorphism (CpG-SNP), is associated with higher PEAR1 expression in platelets and endothelial cells than the minor A allele. The molecular mechanism underlying this difference remains elusive. We have characterized the histone modification profiles of the intronic region surrounding rs12041331 and identified H3K4Me1 enhancer-specific enrichment for the region that covers the CpG-SNP. Interestingly, methylation studies revealed that the CpG site is fully methylated in leukocytes of GG carriers. Nuclear protein extracts from megakaryocytes, endothelial cells, vs control HEK-293 cells show a 3-fold higher affinity for the methylated G allele compared with nonmethylated G or A alleles in a gel electrophoretic mobility shift assay. To understand the positive relationship between methylation and gene expression, we studied DNA methylation at 4 different loci of PEAR1 during in vitro megakaryopoiesis. During differentiation, the CpG-SNP remained fully methylated, while we observed rapid methylation increases at the CpG-island overlapping the first 5'-untranslated region exon, paralleling the increased PEAR1 expression. In the same region, A-allele carriers of rs12041331 showed significantly lower DNA methylation at CGI1 compared with GG homozygote. This CpG-island contains binding sites for the methylation-sensitive transcription factor CTCF, whose binding is known to play a role in enhancer activation and/or repression. In conclusion, we report the molecular characterization of the first platelet function-related CpG-SNP, a genetic predisposition that reinforces PEAR1 enhancer activity through allele-specific DNA methylation. PMID:27313330

  11. 21 CFR 331.11 - Listing of specific active ingredients.

    Code of Federal Regulations, 2013 CFR

    2013-04-01

    .... (5) Dihydroxyaluminum sodium carbonate. (b) Bicarbonate-containing active ingredients: Bicarbonate... old and 100 mEq. of bicarbonate ion for persons 60 years or older. (2) Sodium potassium tartrate. (k) Sodium-containing active ingredients: (1) Sodium bicarbonate (or carbonate when used as a component of...

  12. 21 CFR 331.11 - Listing of specific active ingredients.

    Code of Federal Regulations, 2014 CFR

    2014-04-01

    .... (5) Dihydroxyaluminum sodium carbonate. (b) Bicarbonate-containing active ingredients: Bicarbonate... old and 100 mEq. of bicarbonate ion for persons 60 years or older. (2) Sodium potassium tartrate. (k) Sodium-containing active ingredients: (1) Sodium bicarbonate (or carbonate when used as a component of...

  13. 21 CFR 331.11 - Listing of specific active ingredients.

    Code of Federal Regulations, 2010 CFR

    2010-04-01

    .... (5) Dihydroxyaluminum sodium carbonate. (b) Bicarbonate-containing active ingredients: Bicarbonate... old and 100 mEq. of bicarbonate ion for persons 60 years or older. (2) Sodium potassium tartrate. (k) Sodium-containing active ingredients: (1) Sodium bicarbonate (or carbonate when used as a component of...

  14. 21 CFR 331.11 - Listing of specific active ingredients.

    Code of Federal Regulations, 2012 CFR

    2012-04-01

    .... (5) Dihydroxyaluminum sodium carbonate. (b) Bicarbonate-containing active ingredients: Bicarbonate... old and 100 mEq. of bicarbonate ion for persons 60 years or older. (2) Sodium potassium tartrate. (k) Sodium-containing active ingredients: (1) Sodium bicarbonate (or carbonate when used as a component of...

  15. 21 CFR 331.11 - Listing of specific active ingredients.

    Code of Federal Regulations, 2011 CFR

    2011-04-01

    .... (5) Dihydroxyaluminum sodium carbonate. (b) Bicarbonate-containing active ingredients: Bicarbonate... old and 100 mEq. of bicarbonate ion for persons 60 years or older. (2) Sodium potassium tartrate. (k) Sodium-containing active ingredients: (1) Sodium bicarbonate (or carbonate when used as a component of...

  16. Specific cerebellar activation during Braille reading in blind subjects.

    PubMed

    Gizewski, Elke R; Timmann, Dagmar; Forsting, Michael

    2004-07-01

    The traditional view that the cerebellum is involved only in the control of movements has been changed recently. It has been suggested that the human cerebellum is involved in cognition and language. Likewise, besides cortical activity in sensorimotor and visual areas, an increased global activation of the cerebellum has been revealed during Braille reading in blind subjects. Our purpose was to investigate whether there is cerebellar activation during Braille reading by blind subjects other than sensorimotor activation related to finger movements. Early blind and normal sighted subjects were studied with functional magnetic resonance imaging (fMRI) during Braille reading, tactile discrimination of nonsense dots, dots forming symbols, and finger tapping. The experiments were done in block design. Echo planar imaging sequences were carried out on a 1.5-T MR scanner. All blind individuals reading Braille showed robust activation of the posterior and lateral aspects of cerebellar hemispheral lobules Crus I bilaterally but more predominately on the right side. Additionally, activation was present in the medial cerebellum within lobules IV, V, and VIIIA, predominantly on the right. Discriminating nonsense dots did not reveal any activation of Crus I, but did reveal activation within the medial part of lobules IV, V, and VIIIA, predominately on the right. Analysis of sighted subjects during reading of printed text revealed activation of the posterolateral cerebellar hemisphere in Crus I bilaterally, predominantly on the right. Tactile analysis of dots representing symbols revealed an activation in lobules IV and VIII and in right Crus II but not in Crus I. In conclusion, parts of cerebellar activation during Braille reading in blind subjects (i.e., within lobules IV, V, and VIII) overlap with the known hand representation within the cerebellum and are likely related to the sensorimotor part of the task. Cerebellar activation during Braille reading within bilateral Crus I

  17. 10 CFR 810.8 - Activities requiring specific authorization.

    Code of Federal Regulations, 2010 CFR

    2010-01-01

    ... of the following activities with respect to any foreign country. (1) Designing production reactors... (enrichment), chemical processing of irradiated SNM (reprocessing), fabrication of nuclear fuel containing... reactors, accelerator-driven subcritical assembly systems, or facilities; (3) Designing,...

  18. The Saccharomyces cerevisiae PUT3 activator protein associates with proline-specific upstream activation sequences.

    PubMed Central

    Siddiqui, A H; Brandriss, M C

    1989-01-01

    The PUT1 and PUT2 genes encoding the enzymes of the proline utilization pathway of Saccharomyces cerevisiae are induced by proline and activated by the product of the PUT3 gene. Two upstream activation sequences (UASs) in the PUT1 promoter were identified by homology to the PUT2 UAS. Deletion analysis of the two PUT1 UASs showed that they were functionally independent and additive in producing maximal levels of gene expression. The consensus PUT UAS is a 21-base-pair partially palindromic sequence required in vivo for induction of both genes. The results of a gel mobility shift assay demonstrated that the proline-specific UAS is the binding site of a protein factor. In vitro complex formation was observed in crude extracts of yeast strains carrying either a single genomic copy of the PUT3 gene or the cloned PUT3 gene on a 2 microns plasmid, and the binding was dosage dependent. DNA-binding activity was not observed in extracts of strains carrying either a put3 mutation that caused a noninducible (Put-) phenotype or a deletion of the gene. Wild-type levels of complex formation were observed in an extract of a strain carrying an allele of PUT3 that resulted in a constitutive (Put+) phenotype. Extracts from a strain carrying a PUT3-lacZ gene fusion formed two complexes of slower mobility than the wild-type complex. We conclude that the PUT3 product is either a DNA-binding protein or part of a DNA-binding complex that recognizes the UASs of both PUT1 and PUT2. Binding was observed in extracts of a strain grown in the presence or absence of proline, demonstrating the constitutive nature of the DNA-protein interaction. Images PMID:2689862

  19. Sequence-specific DNA primer effects on telomerase polymerization activity.

    PubMed Central

    Lee, M S; Blackburn, E H

    1993-01-01

    The ribonucleoprotein enzyme telomerase synthesizes one strand of telomeric DNA by copying a template sequence within the RNA moiety of the enzyme. Kinetic studies of this polymerization reaction were used to analyze the mechanism and properties of the telomerase from Tetrahymena thermophila. This enzyme synthesizes TTGGGG repeats, the telomeric DNA sequence of this species, by elongating a DNA primer whose 3' end base pairs with the template-forming domain of the RNA. The enzyme was found to act nonprocessively with short (10- to 12-nucleotide) primers but to become processive as TTGGGG repeats were added. Variation of the 5' sequences of short primers with a common 3' end identified sequence-specific effects which are distinct from those involving base pairing of the 3' end of the primer with the RNA template and which can markedly induce enzyme activity by increasing the catalytic rate of the telomerase polymerization reaction. These results identify an additional mechanistic basis for telomere and DNA end recognition by telomerase in vivo. Images PMID:8413255

  20. The effect of trypsin and chymotrypsin on the bactericidal activity and specific antibody activity of bovine colostrum.

    PubMed Central

    Brock, J H; Arzabe, R; Piñeiro, A; Olivito, A M

    1977-01-01

    Digestion of bovine colostral whey with trypsin or chymotrypsin caused a progressive loss of the complement-mediated bactericidal activity of naturally-occurring colostral antibodies of E. coli 0111. Bactericidal activity was associated primarily with IgG1 immunoglobulin and to a lesser extent with IgM. Chymotrypsin preferentially attacked IgM, destroying its antibacterial activity and producing an apparent decrease in its mol wt. Trypsin preferentially attacked IgG1, but loss of antibacterial activity was in this case not accompanied by a decrease in molecular weight. Using colostral whey with antiperoxidase activity it was shown that the kinetics of loss of specific antibody activity were similar to those of loss of bactericidal activity. It is therefore suggested that trypsin may cause a loss of specific antibody activity of colostral IgG1 without cleaving the immunoglobulin molecule. PMID:321342

  1. 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.

  2. Eukaryote-specific insertion elements control human ARGONAUTE slicer activity.

    PubMed

    Nakanishi, Kotaro; Ascano, Manuel; Gogakos, Tasos; Ishibe-Murakami, Satoko; Serganov, Artem A; Briskin, Daniel; Morozov, Pavel; Tuschl, Thomas; Patel, Dinshaw J

    2013-06-27

    We have solved the crystal structure of human ARGONAUTE1 (hAGO1) bound to endogenous 5'-phosphorylated guide RNAs. To identify changes that evolutionarily rendered hAGO1 inactive, we compared our structure with guide-RNA-containing and cleavage-active hAGO2. Aside from mutation of a catalytic tetrad residue, proline residues at positions 670 and 675 in hAGO1 introduce a kink in the cS7 loop, forming a convex surface within the hAGO1 nucleic-acid-binding channel near the inactive catalytic site. We predicted that even upon restoration of the catalytic tetrad, hAGO1-cS7 sterically hinders the placement of a fully paired guide-target RNA duplex into the endonuclease active site. Consistent with this hypothesis, reconstitution of the catalytic tetrad with R805H led to low-level hAGO1 cleavage activity, whereas combining R805H with cS7 substitutions P670S and P675Q substantially augmented hAGO1 activity. Evolutionary amino acid changes to hAGO1 were readily reversible, suggesting that loading of guide RNA and pairing of seed-based miRNA and target RNA constrain its sequence drift.

  3. 29 CFR 1630.16 - Specific activities permitted.

    Code of Federal Regulations, 2011 CFR

    2011-07-01

    ..., or society is permitted to give preference in employment to individuals of a particular religion to... society of its activities. A religious entity may require that all applicants and employees conform to the...) Regulation of alcohol and drugs. A covered entity: (1) May prohibit the illegal use of drugs and the use...

  4. 29 CFR 1630.16 - Specific activities permitted.

    Code of Federal Regulations, 2013 CFR

    2013-07-01

    ..., or society is permitted to give preference in employment to individuals of a particular religion to... society of its activities. A religious entity may require that all applicants and employees conform to the...) Regulation of alcohol and drugs. A covered entity: (1) May prohibit the illegal use of drugs and the use...

  5. 29 CFR 1630.16 - Specific activities permitted.

    Code of Federal Regulations, 2014 CFR

    2014-07-01

    ..., or society is permitted to give preference in employment to individuals of a particular religion to... society of its activities. A religious entity may require that all applicants and employees conform to the...) Regulation of alcohol and drugs. A covered entity: (1) May prohibit the illegal use of drugs and the use...

  6. 29 CFR 1630.16 - Specific activities permitted.

    Code of Federal Regulations, 2012 CFR

    2012-07-01

    ..., or society is permitted to give preference in employment to individuals of a particular religion to... society of its activities. A religious entity may require that all applicants and employees conform to the...) Regulation of alcohol and drugs. A covered entity: (1) May prohibit the illegal use of drugs and the use...

  7. 29 CFR 1630.16 - Specific activities permitted.

    Code of Federal Regulations, 2010 CFR

    2010-07-01

    ..., or society is permitted to give preference in employment to individuals of a particular religion to... society of its activities. A religious entity may require that all applicants and employees conform to the... of alcohol and drugs. A covered entity: (1) May prohibit the illegal use of drugs and the use...

  8. 50 CFR 635.32 - Specifically authorized activities.

    Code of Federal Regulations, 2012 CFR

    2012-10-01

    ... arrangement; a copy of the High Seas Fishing Compliance Act Permit pursuant to 50 CFR 300.10; documentation... permit, display permit, chartering permit, or shark research permit issued in response to requests for... research permits. For scientific research activities involving the capture of Atlantic sharks,...

  9. 50 CFR 635.32 - Specifically authorized activities.

    Code of Federal Regulations, 2014 CFR

    2014-10-01

    ... arrangement; a copy of the High Seas Fishing Compliance Act Permit pursuant to 50 CFR 300.10; documentation... permit, display permit, chartering permit, or shark research permit issued in response to requests for... research permits. For scientific research activities involving the capture of Atlantic sharks,...

  10. 50 CFR 635.32 - Specifically authorized activities.

    Code of Federal Regulations, 2013 CFR

    2013-10-01

    ... arrangement; a copy of the High Seas Fishing Compliance Act Permit pursuant to 50 CFR 300.10; documentation... permit, display permit, chartering permit, or shark research permit issued in response to requests for... research permits. For scientific research activities involving the capture of Atlantic sharks,...

  11. The protein structures that shape caspase activity, specificity, activation and inhibition

    PubMed Central

    Fuentes-Prior, Pablo; Salvesen, Guy S.

    2004-01-01

    The death morphology commonly known as apoptosis results from a post-translational pathway driven largely by specific limited proteolysis. In the last decade the structural basis for apoptosis regulation has moved from nothing to ‘quite good’, and we now know the fundamental structures of examples from the initiator phase, the pre-mitochondrial regulator phase, the executioner phase, inhibitors and their antagonists, and even the structures of some substrates. The field is as well advanced as the best known of proteolytic pathways, the coagulation cascade. Fundamentally new mechanisms in protease regulation have been disclosed. Structural evidence suggests that caspases have an unusual catalytic mechanism, and that they are activated by apparently unrelated events, depending on which position in the apoptotic pathway they occupy. Some naturally occurring caspase inhibitors have adopted classic inhibition strategies, but other have revealed completely novel mechanisms. All of the structural and mechanistic information can, and is, being applied to drive therapeutic strategies to combat overactivation of apoptosis in degenerative disease, and underactivation in neoplasia. We present a comprehensive review of the caspases, their regulators and inhibitors from a structural and mechanistic point of view, and with an aim to consolidate the many threads that define the rapid growth of this field. PMID:15450003

  12. Nanoliposomes containing Eucalyptus citriodora as antibiotic with specific antimicrobial activity.

    PubMed

    Lin, Lin; Cui, Haiying; Zhou, Hui; Zhang, Xuejing; Bortolini, Christian; Chen, Menglin; Liu, Lei; Dong, Mingdong

    2015-02-14

    Bacterial infections are a serious issue for public health and represent one of the major challenges of modern medicine. In this work, a selective antimicrobial strategy based on triggering of pore-forming toxin, which is secreted by infective bacteria, was designed to fight Staphylococcus aureus. The antimicrobial activity is realized by employing Eucalyptus citriodora oil as antibiotic which in this study is encapsulated in nanoliposomes.

  13. Nanoliposomes containing Eucalyptus citriodora as antibiotic with specific antimicrobial activity.

    PubMed

    Lin, Lin; Cui, Haiying; Zhou, Hui; Zhang, Xuejing; Bortolini, Christian; Chen, Menglin; Liu, Lei; Dong, Mingdong

    2015-02-14

    Bacterial infections are a serious issue for public health and represent one of the major challenges of modern medicine. In this work, a selective antimicrobial strategy based on triggering of pore-forming toxin, which is secreted by infective bacteria, was designed to fight Staphylococcus aureus. The antimicrobial activity is realized by employing Eucalyptus citriodora oil as antibiotic which in this study is encapsulated in nanoliposomes. PMID:25573466

  14. Repetitive Transcranial Magnetic Stimulation Activates Specific Regions in Rat Brain

    NASA Astrophysics Data System (ADS)

    Ji, Ru-Rong; Schlaepfer, Thomas E.; Aizenman, Carlos D.; Epstein, Charles M.; Qiu, Dike; Huang, Justin C.; Rupp, Fabio

    1998-12-01

    Repetitive transcranial magnetic stimulation (rTMS) is a noninvasive technique to induce electric currents in the brain. Although rTMS is being evaluated as a possible alternative to electroconvulsive therapy for the treatment of refractory depression, little is known about the pattern of activation induced in the brain by rTMS. We have compared immediate early gene expression in rat brain after rTMS and electroconvulsive stimulation, a well-established animal model for electroconvulsive therapy. Our result shows that rTMS applied in conditions effective in animal models of depression induces different patterns of immediate-early gene expression than does electroconvulsive stimulation. In particular, rTMS evokes strong neural responses in the paraventricular nucleus of the thalamus (PVT) and in other regions involved in the regulation of circadian rhythms. The response in PVT is independent of the orientation of the stimulation probe relative to the head. Part of this response is likely because of direct activation, as repetitive magnetic stimulation also activates PVT neurons in brain slices.

  15. Target cell-specific modulation of neuronal activity by astrocytes

    NASA Astrophysics Data System (ADS)

    Kozlov, A. S.; Angulo, M. C.; Audinat, E.; Charpak, S.

    2006-06-01

    Interaction between astrocytes and neurons enriches the behavior of brain circuits. By releasing glutamate and ATP, astrocytes can directly excite neurons and modulate synaptic transmission. In the rat olfactory bulb, we demonstrate that the release of GABA by astrocytes causes long-lasting and synchronous inhibition of mitral and granule cells. In addition, astrocytes release glutamate, leading to a selective activation of granule-cell NMDA receptors. Thus, by releasing excitatory and inhibitory neurotransmitters, astrocytes exert a complex modulatory control on the olfactory network. glutamate | GABA | inhibition | olfactory bulb | synchronization

  16. Are the correlates of active school transport context-specific?

    PubMed Central

    Larouche, R; Sarmiento, O L; Broyles, S T; Denstel, K D; Church, T S; Barreira, T V; Chaput, J-P; Fogelholm, M; Hu, G; Kuriyan, R; Kurpad, A; Lambert, E V; Maher, C; Maia, J; Matsudo, V; Olds, T; Onywera, V; Standage, M; Tremblay, M S; Tudor-Locke, C; Zhao, P; Katzmarzyk, P T

    2015-01-01

    OBJECTIVES: Previous research consistently indicates that children who engage in active school transport (AST) are more active than their peers who use motorized modes (car or bus). However, studies of the correlates of AST have been conducted predominantly in high-income countries and have yielded mixed findings. Using data from a heterogeneous sample of 12 country sites across the world, we investigated the correlates of AST in 9–11-year olds. METHODS: The analytical sample comprised 6555 children (53.8% girls), who reported their main travel mode to school and the duration of their school trip. Potential individual and neighborhood correlates of AST were assessed with a parent questionnaire adapted from previously validated instruments. Multilevel generalized linear mixed models (GLMM) were used to examine the associations between individual and neighborhood variables and the odds of engaging in AST while controlling for the child's school. Site moderated the relationship of seven of these variables with AST; therefore we present analyses stratified by site. RESULTS: The prevalence of AST varied from 5.2 to 79.4% across sites and the school-level intra-class correlation ranged from 0.00 to 0.56. For each site, the final GLMM included a different set of correlates of AST. Longer trip duration (that is, ⩾16 min versus ⩽15 min) was associated with lower odds of AST in eight sites. Other individual and neighborhood factors were associated with AST in three sites or less. CONCLUSIONS: Our results indicate wide variability in the prevalence and correlates of AST in a large sample of children from twelve geographically, economically and culturally diverse country sites. This suggests that AST interventions should not adopt a ‘one size fits all' approach. Future research should also explore the association between psychosocial factors and AST in different countries. PMID:27152191

  17. Threshold occupancy and specific cation binding modes in the hammerhead ribozyme active site are required for active conformation

    PubMed Central

    Lee, Tai-Sung; Giambaşu, George M.; Sosa, Carlos P.; Martick, Monika; Scott, William G.; York, Darrin M.

    2009-01-01

    The relationship between formation of active in-line attack conformations and monovalent (Na+) and divalent (Mg2+) metal ion binding in the hammerhead ribozyme has been explored with molecular dynamics simulations. To stabilize repulsions between negatively charged groups, different requirements of threshold occupancy of metal ions were observed in the reactant and activated precursor states both in the presence or absence of a Mg2+ in the active site. Specific bridging coordination patterns of the ions are correlated with the formation of active in-line attack conformations and can be accommodated in both cases. Furthermore, simulation results suggest that the hammerhead ribozyme folds to form an electronegative recruiting pocket that attracts high local concentrations of positive charge. The present simulations help to reconcile experiments that probe the metal ion sensitivity of hammerhead ribozyme catalysis and support the supposition that Mg2+, in addition to stabilizing active conformations, plays a specific chemical role in catalysis. PMID:19265710

  18. Active medulloblastoma enhancers reveal subgroup-specific cellular origins.

    PubMed

    Lin, Charles Y; Erkek, Serap; Tong, Yiai; Yin, Linlin; Federation, Alexander J; Zapatka, Marc; Haldipur, Parthiv; Kawauchi, Daisuke; Risch, Thomas; Warnatz, Hans-Jörg; Worst, Barbara C; Ju, Bensheng; Orr, Brent A; Zeid, Rhamy; Polaski, Donald R; Segura-Wang, Maia; Waszak, Sebastian M; Jones, David T W; Kool, Marcel; Hovestadt, Volker; Buchhalter, Ivo; Sieber, Laura; Johann, Pascal; Chavez, Lukas; Gröschel, Stefan; Ryzhova, Marina; Korshunov, Andrey; Chen, Wenbiao; Chizhikov, Victor V; Millen, Kathleen J; Amstislavskiy, Vyacheslav; Lehrach, Hans; Yaspo, Marie-Laure; Eils, Roland; Lichter, Peter; Korbel, Jan O; Pfister, Stefan M; Bradner, James E; Northcott, Paul A

    2016-02-01

    Medulloblastoma is a highly malignant paediatric brain tumour, often inflicting devastating consequences on the developing child. Genomic studies have revealed four distinct molecular subgroups with divergent biology and clinical behaviour. An understanding of the regulatory circuitry governing the transcriptional landscapes of medulloblastoma subgroups, and how this relates to their respective developmental origins, is lacking. Here, using H3K27ac and BRD4 chromatin immunoprecipitation followed by sequencing (ChIP-seq) coupled with tissue-matched DNA methylation and transcriptome data, we describe the active cis-regulatory landscape across 28 primary medulloblastoma specimens. Analysis of differentially regulated enhancers and super-enhancers reinforced inter-subgroup heterogeneity and revealed novel, clinically relevant insights into medulloblastoma biology. Computational reconstruction of core regulatory circuitry identified a master set of transcription factors, validated by ChIP-seq, that is responsible for subgroup divergence, and implicates candidate cells of origin for Group 4. Our integrated analysis of enhancer elements in a large series of primary tumour samples reveals insights into cis-regulatory architecture, unrecognized dependencies, and cellular origins.

  19. Expanding Test Specifications with Rhetorical Genre Studies and Activity Theory Analyses

    ERIC Educational Resources Information Center

    Kennedy, Lauren Culzean

    2007-01-01

    This research paper describes the benefits of using an activity-based rhetorical perspective to develop English for specific purposes (ESP) test specifications. This approach expands the potential of ESP test specifications to analyze and describe target language use (TLU) situations, TLU tasks, and ESP test tasks. Multiple activity systems are…

  20. Curcumin inhibits srebp-2 expression in activated hepatic stellate cells in vitro by reducing the activity of specificity protein-1.

    PubMed

    Kang, Qiaohua; Chen, Anping

    2009-12-01

    Elevated levels of cholesterol/low-density lipoprotein (LDL) are a risk factor for the development of nonalcoholic steatohepatitis and its associated hepatic fibrosis. However, underlying mechanisms remain elusive. We previously reported that curcumin induced gene expression of peroxisome proliferator-activated receptor (PPAR)-gamma and stimulated its activity, leading to the inhibition of the activation of hepatic stellate cells (HSCs), the major effector cells during hepatic fibrogenesis. We recently showed that curcumin suppressed gene expression of LDL receptor in activated HSCs in vitro by repressing gene expression of the transcription factor sterol regulatory element binding protein-2 (SREBP-2), leading to the reduction in the level of intracellular cholesterol in HSCs and to the attenuation of the stimulatory effects of LDL on HSCs activation. The current study aimed at exploring molecular mechanisms by which curcumin inhibits srebp-2 expression in HSCs. Promoter deletion assays, mutagenesis assays, and EMSAs localize a specificity protein-1 (SP-1) binding GC-box in the srebp-2 promoter, which is responsible for enhancing the promoter activity and responding to curcumin in HSCs. Curcumin suppresses gene expression of SP-1 and reduces its trans-activation activity, which are mediated by the activation of PPARgamma. The inhibitory effect of curcumin on SP-1 binding to the GC-box is confirmed by chromatin immuno-precipitation. In summary, our results demonstrate that curcumin inhibits srebp-2 expression in cultured HSCs by activating PPARgamma and reducing the SP-1 activity, leading to the repression of ldlr expression. These results provide novel insights into molecular mechanisms by which curcumin inhibits LDL-induced HSC activation.

  1. Specification of High Activity Gamma-Ray Sources.

    ERIC Educational Resources Information Center

    International Commission on Radiation Units and Measurements, Washington, DC.

    The report is concerned with making recommendations for the specifications of gamma ray sources, which relate to the quantity of radioactive material and the radiation emitted. Primary consideration is given to sources in teletherapy and to a lesser extent those used in industrial radiography and in irradiation units used in industry and research.…

  2. Sensitivity and Specificity of Hypnosis Effects on Gastric Myoelectrical Activity

    PubMed Central

    Enck, Paul; Weimer, Katja; Muth, Eric R.; Zipfel, Stephan; Martens, Ute

    2013-01-01

    Objectives The effects of hypnosis on physiological (gastrointestinal) functions are incompletely understood, and it is unknown whether they are hypnosis-specific and gut-specific, or simply unspecific effects of relaxation. Design Sixty-two healthy female volunteers were randomly assigned to either a single session of hypnotic suggestion of ingesting an appetizing meal and an unappetizing meal, or to relax and concentrate on having an appetizing or unappetizing meal, while the electrogastrogram (EGG) was recorded. At the end of the session, participants drank water until they felt full, in order to detect EGG-signal changes after ingestion of a true gastric load. During both conditions participants reported their subjective well-being, hunger and disgust at several time points. Results Imagining eating food induced subjective feelings of hunger and disgust as well as changes in the EGG similar to, but more pronounced than those seen with a real gastric water load during both hypnosis and relaxation conditions. These effects were more pronounced when imagining an appetizing meal than with an unappetizing meal. There was no significant difference between the hypnosis and relaxation conditions. Conclusion Imagination with and without hypnosis exhibits similar changes in subjective and objective measures in response to imagining an appetizing and an unappetizing food, indicating high sensitivity but low specificity. PMID:24358287

  3. Energy Landscape Topography Reveals the Underlying Link Between Binding Specificity and Activity of Enzymes

    PubMed Central

    Chu, Wen-Ting; Wang, Jin

    2016-01-01

    Enzyme activity (often quantified by kcat/Km) is the main function of enzyme when it is active against the specific substrate. Higher or lower activities are highly desired for the design of novel enzyme and drug resistance. However, it is difficult to measure the activities of all possible variants and find the “hot-spot” within the limit of experimental time. In this study, we explore the underlying energy landscape of enzyme-substrate interactions and introduce the intrinsic specificity ratio (ISR), which reflects the landscape topography. By studying two concrete systems, we uncover the statistical correlation between the intrinsic specificity and the enzyme activity kcat/Km. This physics-based concept and method show that the energy landscape topography is valuable for understanding the relationship between enzyme specificity and activity. In addition, it can reveal the underlying mechanism of enzyme-substrate actions and has potential applications on enzyme design. PMID:27298067

  4. Energy Landscape Topography Reveals the Underlying Link Between Binding Specificity and Activity of Enzymes

    NASA Astrophysics Data System (ADS)

    Chu, Wen-Ting; Wang, Jin

    2016-06-01

    Enzyme activity (often quantified by kcat/Km) is the main function of enzyme when it is active against the specific substrate. Higher or lower activities are highly desired for the design of novel enzyme and drug resistance. However, it is difficult to measure the activities of all possible variants and find the “hot-spot” within the limit of experimental time. In this study, we explore the underlying energy landscape of enzyme-substrate interactions and introduce the intrinsic specificity ratio (ISR), which reflects the landscape topography. By studying two concrete systems, we uncover the statistical correlation between the intrinsic specificity and the enzyme activity kcat/Km. This physics-based concept and method show that the energy landscape topography is valuable for understanding the relationship between enzyme specificity and activity. In addition, it can reveal the underlying mechanism of enzyme-substrate actions and has potential applications on enzyme design.

  5. Specific activities and DOE-STD-1027-92 hazard Category 2 thresholds, LANL fact sheet

    SciTech Connect

    Clow, J.; DeVore, R.; Elder, J.; Heindel, G.; Inkret, W.; Miller, G.

    1994-11-01

    Data tables are presented to provide consistency in safety analysis work at the Los Alamos National Laboratory. Included are calculated specific activities and calculated hazard classification Category 2 threshold quantities for radionuclides listed in DOE-STD-1027-92. Some calculated threshold quantities differ from the DOE thresholds. Calculated specific activities are also presented for plutonium material types or mixtures.

  6. Activation of defence reactions in Solanaceae: where is the specificity?

    PubMed

    Desender, Sabine; Andrivon, Didier; Val, Florence

    2007-01-01

    When a potential pathogen attempts to infect a plant, biochemical and molecular communication takes place and leads to the induction of plant defence mechanisms. In the case of efficient defence, visible symptoms are restricted and the pathogen does not multiply (incompatible interaction); when defence is inefficient, the plant becomes rapidly infected (compatible interaction). During the last 30 years, a growing body of knowledge on plant-pathogen interactions has been gathered, and a large number of studies investigate the induction of various plant defence reactions by pathogens or by pathogen-derived compounds. However, as most papers focus on incompatible interactions, there is still a lack of understanding about the similarities and differences between compatible and incompatible situations. This review targets the question of specificity in Solanaceae-pathogen interactions, by comparing defence patterns in plants challenged with virulent or avirulent pathogens (or with pathogen-associated molecular patterns from these). A special emphasis is made on analysing whether defence reactions in Solanaceae depend primarily on the type of elicitor, on the plant genotype/species, or on the type of interaction (compatible or incompatible).

  7. Comparison of four specific dynamic office chairs with a conventional office chair: impact upon muscle activation, physical activity and posture.

    PubMed

    Ellegast, Rolf P; Kraft, Kathrin; Groenesteijn, Liesbeth; Krause, Frank; Berger, Helmut; Vink, Peter

    2012-03-01

    Prolonged and static sitting postures provoke physical inactivity at VDU workplaces and are therefore discussed as risk factors for the musculoskeletal system. Manufacturers have designed specific dynamic office chairs featuring structural elements which promote dynamic sitting and therefore physical activity. The aim of the present study was to evaluate the effects of four specific dynamic chairs on erector spinae and trapezius EMG, postures/joint angles and physical activity intensity (PAI) compared to those of a conventional standard office chair. All chairs were fitted with sensors for measurement of the chair parameters (backrest inclination, forward and sideward seat pan inclination), and tested in the laboratory by 10 subjects performing 7 standardized office tasks and by another 12 subjects in the field during their normal office work. Muscle activation revealed no significant differences between the specific dynamic chairs and the reference chair. Analysis of postures/joint angles and PAI revealed only a few differences between the chairs, whereas the tasks performed strongly affected the measured muscle activation, postures and kinematics. The characteristic dynamic elements of each specific chair yielded significant differences in the measured chair parameters, but these characteristics did not appear to affect the sitting dynamics of the subjects performing their office tasks.

  8. Context Specificity of Stress-activated Mitogen-activated Protein (MAP) Kinase Signaling: The Story as Told by Caenorhabditis elegans.

    PubMed

    Andrusiak, Matthew G; Jin, Yishi

    2016-04-01

    Stress-associated p38 and JNK mitogen-activated protein (MAP) kinase signaling cascades trigger specific cellular responses and are involved in multiple disease states. At the root of MAP kinase signaling complexity is the differential use of common components on a context-specific basis. The roundwormCaenorhabditis eleganswas developed as a system to study genes required for development and nervous system function. The powerful genetics ofC. elegansin combination with molecular and cellular dissections has led to a greater understanding of how p38 and JNK signaling affects many biological processes under normal and stress conditions. This review focuses on the studies revealing context specificity of different stress-activated MAPK components inC. elegans.

  9. Context Specificity of Stress-activated Mitogen-activated Protein (MAP) Kinase Signaling: The Story as Told by Caenorhabditis elegans.

    PubMed

    Andrusiak, Matthew G; Jin, Yishi

    2016-04-01

    Stress-associated p38 and JNK mitogen-activated protein (MAP) kinase signaling cascades trigger specific cellular responses and are involved in multiple disease states. At the root of MAP kinase signaling complexity is the differential use of common components on a context-specific basis. The roundwormCaenorhabditis eleganswas developed as a system to study genes required for development and nervous system function. The powerful genetics ofC. elegansin combination with molecular and cellular dissections has led to a greater understanding of how p38 and JNK signaling affects many biological processes under normal and stress conditions. This review focuses on the studies revealing context specificity of different stress-activated MAPK components inC. elegans. PMID:26907690

  10. Chronic stress and moderate physical exercise prompt widespread common activation and limited differential activation in specific brain regions.

    PubMed

    Kim, Tae-Kyung; Han, Pyung-Lim

    2016-10-01

    Chronic stress in rodents produces depressive behaviors, whereas moderate physical exercise counteracts stress-induced depressive behaviors. Chronic stress and physical exercise appear to produce such opposing effects by changing the neural activity of specific brain regions. However, the detailed mechanisms through which the two different types of stimuli regulate brain function in opposite directions are not clearly understood. In the present study, we attempted to explore the neuroanatomical substrates mediating stress-induced behavioral changes and anti-depressant effects of exercise by examining stimulus-dependent c-Fos induction in the brains of mice that were exposed to repeated stress or exercise in a scheduled manner. Systematic and integrated analyses of c-Fos expression profiles indicated that various brain areas, including the prelimbic cortex, lateral septal area, and paraventricular nuclei of hypothalamus were commonly and strongly activated by both stress and exercise, while the lateral habenula and hippocampus were identified as being preferentially activated by stress and exercise, respectively. Exercise-dependent c-Fos expression in all regions examined in the brain occurred in both glutamatergic and GABAergic neurons. These results suggest that chronic stress and moderate exercise produce counteractive effects on mood behaviors, along with prompting widespread common activation and limited differential activation in specific brain regions. PMID:27539656

  11. Chronic stress and moderate physical exercise prompt widespread common activation and limited differential activation in specific brain regions.

    PubMed

    Kim, Tae-Kyung; Han, Pyung-Lim

    2016-10-01

    Chronic stress in rodents produces depressive behaviors, whereas moderate physical exercise counteracts stress-induced depressive behaviors. Chronic stress and physical exercise appear to produce such opposing effects by changing the neural activity of specific brain regions. However, the detailed mechanisms through which the two different types of stimuli regulate brain function in opposite directions are not clearly understood. In the present study, we attempted to explore the neuroanatomical substrates mediating stress-induced behavioral changes and anti-depressant effects of exercise by examining stimulus-dependent c-Fos induction in the brains of mice that were exposed to repeated stress or exercise in a scheduled manner. Systematic and integrated analyses of c-Fos expression profiles indicated that various brain areas, including the prelimbic cortex, lateral septal area, and paraventricular nuclei of hypothalamus were commonly and strongly activated by both stress and exercise, while the lateral habenula and hippocampus were identified as being preferentially activated by stress and exercise, respectively. Exercise-dependent c-Fos expression in all regions examined in the brain occurred in both glutamatergic and GABAergic neurons. These results suggest that chronic stress and moderate exercise produce counteractive effects on mood behaviors, along with prompting widespread common activation and limited differential activation in specific brain regions.

  12. The Activity Chain Safety and Liveness Specification of Composite Web Services

    NASA Astrophysics Data System (ADS)

    Chen, Bo; Huang, Xiaomei

    Web service composition is most impressing method for development and deployment of e-business. Description and modeling the behavior requirements of composite Web services for users and verifying composite Web service compliance to specific requirements is an important key in design of services. But most work does not address the issue of how to model the requirements that the BPEL4WS processes are supposed to satisfy. The specifications in verification works are general temporal relation based on activity or scenario in essence. Distinguish with these work, we propose a novel concept of behavior specification based on activity chain in which granularity is between activity and scenario. Chain existence mode, chain absence mode are designed to express such behavioral requirements based on activity chain that is similar with safety or liveness specification based on activity respectively. Encode them on Labeled Transition System LTS and then give them exact operation semantics. Finally, an example is illustrated.

  13. Characterization of Fus3 localization: active Fus3 localizes in complexes of varying size and specific activity.

    PubMed

    Choi, K Y; Kranz, J E; Mahanty, S K; Park, K S; Elion, E A

    1999-05-01

    The MAP kinase Fus3 regulates many different signal transduction outputs that govern the ability of Saccharomyces cerevisiae haploid cells to mate. Here we characterize Fus3 localization and association with other proteins. By indirect immunofluorescence, Fus3 localizes in punctate spots throughout the cytoplasm and nucleus, with slightly enhanced nuclear localization after pheromone stimulation. This broad distribution is consistent with the critical role Fus3 plays in mating and contrasts that of Kss1, which concentrates in the nucleus and is not required for mating. The majority of Fus3 is soluble and not bound to any one protein; however, a fraction is stably bound to two proteins of approximately 60 and approximately 70 kDa. Based on fractionation and gradient density centrifugation properties, Fus3 exists in a number of complexes, with its activity critically dependent upon association with other proteins. In the presence of alpha factor, nearly all of the active Fus3 localizes in complexes of varying size and specific activity, whereas monomeric Fus3 has little activity. Fus3 has highest specific activity within a 350- to 500-kDa complex previously shown to contain Ste5, Ste11, and Ste7. Ste5 is required for Fus3 to exist in this complex. Upon alpha factor withdrawal, a pool of Fus3 retains activity for more than one cell cycle. Collectively, these results support Ste5's role as a tether and suggest that association of Fus3 in complexes in the presence of pheromone may prevent inactivation in addition to enhancing activation. PMID:10233162

  14. Activation of enhancer elements by the homeobox gene Cdx2 is cell line specific.

    PubMed Central

    Taylor, J K; Levy, T; Suh, E R; Traber, P G

    1997-01-01

    Cdx2 is a caudal-related homeodomain transcription factor that is expressed in complex patterns during mouse development and at high levels in the intestinal epithelium of adult mice. Cdx2 activates transcription of intestinal gene promoters containing specific binding sites. Moreover, Cdx2 has been shown to induce intestinal differentiation in cell lines. In this study, we show that Cdx2 is able to bind to two well defined enhancer elements in the HoxC8 gene. We then demonstrate that Cdx2 is able to activate transcription of heterologous promoters when its DNA binding element is placed in an enhancer context. Furthermore, the ability to activate enhancer elements is cell-line dependent. When the Cdx2 activation domain was linked to the Gal4 DNA binding domain, the chimeric protein was able to activate Gal4 enhancer constructs in an intestinal cell line, but was unable to activate transcription in NIH3T3 cells. These data suggest that there are cell-specific factors that allow the Cdx2 activation domain to function in the activation of enhancer elements. We hypothesize that either a co-activator protein or differential phosphorylation of the activation domain may be the mechanism for intestinal cell line-specific function of Cdx2 and possibly in other tissues in early development. PMID:9171078

  15. Future of low specific activity molybdenum-99/technetium-99m generator.

    PubMed

    Mushtaq, A

    2012-10-01

    In last few years, the shortage of molybdenum-99 (99Mo) was felt in the developed and developing countries hospitals, where diagnostic nuclear medicine is practiced. To overcome the shortage of 99Mo various routes of its production by accelerators and reactors generating low and high specific activity products have been planned. High specific activity 99Mo obtained by fission of uranium-235 (235U) has completely dominated in the manufacturing of technetium-99m (99mTc) generators in last 3-4 decades, but due to proliferation and dirty bomb, issues non fission routes of 99Mo production are emphasized. Future of low specific activity 99Mo is discussed.

  16. Active specific immunotherapy using the immune reaction of a low-dose irradiated tumor tissue. [Mice

    SciTech Connect

    Ogawa, Y.; Imanaka, K.; Ashida, C.; Takashima, H.; Imajo, Y.; Kimura, S.

    1983-04-01

    Active specific immunotherapy using the immune reaction of a low-dose irradiated tumor tissue was studied on the transplanted MM46 tumor of female C3H/He mice after radiotherapy. MM46 tumor cells were inoculated into the right hind paws of mice. On the 5th day, irradiation with the dose irradiated tumor tissue (2000 rad on the fifth day), were injected into the left hind paws of the tumor-bearing mice. Effectiveness of this active specific immunotherapy against tumor was evaluated by the regression of tumor and survival rate of mice. Tumor was markedly regressed and survival rate was significantly increased by the active specific immunitherapy.

  17. Activators of peroxisome proliferator-activated receptor gamma have depot-specific effects on human preadipocyte differentiation.

    PubMed Central

    Adams, M; Montague, C T; Prins, J B; Holder, J C; Smith, S A; Sanders, L; Digby, J E; Sewter, C P; Lazar, M A; Chatterjee, V K; O'Rahilly, S

    1997-01-01

    Activation of peroxisome proliferator-activated receptor (PPAR) gamma, a nuclear receptor highly expressed in adipocytes, induces the differentiation of murine preadipocyte cell lines. Recently, thiazolidinediones (TZDs), a novel class of insulin-sensitizing compounds effective in the treatment of non-insulin-dependent diabetes mellitus (NIDDM) have been shown to bind to PPARgamma with high affinity. We have examined the effects of these compounds on the differentiation of human preadipocytes derived from subcutaneous (SC) and omental (Om) fat. Assessed by lipid accumulation, glycerol 3-phosphate dehydrogenase activity, and mRNA levels, subcultured preadipocytes isolated from either SC or Om depots did not differentiate in defined serum-free medium. Addition of TZDs (BRL49653 or troglitazone) or 15-deoxyDelta12,14prostaglandin J2 (a natural PPARgamma ligand) enhanced markedly the differentiation of preadipocytes from SC sites, assessed by all three criteria. The rank order of potency of these agents in inducing differentiation matched their ability to activate transcription via human PPARgamma. In contrast, preadipocytes from Om sites in the same individuals were refractory to TZDs, although PPARgamma was expressed at similar levels in both depots. The mechanism of this depot-specific TZD response is unknown. However, given the association between Om adiposity and NIDDM, the site-specific responsiveness of human preadipocytes to TZDs may be involved in the beneficial effects of these compounds on in vivo insulin sensitivity. PMID:9399962

  18. Angiotensin II and norepinephrine activate specific calcineurin-dependent NFAT transcription factor isoforms in cardiomyocytes.

    PubMed

    Lunde, Ida G; Kvaløy, Heidi; Austbø, Bjørg; Christensen, Geir; Carlson, Cathrine R

    2011-11-01

    Norepinephrine (NE) and angiotensin II (ANG II) are primary effectors of the sympathetic adrenergic and the renin-angiotensin-aldosterone systems, mediating hypertrophic, apoptotic, and fibrotic events in the myocardium. As NE and ANG II have been shown to affect intracellular calcium in cardiomyocytes, we hypothesized that they activate the calcium-sensitive, prohypertrophic calcineurin-nuclear factor of activated T-cell (NFATc) signaling pathway. More specifically, we have investigated isoform-specific activation of NFAT in NE- and ANG II-stimulated cardiomyocytes, as it is likely that each of the four calcineurin-dependent isoforms, c1-c4, play specific roles. We have stimulated neonatal ventriculocytes from C57/B6 and NFAT-luciferase reporter mice with ANG II or NE and quantified NFAT activity by luciferase activity and phospho-immunoblotting. ANG II and NE increased calcineurin-dependent NFAT activity 2.4- and 1.9-fold, measured as luciferase activity after 24 h of stimulation, and induced protein synthesis, measured by radioactive leucine incorporation after 24 and 72 h. To optimize measurements of NFAT isoforms, we examined the specificity of NFAT antibodies on peptide arrays and by immunoblotting with designed blocking peptides. Western analyses showed that both effectors activate NFATc1 and c4, while NFATc2 activity was regulated by NE only, as measured by phospho-NFAT levels. Neither ANG II nor NE activated NFATc3. As today's main therapies for heart failure aim at antagonizing the adrenergic and renin-angiotensin-aldosterone systems, understanding their intracellular actions is of importance, and our data, through validating a method for measuring myocardial NFATs, indicate that ANG II and NE activate specific NFATc isoforms in cardiomyocytes.

  19. Identification of specific activities associated with fall-related injuries, active component, U.S. Army, 2011.

    PubMed

    Hauschild, Veronique D; Schuh, Anna; Taylor, Bonnie J; Canham-Chervak, Michelle; Jones, Bruce H

    2016-06-01

    Although falls continue to be a leading mechanism of serious injuries among military populations, interventions must target activities or hazards that can be controlled or managed. This project aimed to identify activities most frequently associated with Army soldier fall-related injuries to prioritize prevention strategies for this substantial health burden. Narrative data from Army safety, medical evacuation, and casualty reporting systems were reviewed to select incidents meeting inclusion criteria and assign established codes. Nondeployed (n=988) and deployed (n=254) injury rates were not statistically different (2.20 per 1,000 non-deployed person-years [p-yrs], 2.21 per 1,000 deployed p-yrs, respectively). More than 75% of injuries were temporarily disabling fractures, sprains, and strains, primarily to lower extremities. The most frequent activities associated with non-deployed fall injuries were sports (e.g., snowboarding and basketball; 22%), parachuting (20%), walking/marching (19%), and climbing (15%). Ice and snow were the leading hazard (43%). The most common associated activities among deployed soldiers were occupational tasks (53%), walking/patrolling (24%), climbing (23%), and sports (17%). Specific interventions that target the activities and hazards identified in this investigation are suggested as priorities to reduce Army fall-related injuries. PMID:27362343

  20. Identification of specific activities associated with fall-related injuries, active component, U.S. Army, 2011.

    PubMed

    Hauschild, Veronique D; Schuh, Anna; Taylor, Bonnie J; Canham-Chervak, Michelle; Jones, Bruce H

    2016-06-01

    Although falls continue to be a leading mechanism of serious injuries among military populations, interventions must target activities or hazards that can be controlled or managed. This project aimed to identify activities most frequently associated with Army soldier fall-related injuries to prioritize prevention strategies for this substantial health burden. Narrative data from Army safety, medical evacuation, and casualty reporting systems were reviewed to select incidents meeting inclusion criteria and assign established codes. Nondeployed (n=988) and deployed (n=254) injury rates were not statistically different (2.20 per 1,000 non-deployed person-years [p-yrs], 2.21 per 1,000 deployed p-yrs, respectively). More than 75% of injuries were temporarily disabling fractures, sprains, and strains, primarily to lower extremities. The most frequent activities associated with non-deployed fall injuries were sports (e.g., snowboarding and basketball; 22%), parachuting (20%), walking/marching (19%), and climbing (15%). Ice and snow were the leading hazard (43%). The most common associated activities among deployed soldiers were occupational tasks (53%), walking/patrolling (24%), climbing (23%), and sports (17%). Specific interventions that target the activities and hazards identified in this investigation are suggested as priorities to reduce Army fall-related injuries.

  1. Dissecting signaling through activation of specific Src-effector complexes in vivo

    PubMed Central

    Karginov, A.V.; Tsygankov, D.; Berginski, M.; Chu, P.-H.; Trudeau, E.D.; Yi, J.J.; Gomez, S.; Elston, T.C.; Hahn, K.M.

    2014-01-01

    We describe an approach to selectively activate a kinase in a specific protein complex or at a specific subcellular location within living cells, and within minutes. This reveals the effects of specific kinase pathways without time for genetic compensation. The new technique, dubbed RapRTAP (rapamycin regulated targeted activation of pathways) was used to dissect the role of Src kinase interactions with FAK and p130Cas in cell motility and morphodynamics. The overall effects of Src activation on cell morphology and adhesion dynamics were first quantified, without restricting effector access. Subsets of Src induced behaviors were then attributed to specific interactions between Src and the two downstream proteins. Activation of Src in the cytoplasm versus at the cell membrane also produced distinct phenotypes. The conserved nature of the kinase site modified for RapRTAP indicates that the technique can be applied to many kinases. PMID:24609359

  2. Estimation of specific activity of 177Lu by 'saturation assay' principle using DOTA as ligand.

    PubMed

    Pillai, Ambikalmajan M R; Chakraborty, Sudipta; Das, Tapas

    2015-01-01

    Lutetium-177 is a widely used therapeutic radionuclide in targeted therapy and it is important to know its specific activity at the time of radiopharmaceutical preparation, especially for radiolabeling peptides. However, there are no direct methods for the experimental determination of the specific activity which can be readily applied in a hospital radiopharmacy. A new technique based on the 'saturation assay' principle using DOTA as the binding agent for the estimation of specific activity of (177)Lu is reported. The studies demonstrate the proof of principle of this new assay technique. The method is general and can be modified and applied for the estimation of specific activity of other metallic radionuclides by using DOTA or other suitable chelating agents.

  3. Virus-specific T cells engineered to coexpress tumor-specific receptors: persistence and antitumor activity in individuals with neuroblastoma.

    PubMed

    Pule, Martin A; Savoldo, Barbara; Myers, G Doug; Rossig, Claudia; Russell, Heidi V; Dotti, Gianpietro; Huls, M Helen; Liu, Enli; Gee, Adrian P; Mei, Zhuyong; Yvon, Eric; Weiss, Heidi L; Liu, Hao; Rooney, Cliona M; Heslop, Helen E; Brenner, Malcolm K

    2008-11-01

    Cytotoxic T lymphocytes (CTLs) directed to nonviral tumor-associated antigens do not survive long term and have limited antitumor activity in vivo, in part because such tumor cells typically lack the appropriate costimulatory molecules. We therefore engineered Epstein-Barr virus (EBV)-specific CTLs to express a chimeric antigen receptor directed to the diasialoganglioside GD2, a nonviral tumor-associated antigen expressed by human neuroblastoma cells. We reasoned that these genetically engineered lymphocytes would receive optimal costimulation after engagement of their native receptors, enhancing survival and antitumor activity mediated through their chimeric receptors. Here we show in individuals with neuroblastoma that EBV-specific CTLs expressing a chimeric GD2-specific receptor indeed survive longer than T cells activated by the CD3-specific antibody OKT3 and expressing the same chimeric receptor but lacking virus specificity. Infusion of these genetically modified cells seemed safe and was associated with tumor regression or necrosis in half of the subjects tested. Hence, virus-specific CTLs can be modified to function as tumor-directed effector cells.

  4. Virus-specific T cells engineered to coexpress tumor-specific receptors: persistence and antitumor activity in individuals with neuroblastoma

    PubMed Central

    Pule, Martin A; Savoldo, Barbara; Myers, G Doug; Rossig, Claudia; Russell, Heidi V; Dotti, Gianpietro; Huls, M Helen; Liu, Enli; Gee, Adrian P; Mei, Zhuyong; Yvon, Eric; Weiss, Heidi L; Liu, Hao; Rooney, Cliona M; Heslop, Helen E; Brenner, Malcolm K

    2009-01-01

    Cytotoxic T lymphocytes (CTLs) directed to nonviral tumor–associated antigens do not survive long term and have limited antitumor activity in vivo, in part because such tumor cells typically lack the appropriate costimulatory molecules. We therefore engineered Epstein-Barr virus (EBV)-specific CTLs to express a chimeric antigen receptor directed to the diasialoganglioside GD2, a nonviral tumor–associated antigen expressed by human neuroblastoma cells. We reasoned that these genetically engineered lymphocytes would receive optimal costimulation after engagement of their native receptors, enhancing survival and antitumor activity mediated through their chimeric receptors. Here we show in individuals with neuroblastoma that EBV-specific CTLs expressing a chimeric GD2-specific receptor indeed survive longer than T cells activated by the CD3-specific antibody OKT3 and expressing the same chimeric receptor but lacking virus specificity. Infusion of these genetically modified cells seemed safe and was associated with tumor regression or necrosis in half of the subjects tested. Hence, virus-specific CTLs can be modified to function as tumor-directed effector cells. PMID:18978797

  5. Rapid, specific, no-wash, far-red fluorogen activation in subcellular compartments by targeted fluorogen activating proteins.

    PubMed

    Telmer, Cheryl A; Verma, Richa; Teng, Haibing; Andreko, Susan; Law, Leann; Bruchez, Marcel P

    2015-05-15

    Live cell imaging requires bright photostable dyes that can target intracellular organelles and proteins with high specificity in a no-wash protocol. Organic dyes possess the desired photochemical properties and can be covalently linked to various protein tags. The currently available fluorogenic dyes are in the green/yellow range where there is high cellular autofluorescence and the near-infrared (NIR) dyes need to be washed out. Protein-mediated activation of far-red fluorogenic dyes has the potential to address these challenges because the cell-permeant dye is small and nonfluorescent until bound to its activating protein, and this binding is rapid. In this study, three single chain variable fragment (scFv)-derived fluorogen activating proteins (FAPs), which activate far-red emitting fluorogens, were evaluated for targeting, brightness, and photostability in the cytosol, nucleus, mitochondria, peroxisomes, and endoplasmic reticulum with a cell-permeant malachite green analog in cultured mammalian cells. Efficient labeling was achieved within 20-30 min for each protein upon the addition of nM concentrations of dye, producing a signal that colocalized significantly with a linked mCerulean3 (mCer3) fluorescent protein and organelle specific dyes but showed divergent photostability and brightness properties dependent on the FAP. These FAPs and the ester of malachite green dye (MGe) can be used as specific, rapid, and wash-free labels for intracellular sites in live cells with far-red excitation and emission properties, useful in a variety of multicolor experiments. PMID:25650487

  6. Self-Efficacy and Social Support as Mediators Between Culturally Specific Dance and Lifestyle Physical Activity

    PubMed Central

    Murrock, Carolyn J.; Madigan, Elizabeth

    2013-01-01

    Culturally specific dance has the potential to generate health benefits but is seldom used even among studies advocating culturally specific interventions. This study examined the components of self-efficacy and social support as mediators between culturally specific dance and lifestyle physical activity in African American women (N = 126). An experimental design compared intervention and control groups for mediating effects of self-efficacy and social support on lifestyle physical activity. Findings indicated that only outcome expectations and social support from friends mediated effects. Culturally specific dance is a first step in encouraging African American women to become more physically active and improve health outcomes. The implications are that culturally specific dance programs can improve health outcomes by including members of underserved populations. PMID:18763475

  7. Cyclotron production of ``very high specific activity'' platinum radiotracers in No Carrier Added form

    NASA Astrophysics Data System (ADS)

    Birattari, C.; Bonardi, M.; Groppi, F.; Gini, L.; Gallorini, M.; Sabbioni, E.; Stroosnijder, M. F.

    2001-12-01

    At the "Radiochemistry Laboratory" of Accelerators and Applied Superconductivity Laboratory, LASA, several production and quality assurance methods for short-lived and high specific activity radionuclides, have been developed. Presently, the irradiations are carried out at the Scanditronix MC40 cyclotron (K=38; p, d, He-4 and He-3) of JRC-Ispra, Italy, of the European Community, while both chemical purity and specific activity determination are carried out at the TRIGA MARK II research reactor of University of Pavia and at LASA itself. In order to optimize the irradiation conditions for platinum radiotracer production, both thin- and thick-target excitation function of natOs(α,xn) nuclear reactions were measured. A very selective radiochemical separation to obtain Pt radiotracers in No Carrier Added form, has been developed. Both real specific activity and chemical purity of radiotracer, have been determined by neutron activation analysis and atomic absorption spectrometry. An Isotopic Dilution Factor (IDF) of the order of 50 is achieved.

  8. Targeted disruption of cocaine-activated accumbens neurons prevents context-specific sensitization

    PubMed Central

    Koya, Eisuke; Golden, Sam A.; Harvey, Brandon K.; Guez, Danielle H.; Berkow, Alexander; Simmons, Danielle E.; Bossert, Jennifer M.; Nair, Sunila G.; Uejima, Jamie L.; Marin, Marcelo T.; Mitchell, Timothy; Farquhar, David; Ghosh, Sukhen; Mattson, Brandi J.; Hope, Bruce T.

    2009-01-01

    Learned associations between effects of abused drugs and the drug administration environment play important roles in drug addiction. Histochemical and electrophysiological studies suggest that these associations are encoded in sparsely distributed nucleus accumbens neurons that are selectively activated by drugs and drug-associated cues. Although correlations between accumbens neuronal activity and responsivity to drugs and drug cues have been observed, no technique exists for selectively manipulating these activated neurons and establishing their causal role in behavioral effects of drugs and drug cues. Here we describe a novel method, termed ‘Daun02-inactivation method’, that selectively inactivates a minority of neurons activated by cocaine in an environment repeatedly paired with cocaine to demonstrate a causal role for these activated neurons in context-specific cocaine-induced psychomotor sensitization in rats. This method provides a new tool to study causal roles of selectively activated neurons in behavioral effects of drugs and drug cues and in other learned behaviors. PMID:19620976

  9. Activity of site-specific endonucleases on complexes of plasmid DNA with multiwalled carbon nanotubes

    NASA Astrophysics Data System (ADS)

    Egorova, V. P.; Krylova, H. V.; Lipnevich, I. V.; Veligura, A. A.; Shulitsky, B. G.; Asayonok, A. A.; Vaskovtsev, E. V.

    2016-08-01

    We have synthesized and investigated structural and functional properties of plasmid DNA complexes with multi-walled carbon nanotubes (MWCNTs) for detection of changes in structural state of the plasmid DNA at its recognition by site-specific endonuclease. It has been also established that the site-specific endonuclease is functionally active on the surface of MWCNTs.

  10. The Role of Specificity, Targeted Learning Activities, and Prior Knowledge for the Effects of Relevance Instructions

    ERIC Educational Resources Information Center

    Roelle, Julian; Lehmkuhl, Nina; Beyer, Martin-Uwe; Berthold, Kirsten

    2015-01-01

    In 2 experiments we examined the role of (a) specificity, (b) the type of targeted learning activities, and (c) learners' prior knowledge for the effects of relevance instructions on learning from instructional explanations. In Experiment 1, we recruited novices regarding the topic of atomic structure (N = 80) and found that "specific"…

  11. Physical activity estimated by the bone-specific physical activity questionnaire is also associated with cardiovascular risk.

    PubMed

    Weeks, Benjamin K; Purvis, Meredith; Beck, Belinda R

    2016-11-01

    The nature of physical activity that benefits bone is traditionally thought to differ from that benefiting cardiovascular health. Accordingly, exercise recommendations for improving bone health and cardiovascular health are largely incongruent. Our aim was to determine the associations between high-impact physical activity participation and both cardiovascular disease risk factors and bone mass. We recruited 94 men and women (age 34.0 ± 13.3 years) to undergo measures of cardiovascular disease risk (BMI, total cholesterol, fasting blood glucose, waist-to-hip ratio, and mean arterial pressure) and dual-energy X-ray absorptiometry (DXA XR-800, Norland) measures of bone mass (femoral neck, lumbar spine, and whole body BMD) and body composition (whole body lean mass and fat mass). Physical activity participation was estimated using the bone-specific physical activity questionnaire (BPAQ). Those in the upper tertile for current BPAQ score exhibited lower total cholesterol, waist-to-hip ratio, and mean arterial pressure than those in the lower tertiles (P < 0.05) with the relationship being mild-to-moderate (r = -0.49 to 0.29, P < 0.01). Those in the upper tertile for BPAQ score also had greater lumbar spine BMD than those in the lower tertile (P = 0.008), with BPAQ score predicting 6% of the variance in BMD (P = 0.02). We conclude that high-impact physical activity as captured by the BPAQ may be beneficial for both bone health and for attenuating cardiovascular disease risk. PMID:26937743

  12. Stressor-specific activation of the hypothalamic-pituitary-adrenocortical axis.

    PubMed

    Pacák, K

    2000-01-01

    New information has accrued from in vivo microdialysis studies about stress-related changes in norepinephrine concentrations in extracellular fluid of the paraventricular nucleus (PVN) and the activation of the hypothalamic-pituitary-adrenocortical (HPA) axis. Our data on the effects of lower brainstem hemisections show that paraventricular noradrenergic terminals are derived mainly from medullary A1 and A2 catecholaminergic cells. The activation of these cells contributes importantly to stress-induced noradrenergic activation in the paraventricular nucleus of conscious animals. The results from brainstem hemisection experiments also indicate that baseline levels and immobilization-induced increments in corticotropin-releasing hormone (CRH) mRNA expression in the PVN depend on ipsilaterally ascending medullary tract. Thus, the prevalent concept that stress-induced noradrenergic activation of the HPA axis depends mainly on activation of locus ceruleus noradrenergic neurons requires re-evaluation. Our new stress concepts favor stressor-specific activation of the HPA axis. The present data also suggest the existence of stressor-specific central pathways that differentially participate in the regulation of sympathoneuronal and adrenomedullary outflows as well as of the activity of the HPA axis. Furthermore, the results are inconsistent with a founding tenet of Selye's stress theory, the doctrine of nonspecificity, which defines stress as the nonspecific response of the body to any demand. We expect that future studies in this area will focus on further examination of the notion of stressor-specific patterns of central neurotransmitter release and elucidate the genetic bases of these patterns.

  13. Recognition of military-specific physical activities with body-fixed sensors.

    PubMed

    Wyss, Thomas; Mäder, Urs

    2010-11-01

    The purpose of this study was to develop and validate an algorithm for recognizing military-specific, physically demanding activities using body-fixed sensors. To develop the algorithm, the first group of study participants (n = 15) wore body-fixed sensors capable of measuring acceleration, step frequency, and heart rate while completing six military-specific activities: walking, marching with backpack, lifting and lowering loads, lifting and carrying loads, digging, and running. The accuracy of the algorithm was tested in these isolated activities in a laboratory setting (n = 18) and in the context of daily military training routine (n = 24). The overall recognition rates during isolated activities and during daily military routine activities were 87.5% and 85.5%, respectively. We conclude that the algorithm adequately recognized six military-specific physical activities based on sensor data alone both in a laboratory setting and in the military training environment. By recognizing type of physical activities this objective method provides additional information on military-job descriptions. PMID:21121495

  14. Specific anion effects on ATPase activity, calmodulin sensitivity, and solubilization of dynein ATPases.

    PubMed

    Blum, J J; Hayes, A

    1984-01-01

    The basal ATPase activity of 30S dynein, whether obtained by extraction of ciliary axonemes with a high (0.5 M NaCl) or low (1 mM Tris-0.1 mM EDTA) ionic strength buffer is increased by NaCl, NaNO3, and Na acetate, with NaNO3 causing the largest increase. The calmodulin-activated ATPase activity of 30S dynein is also increased by addition of NaCl, NaNO3, or Na acetate, but the effects are less pronounced than on basal activity, so that the calmodulin activation ratio (CAR) decreases to 1.0 as salt concentration increases to 0.2 M. These salts also reduce the CAR of 14S dynein ATPase to 1.0 but by strongly inhibiting the calmodulin-activated ATPase activity and only slightly inhibiting the basal activity. Sodium fluoride differs both quantitatively and qualitatively from the other three salts studied. It inhibits the ATPase activity of both 14S and 30S dyneins at concentrations below 5 mM and, by a stronger inhibition of the calmodulin-activated ATPase activities, reduces the CAR to 1.0. Na acetate does not inhibit axonemal ATPase, nor does it interfere with the drop in turbidity caused by ATP and extracts very little protein from the axonemes. NaCl and, especially, NaNO3, cause a slow decrease in A350 of an axonemal suspension and an inhibition of the turbidity response to ATP. NaF, at concentrations comparable to those that inhibit the ATPase activities of the solubilized dyneins, also inhibits axonemal ATPase activity and the turbidity response. Pretreatment of demembranated axonemes with a buffer containing 0.25 M sodium acetate for 5 min followed by extraction for 5 min with a buffer containing 0.5 M NaCl and resolution of the extracted dynein on a sucrose density gradient generally yields a 30S dynein that is activated by calmodulin in a heterogeneous manner, ie, the "light" 30S dynein ATPase fractions are more activated than the "heavy" 30S dynein fractions. These results demonstrate specific anion effects on the basal and calmodulin-activated dynein ATPase

  15. Echicetin Coated Polystyrene Beads: A Novel Tool to Investigate GPIb-Specific Platelet Activation and Aggregation

    PubMed Central

    Petunin, Alexey; Clemetson, Kenneth J.; Gambaryan, Stepan; Walter, Ulrich

    2014-01-01

    von Willebrand factor/ristocetin (vWF/R) induces GPIb-dependent platelet agglutination and activation of αIIbβ3 integrin, which also binds vWF. These conditions make it difficult to investigate GPIb-specific signaling pathways in washed platelets. Here, we investigated the specific mechanisms of GPIb signaling using echicetin-coated polystyrene beads, which specifically activate GPIb. We compared platelet activation induced by echicetin beads to vWF/R. Human platelets were stimulated with polystyrene beads coated with increasing amounts of echicetin and platelet activation by echicetin beads was then investigated to reveal GPIb specific signaling. Echicetin beads induced αIIbβ3-dependent aggregation of washed platelets, while under the same conditions vWF/R treatment led only to αIIbβ3-independent platelet agglutination. The average distance between the echicetin molecules on the polystyrene beads must be less than 7 nm for full platelet activation, while the total amount of echicetin used for activation is not critical. Echicetin beads induced strong phosphorylation of several proteins including p38, ERK and PKB. Synergistic signaling via P2Y12 and thromboxane receptor through secreted ADP and TxA2, respectively, were important for echicetin bead triggered platelet activation. Activation of PKG by the NO/sGC/cGMP pathway inhibited echicetin bead-induced platelet aggregation. Echicetin-coated beads are powerful and reliable tools to study signaling in human platelets activated solely via GPIb and GPIb-triggered pathways. PMID:24705415

  16. Relative Contributions of Specific Activity Histories and Spontaneous Processes to Size Remodeling of Glutamatergic Synapses

    PubMed Central

    Dvorkin, Roman; Ziv, Noam E.

    2016-01-01

    The idea that synaptic properties are defined by specific pre- and postsynaptic activity histories is one of the oldest and most influential tenets of contemporary neuroscience. Recent studies also indicate, however, that synaptic properties often change spontaneously, even in the absence of specific activity patterns or any activity whatsoever. What, then, are the relative contributions of activity history-dependent and activity history-independent processes to changes synapses undergo? To compare the relative contributions of these processes, we imaged, in spontaneously active networks of cortical neurons, glutamatergic synapses formed between the same axons and neurons or dendrites under the assumption that their similar activity histories should result in similar size changes over timescales of days. The size covariance of such commonly innervated (CI) synapses was then compared to that of synapses formed by different axons (non-CI synapses) that differed in their activity histories. We found that the size covariance of CI synapses was greater than that of non-CI synapses; yet overall size covariance of CI synapses was rather modest. Moreover, momentary and time-averaged sizes of CI synapses correlated rather poorly, in perfect agreement with published electron microscopy-based measurements of mouse cortex synapses. A conservative estimate suggested that ~40% of the observed size remodeling was attributable to specific activity histories, whereas ~10% and ~50% were attributable to cell-wide and spontaneous, synapse-autonomous processes, respectively. These findings demonstrate that histories of naturally occurring activity patterns can direct glutamatergic synapse remodeling but also suggest that the contributions of spontaneous, possibly stochastic, processes are at least as great. PMID:27776122

  17. Roles of s3 site residues of nattokinase on its activity and substrate specificity.

    PubMed

    Wu, Shuming; Feng, Chi; Zhong, Jin; Huan, Liandong

    2007-09-01

    Nattokinase (Subtilisin NAT, NK) is a bacterial serine protease with high fibrinolytic activity. To probe their roles on protease activity and substrate specificity, three residues of S3 site (Gly(100), Ser(101) and Leu(126)) were mutated by site-directed mutagenesis. Kinetics parameters of 20 mutants were measured using tetrapeptides as substrates, and their fibrinolytic activities were determined by fibrin plate method. Results of mutation analysis showed that Gly(100) and Ser(101) had reverse steric and electrostatic effects. Residues with bulky or positively charged side chains at position 100 decreased the substrate binding and catalytic activity drastically, while residues with the same characters at position 101 could obviously enhance protease and fibrinolytic activity of NK. Mutation of Leu(126) might impair the structure of the active cleft and drastically decreased the activity of NK. Kinetics studies of the mutants showed that S3 residues were crucial to keep protease activity while they moderately affected substrate specificity of NK. The present study provided some original insight into the P3-S3 interaction in NK and other subtilisins, as well as showed successful protein engineering cases to improve NK as a potential therapeutic agent.

  18. NOTCH activation interferes with cell fate specification in the gastrulating mouse embryo.

    PubMed

    Souilhol, Céline; Perea-Gomez, Aitana; Camus, Anne; Beck-Cormier, Sarah; Vandormael-Pournin, Sandrine; Escande, Marie; Collignon, Jérôme; Cohen-Tannoudji, Michel

    2015-11-01

    NOTCH signalling is an evolutionarily conserved pathway involved in intercellular communication essential for cell fate choices during development. Although dispensable for early aspects of mouse development, canonical RBPJ-dependent NOTCH signalling has been shown to influence lineage commitment during embryonic stem cell (ESC) differentiation. NOTCH activation in ESCs promotes the acquisition of a neural fate, whereas its suppression favours their differentiation into cardiomyocytes. This suggests that NOTCH signalling is implicated in the acquisition of distinct embryonic fates at early stages of mammalian development. In order to investigate in vivo such a role for NOTCH signalling in shaping cell fate specification, we use genetic approaches to constitutively activate the NOTCH pathway in the mouse embryo. Early embryonic development, including the establishment of anterior-posterior polarity, is not perturbed by forced NOTCH activation. By contrast, widespread NOTCH activity in the epiblast triggers dramatic gastrulation defects. These are fully rescued in a RBPJ-deficient background. Epiblast-specific NOTCH activation induces acquisition of neurectoderm identity and disrupts the formation of specific mesodermal precursors including the derivatives of the anterior primitive streak, the mouse organiser. In addition, we show that forced NOTCH activation results in misregulation of NODAL signalling, a major determinant of early embryonic patterning. Our study reveals a previously unidentified role for canonical NOTCH signalling during mammalian gastrulation. It also exemplifies how in vivo studies can shed light on the mechanisms underlying cell fate specification during in vitro directed differentiation.

  19. Controlling nuclear JAKs and STATs for specific gene activation by IFNγ

    PubMed Central

    Noon-Song, Ezra N.; Ahmed, Chulbul M.; Dabelic, Rea; Canton, Johnathan; Johnson, Howard M.

    2011-01-01

    We previously showed that gamma interferon (IFNγ) and its receptor subunit, IFNGR1, interacted with the promoter region of IFNγ-activated genes along with transcription factor STAT1α. Recent studies have suggested that activated Janus kinases pJAK2 and pJAK1 also played a role in gene activation by phosphorylation of histone H3 on tyrosine 41. This study addresses the question of the role of activated JAKs in specific gene activation by IFNγ. We carried out chromatin immunoprecipitation (ChIP) followed by PCR in IFNγ treated WISH cells and showed association of pJAK1, pJAK2, IFNGR1, and STAT1 on the same DNA sequence of the IRF-1 gene promoter. The β-actin gene, which is not activated by IFNγ, did not show this association. The movement of activated JAK to the nucleus and the IRF-1 promoter was confirmed by the combination of nuclear fractionation, confocal microscopy and DNA precipitation analysis using the biotinylated GAS promoter. Activated JAKs in the nucleus was associated with phosphorylated tyrosine 41 on histone H3 in the region of the GAS promoter. Unphosphorylated JAK2 was found to be constitutively present in the nucleus and was capable of undergoing activation in IFNγ treated cells, most likely via nuclear IFNGR1. Association of pJAK2 and IFNGR1 with histone H3 in IFNγ treated cells was demonstrated by histone H3 immunoprecipitation. Unphosphorylated STAT1 protein was associated with histone H3 of untreated cells. IFNγ treatment resulted in its disassociation and then re-association as pSTAT1. The results suggest a novel role for activated JAKs in epigenetic events for specific gene activation. PMID:21689637

  20. Development of isoform-specific sensors of polypeptide GalNAc-transferase activity.

    PubMed

    Song, Lina; Bachert, Collin; Schjoldager, Katrine T; Clausen, Henrik; Linstedt, Adam D

    2014-10-31

    Humans express up to 20 isoforms of GalNAc-transferase (herein T1-T20) that localize to the Golgi apparatus and initiate O-glycosylation. Regulation of this enzyme family affects a vast array of proteins transiting the secretory pathway and diseases arise upon misregulation of specific isoforms. Surprisingly, molecular probes to monitor GalNAc-transferase activity are lacking and there exist no effective global or isoform-specific inhibitors. Here we describe the development of T2- and T3-isoform specific fluorescence sensors that traffic in the secretory pathway. Each sensor yielded little signal when glycosylated but was strongly activated in the absence of its glycosylation. Specificity of each sensor was assessed in HEK cells with either the T2 or T3 enzymes deleted. Although the sensors are based on specific substrates of the T2 and T3 enzymes, elements in or near the enzyme recognition sequence influenced their activity and required modification, which we carried out based on previous in vitro work. Significantly, the modified T2 and T3 sensors were activated only in cells lacking their corresponding isozymes. Thus, we have developed T2- and T3-specific sensors that will be valuable in both the study of GalNAc-transferase regulation and in high-throughput screening for potential therapeutic regulators of specific GalNAc-transferases.

  1. Activation of transcriptional activity of HSE by a novel mouse zinc finger protein ZNFD specifically expressed in testis.

    PubMed

    Xu, Fengqin; Wang, Weiping; Lei, Chen; Liu, Qingmei; Qiu, Hao; Muraleedharan, Vinaydhar; Zhou, Bin; Cheng, Hongxia; Huang, Zhongkai; Xu, Weian; Li, Bichun; Wang, Minghua

    2012-04-01

    Zinc finger proteins (ZFPs) that contain multiple cysteine and/or histidine residues perform important roles in various cellular functions, including transcriptional regulation, cell proliferation, differentiation, and apoptosis. The Cys-Cys-His-His (C(2)H(2)) type of ZFPs are the well-defined members of this super family and are the largest and most complex proteins in eukaryotic genomes. In this study, we identified a novel C(2)H(2) type of zinc finger gene ZNFD from mice which has a 1,002 bp open reading frame and encodes a protein with 333 amino acid residues. The predicted 37.4 kDa protein contains a C(2)H(2) zinc finger domain. ZNFD gene is located on chromosome 18qD1. RT-PCR analysis revealed that the ZNFD gene was specifically expressed in mouse testis but not in other tissues. Subcellular localization analysis demonstrated that ZNFD was localized in the nucleus. Reporter gene assays showed that overexpression of ZNFD in the COS7 cells activates the transcriptional activities of heat shock element (HSE). Overall, these results suggest that ZNFD is a member of the zinc finger transcription factor family and it participates in the transcriptional regulation of HSE. Many heat shock proteins regulated by HSE are involved in testicular development. Therefore, our results suggest that ZNFD may probably participate in the development of mouse testis and function as a transcription activator in HSE-mediated gene expression and signaling pathways.

  2. Phosphatase activity and specific methanogenic activity in an anaerobic reactor treating sludge from a brackish recirculation aquaculture system.

    PubMed

    Zhang, Xuedong; Ferreira, Rui B; Spanjers, Henri; van Lier, Jules B

    2013-01-01

    Anaerobic treatment of high salinity sludge from marine/brackish recirculation aquaculture systems is potentially limited by inhibition of enzymatic activities and cell lysis resulting from high osmotic pressures. To further address these limitations the following investigations were conducted: effect of salinity on phosphatase activity (PA), soluble microbial products (SMP) production, and presence of extracellular polymeric substances (EPS); effect of iron (III) chloride (FeCl3) on PA and specific methanogenic activity (SMA); effect of addition of the compatible solute glycine betaine (GB) and potassium on PA, as well as on SMP and EPS production, all under saline conditions. The results show that salinity has different effects on PA of anaerobes under starvation and feeding conditions. FeCl3 increased the SMA of the sludge by 22.5% at 100 mg FeCl3/L compared with a control group (0 mg FeCl3/L). Furthermore, results of analysis of variance tests show that betaine increased the polysaccharide content of EPS and polypeptide content of SMP. However, addition of 1 mM potassium chloride did not show a significant effect on EPS and SMP composition. In conclusion, anaerobic digestion of salty sludges from a brackish aquaculture recirculation system may not be negatively affected by FeCl3 addition to concentrate waste streams, whereas GB boosts the production of SMP and EPS.

  3. Pyrimidine-specific 5' nucleotidase activity in bovine erythrocytes: effect of phlebotomy and lead poisoning

    SciTech Connect

    George, J.W.; Duncan, J.R.

    1982-01-01

    Erythrocyte pyrimidine-specific 5' nucleotidase (PY5'N) (E.C. 3.1.3.5) was measured in healthy, anemic, and lead-poisoned calves to determine whether low activity of PY5'N is associated with the propensity of cattle to develop basophilic stippling of erythrocytes. Low activity of PY5'N has been associated with basophilic stippling of erythrocytes in persons with inherited hemolytic anemia and with lead poisoning. A radiometric technique, using (/sup 14/C)cytidine monophosphate as the substrate, was used to measured PY5'N activity. The erythrocytes from 4 healthy calves had much lower activity (mean of 7.1 +/- 1.6 nmols of (/sup 14/C)cytidine monophosphate hydrolyzed/min/g of hemoglobin) than has been reported for human erythrocytes. The pH response curve of bovine PY5'N was similar to that of the human enzyme, with maximal activity around pH 7. Experimental hemorrhagic anemia in these calves increased PY5'N activity 6-to 7-fold, with peak activity occurring concomitantly with maximum reticulocytosis. Two of the calves were then given lead per os, and the PY5'N activity decreased within 24 hours to base-line values. In the 2 other calves not given lead, the PY5'N activity declined slowly, but did not reach base-line values after 14 days.

  4. Successful Remembering Elicits Event-Specific Activity Patterns in Lateral Parietal Cortex

    PubMed Central

    Chun, Marvin M.

    2014-01-01

    Remembering a past event involves reactivation of content-specific patterns of neural activity in high-level perceptual regions (e.g., ventral temporal cortex, VTC). In contrast, the subjective experience of vivid remembering is typically associated with increased activity in lateral parietal cortex (LPC)—“retrieval success effects” that are thought to generalize across content types. However, the functional significance of LPC activation during memory retrieval remains a subject of active debate. In particular, theories are divided with respect to whether LPC actively represents retrieved content or if LPC activity only scales with content reactivation elsewhere (e.g., VTC). Here, we report a human fMRI study of visual memory recall (faces vs scenes) in which complementary forms of multivoxel pattern analysis were used to test for and compare content reactivation within LPC and VTC. During recall of visual images, we observed robust reactivation of broad category information (face vs scene) in both VTC and LPC. Moreover, recall-related activity patterns in LPC, but not VTC, differentiated between individual events. Importantly, these content effects were particularly evident in areas of LPC (namely, angular gyrus) in which activity scaled with subjective reports of recall vividness. These findings provide striking evidence that LPC not only signals that memories have been successfully recalled, but actively represents what is being remembered. PMID:24899726

  5. Phase advancement and nucleus-specific timing of thalamocortical activity during slow cortical oscillation

    PubMed Central

    Slézia, Andrea; Hangya, Balázs; Ulbert, István; Acsády, László

    2011-01-01

    The exact timing of cortical afferent activity is instrumental for the correct coding and retrieval of internal and external stimuli. Thalamocortical inputs represent the most significant subcortical pathway to the cortex, but the precise timing and temporal variability of thalamocortical activity is not known. To examine this question, we studied the phase of thalamic action potentials relative to cortical oscillations and established correlations among phase, the nuclear location of the thalamocortical neurons and the frequency of cortical activity. The phase of thalamic action potentials depended on the exact frequency of the slow cortical oscillation both on long (minutes) and short (single wave) time scales. Faster waves were accompanied by phase advancement in both cases. Thalamocortical neurons located in different nuclei fired at significantly different phases of the slow waves but were active at similar phase of spindle oscillations. Different thalamic nuclei displayed distinct burst patterns. Bursts with higher number of action potentials displayed progressive phase advancement in a nucleus-specific manner. Thalamic neurons located along nuclear borders were characterized by mixed burst and phase properties. Our data demonstrate that the temporal relationship between cortical and thalamic activity is not fixed but displays dynamic changes during oscillatory activity. The timing depends on the precise location and exact activity of thalamocortical cells and the ongoing cortical network pattern. This variability of thalamic output and its coupling to cortical activity can enable thalamocortical neurons to actively participate in the coding and retrieval of complex cortical signals. PMID:21228169

  6. Controlling nuclear JAKs and STATs for specific gene activation by IFN{gamma}

    SciTech Connect

    Noon-Song, Ezra N.; Ahmed, Chulbul M.; Dabelic, Rea; Canton, Johnathan; Johnson, Howard M.

    2011-07-08

    Highlights: {yields} Gamma interferon (IFN{gamma}) and its receptor subunit, IFNGR1, interact with the promoter region of IFN{gamma}-associated genes along with transcription factor STAT1{alpha}. {yields} We show that activated Janus kinases pJAK2 and pJAK1 also associate with IFNGR1 in the nucleus. {yields} The activated Janus kinases are responsible for phosphorylation of tyrosine 41 on histone H3, an important epigenetic event for specific gene activation. -- Abstract: We previously showed that gamma interferon (IFN{gamma}) and its receptor subunit, IFNGR1, interacted with the promoter region of IFN{gamma}-activated genes along with transcription factor STAT1{alpha}. Recent studies have suggested that activated Janus kinases pJAK2 and pJAK1 also played a role in gene activation by phosphorylation of histone H3 on tyrosine 41. This study addresses the question of the role of activated JAKs in specific gene activation by IFN{gamma}. We carried out chromatin immunoprecipitation (ChIP) followed by PCR in IFN{gamma} treated WISH cells and showed association of pJAK1, pJAK2, IFNGR1, and STAT1 on the same DNA sequence of the IRF-1 gene promoter. The {beta}-actin gene, which is not activated by IFN{gamma}, did not show this association. The movement of activated JAK to the nucleus and the IRF-1 promoter was confirmed by the combination of nuclear fractionation, confocal microscopy and DNA precipitation analysis using the biotinylated GAS promoter. Activated JAKs in the nucleus was associated with phosphorylated tyrosine 41 on histone H3 in the region of the GAS promoter. Unphosphorylated JAK2 was found to be constitutively present in the nucleus and was capable of undergoing activation in IFN{gamma} treated cells, most likely via nuclear IFNGR1. Association of pJAK2 and IFNGR1 with histone H3 in IFN{gamma} treated cells was demonstrated by histone H3 immunoprecipitation. Unphosphorylated STAT1 protein was associated with histone H3 of untreated cells. IFN

  7. Differential activity of a tissue-specific extinguisher locus in hepatic and nonhepatic cells.

    PubMed Central

    Gourdeau, H; Peterson, T C; Fournier, R E

    1989-01-01

    Tissue-specific extinguisher 1 (Tse-1) is a genetic locus on mouse chromosome 11 that can repress expression of several liver genes in trans. This locus is clearly active in fibroblasts, as hepatoma cells retaining fibroblast chromosome 11 are extinguished for both tyrosine aminotransferase and phosphoenolpyruvate carboxykinase gene expression. To assess the activity of Tse-1 in other tissues, we transferred mouse chromosome 11 from several different cell types into rat hepatoma recipients. Tse-1 was active in nonhepatic cell lines derived from each primary germ layer, but Tse-1 activity was not apparent in hybrids between hepatoma cells and primary mouse hepatocytes. These differences in the genetic activity of murine Tse-1 were apparently heritable in cis. Images PMID:2568581

  8. Differential activity of a tissue-specific extinguisher locus in hepatic and nonhepatic cells.

    PubMed

    Gourdeau, H; Peterson, T C; Fournier, R E

    1989-05-01

    Tissue-specific extinguisher 1 (Tse-1) is a genetic locus on mouse chromosome 11 that can repress expression of several liver genes in trans. This locus is clearly active in fibroblasts, as hepatoma cells retaining fibroblast chromosome 11 are extinguished for both tyrosine aminotransferase and phosphoenolpyruvate carboxykinase gene expression. To assess the activity of Tse-1 in other tissues, we transferred mouse chromosome 11 from several different cell types into rat hepatoma recipients. Tse-1 was active in nonhepatic cell lines derived from each primary germ layer, but Tse-1 activity was not apparent in hybrids between hepatoma cells and primary mouse hepatocytes. These differences in the genetic activity of murine Tse-1 were apparently heritable in cis.

  9. Specific pollen allergen activates eosinophils of the patient with chronic allergic contact urticaria.

    PubMed

    Panaszek, B; Małolepszy, J; Kuryszko, J; Litwa, M

    1994-01-01

    The aim of the study was to evaluate the activation of eosinophils in an unique case of a young man with atopy manifested as chronic pollen contact urticaria. In order to reveal the role of eosinophils in that case, the study was performed by means of monoclonal antibodies EG2 and chemiluminescence. In addition, comparative electron microscopic study of peripheral blood and skin infiltrating eosinophils were performed for which the name ultrastructural morphometric analysis of intracytoplasmic eosinophil granules has been proposed. The results indicated, that 40% of peripheral blood eosinophils were activated spontaneously and they were more active than those in skin infiltrates. Specific pollen allergen caused activation of 100% of peripheral blood eosinophils. The study suggests presence of a systemic pattern of eosinophil activation in atopy. PMID:7487362

  10. Expression and purification of recombinant active prostate-specific antigen from Escherichia coli.

    PubMed

    Jeong, Sujin; Lee, Seong-Wook

    2007-05-01

    Human prostate-specific antigen (PSA), a 33 kDa serine protease with comprehensive homology to glandular kallikrein, is secreted from prostatic tissue into the seminal fluid and enters into the circulation. The level of PSA increases in the serum of patients with prostatic cancer and hence is widely employed as a marker of the disease status. In particular, an enzymatically active PSA that is a form cleaved at the N-terminal seven-amino-acids prosequence, APLILSR, of proPSA may play an important roll in the progression of prostate cancer. Thus, the presence of the active form would selectively discriminate the cancer from benign prostatic hyperplasia. In this study, we developed a convenient purification method for the acquisition of active PSA and proPSA. Recombinant proPSA and active PSA were expressed directly in Escherichia coli, easily and efficiently isolated from inclusion bodies, refolded, and purified. Moreover, the enzymatic activity of the recombinant active PSA was confirmed as serine protease using chromogenic chymotrypsin substrate. This purified active PSA could be further applied to scrutinize the biological or conformational characteristics of the protein and to develop specific diagnostic and/or therapeutic agents against prostate cancer.

  11. Specific quorum sensing-disrupting activity (A QSI) of thiophenones and their therapeutic potential.

    PubMed

    Yang, Qian; Scheie, Anne Aamdal; Benneche, Tore; Defoirdt, Tom

    2015-01-01

    Disease caused by antibiotic resistant pathogens is becoming a serious problem, both in human and veterinary medicine. The inhibition of quorum sensing, bacterial cell-to-cell communication, is a promising alternative strategy to control disease. In this study, we determined the quorum sensing-disrupting activity of 20 thiophenones towards the quorum sensing model bacterium V. harveyi. In order to exclude false positives, we propose a new parameter (AQSI) to describe specific quorum sensing activity. AQSI is defined as the ratio between inhibition of quorum sensing-regulated activity in a reporter strain and inhibition of the same activity when it is independent of quorum sensing. Calculation of AQSI allowed to exclude five false positives, whereas the six most active thiophenones (TF203, TF307, TF319, TF339, TF342 and TF403) inhibited quorum sensing at 0.25 μM, with AQSI higher than 10. Further, we determined the protective effect and toxicity of the thiophenones in a highly controlled gnotobiotic model system with brine shrimp larvae. There was a strong positive correlation between the specific quorum sensing-disrupting activity of the thiophenones and the protection of brine shrimp larvae against pathogenic V. harveyi. Four of the most active quorum sensing-disrupting thiophenones (TF 203, TF319, TF339 and TF342) were considered to be promising since they have a therapeutic potential of at least 10. PMID:26647822

  12. Reaction time-related activity reflecting periodic, task-specific cognitive control.

    PubMed

    Barber, Anita D; Pekar, James J; Mostofsky, Stewart H

    2016-01-01

    Reaction time (RT) is associated with increased amplitude of the Blood Oxygen-Level Dependent (BOLD) response in cognitive control regions. The current study examined whether the Primary Condition (PC) effect and RT-BOLD effect both reflect the same cognitive control processes. In addition, RT-BOLD effects were examined in two Go/No-go tasks with different demands to determine whether RT-related activity is task-dependent, reflecting the recruitment of task-specific cognitive processes. Data simulations showed that RT-related activity could be distinguished from that of the primary condition if it is mean-centered. In that case, RT-related activity reflects periodically-engaged processes rather than "time-on-task" (ToT). RT-related activity was mostly distinct from that of the primary Go contrast, particularly for the perceptual decision task. Therefore, RT effects can reflect additional cognitive processes that are not captured by the PC contrast consistent with a periodic-engagement account. RT-BOLD effects occurred in a separate set of regions for the two tasks. For the task requiring a perceptual decision, RT-related activity occurred within occipital and posterior parietal regions supporting visual attention. For the task requiring a working memory decision, RT-related activity occurred within fronto-parietal regions supporting the maintenance and retrieval of task representations. The findings suggest that RT-related activity reflects task-specific processes that are periodically-engaged, particularly during less demanding tasks. PMID:26318935

  13. A sandwich ELISA for the conformation-specific quantification of the activated form of human Bax.

    PubMed

    Teijido, Oscar; Ganesan, Yogesh Tengarai; Llanos, Raul; Peton, Ashley; Urtecho, Jean-Baptiste; Soprani, Adauri; Villamayor, Aimee; Antonsson, Bruno; Manon, Stéphen; Dejean, Laurent

    2016-03-15

    Bcl-2 family proteins are critical regulators of mitochondrial outer membrane permeabilization (MOMP), which represents the point of no return of apoptotic cell death. The exposure of the Bax N-terminus at the mitochondria reflects Bax activation; and this activated configuration of the Bax protein is associated with MOMP. N-terminal exposure can be detected using specific monoclonal and/or polyclonal antibodies, and the onset of activated Bax has extensively been used as an early marker of apoptosis. The protocols of immunoprecipitation and/or immunocytochemistry commonly used to detect activated Bax are long and tedious, and allow semiquantification of the antigen at best. The sandwich ELISA protocol we developed has a 5 ng/mL detection limit and is highly specific for the activated conformation of Bax. This ELISA allows a rapid quantification of activated human Bax in whole cells and isolated mitochondria protein extracts. These properties grant this assay the potential to further clarify the prognostic and diagnostic value of activated Bax in disorders associated with deregulated apoptotic pathways such as degenerative diseases or cancer. PMID:26748144

  14. Specific quorum sensing-disrupting activity (AQSI) of thiophenones and their therapeutic potential

    PubMed Central

    Yang, Qian; Aamdal Scheie, Anne; Benneche, Tore; Defoirdt, Tom

    2015-01-01

    Disease caused by antibiotic resistant pathogens is becoming a serious problem, both in human and veterinary medicine. The inhibition of quorum sensing, bacterial cell-to-cell communication, is a promising alternative strategy to control disease. In this study, we determined the quorum sensing-disrupting activity of 20 thiophenones towards the quorum sensing model bacterium V. harveyi. In order to exclude false positives, we propose a new parameter (AQSI) to describe specific quorum sensing activity. AQSI is defined as the ratio between inhibition of quorum sensing-regulated activity in a reporter strain and inhibition of the same activity when it is independent of quorum sensing. Calculation of AQSI allowed to exclude five false positives, whereas the six most active thiophenones (TF203, TF307, TF319, TF339, TF342 and TF403) inhibited quorum sensing at 0.25 μM, with AQSI higher than 10. Further, we determined the protective effect and toxicity of the thiophenones in a highly controlled gnotobiotic model system with brine shrimp larvae. There was a strong positive correlation between the specific quorum sensing-disrupting activity of the thiophenones and the protection of brine shrimp larvae against pathogenic V. harveyi. Four of the most active quorum sensing-disrupting thiophenones (TF 203, TF319, TF339 and TF342) were considered to be promising since they have a therapeutic potential of at least 10. PMID:26647822

  15. Specific quorum sensing-disrupting activity (A QSI) of thiophenones and their therapeutic potential.

    PubMed

    Yang, Qian; Scheie, Anne Aamdal; Benneche, Tore; Defoirdt, Tom

    2015-12-09

    Disease caused by antibiotic resistant pathogens is becoming a serious problem, both in human and veterinary medicine. The inhibition of quorum sensing, bacterial cell-to-cell communication, is a promising alternative strategy to control disease. In this study, we determined the quorum sensing-disrupting activity of 20 thiophenones towards the quorum sensing model bacterium V. harveyi. In order to exclude false positives, we propose a new parameter (AQSI) to describe specific quorum sensing activity. AQSI is defined as the ratio between inhibition of quorum sensing-regulated activity in a reporter strain and inhibition of the same activity when it is independent of quorum sensing. Calculation of AQSI allowed to exclude five false positives, whereas the six most active thiophenones (TF203, TF307, TF319, TF339, TF342 and TF403) inhibited quorum sensing at 0.25 μM, with AQSI higher than 10. Further, we determined the protective effect and toxicity of the thiophenones in a highly controlled gnotobiotic model system with brine shrimp larvae. There was a strong positive correlation between the specific quorum sensing-disrupting activity of the thiophenones and the protection of brine shrimp larvae against pathogenic V. harveyi. Four of the most active quorum sensing-disrupting thiophenones (TF 203, TF319, TF339 and TF342) were considered to be promising since they have a therapeutic potential of at least 10.

  16. Relationship between methanogenic cofactor content and maximum specific methanogenic activity of anaerobic granular sludges

    SciTech Connect

    Gorris, L.G.; de Kok, T.M.; Kroon, B.M.; van der Drift, C.; Vogels, G.D.

    1988-05-01

    In this study we investigated whether a relationship exists between the methanogenic activity and the content of specific methanogenic cofactors of granular sludges cultured on different combinations of volatile fatty acids in upflow anaerobic sludge blanket or fluidized-bed reactors. Significant correlations were measured in both cases between the contents of coenzyme F/sub 420/-2 or methanopterin and the maximum specific methanogenic activities on propionate, butyrate, and hydrogen, but not acetate. For both sludges the content of sarcinapterin appeared to be correlated with methanogenic activities on propionate, butyrate, and acetate, but not hydrogen. Similar correlations were measured with regard to the total content of coenzyme F/sub 420/-4 and F/sub 420/-5 sludges from fluidized-bed reactors. The results indicate that the contents of specific methanogenic cofactors measured in anaerobic granular sludges can be used to estimate the hydrogenotrophic or acetotrophic methanogenic potential of these sludges.

  17. Future of low specific activity molybdenum-99/technetium-99m generator.

    PubMed

    Mushtaq, A

    2012-10-01

    In last few years, the shortage of molybdenum-99 (99Mo) was felt in the developed and developing countries hospitals, where diagnostic nuclear medicine is practiced. To overcome the shortage of 99Mo various routes of its production by accelerators and reactors generating low and high specific activity products have been planned. High specific activity 99Mo obtained by fission of uranium-235 (235U) has completely dominated in the manufacturing of technetium-99m (99mTc) generators in last 3-4 decades, but due to proliferation and dirty bomb, issues non fission routes of 99Mo production are emphasized. Future of low specific activity 99Mo is discussed. PMID:22642420

  18. Catalase activity of different Candida species after exposition to specific antiserum

    PubMed Central

    Miyasaka, Natália R.S.; Unterkircher, Carmelinda S.; Shimizu, Mario T.

    2008-01-01

    Antisera were developed in rabbits after challenge with intracellular antigens of Candida albicans, C. tropicalis and C. parapsilosis. Microorganism catalase has been correlated with virulence, resistance to drugs and immunogenicity. The intracellular catalase is consistently present in strains of Candida and in this paper, the enzyme activity was analysed by PAGE after exposition to antisera. The catalases of C. albicans, C. parapsilosis and C. tropicalis were immunogenic and differed in their binding to specific antibodies raised in rabbits. Tests of cross-reactivity between different Candida species showed that when antiserum from C. albicans immunized rabbit was incubated with intracellular extracts of these three Candida species, the catalases activities were abolished. However, the antisera from C. parapsilosis or C. tropicalis immunized rabbits did not affect the catalase activity of C. albicans; the enzyme of C. albicans was inactivated only by the antiserum to the catalase of own C. albicans. The antiserum to the catalase of C. tropicalis was species-specific and did not cross-react with catalases of C. albicans and C. parapsilosis. The activities of Aspergillus niger and bovine catalases were not affected by the antiserum from any Candida immunized rabbits. This report is a preliminary study of specific antisera that react against intracellular catalase of Candida sp. and neutralize the enzymatic activity. Further study is necessary to develop species-specific antibody once differences in the susceptibility of the Candida species to commonly used antifungal drugs make identification to the species level important. PMID:24031174

  19. A chromatin activity based chemoproteomic approach reveals a transcriptional repressome for gene-specific silencing

    PubMed Central

    Liu, Cui; Yu, Yanbao; Liu, Feng; Wei, Xin; Wrobel, John A.; Gunawardena, Harsha P.; Zhou, Li; Jin, Jian; Chen, Xian

    2015-01-01

    Immune cells develop endotoxin tolerance (ET) after prolonged stimulation. ET increases the level of a repression mark H3K9me2 in the transcriptional-silent chromatin specifically associated with pro-inflammatory genes. However, it is not clear what proteins are functionally involved in this process. Here we show that a novel chromatin activity based chemoproteomic (ChaC) approach can dissect the functional chromatin protein complexes that regulate ET-associated inflammation. Using UNC0638 that binds the enzymatically active H3K9-specific methyltransferase G9a/GLP, ChaC reveals that G9a is constitutively active at a G9a-dependent mega-dalton repressome in primary endotoxin-tolerant macrophages. G9a/GLP broadly impacts the ET-specific reprogramming of the histone code landscape, chromatin remodeling, and the activities of select transcription factors. We discover that the G9a-dependent epigenetic environment promotes the transcriptional repression activity of c-Myc for gene-specific co-regulation of chronic inflammation. ChaC may be also applicable to dissect other functional protein complexes in the context of phenotypic chromatin architectures. PMID:25502336

  20. Molecular profiling of tumor-specific TH1 cells activated in vivo

    PubMed Central

    Lorvik, Kristina Berg; Haabeth, Ole Audun Werner; Clancy, Trevor; Bogen, Bjarne; Corthay, Alexandre

    2013-01-01

    The central role of tumor-specific TH1 cells in anticancer immune responses is becoming increasingly appreciated. However, little is known about how these cells are generated in vivo. Here, we used flow cytometry and gene expression microarrays to characterize the primary activation and TH1 differentiation of naïve tumor-specific CD4+ T cells in a mouse model of cancer immunosurveillance. We took advantage of T-cell receptor-transgenic mice in which CD4+ T cells recognize a tumor-specific antigen secreted by MHC class II-negative MOPC315 myeloma cells. Cancer cells were injected subcutaneously and T-cell activation was analyzed in draining lymph nodes and at the incipient tumor site 8 d later. Upon activation and migration to incipient tumor sites, tumor-specific CD4+ T cells exhibited the upregulation of 29 cell-surface molecules (CD2, CD5, CD11a, CD18, CD25, CD28, CD44, CD45, CD49d, CD51, CD54, CD69, CD71, CD83, CD86, CD90, CD95, CD102, CD122, CD153, CD166, CD200, CD249, CD254, CD274, CD279, Ly6C, MHC class I and CCR7) and the downregulation of five (CD27, CD31, CD45RB, CD62L and CD126). Activated CD4+ T cells produced interferon γ, a cytokine consistent with a TH1-polarized response, tumor necrosis factor α as well as interleukin (IL)-2, IL-3 and IL-10. The activation of naïve tumor-specific CD4+ T cells in draining lymph nodes resulted in the upregulation of 609 genes and the downregulation of 284 genes. The bioinformatic analysis of differentially expressed genes identified functional pathways related to tumor-specific TH1 cell activation. This study may represent a useful resource to guide the development of TH1-based immunotherapies against cancer. PMID:23762808

  1. Tissue-specific inhibition and recovery of esterase activities in Lumbricus terrestris experimentally exposed to chlorpyrifos.

    PubMed

    Vejares, Sandra González; Sabat, Pablo; Sanchez-Hernandez, Juan C

    2010-04-01

    Exposure and effect assessment of organophosphate (OP) pesticides generally involves the use of cholinesterase (ChE) inhibition. In earthworm, this enzyme activity is often measured in homogenates from the whole organism. Here we examine the tissue-specific response of ChE and carboxylesterase (CE) activities in Lumbricus terrestris experimentally exposed to chlorpyrifos-spiked field soils. Esterases were measured in different gut segments and in the seminal vesicles of earthworms following acute exposure (2 d) to the OP and during 35d of a recovery period. We found that inhibition of both esterase activities was dependent on the tissue. Cholinesterase activity decreased in the pharynx, crop, foregut and seminal vesicles in a concentration-dependent way, whereas CE activity (4-nitrophenyl valerate) was strongly inhibited in these tissues. Gizzard CE activity was not inhibited by the OP, even an increase of enzyme activity was evident during the recovery period. These results suggest that both esterases should be determined jointly in selected tissues of earthworms. Moreover, the high levels of gut CE activity and its inhibition and recovery dynamic following OP exposure suggest that this esterase could play an important role as an enzymatic barrier against OP uptake from the ingested contaminated soil. PMID:20045489

  2. Behavioral state-specific inhibitory postsynaptic potentials impinge on cat lumbar motoneurons during active sleep.

    PubMed

    Morales, F R; Boxer, P; Chase, M H

    1987-11-01

    High-gain intracellular records were obtained from lumbar motoneurons in intact, undrugged cats during naturally occurring states of wakefulness, quiet sleep, and active sleep. Spontaneous, discrete, inhibitory postsynaptic potentials (IPSPs) were found to impinge on lumbar motoneurons during all states of sleep and wakefulness. IPSPs which occurred during wakefulness and quiet sleep were of relatively low amplitude and had a low frequency of occurrence. During the state of active sleep there occurred a great increase in inhibitory input. This was the result of the appearance of large-amplitude IPSPs and of an increase in the frequency of low-amplitude IPSPs which were indistinguishable from those recorded during wakefulness and quiet sleep. In addition to a difference in amplitude, the time course of the large IPSPs recorded during active sleep further differentiated them from the smaller IPSPs recorded during wakefulness, quiet sleep, and active sleep; i.e., their rise-time and half-width were of longer duration and their rate-of-rise was significantly faster. We suggest that the large, active sleep-specific IPSPs reflect the activity of a group of inhibitory interneurons which are inactive during wakefulness and quiet sleep and which discharge during active sleep. These as yet unidentified interneurons would then serve as the last link in the brain stem-spinal cord inhibitory system which is responsible for producing muscle atonia during the state of active sleep. PMID:3666087

  3. Cooperative activation of tissue-specific genes by pRB and E2F1.

    PubMed

    Flowers, Stephen; Xu, Fuhua; Moran, Elizabeth

    2013-04-01

    The retinoblastoma tumor suppressor protein pRB is conventionally regarded as an inhibitor of the E2F family of transcription factors. Conversely, pRB is also recognized as an activator of tissue-specific gene expression along various lineages including osteoblastogenesis. During osteoblast differentiation, pRB directly targets Alpl and Bglap, which encode the major markers of osteogenesis alkaline phosphatase and osteocalcin. Surprisingly, p130 and repressor E2Fs were recently found to cooccupy and repress Alpl and Bglap in proliferating osteoblast precursors before differentiation. This raises the further question of whether these genes convert to E2F activation targets when differentiation begins, which would constitute a remarkable situation wherein pRB and E2F would be cotargeting genes for activation. Chromatin immunoprecipitation analysis in an osteoblast differentiation model shows that Alpl and Bglap are indeed targeted by an activator E2F, i.e., is E2F1. Promoter occupation of Alpl and Bglap by E2F1 occurs specifically during activation, and depletion of E2F1 severely impairs their induction. Mechanistically, promoter occupation by E2F1 and pRB is mutually dependent, and without this cooperative effect, activation steps previously shown to be dependent on pRB, including recruitment of RNA polymerase II, are impaired. Myocyte- and adipocyte-specific genes are also cotargeted by E2F1 and pRB during differentiation along their respective lineages. The finding that pRB and E2F1 cooperate to activate expression of tissue-specific genes is a paradigm distinct from the classical concept of pRB as an inhibitor of E2F1, but is consistent with the observed roles of these proteins in physiological models.

  4. RhoGEF Specificity Mutants Implicate RhoA as a Target for Dbs Transforming Activity

    PubMed Central

    Cheng, Li; Rossman, Kent L.; Mahon, Gwendolyn M.; Worthylake, David K.; Korus, Malgorzata; Sondek, John; Whitehead, Ian P.

    2002-01-01

    Dbs is a Rho-specific guanine nucleotide exchange factor (RhoGEF) that exhibits transforming activity when overexpressed in NIH 3T3 mouse fibroblasts. Like many RhoGEFs, the in vitro catalytic activity of Dbs is not limited to a single substrate. It can catalyze the exchange of GDP for GTP on RhoA and Cdc42, both of which are expressed in most cell types. This lack of substrate specificity, which is relatively common among members of the RhoGEF family, complicates efforts to determine the molecular basis of their transforming activity. We have recently determined crystal structures of several RhoGEFs bound to their cognate GTPases and have used these complexes to predict structural determinants dictating the specificities of coupling between RhoGEFs and GTPases. Guided by this information, we mutated Dbs to alter significantly its relative exchange activity for RhoA versus Cdc42 and show that the transformation potential of Dbs correlates with exchange on RhoA but not Cdc42. Supporting this conclusion, oncogenic Dbs activates endogenous RhoA but not endogenous Cdc42 in NIH 3T3 cells. Similarly, a competitive inhibitor that blocks RhoA activation also blocks Dbs-mediated transformation. In conclusion, this study highlights the usefulness of specificity mutants of RhoGEFs as tools to genetically dissect the multiple signaling pathways potentially activated by overexpressed or oncogenic RhoGEFs. These ideas are exemplified for Dbs, which is strongly implicated in the transformation of NIH 3T3 cells via RhoA and not Cdc42. PMID:12215546

  5. Mercury specifically induces LINE-1 activity in a human neuroblastoma cell line.

    PubMed

    Habibi, Laleh; Shokrgozar, Mohammad Ali; Tabrizi, Mina; Modarressi, Mohammad Hossein; Akrami, Seyed Mohammad

    2014-01-01

    L1 retro-elements comprise 17% of the human genome. Approximately 100 copies of these autonomous mobile elements are active in our DNA and can cause mutations, gene disruptions, and genomic instability. Therefore, human cells control the activities of L1 elements, in order to prevent their deleterious effects through different mechanisms. However, some toxic agents increase the retrotransposition activity of L1 elements in somatic cells. In order to identify specific effects of neurotoxic metals on L1 activity in neuronal cells, we studied the effects of mercury and cobalt on L1-retroelement activity by measuring levels of cellular transcription, protein expression, and genomic retrotransposition in a neuroblastoma cell line compared with the effects in three non-neuronal cell lines. Our results show that mercury increased the expression of L1 RNA, the activity of the L1 5'UTR, and L1 retrotransposition exclusively in the neuroblastoma cell line but not in non-neuronal cell lines. However, cobalt increased the expression of L1 RNA in neuroblastoma cells, HeLa cells, and wild-type human fibroblasts, and also increased the activity of the L1 5'UTR as well as the SV40 promoter in HeLa cells but not in neuroblastoma cells. Exposure to cobalt did not result in increased retrotransposition activity in HeLa cells or neuroblastoma cells. We conclude that non-toxic levels of the neurotoxic agent mercury could influence DNA by increasing L1 activities, specifically in neuronal cells, and may make these cells susceptible to neurodegeneration over time.

  6. Correlates of children's time-specific physical activity: a review of the literature.

    PubMed

    Stanley, Rebecca M; Ridley, Kate; Dollman, James

    2012-01-01

    area and a need for further investigation into time-specific physical activity behaviour so that interventions designed for these specific periods can target the important correlates.

  7. Cancer-specific binary expression system activated in mice by bacteriophage HK022 Integrase

    PubMed Central

    Elias, Amer; Spector, Itay; Sogolovsky-Bard, Ilana; Gritsenko, Natalia; Rask, Lene; Mainbakh, Yuli; Zilberstein, Yael; Yagil, Ezra; Kolot, Mikhail

    2016-01-01

    Binary systems based on site-specific recombination have been used for tumor specific transcription targeting of suicide genes in animal models. In these binary systems a site specific recombinase or integrase that is expressed from a tumor specific promoter drives tumor specific expression of a cytotoxic gene. In the present study we developed a new cancer specific binary expression system activated by the Integrase (Int) of the lambdoid phage HK022. We demonstrate the validity of this system by the specific expression of a luciferase (luc) reporter in human embryonic kidney 293T (HEK293T) cells and in a lung cancer mouse model. Due to the absence viral vectors and of cytotoxicity the Int based binary system offers advantages over previously described counterparts and may therefore be developed into a safer cancer cell killing system. PMID:27117628

  8. 2,4-Dichlorophenol hydroxylase for chlorophenol removal: Substrate specificity and catalytic activity.

    PubMed

    Ren, Hejun; Li, Qingchao; Zhan, Yang; Fang, Xuexun; Yu, Dahai

    2016-01-01

    Chlorophenols (CPs) are common environmental pollutants. As such, different treatments have been assessed to facilitate their removal. In this study, 2,4-dichlorophenol (2,4-DCP) hydroxylase was used to systematically investigate the activity and removal ability of 19CP congeners at 25 and 0 °C. Results demonstrated that 2,4-DCP hydroxylase exhibited a broad substrate specificity to CPs. The activities of 2,4-DCP hydroxylase against specific CP congeners, including 3-CP, 2,3,6-trichlorophenol, 2-CP, and 2,3-DCP, were higher than those against 2,4-DCP, which is the preferred substrate of previously reported 2,4-DCP hydroxylase. To verify whether cofactors are necessary to promote hydroxylase activity against CP congeners, we added FAD and found that the added FAD induced a 1.33-fold to 5.13-fold significant increase in hydroxylase activity against different CP congeners. The metabolic pathways of the CP degradation in the enzymatic hydroxylation step were preliminarily proposed on the basis of the analyses of the enzymatic activities against 19CP congeners. We found that the high activity and removal rate of 2,4-DCP hydroxylase against CPs at 0 °C enhance the low-temperature-adaptability of this enzyme to the CP congeners; as such, the proposed removal process may be applied to biochemical, bioremediation, and industrial processes, particularly in cold environments.

  9. Cardiac-Specific YAP Activation Improves Cardiac Function and Survival in an Experimental Murine MI Model

    PubMed Central

    Lin, Zhiqiang; von Gise, Alexander; Zhou, Pingzhu; Gu, Fei; Ma, Qing; Jiang, Jiangming; Yau, Allan L.; Buck, Jessica N.; Gouin, Katryna A.; van Gorp, Pim R. R.; Zhou, Bin; Chen, Jinghai; Seidman, Jonathan G.; Wang, Da-zhi; Pu, William T.

    2014-01-01

    Rationale Yes-Associated Protein (YAP), the terminal effector of the Hippo signaling pathway, is crucial for regulating embryonic cardiomyocyte (CM) proliferation. Objective We hypothesized that YAP activation after myocardial infarction would preserve cardiac function and improve survival. Methods and Results We used a cardiac-specific, inducible expression system to activate YAP in adult mouse heart. Activation of YAP in adult heart promoted CM proliferation and did not deleteriously affect heart function. Furthermore, YAP activation after myocardial infarction (MI) preserved heart function and reduced infarct size. Using adeno-associated virus subtype 9 (AAV9) as a delivery vector, we expressed human YAP in the adult murine myocardium immediately after MI. We found that AAV9:hYAP significantly improved cardiac function and mouse survival. AAV9:hYAP did not exert its salutary effects by reducing CM apoptosis. Rather, AAV9:hYAP stimulated adult CM proliferation. Gene expression profiling indicated that AAV9:hYAP stimulated expression of cell cycle genes and promoted a less mature cardiac gene expression signature. Conclusions Cardiac specific YAP activation after MI mitigated myocardial injury, improved cardiac function, and enhanced survival. These findings suggest that therapeutic activation of YAP or its downstream targets, potentially through AAV-mediated gene therapy, may be a strategy to improve outcome after MI. PMID:24833660

  10. Specificity of archaeal caspase activity in the extreme halophile Haloferax volcanii.

    PubMed

    Seth-Pasricha, Mansha; Bidle, Kelly A; Bidle, Kay D

    2013-04-01

    Caspase-like proteases are key initiators and executioners of programmed cell death (PCD), which is initiated by environmental stimuli and manifests in organisms ranging from unicellular microbes to higher eukaryotes. Archaea had been absent from the caspase inheritance discussion due to a lack of gene homologues. We recently demonstrated extremely high, basal caspase-like catalytic activity in the model haloarcheon, Haloferax volcanii, which was linked to the cellular stress response and was widespread among diverse Archaea. Here, we rigorously tested the catalytic specificity of the observed archaeal caspase-like activities using hydrolytic assays with a diverse suite of protease substrates and inhibitors compared with known model serine and cysteine proteases (trypsin, cathepsin, papain, and human caspase-8). Our experiments demonstrate that exponentially growing H. volcanii possesses a highly specific caspase-like activity that most closely resembles caspase-4, is preferentially inhibited by the pancaspase inhibitor, zVAD-FMK, and has no crossreactivity with other known protease families. Our findings firmly root the extremely high levels of caspase-like activity as the dominant proteolytic activity in this extreme haloarcheaon, thereby providing further support for housekeeping functions in Haloarchaea. Given the deep archaeal roots of eukaryotes, we suggest that this activity served as a foundation for stress pathways in higher organisms.

  11. Site-specific phosphorylation and microtubule dynamics control Pyrin inflammasome activation.

    PubMed

    Gao, Wenqing; Yang, Jieling; Liu, Wang; Wang, Yupeng; Shao, Feng

    2016-08-16

    Pyrin, encoded by the MEFV gene, is best known for its gain-of-function mutations causing familial Mediterranean fever (FMF), an autoinflammatory disease. Pyrin forms a caspase-1-activating inflammasome in response to inactivating modifications of Rho GTPases by various bacterial toxins or effectors. Pyrin-mediated innate immunity is unique in that it senses bacterial virulence rather than microbial molecules, but its mechanism of activation is unknown. Here we show that Pyrin was phosphorylated in bone marrow-derived macrophages and dendritic cells. We identified Ser-205 and Ser-241 in mouse Pyrin whose phosphorylation resulted in inhibitory binding by cellular 14-3-3 proteins. The two serines underwent dephosphorylation upon toxin stimulation or bacterial infection, triggering 14-3-3 dissociation, which correlated with Pyrin inflammasome activation. We developed antibodies specific for phosphorylated Ser-205 and Ser-241, which confirmed the stimuli-induced dephosphorylation of endogenous Pyrin. Mutational analyses indicated that both phosphorylation and signal-induced dephosphorylation of Ser-205/241 are important for Pyrin activation. Moreover, microtubule drugs, including colchicine, commonly used to treat FMF, effectively blocked activation of the Pyrin inflammasome. These drugs did not affect Pyrin dephosphorylation and 14-3-3 dissociation but inhibited Pyrin-mediated apoptosis-associated Speck-like protein containing CARD (ASC) aggregation. Our study reveals that site-specific (de)phosphorylation and microtubule dynamics critically control Pyrin inflammasome activation, illustrating a fine and complex mechanism in cytosolic immunity. PMID:27482109

  12. Tissue specificity of enhancer and promoter activities of a HERV-K(HML-2) LTR.

    PubMed

    Ruda, V M; Akopov, S B; Trubetskoy, D O; Manuylov, N L; Vetchinova, A S; Zavalova, L L; Nikolaev, L G; Sverdlov, E D

    2004-08-01

    Transient expression of a luciferase reporter gene was used to evaluate tissue-specific promoter and enhancer activities of a solitary extraviral long terminal repeat (LTR) of the human endogenous retrovirus K (HERV-K) in several human and CHO cell lines. The promoter activity of the LTR varied from virtually not detectable (GS and Jurkat cells) to as high as that of the SV40 early promoter (Tera-1 human testicular embryonal carcinoma cells). The negative regulatory element (NRE) of the LTR retained its activity in all cell lines where the LTR could act as a promoter, and was also capable of binding host cell nuclear proteins. The enhancer activity of the LTR towards the SV40 early promoter was detected only in Tera-1 cells and was not observed in a closely related human testicular embryonal carcinoma cell line of different origin, NT2/D1. A comparison of proteins bound to central part of the LTR in nuclear extracts from Tera-1 and NT2/D1 by electrophoretic mobility shift assay revealed striking differences that could be determined by different LTR enhancer activities in these cells. Tissue specificity of the SV40 early promoter activity was also revealed.

  13. Orexin-dependent activation of layer VIb enhances cortical network activity and integration of non-specific thalamocortical inputs.

    PubMed

    Hay, Y Audrey; Andjelic, Sofija; Badr, Sammy; Lambolez, Bertrand

    2015-11-01

    Neocortical layer VI is critically involved in thalamocortical activity changes during the sleep/wake cycle. It receives dense projections from thalamic nuclei sensitive to the wake-promoting neuropeptides orexins, and its deepest part, layer VIb, is the only cortical lamina reactive to orexins. This convergence of wake-promoting inputs prompted us to investigate how layer VIb can modulate cortical arousal, using patch-clamp recordings and optogenetics in rat brain slices. We found that the majority of layer VIb neurons were excited by nicotinic agonists and orexin through the activation of nicotinic receptors containing α4-α5-β2 subunits and OX2 receptor, respectively. Specific effects of orexin on layer VIb neurons were potentiated by low nicotine concentrations and we used this paradigm to explore their intracortical projections. Co-application of nicotine and orexin increased the frequency of excitatory post-synaptic currents in the ipsilateral cortex, with maximal effect in infragranular layers and minimal effect in layer IV, as well as in the contralateral cortex. The ability of layer VIb to relay thalamocortical inputs was tested using photostimulation of channelrhodopsin-expressing fibers from the orexin-sensitive rhomboid nucleus in the parietal cortex. Photostimulation induced robust excitatory currents in layer VIa neurons that were not pre-synaptically modulated by orexin, but exhibited a delayed, orexin-dependent, component. Activation of layer VIb by orexin enhanced the reliability and spike-timing precision of layer VIa responses to rhomboid inputs. These results indicate that layer VIb acts as an orexin-gated excitatory feedforward loop that potentiates thalamocortical arousal.

  14. Nitrite-Specific Active Transport System of the Cyanobacterium Synechococcus sp. Strain PCC 7942

    PubMed Central

    Maeda, Shin-ichi; Okamura, Masato; Kobayashi, Masaki; Omata, Tatsuo

    1998-01-01

    Studies on the nitrite uptake capability of a mutant of Synechococcus sp. strain PCC 7942 lacking the ATP-binding cassette-type nitrate-nitrite-bispecific transporter revealed the occurrence of a nitrite-specific active transport system with an apparent Km (NO2−) of about 20 μM. Similar to the nitrate-nitrite-bispecific transporter, the nitrite-specific transporter was reversibly inhibited by ammonium in the medium. PMID:9852027

  15. MAPK specificity in the yeast pheromone response independent of transcriptional activation.

    PubMed

    Breitkreutz, A; Boucher, L; Tyers, M

    2001-08-21

    The mechanisms whereby different external cues stimulate the same mitogen-activated protein kinase (MAPK) cascade, yet trigger an appropriately distinct biological response, epitomize the conundrum of specificity in cell signaling. In yeast, shared upstream components of the mating pheromone and filamentous growth pathways activate two related MAPKs, Fus3 and Kss1, which in turn regulate programs of gene expression via the transcription factor Ste12. As fus3, but not kss1, strains are impaired for mating, Fus3 exhibits specificity for the pheromone response. To account for this specificity, it has been suggested that Fus3 physically occludes Kss1 from pheromone-activated signaling complexes, which are formed on the scaffold protein Ste5. However, we find that genome-wide expression profiles of pheromone-treated wild-type, fus3, and kss1 deletion strains are highly correlated for all induced genes and, further, that two catalytically inactive versions of Fus3 fail to abrogate the pheromone-induced transcriptional response. Consistently, Fus3 and Kss1 kinase activity is induced to an equivalent extent in pheromone-treated cells. In contrast, both in vivo and in an in vitro-reconstituted MAPK system, Fus3, but not Kss1, exhibits strong substrate selectivity toward Far1, a bifunctional protein required for polarization and G(1) arrest. This effect accounts for the failure to repress G(1)-S specific transcription in fus3 strains and, in part, explains the mating defect of such strains. MAPK specificity in the pheromone response evidently occurs primarily at the substrate level, as opposed to specific kinase activation by dedicated signaling complexes. PMID:11525741

  16. Electrochemical assay of active prostate-specific antigen (PSA) using ferrocene-functionalized peptide probes

    SciTech Connect

    Zhao, Ning; He, Yuqing; Mao, Xun; Sun, Yuhan; Zhang, Xibao; Li, Chen-Zhong; Lin, Yuehe; Liu, Guodong

    2010-03-24

    This paper presents a novel approach to electrochemically determine enzymatically active PSA using ferrocene-functionalized helix peptide (CHSSLKQK). The principle of electrochemical measurement is based on the specific proteolytic cleavage events of the FC-peptide on the gold electrode surface in the presence of PSA, resulting the change of the current signal of the electrode. The percentage of the decreased current is linear with the concentration of active PSA at the range of 0.5-40 ng/mL with a detection limit of 0.2 ng/mL. The direct transduction of peptide cleavage events into an electrical signal provides a simple, sensitive method for detecting the enzymatic activity of PSA and determining the active PSA concentration.

  17. Transient activation of specific neurons in mice by selective expression of the capsaicin receptor

    PubMed Central

    Güler, Ali D.; Rainwater, Aundrea; Parker, Jones G.; Jones, Graham L.; Argilli, Emanuela; Arenkiel, Benjamin R.; Ehlers, Michael D.; Bonci, Antonello; Zweifel, Larry s.; Palmiter, Richard D.

    2013-01-01

    The ability to control the electrical activity of a neuronal subtype is a valuable tool in deciphering the role of discreet cell populations in complex neural circuits. Recent techniques that allow remote control of neurons are either labor intensive and invasive or indirectly coupled to neural electrical potential with low temporal resolution. Here we show the rapid, reversible and direct activation of genetically identified neuronal subpopulations by generating two inducible transgenic mouse models. Confined expression of the capsaicin receptor, TRPV1, allows cell-specific activation after peripheral or oral delivery of ligand in freely moving mice. Capsaicin-induced activation of dopaminergic or serotonergic neurons reversibly alters both physiological and behavioural responses within minutes, and lasts ~10 min. These models showcase a robust and remotely controllable genetic tool that modulates a distinct cell population without the need for invasive and labour-intensive approaches. PMID:22434189

  18. Elimination of dendritic spines with long-term memory is specific to active circuits

    PubMed Central

    Sanders, Jeff; Cowansage, Kiriana; Baumgärtel, Karsten; Mayford, Mark

    2012-01-01

    Structural changes in brain circuits active during learning are thought to be important for long-term memory storage. If these changes support long-term information storage they might be expected to be present at distant timepoints after learning, as well as specific to the circuit activated with learning, and sensitive to the contingencies of the behavioral paradigm. Here, we show such changes in the hippocampus as a result of contextual fear conditioning. There were significantly fewer spines specifically on active neurons of fear-conditioned mice. This spine loss did not occur in homecage mice or in mice exposed to the training context alone. Mice exposed to unpaired shocks showed a generalized reduction in spines. These learning-related changes in spine density could reflect a direct mechanism of encoding or alternately could reflect a compensatory adaptation to previously described enhancement in transmission due to glutamate receptor insertion. PMID:22956846

  19. Tonotopic and Field-Specific Representation of Long-Lasting Sustained Activity in Rat Auditory Cortex

    PubMed Central

    Shiramatsu, Tomoyo I.; Noda, Takahiro; Akutsu, Kan; Takahashi, Hirokazu

    2016-01-01

    Cortical information processing of the onset, offset, and continuous plateau of an acoustic stimulus should play an important role in acoustic object perception. To date, transient activities responding to the onset and offset of a sound have been well investigated and cortical subfields and topographic representation in these subfields, such as place code of sound frequency, have been well characterized. However, whether these cortical subfields with tonotopic representation are inherited in the sustained activities that follow transient activities and persist during the presentation of a long-lasting stimulus remains unknown, because sustained activities do not exhibit distinct, reproducible, and time-locked responses in their amplitude to be characterized by grand averaging. To address this gap in understanding, we attempted to decode sound information from densely mapped sustained activities in the rat auditory cortex using a sparse parameter estimation method called sparse logistic regression (SLR), and investigated whether and how these activities represent sound information. A microelectrode array with a grid of 10 × 10 recording sites within an area of 4.0 mm × 4.0 mm was implanted in the fourth layer of the auditory cortex in rats under isoflurane anesthesia. Sustained activities in response to long-lasting constant pure tones were recorded. SLR then was applied to discriminate the sound-induced band-specific power or phase-locking value from those of spontaneous activities. The highest decoding performance was achieved in the high-gamma band, indicating that cortical inhibitory interneurons may contribute to the sparse tonotopic representation in sustained activities by mediating synchronous activities. The estimated parameter in the SLR decoding revealed that the informative recording site had a characteristic frequency close to the test frequency. In addition, decoding of the four test frequencies demonstrated that the decoding performance of the SLR

  20. Tonotopic and Field-Specific Representation of Long-Lasting Sustained Activity in Rat Auditory Cortex.

    PubMed

    Shiramatsu, Tomoyo I; Noda, Takahiro; Akutsu, Kan; Takahashi, Hirokazu

    2016-01-01

    Cortical information processing of the onset, offset, and continuous plateau of an acoustic stimulus should play an important role in acoustic object perception. To date, transient activities responding to the onset and offset of a sound have been well investigated and cortical subfields and topographic representation in these subfields, such as place code of sound frequency, have been well characterized. However, whether these cortical subfields with tonotopic representation are inherited in the sustained activities that follow transient activities and persist during the presentation of a long-lasting stimulus remains unknown, because sustained activities do not exhibit distinct, reproducible, and time-locked responses in their amplitude to be characterized by grand averaging. To address this gap in understanding, we attempted to decode sound information from densely mapped sustained activities in the rat auditory cortex using a sparse parameter estimation method called sparse logistic regression (SLR), and investigated whether and how these activities represent sound information. A microelectrode array with a grid of 10 × 10 recording sites within an area of 4.0 mm × 4.0 mm was implanted in the fourth layer of the auditory cortex in rats under isoflurane anesthesia. Sustained activities in response to long-lasting constant pure tones were recorded. SLR then was applied to discriminate the sound-induced band-specific power or phase-locking value from those of spontaneous activities. The highest decoding performance was achieved in the high-gamma band, indicating that cortical inhibitory interneurons may contribute to the sparse tonotopic representation in sustained activities by mediating synchronous activities. The estimated parameter in the SLR decoding revealed that the informative recording site had a characteristic frequency close to the test frequency. In addition, decoding of the four test frequencies demonstrated that the decoding performance of the SLR

  1. Meditation-specific prefrontal cortical activation during acem meditation: an fMRI study.

    PubMed

    Davanger, Svend; Ellingsen, Oyvind; Holen, Are; Hugdahl, Kenneth

    2010-08-01

    Some of the most popular meditation practices emphasize a relaxed focus of attention in which thoughts, images, sensations, and emotions may emerge and pass freely without actively controlling or pursuing them. Several recent studies show that meditation activates frontal brain areas associated with attention focusing and physical relaxation. The objective of the present study was to assess whether brain activation during relaxed focusing on a meditation sound could be distinguished from similar, concentrative control tasks. Brain activation was measured with functional magnetic resonance imaging (fMRI) in experienced practitioners of Acem meditation. Bilateral areas of the inferior frontal gyrus (BA47) were significantly more activated during repetition of a meditation sound than during concentrative meditation-like cognitive tasks. Meditation-specific brain activation did not habituate over time, but increased in strength with continuous meditation bouts. These observations suggest that meditation with a relaxed focus of attention may activate distinct areas of the prefrontal cortex, with implications for the understanding of neurobiological correlates of meditation.

  2. Puromycin insensitive leucyl-specific aminopeptidase (PILSAP) affects RhoA activation in endothelial cells.

    PubMed

    Suzuki, Takahiro; Abe, Mayumi; Miyashita, Hiroki; Kobayashi, Toshimitsu; Sato, Yasufumi

    2007-06-01

    Puromycin insensitive leucyl-specific aminopeptidase (PILSAP) expressed in endothelial cells (ECs) plays an important role in angiogenesis due to its involvement in migration, proliferation and network formation. Here we examined the biological function of PILSAP with respect to EC morphogenesis and the related intracellular signaling for this process. When mouse endothelial MSS31 cells were cultured, a dominant negative PILSAP mutant converted cell shape to disk-like morphology, blocked stress fiber formation, and augmented membrane ruffling in random directions. These phenotypic changes led us to test whether PILSAP affected activities of Rho family small G-proteins. Abrogation of PILSAP enzymatic activity or its expression attenuated RhoA but not Rac1 activation during cell adhesion. This attenuation of RhoA activation was also evident when G-protein coupled receptors such as proteinase-activated receptor or lysophosphatidic acid receptor were activated in ECs. These results indicate that PILSAP affects RhoA activation and that influences the proper function of ECs.

  3. The length of a lantibiotic hinge region has profound influence on antimicrobial activity and host specificity.

    PubMed

    Zhou, Liang; van Heel, Auke J; Kuipers, Oscar P

    2015-01-01

    Lantibiotics are ribosomally synthesized (methyl)lanthionine containing peptides which can efficiently inhibit the growth of Gram-positive bacteria. As lantibiotics kill bacteria efficiently and resistance to them is difficult to be obtained, they have the potential to be used in many applications, e.g., in pharmaceutical industry or food industry. Nisin can inhibit the growth of Gram-positive bacteria by binding to lipid II and by making pores in their membrane. The C-terminal part of nisin is known to play an important role during translocation over the membrane and forming pore complexes. However, as the thickness of bacterial membranes varies between different species and environmental conditions, this property could have an influence on the pore forming activity of nisin. To investigate this, the so-called "hinge region" of nisin (residues NMK) was engineered to vary from one to six amino acid residues and specific activity against different indicators was compared. Antimicrobial activity in liquid culture assays showed that wild type nisin is most active, while truncation of the hinge region dramatically reduced the activity of the peptide. However, one or two amino acids extensions showed only slightly reduced activity against most indicator strains. Notably, some variants (+2, +1, -1, -2) exhibited higher antimicrobial activity than nisin in agar well diffusion assays against Lactococcus lactis MG1363, Listeria monocytogenes, Enterococcus faecalis VE14089, Bacillus sporothermodurans IC4 and Bacillus cereus 4153 at certain temperatures.

  4. Prefusion F-specific antibodies determine the magnitude of RSV neutralizing activity in human sera.

    PubMed

    Ngwuta, Joan O; Chen, Man; Modjarrad, Kayvon; Joyce, M Gordon; Kanekiyo, Masaru; Kumar, Azad; Yassine, Hadi M; Moin, Syed M; Killikelly, April M; Chuang, Gwo-Yu; Druz, Aliaksandr; Georgiev, Ivelin S; Rundlet, Emily J; Sastry, Mallika; Stewart-Jones, Guillaume B E; Yang, Yongping; Zhang, Baoshan; Nason, Martha C; Capella, Cristina; Peeples, Mark E; Ledgerwood, Julie E; McLellan, Jason S; Kwong, Peter D; Graham, Barney S

    2015-10-14

    Respiratory syncytial virus (RSV) is estimated to claim more lives among infants <1 year old than any other single pathogen, except malaria, and poses a substantial global health burden. Viral entry is mediated by a type I fusion glycoprotein (F) that transitions from a metastable prefusion (pre-F) to a stable postfusion (post-F) trimer. A highly neutralization-sensitive epitope, antigenic site Ø, is found only on pre-F. We determined what fraction of neutralizing (NT) activity in human sera is dependent on antibodies specific for antigenic site Ø or other antigenic sites on F in healthy subjects from ages 7 to 93 years. Adsorption of individual sera with stabilized pre-F protein removed >90% of NT activity and depleted binding antibodies to both F conformations. In contrast, adsorption with post-F removed ~30% of NT activity, and binding antibodies to pre-F were retained. These findings were consistent across all age groups. Protein competition neutralization assays with pre-F mutants in which sites Ø or II were altered to knock out binding of antibodies to the corresponding sites showed that these sites accounted for ~35 and <10% of NT activity, respectively. Binding competition assays with monoclonal antibodies (mAbs) indicated that the amount of site Ø-specific antibodies correlated with NT activity, whereas the magnitude of binding competed by site II mAbs did not correlate with neutralization. Our results indicate that RSV NT activity in human sera is primarily derived from pre-F-specific antibodies, and therefore, inducing or boosting NT activity by vaccination will be facilitated by using pre-F antigens that preserve site Ø.

  5. Prefusion F–specific antibodies determine the magnitude of RSV neutralizing activity in human sera

    PubMed Central

    Ngwuta, Joan O.; Chen, Man; Modjarrad, Kayvon; Joyce, M. Gordon; Kanekiyo, Masaru; Kumar, Azad; Yassine, Hadi M.; Moin, Syed M.; Killikelly, April M.; Chuang, Gwo-Yu; Druz, Aliaksandr; Georgiev, Ivelin S.; Rundlet, Emily J.; Sastry, Mallika; Stewart-Jones, Guillaume B. E.; Yang, Yongping; Zhang, Baoshan; Nason, Martha C.; Capella, Cristina; Peeples, Mark E.; Ledgerwood, Julie E.; McLellan, Jason S.; Kwong, Peter D.; Graham, Barney S.

    2015-01-01

    Respiratory syncytial virus (RSV) is estimated to claim more lives among infants <1 year old than any other single pathogen, except malaria, and poses a substantial global health burden. Viral entry is mediated by a type I fusion glycoprotein (F) that transitions from a metastable prefusion (pre-F) to a stable postfusion (post-F) trimer. A highly neutralization-sensitive epitope, antigenic site Ø, is found only on pre-F. We determined what fraction of neutralizing (NT) activity in human sera is dependent on antibodies specific for antigenic site Ø or other antigenic sites on F in healthy subjects from ages 7 to 93 years. Adsorption of individual sera with stabilized pre-F protein removed >90% of NT activity and depleted binding antibodies to both F conformations. In contrast, adsorption with post-F removed ~30% of NT activity, and binding antibodies to pre-F were retained. These findings were consistent across all age groups. Protein competition neutralization assays with pre-F mutants in which sites Ø or II were altered to knock out binding of antibodies to the corresponding sites showed that these sites accounted for ~35 and <10% of NT activity, respectively. Binding competition assays with monoclonal antibodies (mAbs) indicated that the amount of site Ø–specific antibodies correlated with NT activity, whereas the magnitude of binding competed by site II mAbs did not correlate with neutralization. Our results indicate that RSV NT activity in human sera is primarily derived from pre-F–specific antibodies, and therefore, inducing or boosting NT activity by vaccination will be facilitated by using pre-F antigens that preserve site Ø. PMID:26468324

  6. The calculation of surface orbital energies for specific types of active sites on dispersed metal catalysts

    SciTech Connect

    Augustine, R.L.; Lahanas, K.M.; Cole, F.

    1992-11-01

    An angular overlap calculation has been used to determine the s, p, and d orbital energy levels of the different types of surface sites present on dispersed metal catalysts. These data can permit a Frontier Molecular Orbital treatment of specific site activities as long as the surface orbital availability for overlap with adsorbed substrates is considered along with its energy value and symmetry.

  7. The calculation of surface orbital energies for specific types of active sites on dispersed metal catalysts

    SciTech Connect

    Augustine, R.L.; Lahanas, K.M.; Cole, F.

    1992-01-01

    An angular overlap calculation has been used to determine the s, p, and d orbital energy levels of the different types of surface sites present on dispersed metal catalysts. These data can permit a Frontier Molecular Orbital treatment of specific site activities as long as the surface orbital availability for overlap with adsorbed substrates is considered along with its energy value and symmetry.

  8. Are There Gender-Specific Risk Factors for Suicidal Activity among Patients with Schizophrenia and Depression?

    ERIC Educational Resources Information Center

    Kaplan, Kalman J.; Harrow, Martin; Faull, Robert N.

    2012-01-01

    Are there gender-specific risk factors for suicidal activity among patients with schizophrenia and depression? A total of 74 schizophrenia patients (51 men, 23 women) and 77 unipolar nonpsychotic depressed patients (26 men, 51 women) from the Chicago Follow-up Study were studied prospectively at 2 years posthospitalization and again at 7.5 years.…

  9. Specific and Nonspecific Neural Activity during Selective Processing of Visual Representations in Working Memory

    ERIC Educational Resources Information Center

    Oh, Hwamee; Leung, Hoi-Chung

    2010-01-01

    In this fMRI study, we investigated prefrontal cortex (PFC) and visual association regions during selective information processing. We recorded behavioral responses and neural activity during a delayed recognition task with a cue presented during the delay period. A specific cue ("Face" or "Scene") was used to indicate which one of the two…

  10. Context Fear Learning Specifically Activates Distinct Populations of Neurons in Amygdala and Hypothalamus

    ERIC Educational Resources Information Center

    Trogrlic, Lidia; Wilson, Yvette M.; Newman, Andrew G.; Murphy, Mark

    2011-01-01

    The identity and distribution of neurons that are involved in any learning or memory event is not known. In previous studies, we identified a discrete population of neurons in the lateral amygdala that show learning-specific activation of a c-"fos"-regulated transgene following context fear conditioning. Here, we have extended these studies to…

  11. Transportation impact analysis for the shipment of low specific activity nitric acid. Revisison 1

    SciTech Connect

    Green, J.R.

    1995-05-16

    This is in support of the Plutonium-Uranium Extraction (PUREX) Facility Low Specific Activity (LSA) Nitric Acid Shipment Environmental Assessment. It analyzes potential toxicological and radiological risks associated with transportation of PUREX Facility LSA Nitric Acid from the Hanford Site to Portsmouth VA, Baltimore MD, and Port Elizabeth NJ.

  12. Context-Specific Associations of Physical Activity and Sedentary Behavior With Cognition in Children

    PubMed Central

    Aggio, Daniel; Smith, Lee; Fisher, Abigail; Hamer, Mark

    2016-01-01

    In the present study, we investigated how overall and specific domains of physical activity and sedentary behavior at the age of 7 years were associated with cognition at the age of 11 years in 8,462 children from the Millennium Cohort Study. Data were collected from 2001 to 2013. Participation in domains of physical activity and sedentary behavior at 7 years of age were reported. Activity levels were also measured objectively. Cognition was assessed using the British Ability Scales. General linear models were used to assess longitudinal associations of physical activity and sedentary behavior, measured both objectively and via self-report, with cognition. Analyses were adjusted for prespecified covariates. Sports/physical activity club attendance (B = 0.6, 95% confidence interval (CI): 0.2, 1.1), doing homework (B = 0.5, 95% CI: 0.0, 0.9), and objectively measured sedentary time (B = 0.8, 95% CI: 0.1, 1.4) at age 7 years were positively associated with cognition at age 11 years in final the models. Television viewing was negatively associated with cognition (B = −1.7, 95% CI: −2.4, −1.0), although the association was attenuated to the null after adjustments for baseline cognition. Objectively measured light physical activity was inversely associated with cognition (B = −0.7, 95% CI: −1.3, −0.1). Moderate-to-vigorous physical activity was also inversely associated with cognition in girls only (B = −1.1, 95% CI: −2.0, −0.3). Associations of physical activity and sedentary behavior with cognition appear to be context-specific in young people. PMID:27226249

  13. Context-Specific Associations of Physical Activity and Sedentary Behavior With Cognition in Children.

    PubMed

    Aggio, Daniel; Smith, Lee; Fisher, Abigail; Hamer, Mark

    2016-06-15

    In the present study, we investigated how overall and specific domains of physical activity and sedentary behavior at the age of 7 years were associated with cognition at the age of 11 years in 8,462 children from the Millennium Cohort Study. Data were collected from 2001 to 2013. Participation in domains of physical activity and sedentary behavior at 7 years of age were reported. Activity levels were also measured objectively. Cognition was assessed using the British Ability Scales. General linear models were used to assess longitudinal associations of physical activity and sedentary behavior, measured both objectively and via self-report, with cognition. Analyses were adjusted for prespecified covariates. Sports/physical activity club attendance (B = 0.6, 95% confidence interval (CI): 0.2, 1.1), doing homework (B = 0.5, 95% CI: 0.0, 0.9), and objectively measured sedentary time (B = 0.8, 95% CI: 0.1, 1.4) at age 7 years were positively associated with cognition at age 11 years in final the models. Television viewing was negatively associated with cognition (B = -1.7, 95% CI: -2.4, -1.0), although the association was attenuated to the null after adjustments for baseline cognition. Objectively measured light physical activity was inversely associated with cognition (B = -0.7, 95% CI: -1.3, -0.1). Moderate-to-vigorous physical activity was also inversely associated with cognition in girls only (B = -1.1, 95% CI: -2.0, -0.3). Associations of physical activity and sedentary behavior with cognition appear to be context-specific in young people. PMID:27226249

  14. Specific induction of endogenous viral restriction factors using CRISPR/Cas-derived transcriptional activators

    PubMed Central

    Bogerd, Hal P.; Kornepati, Anand V. R.; Marshall, Joy B.; Kennedy, Edward M.; Cullen, Bryan R.

    2015-01-01

    Whereas several mammalian proteins can restrict the replication of HIV-1 and other viruses, these are often not expressed in relevant target cells. A potential method to inhibit viral replication might therefore be to use synthetic transcription factors to induce restriction factor expression. In particular, mutants of the RNA-guided DNA binding protein Cas9 that have lost their DNA cleavage activity could be used to recruit transcription activation domains to specific promoters. However, initial experiments revealed only weak activation unless multiple promoter-specific single guide RNAs (sgRNAs) were used. Recently, the recruitment of multiple transcription activation domains by a single sgRNA, modified to contain MS2-derived stem loops that recruit fusion proteins consisting of the MS2 coat protein linked to transcription activation domains, was reported to induce otherwise silent cellular genes. Here, we demonstrate that such “synergistic activation mediators” can induce the expression of two restriction factors, APOBEC3G (A3G) and APOBEC3B (A3B), in human cells that normally lack these proteins. We observed modest activation of endogenous A3G or A3B expression using single sgRNAs but high expression when two sgRNAs were used. Whereas the induced A3G and A3B proteins both blocked infection by an HIV-1 variant lacking a functional vif gene by inducing extensive dC-to-dU editing, only the induced A3B protein inhibited wild-type HIV-1. These data demonstrate that Cas9-derived transcriptional activators have the potential to be used for screens for endogenous genes that affect virus replication and raise the possibility that synthetic transcription factors might prove clinically useful if efficient delivery mechanisms could be developed. PMID:26668372

  15. Differential Neural Activity during Search of Specific and General Autobiographical Memories elicited by Musical Cues

    PubMed Central

    Ford, Jaclyn Hennessey; Addis, Donna Rose; Giovanello, Kelly S.

    2011-01-01

    Previous neuroimaging studies that have examined autobiographical memory specificity have utilized retrieval cues associated with prior searches of the event, potentially changing the retrieval processes being investigated. In the current study, musical cues were used to naturally elicit memories from multiple levels of specificity (i.e., lifetime period, general event, and event-specific). Sixteen young adults participated in a neuroimaging study in which they retrieved autobiographical memories associated with musical cues. These musical cues led to the retrieval of highly emotional memories that had low levels of prior retrieval. Retrieval of all autobiographical memory levels was associated with activity in regions in the autobiographical memory network, specifically the ventromedial prefrontal cortex, posterior cingulate, and right medial temporal lobe. Owing to the use of music, memories from varying levels of specificity were retrieved, allowing for comparison of event memory and abstract personal knowledge, as well as comparison of specific and general event memory. Dorsolateral and dorsomedial prefrontal regions were engaged during event retrieval relative to personal knowledge retrieval, and retrieval of specific event memories was associated with increased activity in the bilateral medial temporal lobe and dorsomedial prefrontal cortex relative to retrieval of general event memories. These results suggest that the initial search processes for memories of different specificity levels preferentially engage different components of the autobiographical memory network. The potential underlying causes of these neural differences are discussed. PMID:21600227

  16. Accuracy of Optimized Branched Algorithms to Assess Activity-Specific PAEE

    PubMed Central

    Edwards, Andy G.; Hill, James O.; Byrnes, William C.; Browning, Raymond C.

    2009-01-01

    PURPOSE To assess the activity-specific accuracy achievable by branched algorithm (BA) analysis of simulated daily-living physical activity energy expenditure (PAEE) within a sedentary population. METHODS Sedentary men (n=8) and women (n=8) first performed a treadmill calibration protocol, during which heart rate (HR), accelerometry (ACC), and PAEE were measured in 1-minute epochs. From these data, HR-PAEE, and ACC-PAEE regressions were constructed and used in each of six analytic models to predict PAEE from ACC and HR data collected during a subsequent simulated daily-living protocol. Criterion PAEE was measured during both protocols via indirect calorimetry. The accuracy achieved by each model was assessed by the root mean square of the difference between model-predicted daily–living PAEE and the criterion daily-living PAEE (expressed here as % of mean daily living PAEE). RESULTS Across the range of activities an unconstrained post hoc optimized branched algorithm best predicted criterion PAEE. Estimates using individual calibration were generally more accurate than those using group calibration (14 vs. 16 % error, respectively). These analyses also performed well within each of the six daily-living activities, but systematic errors appeared for several of those activities, which may be explained by an inability of the algorithm to simultaneously accommodate a heterogeneous range of activities. Analyses of between mean square error by subject and activity suggest that optimization involving minimization of RMS for total daily-living PAEE is associated with decreased error between subjects but increased error between activities. CONCLUSION The performance of post hoc optimized branched algorithms may be limited by heterogeneity in the daily-living activities being performed. PMID:19952842

  17. Promiscuous activity of ER glucosidase II discovered through donor specificity analysis of UGGT

    SciTech Connect

    Miyagawa, Atsushi; Totani, Kiichiro; Matsuo, Ichiro; Ito, Yukishige

    2010-12-17

    Research highlights: {yields} UGGT has a narrow donor specificity. {yields} UGGT gave several non-natural high-mannose-type glycans. {yields} G-II has a promiscuous activity as broad specificity hexosidase. -- Abstract: In glycoprotein quality control system in the endoplasmic reticulum (ER), UGGT (UDP-glucose:glycoprotein glucosyltransferase) and glucosidase II (G-II) play key roles. UGGT serves as a glycoprotein folding sensor by virtue of its unique specificity to glucosylate glycoproteins at incompletely folded stage. By using various UDP-Glc analogues, we first analyzed donor specificity of UGGT, which was proven to be rather narrow. However, marginal activity was observed with UDP-galactose and UDP-glucuronic acid as well as with 3-, 4- and 6-deoxy glucose analogues to give corresponding transfer products. Intriguingly, G-II smoothly converted all of them back to Man{sub 9}GlcNAc{sub 2}, providing an indication that G-II has a promiscuous activity as a broad specificity hexosidase.

  18. Real-time PCR detection of telomerase activity using specific molecular beacon probes.

    PubMed

    Kong, Deming; Jin, Yawei; Yin, Yuji; Mi, Huaifeng; Shen, Hanxi

    2007-06-01

    Telomerase is a potentially important biomarker and a prognostic indicator of cancer. Several techniques for assessing telomerase activity, including the telomeric repeat amplification protocol (TRAP) and its modified versions, have been developed. Of these methods, real-time quantitative TRAP (RTQ-TRAP) is considered the most promising. In this work, a novel RTQ-TRAP method is developed in which a telomeric repeats-specific molecular beacon is used. The use of the molecular beacon can improve the specificity of the RTQ-TRAP assay, making the method suitable for studying the overall processivity results and the turnover rate of telomerase. In addition, the real-time, closed-tube protocol used obviates the need for post-amplification procedures, reduces the risk of carryover contamination, and supports high throughput. Its performance in synthetic telomerase products and cell extracts suggests that the developed molecular beacon assay can further enhance the clinical utility of telomerase activity as a biomarker/indicator in cancer diagnosis and prognosis. The method also provides a novel approach to the specific detection of some particular gene sequences to which sequence-specific fluorogenic probes cannot be applied directly. Figure Real-time PCR detection of telomerase activity using specific molecular beacon probes.

  19. Screening of DUB activity and specificity by MALDI-TOF mass spectrometry.

    PubMed

    Ritorto, Maria Stella; Ewan, Richard; Perez-Oliva, Ana B; Knebel, Axel; Buhrlage, Sara J; Wightman, Melanie; Kelly, Sharon M; Wood, Nicola T; Virdee, Satpal; Gray, Nathanael S; Morrice, Nicholas A; Alessi, Dario R; Trost, Matthias

    2014-01-01

    Deubiquitylases (DUBs) are key regulators of the ubiquitin system which cleave ubiquitin moieties from proteins and polyubiquitin chains. Several DUBs have been implicated in various diseases and are attractive drug targets. We have developed a sensitive and fast assay to quantify in vitro DUB enzyme activity using matrix-assisted laser desorption/ionization time-of-flight (MALDI-TOF) mass spectrometry. Unlike other current assays, this method uses unmodified substrates, such as diubiquitin topoisomers. By analysing 42 human DUBs against all diubiquitin topoisomers we provide an extensive characterization of DUB activity and specificity. Our results confirm the high specificity of many members of the OTU and JAB/MPN/Mov34 metalloenzyme DUB families and highlight that all USPs tested display low linkage selectivity. We also demonstrate that this assay can be deployed to assess the potency and specificity of DUB inhibitors by profiling 11 compounds against a panel of 32 DUBs. PMID:25159004

  20. Synthesis of high specific activity (1- sup 3 H) farnesyl pyrophosphate

    SciTech Connect

    Saljoughian, M.; Morimoto, H.; Williams, P.G.

    1991-08-01

    The synthesis of tritiated farnesyl pyrophosphate with high specific activity is reported. trans-trans Farnesol was oxidized to the corresponding aldehyde followed by reduction with lithium aluminium tritide (5%-{sup 3}H) to give trans-trans (1-{sup 3}H)farnesol. The specific radioactivity of the alcohol was determined from its triphenylsilane derivative, prepared under very mild conditions. The tritiated alcohol was phosphorylated by initial conversion to an allylic halide, and subsequent treatment of the halide with tris-tetra-n-butylammonium hydrogen pyrophosphate. The hydride procedure followed in this work has advantages over existing methods for the synthesis of tritiated farnesyl pyrophosphate, with the possibility of higher specific activity and a much higher yield obtained. 10 refs., 3 figs.

  1. Screening of DUB activity and specificity by MALDI-TOF mass spectrometry

    PubMed Central

    Ritorto, Maria Stella; Ewan, Richard; Perez-Oliva, Ana B.; Knebel, Axel; Buhrlage, Sara J.; Wightman, Melanie; Kelly, Sharon M.; Wood, Nicola T.; Virdee, Satpal; Gray, Nathanael S.; Morrice, Nicholas A.; Alessi, Dario R.; Trost, Matthias

    2014-01-01

    Deubiquitylases (DUBs) are key regulators of the ubiquitin system which cleave ubiquitin moieties from proteins and polyubiquitin chains. Several DUBs have been implicated in various diseases and are attractive drug targets. We have developed a sensitive and fast assay to quantify in vitro DUB enzyme activity using matrix-assisted laser desorption/ionization time-of-flight (MALDI-TOF) mass spectrometry. Unlike other current assays, this method uses unmodified substrates, such as diubiquitin topoisomers. By analysing 42 human DUBs against all diubiquitin topoisomers we provide an extensive characterization of DUB activity and specificity. Our results confirm the high specificity of many members of the OTU and JAB/MPN/Mov34 metalloenzyme DUB families and highlight that all USPs tested display low linkage selectivity. We also demonstrate that this assay can be deployed to assess the potency and specificity of DUB inhibitors by profiling 11 compounds against a panel of 32 DUBs. PMID:25159004

  2. Screening of DUB activity and specificity by MALDI-TOF mass spectrometry.

    PubMed

    Ritorto, Maria Stella; Ewan, Richard; Perez-Oliva, Ana B; Knebel, Axel; Buhrlage, Sara J; Wightman, Melanie; Kelly, Sharon M; Wood, Nicola T; Virdee, Satpal; Gray, Nathanael S; Morrice, Nicholas A; Alessi, Dario R; Trost, Matthias

    2014-08-27

    Deubiquitylases (DUBs) are key regulators of the ubiquitin system which cleave ubiquitin moieties from proteins and polyubiquitin chains. Several DUBs have been implicated in various diseases and are attractive drug targets. We have developed a sensitive and fast assay to quantify in vitro DUB enzyme activity using matrix-assisted laser desorption/ionization time-of-flight (MALDI-TOF) mass spectrometry. Unlike other current assays, this method uses unmodified substrates, such as diubiquitin topoisomers. By analysing 42 human DUBs against all diubiquitin topoisomers we provide an extensive characterization of DUB activity and specificity. Our results confirm the high specificity of many members of the OTU and JAB/MPN/Mov34 metalloenzyme DUB families and highlight that all USPs tested display low linkage selectivity. We also demonstrate that this assay can be deployed to assess the potency and specificity of DUB inhibitors by profiling 11 compounds against a panel of 32 DUBs.

  3. Diversity between mammalian tolloid proteinases: Oligomerisation and non-catalytic domains influence activity and specificity

    PubMed Central

    Bayley, Christopher P.; Ruiz Nivia, Hilda D.; Dajani, Rana; Jowitt, Thomas A.; Collins, Richard F.; Rada, Heather; Bird, Louise E.; Baldock, Clair

    2016-01-01

    The mammalian tolloid family of metalloproteinases is essential for tissue patterning and extracellular matrix assembly. The four members of the family: bone morphogenetic protein-1 (BMP-1), mammalian tolloid (mTLD), tolloid-like (TLL)-1 and TLL-2 differ in their substrate specificity and activity levels, despite sharing similar domain organization. We have previously described a model of substrate exclusion by dimerisation to explain differences in the activities of monomeric BMP-1 and dimers of mTLD and TLL-1. Here we show that TLL-2, the least active member of the tolloid family, is predominantly monomeric in solution, therefore it appears unlikely that substrate exclusion via dimerisation is a mechanism for regulating TLL-2 activity. X-ray scattering and electron microscopy structural and biophysical analyses reveal an elongated shape for the monomer and flexibility in the absence of calcium. Furthermore, we show that TLL-2 can cleave chordin in vitro, similar to other mammalian tolloids, but truncated forms of TLL-2 mimicking BMP-1 are unable to cleave chordin. However, both the N- and C-terminal non-catalytic domains from all mammalian tolloids bind chordin with high affinity. The mechanisms underlying substrate specificity and activity in the tolloid family are complex with variation between family members and depend on both multimerisation and substrate interaction. PMID:26902455

  4. Korean immigrant women's physical activity experience: a situation-specific theory.

    PubMed

    Im, Eun-Ok; Chang, Sun Ju; Nguyen, Giang; Stringer, Lynn; Chee, Wonshik; Chee, Eunice

    2015-01-01

    To develop successful physical activity promotion programs for midlife immigrant women, especially for Korean immigrant midlife women, concrete theoretical bases are needed. However, virtually no theoretical frameworks and/or theories exist that can explain the influences of immigration transition on the physical activity experience of midlife immigrant women in general or Korean immigrant midlife women in specific. The purpose of this article is to present a situation-specific theory on physical activity experience of Korean immigrant midlife women (SPAKIM) with its development process. An integrative approach was used to develop the theory based on the midlife women's attitudes toward physical activity (MAPA) theory, the transitions theory, a review of the relevant literature, and two studies on midlife women's attitudes toward physical activity. The proposed theory includes nature of transitions, nonmodifiable and modifiable transition conditions, contexts of daily life, patterns of response, and nursing therapeutics as major concepts, and each major concept includes several related subconcepts. Because several concepts of the theory were developed mainly based on the literature review, the major concepts and related subconcepts need to be further developed and evaluated in future studies.

  5. Cardiac Myocyte-Specific AHR Activation Phenocopies TCDD-Induced Toxicity in Zebrafish

    PubMed Central

    Lanham, Kevin A.; Plavicki, Jessica; Peterson, Richard E.; Heideman, Warren

    2014-01-01

    Exposure of zebrafish embryos to 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) activates the zebrafish aryl hydrocarbon receptor 2 (AHR) to produce developmental and cardiovascular toxicity. AHR is found in the heart; however, AHR activation by TCDD is not confined to the heart and occurs throughout the organism. In order to understand the cause of cardiotoxicity, we constructed a constitutively active AHR (caAHR) based on the zebrafish AHR2 and expressed it specifically in cardiomyocytes. We show that AHR activation within the cardiomyocytes can account for the heart failure induced by TCDD. Expression of the caAHR within the heart produced cardiac malformations, loss of circulation, and pericardial edema. The heart-specific activation of AHR reproduced several other well-characterized endpoints of TCDD toxicity outside of the cardiovascular system, including defects in swim bladder and craniofacial development. This work identifies a single cellular site of TCDD action, the myocardial cell, that can account for the severe cardiovascular collapse observed following early life stage exposure to TCDD, and contributes to other forms of toxicity. PMID:25037585

  6. Activation of epitope-specific memory cytotoxic T lymphocyte responses by synthetic peptides.

    PubMed

    Reali, E; Guerrini, R; Giori, B; Borghi, M; Marastoni, M; Tomatis, R; Traniello, S; Masucci, M G; Gavioli, R

    1996-08-01

    Cytotoxic T lymphocytes (CTL) recognize antigens as short peptides selected for presentation by their ability to bind to MHC class I molecules. Polyclonal Epstein-Barr virus (EBV)-specific memory CTL responses, reactivated from blood lymphocytes of HLA-A11-positive individuals by stimulation with the autologous EBV-transformed lymphoblastoid cell line (LCL), are often dominated by reactivites directed to the peptide epitope IVTDFSVIK (IVT), corresponding to amino acids 416-424 of EBV nuclear antigen-4 (EBNA4). We now report the selective activation of IVT-specific CTL by stimulation of lymphocytes with the corresponding synthetic peptide. A more than 10-fold increase in frequency of CTL clones with this specificity (from 8% to 96%) was obtained when the peptide was presented by HLA-A11-transfected T2 cells (T2/A11). Titration of synthetic peptide in cytotoxic assay demonstrated that clones activated under these conditions are as efficient as clones activated by conventional LCL stimulations. Induction of memory CTL responses required low surface density of MHC: peptide complexes, since reactivation was achieved by stimulation with T2/A11 cells pulsed with concentrations of peptide that are suboptimal for induction of target cell lysis. This protocol of activation revealed the presence of IVT-specific CTL precursors in a donor that failed to mount an IVT-specific response upon stimulation with the autologous B95.8 virus-transformed LCL. The results suggest that stimulation with synthetic peptide epitopes can be efficiently used for induction of memory CTL responses, and may be particularly helpful for the selective expansion of subdominant CTL specificities.

  7. The capsid proteins of Aleutian mink disease virus activate caspases and are specifically cleaved during infection.

    PubMed

    Cheng, Fang; Chen, Aaron Yun; Best, Sonja M; Bloom, Marshall E; Pintel, David; Qiu, Jianming

    2010-03-01

    Aleutian mink disease virus (AMDV) is currently the only known member of the genus Amdovirus in the family Parvoviridae. It is the etiological agent of Aleutian disease of mink. We have previously shown that a small protein with a molecular mass of approximately 26 kDa was present during AMDV infection and following transfection of capsid expression constructs (J. Qiu, F. Cheng, L. R. Burger, and D. Pintel, J. Virol. 80:654-662, 2006). In this study, we report that the capsid proteins were specifically cleaved at aspartic acid residue 420 (D420) during virus infection, resulting in the previously observed cleavage product. Mutation of a single amino acid residue at D420 abolished the specific cleavage. Expression of the capsid proteins alone in Crandell feline kidney (CrFK) cells reproduced the cleavage of the capsid proteins in virus infection. More importantly, capsid protein expression alone induced active caspases, of which caspase-10 was the most active. Active caspases, in turn, cleaved capsid proteins in vivo. Our results also showed that active caspase-7 specifically cleaved capsid proteins at D420 in vitro. These results suggest that viral capsid proteins alone induce caspase activation, resulting in cleavage of capsid proteins. We also provide evidence that AMDV mutants resistant to caspase-mediated capsid cleavage increased virus production approximately 3- to 5-fold in CrFK cells compared to that produced from the parent virus AMDV-G at 37 degrees C but not at 31.8 degrees C. Collectively, our results indicate that caspase activity plays multiple roles in AMDV infection and that cleavage of the capsid proteins might have a role in regulating persistent infection of AMDV. PMID:20042496

  8. Ascites Specific Inhibition of CD1d-Mediated Activation of NKT cells

    PubMed Central

    Webb, Tonya J.; Giuntoli, Robert L.; Rogers, Ophelia; Schneck, Jonathan; Oelke, Mathias

    2009-01-01

    Purpose Natural killer T (NKT) cells recognize lipid antigen presented by CD1 molecules. NKT cells can both directly, through cytotoxicity, and indirectly, through activation of other effector cells, mediate anti-tumor immunity. However, it has been shown that tumor associated lipids are frequently shed into the tumor microenvironment, which can mediate immunosuppressive activity. Given that ovarian cancer associated ascites has been reported to have increased levels of gangliosides, we examined the effect of tumor associated and other ascites on CD1d-mediated antigen presentation to NKT cells. Experimental Design To investigate the effects of ascites on NKT cell activation, we pretreated CD1d-expressing cells with the ascites and measured their ability to stimulate cytokine production in NKT cells. To determine whether antigen processing or editing was necessary, CD1d-Ig-based artificial Antigen Presenting Cells (aAPC) were also incubated with ascites. In addition, to examine specificity, we analyzed whether ascites fluid could influence the activation of classical CD8+ T cells. Results Pretreatment of CD1d-expressing cells with ascites from the majority of patients inhibited the cells’ ability to stimulate/activate NKT cells in a dose-dependent manner. Ascites treatment also partially blocked the ability of α-GalCer loaded CD1d-Ig-based artificial Antigen Presenting Cells (aAPC) to activate NKT cells. In addition, our data demonstrate that treatment with ascites does not inhibit HLA-A2 mediated activation of classical CD8+ T cells. Conclusions Together, these data suggest that ovarian and other cancers may have developed immune evasion mechanisms specifically targeting the CD1/NKT cell system. PMID:19047090

  9. Wobble Pairs of the HDV Ribozyme Play Specific Roles in Stabilization of Active Site Dynamics

    PubMed Central

    Sripathi, Kamali N.; Banáš, Pavel; Reblova, Kamila; Šponer, Jiři; Otyepka, Michal

    2015-01-01

    The hepatitis delta virus (HDV) is the only known human pathogen whose genome contains a catalytic RNA motif (ribozyme). The overall architecture of the HDV ribozyme is that of a double-nested pseudoknot, with two GU pairs flanking the active site. Although extensive studies have shown that mutation of either wobble results in decreased catalytic activity, little work has focused on linking these mutations to specific structural effects on catalytic fitness. Here we use molecular dynamics simulations based on an activated structure to probe the active site dynamics as a result of wobble pair mutations. In both wild-type and mutant ribozymes, the in-line fitness of the active site (as a measure of catalytic proficiency) strongly depends on the presence of a C75(N3H3+)N1(O5′) hydrogen bond, which positions C75 as the general acid for the reaction. Our mutational analyses show that each GU wobble supports catalytically fit conformations in distinct ways; the reverse G25U20 wobble promotes high in-line fitness, high occupancy of the C75(N3H3+)G1(O5′) general-acid hydrogen bond and stabilization of the G1U37 wobble, while the G1U37 wobble acts more locally by stabilizing high in-line fitness and the C75(N3H3+)G1(O5′) hydrogen bond. We also find that stable type I A-minor and P1.1 hydrogen bonding above and below the active site, respectively, prevent local structural disorder from spreading and disrupting global conformation. Taken together, our results define specific, often redundant architectural roles for several structural motifs of the HDV ribozyme active site, expanding the known roles of these motifs within all HDV-like ribozymes and other structured RNAs. PMID:25631765

  10. Production of 191Pt radiotracer with high specific activity for the development of preconcentration procedures

    NASA Astrophysics Data System (ADS)

    Parent, M.; Strijckmans, K.; Cornelis, R.; Dewaele, J.; Dams, R.

    1994-04-01

    A radiotracer of Pt with suitable nuclear characteristics and high specific activity (i.e. activity to mass ratio) is a powerful tool when developing preconcentration methods for the determination of base-line levels of Pt in e.g. environmental and biological samples. Two methods were developed for the production of 191Pt with high specific activity and radionuclidic purity: (1) via the 190Pt(n, γ) 191Pt reaction by neutron irradiation of enriched Pt in a nuclear reactor at high neutron fluence rate and (2) via the 191Ir(p, n) 191Pt reaction by proton irradiation of natural Ir with a cyclotron, at an experimentally optimized proton energy. For the latter method it was necessary to separate Pt from the Ir matrix. For that reason either liquid-liquid extraction with dithizone or adsorption chromatography were used. The yields, the specific activities and the radionuclidic purities were experimentally determined as a function of the proton energy and compared to the former method. The half-life of 191Pt was accurately determined to be 2.802 ± 0.025 d.

  11. Preparation and quantification of 3'-phosphoadenosine 5'-phospho(35S)sulfate with high specific activity

    SciTech Connect

    Vargas, F.

    1988-07-01

    The synthesis and quantitation of the sulfate donor 3'-phosphoadenosine 5'-phospho(35S)sulfate (PAP35S), prepared from inorganic (35S)sulfate and ATP, were studied. An enzymatic transfer method based upon the quantitative transfer of (35S)sulfate from PAP35S to 2-naphthol and 4-methylumbelliferone by the action of phenolsulfotransferase activity from rat brain cytosol was also developed. The 2-naphthyl(35S)sulfate or 35S-methylumbelliferone sulfate formed was isolated by polystyrene bead chromatography. This method allows the detection of between 0.1 pmol and 1 nmol/ml of PAP35S. PAP35S of high specific activity (75 Ci/mmol) was prepared by incubating ATP and carrier-free Na2 35SO4 with a 100,000g supernatant fraction from rat spleen. The product was purified by ion-exchange chromatography. The specific activity and purity of PAP35S were estimated by examining the ratios of Km values for PAP35S of the tyrosyl protein sulfotransferase present in microsomes from rat cerebral cortex. The advantage and applications of these methods for the detection of femtomole amounts, and the synthesis of large scale quantities of PAP35S with high specific activity are discussed.

  12. Activation of the amino acid response modulates lineage specification during differentiation of murine embryonic stem cells

    PubMed Central

    Shan, Jixiu; Hamazaki, Takashi; Tang, Tiffany A.; Terada, Naohiro

    2013-01-01

    In somatic cells, a collection of signaling pathways activated by amino acid limitation have been identified and referred to as the amino acid response (AAR). Despite the importance of possible detrimental effects of nutrient limitation during in vitro culture, the AAR has not been investigated in embryonic stem cells (ESC). AAR activation caused the expected increase in transcription factors that mediate specific AAR pathways, as well as the induction of asparagine synthetase, a terminal AAR target gene. Neither AAR activation nor stable knockdown of activating transcription factor (Atf) 4, a transcriptional mediator of the AAR, adversely affected ESC self-renewal or pluripotency. Low-level induction of the AAR over a 12-day period of embryoid body differentiation did alter lineage specification such that the primitive endodermal, visceral endodermal, and endodermal lineages were favored, whereas mesodermal and certain ectodermal lineages were suppressed. Knockdown of Atf4 further enhanced the AAR-induced increase in endodermal formation, suggesting that this phenomenon is mediated by an Atf4-independent mechanism. Collectively, the results indicate that, during differentiation of mouse embryoid bodies in culture, the availability of nutrients, such as amino acids, can influence the formation of specific cell lineages. PMID:23736538

  13. Cardiac-Specific Activation of IKK2 Leads to Defects in Heart Development and Embryonic Lethality.

    PubMed

    Kraut, Bärbel; Maier, Harald J; Kókai, Enikö; Fiedler, Katja; Boettger, Thomas; Illing, Annett; Kostin, Sawa; Walther, Paul; Braun, Thomas; Wirth, Thomas

    2015-01-01

    The transcription factor NF-κB has been associated with a range of pathological conditions of the heart, mainly based on its function as a master regulator of inflammation and pro-survival factor. Here, we addressed the question what effects activation of NF-κB can have during murine heart development. We expressed a constitutively active (CA) mutant of IKK2, the kinase activating canonical NF-κB signaling, specifically in cardiomyocytes under the control of the α-myosin heavy chain promoter. Expression of IKK2-CA resulted in embryonic lethality around E13. Embryos showed defects in compact zone formation and the contractile apparatus, and overall were characterized by widespread inflammation with infiltration of myeloid cells. Gene expression analysis suggested an interferon type I signature, with increased expression of interferon regulatory factors. While apoptosis of cardiomyocytes was only increased at later stages, their proliferation was decreased early on, providing an explanation for the disturbed compact zone formation. Mechanistically, this could be explained by activation of the JAK/STAT axis and increased expression of the cell cycle inhibitor p21. A rescue experiment with an IκBα superrepressor demonstrated that the phenotype was dependent on NF-κB. We conclude that activation of NF-κB is detrimental during normal heart development due to excessive activation of pro-inflammatory pathways.

  14. Enhancement of Chaperone Activity of Plant-Specific Thioredoxin through γ-Ray Mediated Conformational Change.

    PubMed

    Lee, Seung Sik; Jung, Hyun Suk; Park, Soo-Kwon; Lee, Eun Mi; Singh, Sudhir; Lee, Yuno; Lee, Kyun Oh; Lee, Sang Yeol; Chung, Byung Yeoup

    2015-11-13

    AtTDX, a thioredoxin-like plant-specific protein present in Arabidopsis is a thermo-stable and multi-functional enzyme. This enzyme is known to act as a thioredoxin and as a molecular chaperone depending upon its oligomeric status. The present study examines the effects of γ-irradiation on the structural and functional changes of AtTDX. Holdase chaperone activity of AtTDX was increased and reached a maximum at 10 kGy of γ-irradiation and declined subsequently in a dose-dependent manner, together with no effect on foldase chaperone activity. However, thioredoxin activity decreased gradually with increasing irradiation. Electrophoresis and size exclusion chromatography analysis showed that AtTDX had a tendency to form high molecular weight (HMW) complexes after γ-irradiation and γ-ray-induced HMW complexes were tightly associated with a holdase chaperone activity. The hydrophobicity of AtTDX increased with an increase in irradiation dose till 20 kGy and thereafter decreased further. Analysis of the secondary structures of AtTDX using far UV-circular dichroism spectra revealed that the irradiation remarkably increased the exposure of β-sheets and random coils with a dramatic decrease in α-helices and turn elements in a dose-dependent manner. The data of the present study suggest that γ-irradiation may be a useful tool for increasing holdase chaperone activity without adversely affecting foldase chaperone activity of thioredoxin-like proteins.

  15. Enhancement of Chaperone Activity of Plant-Specific Thioredoxin through γ-Ray Mediated Conformational Change.

    PubMed

    Lee, Seung Sik; Jung, Hyun Suk; Park, Soo-Kwon; Lee, Eun Mi; Singh, Sudhir; Lee, Yuno; Lee, Kyun Oh; Lee, Sang Yeol; Chung, Byung Yeoup

    2015-01-01

    AtTDX, a thioredoxin-like plant-specific protein present in Arabidopsis is a thermo-stable and multi-functional enzyme. This enzyme is known to act as a thioredoxin and as a molecular chaperone depending upon its oligomeric status. The present study examines the effects of γ-irradiation on the structural and functional changes of AtTDX. Holdase chaperone activity of AtTDX was increased and reached a maximum at 10 kGy of γ-irradiation and declined subsequently in a dose-dependent manner, together with no effect on foldase chaperone activity. However, thioredoxin activity decreased gradually with increasing irradiation. Electrophoresis and size exclusion chromatography analysis showed that AtTDX had a tendency to form high molecular weight (HMW) complexes after γ-irradiation and γ-ray-induced HMW complexes were tightly associated with a holdase chaperone activity. The hydrophobicity of AtTDX increased with an increase in irradiation dose till 20 kGy and thereafter decreased further. Analysis of the secondary structures of AtTDX using far UV-circular dichroism spectra revealed that the irradiation remarkably increased the exposure of β-sheets and random coils with a dramatic decrease in α-helices and turn elements in a dose-dependent manner. The data of the present study suggest that γ-irradiation may be a useful tool for increasing holdase chaperone activity without adversely affecting foldase chaperone activity of thioredoxin-like proteins. PMID:26580605

  16. Enhancement of Chaperone Activity of Plant-Specific Thioredoxin through γ-Ray Mediated Conformational Change

    PubMed Central

    Lee, Seung Sik; Jung, Hyun Suk; Park, Soo-Kwon; Lee, Eun Mi; Singh, Sudhir; Lee, Yuno; Lee, Kyun Oh; Lee, Sang Yeol; Chung, Byung Yeoup

    2015-01-01

    AtTDX, a thioredoxin-like plant-specific protein present in Arabidospis is a thermo-stable and multi-functional enzyme. This enzyme is known to act as a thioredoxin and as a molecular chaperone depending upon its oligomeric status. The present study examines the effects of γ-irradiation on the structural and functional changes of AtTDX. Holdase chaperone activity of AtTDX was increased and reached a maximum at 10 kGy of γ-irradiation and declined subsequently in a dose-dependent manner, together with no effect on foldase chaperone activity. However, thioredoxin activity decreased gradually with increasing irradiation. Electrophoresis and size exclusion chromatography analysis showed that AtTDX had a tendency to form high molecular weight (HMW) complexes after γ-irradiation and γ-ray-induced HMW complexes were tightly associated with a holdase chaperone activity. The hydrophobicity of AtTDX increased with an increase in irradiation dose till 20 kGy and thereafter decreased further. Analysis of the secondary structures of AtTDX using far UV-circular dichroism spectra revealed that the irradiation remarkably increased the exposure of β-sheets and random coils with a dramatic decrease in α-helices and turn elements in a dose-dependent manner. The data of the present study suggest that γ-irradiation may be a useful tool for increasing holdase chaperone activity without adversely affecting foldase chaperone activity of thioredoxin-like proteins. PMID:26580605

  17. [Evaluation of specific activity of preparations of allergens from synanthropic insects].

    PubMed

    Berzhets, V M; Radikova, O V; Khlgatian, S V; Berzhets, A I; Kropotova, I S

    2006-01-01

    Physical, chemical and immunobiological characteristics of allergens from synanthropic insects were studied by tests for anaphylaxis, indirect degranulation of mast cells test and ELISA. Sera from 20 patients with severe and intermediate atopic asthma with sensiblization to common allergens have been studied. All extracts of allergens from synanthropic insects (german cockroach, oriental cockroach, american cockroach, speckled feeder cockroach, cricket, common house fly, brown house moth, confused flour beetle, rice weevil, grain weevil) have specific activity. Extracts of allergens from common house fly, brown house moth, german cockroach and oriental cockroach had the strongest allergenic activity as measured by ELISA. Obtained allergens can be used for insect allergy diagnostics.

  18. Dictyostelium discoideum contains three inositol monophosphatase activities with different substrate specificities and sensitivities to lithium.

    PubMed Central

    Van Dijken, P; Bergsma, J C; Hiemstra, H S; De Vries, B; Van Der Kaay, J; Van Haastert, P J

    1996-01-01

    The small ion lithium, a very effective agent in the treatment of manic depressive patients, inhibits the mammalian enzyme inositol monophosphatase, which is proposed as the biological target for the effects of lithium. In this study we investigated Dictyostelium discoideum inositol monophosphatase activity. Partial purification of the proteins in the soluble cell fraction using anion-exchange chromatography revealed the presence of at least three enzyme activities capable of degrading inositol monophosphate isomers. The first activity was similar to the monophosphatase found in mammalian cells, as it degraded Ins(4)P, Ins(1)P and to a lesser extent Ins(3)P, was dependent on MgCl2 and inhibited by LiCl in a uncompetitive [corrected] manner. The second enzyme activity was specific for Ins(4)P; the enzyme activity was not dependent on MgCl2 and not inhibited by LiCl. The third monophosphatase activity degraded especially Ins(3)P, but also Ins(4)P and Ins(1)P; increasing concentrations of MgCl2 inhibited this enzyme activity, whereas LiCl had no effect. In vivo, LiCl induces a reduction of inositol levels by about 20%. In [3H]inositol-labelled cells LiCl causes a 6-fold increase in the radioactivity of [3H]Ins(1)P, a doubling of [3H]Ins(4)P and a slight decrease in the radioactivity in [3H]Ins(3)P. These data indicate that the biological effects of lithium in Dictyostelium are not due to depletion of the inositol pool by inhibition of inositol monophosphatase activity. PMID:8670062

  19. Comprehensive analysis of the specificity of transcription activator-like effector nucleases

    PubMed Central

    Juillerat, Alexandre; Dubois, Gwendoline; Valton, Julien; Thomas, Séverine; Stella, Stefano; Maréchal, Alan; Langevin, Stéphanie; Benomari, Nassima; Bertonati, Claudia; Silva, George H.; Daboussi, Fayza; Epinat, Jean-Charles; Montoya, Guillermo; Duclert, Aymeric; Duchateau, Philippe

    2014-01-01

    A key issue when designing and using DNA-targeting nucleases is specificity. Ideally, an optimal DNA-targeting tool has only one recognition site within a genomic sequence. In practice, however, almost all designer nucleases available today can accommodate one to several mutations within their target site. The ability to predict the specificity of targeting is thus highly desirable. Here, we describe the first comprehensive experimental study focused on the specificity of the four commonly used repeat variable diresidues (RVDs; NI:A, HD:C, NN:G and NG:T) incorporated in transcription activator-like effector nucleases (TALEN). The analysis of >15 500 unique TALEN/DNA cleavage profiles allowed us to monitor the specificity gradient of the RVDs along a TALEN/DNA binding array and to present a specificity scoring matrix for RVD/nucleotide association. Furthermore, we report that TALEN can only accommodate a relatively small number of position-dependent mismatches while maintaining a detectable activity at endogenous loci in vivo, demonstrating the high specificity of these molecular tools. We thus envision that the results we provide will allow for more deliberate choices of DNA binding arrays and/or DNA targets, extending our engineering capabilities. PMID:24569350

  20. The maximum specific hydrogen-producing activity of anaerobic mixed cultures: definition and determination.

    PubMed

    Mu, Yang; Yang, Hou-Yun; Wang, Ya-Zhou; He, Chuan-Shu; Zhao, Quan-Bao; Wang, Yi; Yu, Han-Qing

    2014-06-10

    Fermentative hydrogen production from wastes has many advantages compared to various chemical methods. Methodology for characterizing the hydrogen-producing activity of anaerobic mixed cultures is essential for monitoring reactor operation in fermentative hydrogen production, however there is lack of such kind of standardized methodologies. In the present study, a new index, i.e., the maximum specific hydrogen-producing activity (SHAm) of anaerobic mixed cultures, was proposed, and consequently a reliable and simple method, named SHAm test, was developed to determine it. Furthermore, the influences of various parameters on the SHAm value determination of anaerobic mixed cultures were evaluated. Additionally, this SHAm assay was tested for different types of substrates and bacterial inocula. Our results demonstrate that this novel SHAm assay was a rapid, accurate and simple methodology for determining the hydrogen-producing activity of anaerobic mixed cultures. Thus, application of this approach is beneficial to establishing a stable anaerobic hydrogen-producing system.

  1. The maximum specific hydrogen-producing activity of anaerobic mixed cultures: definition and determination

    NASA Astrophysics Data System (ADS)

    Mu, Yang; Yang, Hou-Yun; Wang, Ya-Zhou; He, Chuan-Shu; Zhao, Quan-Bao; Wang, Yi; Yu, Han-Qing

    2014-06-01

    Fermentative hydrogen production from wastes has many advantages compared to various chemical methods. Methodology for characterizing the hydrogen-producing activity of anaerobic mixed cultures is essential for monitoring reactor operation in fermentative hydrogen production, however there is lack of such kind of standardized methodologies. In the present study, a new index, i.e., the maximum specific hydrogen-producing activity (SHAm) of anaerobic mixed cultures, was proposed, and consequently a reliable and simple method, named SHAm test, was developed to determine it. Furthermore, the influences of various parameters on the SHAm value determination of anaerobic mixed cultures were evaluated. Additionally, this SHAm assay was tested for different types of substrates and bacterial inocula. Our results demonstrate that this novel SHAm assay was a rapid, accurate and simple methodology for determining the hydrogen-producing activity of anaerobic mixed cultures. Thus, application of this approach is beneficial to establishing a stable anaerobic hydrogen-producing system.

  2. Specificity of MAP kinase signaling in yeast differentiation involves transient versus sustained MAPK activation.

    PubMed

    Sabbagh, W; Flatauer, L J; Bardwell, A J; Bardwell, L

    2001-09-01

    Signals transmitted by common components often elicit distinct (yet appropriate) outcomes. In yeast, two developmental options-mating and invasive growth-are both regulated by the same MAP kinase cascade. Specificity has been thought to result from specialized roles for the two MAP kinases, Kss1 and Fus3, and because Fus3 prevents Kss1 from gaining access to the mating pathway. Kss1 has been thought to participate in mating only when Fus3 is absent. Instead, we show that Kss1 is rapidly phosphorylated and potently activated by mating pheromone in wild-type cells, and that this is required for normal pheromone-induced gene expression. Signal identity is apparently maintained because active Fus3 limits the extent of Kss1 activation, thereby preventing inappropriate signal crossover. PMID:11583629

  3. Subfamily-Specific Fluorescent Probes for Cysteine Proteases Display Dynamic Protease Activities during Seed Germination1

    PubMed Central

    Lu, Haibin; Chandrasekar, Balakumaran; Oeljeklaus, Julian; Misas-Villamil, Johana C.; Wang, Zheming; Shindo, Takayuki; Bogyo, Matthew; Kaiser, Markus; van der Hoorn, Renier A.L.

    2015-01-01

    Cysteine proteases are an important class of enzymes implicated in both developmental and defense-related programmed cell death and other biological processes in plants. Because there are dozens of cysteine proteases that are posttranslationally regulated by processing, environmental conditions, and inhibitors, new methodologies are required to study these pivotal enzymes individually. Here, we introduce fluorescence activity-based probes that specifically target three distinct cysteine protease subfamilies: aleurain-like proteases, cathepsin B-like proteases, and vacuolar processing enzymes. We applied protease activity profiling with these new probes on Arabidopsis (Arabidopsis thaliana) protease knockout lines and agroinfiltrated leaves to identify the probe targets and on other plant species to demonstrate their broad applicability. These probes revealed that most commercially available protease inhibitors target unexpected proteases in plants. When applied on germinating seeds, these probes reveal dynamic activities of aleurain-like proteases, cathepsin B-like proteases, and vacuolar processing enzymes, coinciding with the remobilization of seed storage proteins. PMID:26048883

  4. Antiviral activity and specific modes of action of bacterial prodigiosin against Bombyx mori nucleopolyhedrovirus in vitro.

    PubMed

    Zhou, Wei; Zeng, Cheng; Liu, RenHua; Chen, Jie; Li, Ru; Wang, XinYan; Bai, WenWen; Liu, XiaoYuan; Xiang, TingTing; Zhang, Lin; Wan, YongJi

    2016-05-01

    Prodigiosin, the tripyrrole red pigment, is a bacterial secondary metabolite with multiple bioactivities; however, the antiviral activity has not been reported yet. In the present study, we found the antiviral activity of bacterial prodigiosin on Bombyx mori nucleopolyhedrovirus (BmNPV)-infected cells in vitro, with specific modes of action. Prodigiosin at nontoxic concentrations selectively killed virus-infected cells, inhibited viral gene transcription, especially viral early gene ie-1, and prevented virus-mediated membrane fusion. Under prodigiosin treatment, both progeny virus production and viral DNA replication were significantly inhibited. Fluorescent assays showed that prodigiosin predominantly located in cytoplasm which suggested it might interact with cytoplasm factors to inhibit virus replication. In conclusion, the present study clearly indicates that prodigiosin possesses significant antiviral activity against BmNPV.

  5. Autocatalytic activity and substrate specificity of the pestivirus N-terminal protease N{sup pro}

    SciTech Connect

    Gottipati, Keerthi; Acholi, Sudheer; Ruggli, Nicolas; Choi, Kyung H.

    2014-03-15

    Pestivirus N{sup pro} is the first protein translated in the viral polypeptide, and cleaves itself off co-translationally generating the N-terminus of the core protein. Once released, N{sup pro} blocks the host's interferon response by inducing degradation of interferon regulatory factor-3. N{sup pro'}s intracellular autocatalytic activity and lack of trans-activity have hampered in vitro cleavage studies to establish its substrate specificity and the roles of individual residues. We constructed N{sup pro}-GFP fusion proteins that carry the authentic cleavage site and determined the autoproteolytic activities of N{sup pro} proteins containing substitutions at the predicted catalytic sites Glu22 and Cys69, at Arg100 that forms a salt bridge with Glu22, and at the cleavage site Cys168. Contrary to previous reports, we show that N{sup pro'}s catalytic activity does not involve Glu22, which may instead be involved in protein stability. Furthermore, N{sup pro} does not have specificity for Cys168 at the cleavage site even though this residue is conserved throughout the pestivirus genus. - Highlights: • N{sup pro'}s autoproteolysis is studied using N{sup pro}-GFP fusion proteins. • N-terminal 17 amino acids are dispensable without loss of protease activity. • The putative catalytic residue Glu22 is not involved in protease catalysis. • No specificity for Cys168 at the cleavage site despite evolutionary conservation. • N{sup pro} prefers small amino acids with non-branched beta carbons at the P1 position.

  6. Response of serotonergic caudal raphe neurons in relation to specific motor activities in freely moving cats.

    PubMed

    Veasey, S C; Fornal, C A; Metzler, C W; Jacobs, B L

    1995-07-01

    Serotonergic neuronal responses during three specific motor activities were studied in nuclei raphe obscurus (NRO) and raphe pallidus (NRP) of freely moving cats by means of extracellular single-unit recordings. Responses to treadmill-induced locomotion were primarily excitatory, with 21 of 24 neurons displaying increased firing rates, directly related to treadmill speed. Individual regression analyses determined three response patterns: maximal activation at low speed (0.25 m/sec), augmentation of neuronal activity only at high treadmill speed (0.77 m/sec), and a linear increase. A smaller fraction of NRO and NRP serotonergic neurons (6 of 27) also responded to hypercarbic ventilatory challenge with increased firing rates. The magnitude of neuronal response was dependent upon the fraction of inspired CO2 and was related to ventilatory motor output, specifically, inspiratory amplitude. A subgroup of neurons responsive to hypercarbia in wakefulness demonstrated significant reductions in neuronal response to hypercarbia in slow-wave sleep. Finally, unit activity for 12 of 29 cells increased in response to spontaneous feeding, displaying two distinct patterns of neuronal response in relation to onset and termination of feeding: rapid activation and deactivation versus a gradual increase and decrease. More than half of the cells studied under all three conditions were responsive to more than one motor task. These results indicate that serotonergic caudal raphe neurons are responsive to specific motor system challenges, with many neurons responsive to multiple motor tasks, and that the responsiveness of serotonergic neurons to at least one motor task, hypercarbic ventilatory challenge, is state dependent.

  7. Mammary gland-specific expression of biologically active human osteoprotegerin in transgenic mice.

    PubMed

    Sung, Yoon-Young; Lee, Chul-Sang

    2013-03-01

    Osteoprotegerin (OPG) is a secreted glycoprotein that regulates bone resorption by inhibiting differentiation and activation of osteoclast, thereby potentially useful for the treatment of many bone diseases associated with increased bone loss. In this study, we designed a novel cDNA expression cassette by modifying the potent and mammary gland-specific goat β-casein/hGH hybrid gene construct and examined human OPG (hOPG) cDNA expression in transgenic mice. Six transgenic mice all successfully expressed hOPG in their milk at the level of 0.06-2,000 µg/ml. An estimated molecular weight of the milk hOPG was 55 kDa in SDS-PAGE, which is the same as a naturally glycosylated monomer. This hOPG expression was highly specific to the mammary glands of transgenic mice. hOPG mRNA was not detected in any organs analyzed except mammary gland. Functional integrity of milk hOPG was evaluated by TRAP (tartrate-resistant acid phosphatase) activity assay in bone marrow cell cultures. OPG ligand (OPG-L) treatment increased TRAP activity by two fold but it was completely abolished by co-treatment with transgenic milk containing hOPG. Taken together, our novel cDNA expression cassette could direct an efficient expression of biologically active hOPG, a potential candidate pharmaceutical for bone diseases, only in the mammary gland of transgenic mice.

  8. Increased CD28 serum levels are not associated with specific clinical activity in systemic lupus erythematosus.

    PubMed

    Brambila-Tapia, Aniel J L; Gámez-Nava, Jorge I; Salazar-Páramo, Mario; Munoz-Valle, José F; González-López, Laura; Llamas-Covarrubias, Mara A; Gutiérrez-Urena, Sergio R; Vázquez-Del Mercado, Mónica; Dávalos-Rodríguez, Ingrid P

    2011-10-01

    CD28 is one of the main activator receptors involved in systemic lupus erythematosus (SLE) pathogenesis, and its expression and serum levels are significantly higher in patients with SLE and other autoimmune diseases than in healthy controls (HC). However, it is unknown whether this increase is associated with specific organ damage. Therefore, our objective was to measure the CD28 levels in serum from SLE and HC groups to confirm the CD28 serum levels increase, as reported previously, and to determine whether this increase was associated with specific organ activity and the SLE Disease Activity Index (SLEDAI). Forty SLE patients and 40 matched HC were included, and the age, disease duration, SLEDAI and Mexican SLEDAI were recorded for the SLE group. CD28 serum levels were measured by ELISA. There was a statistically significant increase in the CD28 serum levels of SLE patients compared to controls (p = 0.039); however, we did not find any significant correlation with disease activity indices or organ involvement, although we found a significant but low correlation with C3. Our results and a review of the literature suggest that the increase in CD28 serum levels may be the result of CD28 gene overexpression, which could be related to the decrease in CD28+ T cells, T-cell hyporesponsiveness and immune impairment that occurs in SLE.

  9. Lysine-specific demethylase 1 promotes brown adipose tissue thermogenesis via repressing glucocorticoid activation.

    PubMed

    Zeng, Xing; Jedrychowski, Mark P; Chen, Yi; Serag, Sara; Lavery, Gareth G; Gygi, Steve P; Spiegelman, Bruce M

    2016-08-15

    Brown adipocytes display phenotypic plasticity, as they can switch between the active states of fatty acid oxidation and energy dissipation versus a more dormant state. Cold exposure or β-adrenergic stimulation favors the active thermogenic state, whereas sympathetic denervation or glucocorticoid administration promotes more lipid accumulation. Our understanding of the molecular mechanisms underlying these switches is incomplete. Here we found that LSD1 (lysine-specific demethylase 1), a histone demethylase, regulates brown adipocyte metabolism in two ways. On the one hand, LSD1 associates with PRDM16 to repress expression of white fat-selective genes. On the other hand, LSD1 represses HSD11B1 (hydroxysteroid 11-β-dehydrogenase isozyme 1), a key glucocorticoid-activating enzyme, independently from PRDM16. Adipose-specific ablation of LSD1 impaired mitochondrial fatty acid oxidation capacity of the brown adipose tissue, reduced whole-body energy expenditure, and increased fat deposition, which can be significantly alleviated by simultaneously deleting HSD11B1. These findings establish a novel regulatory pathway connecting histone modification and hormone activation with mitochondrial oxidative capacity and whole-body energy homeostasis. PMID:27566776

  10. Characterization and activity enhancement of the phloem-specific pumpkin PP2 gene promoter.

    PubMed

    Guo, Hongnian; Chen, Xiaoying; Zhang, Haili; Fang, Rongxiang; Yuan, Zhengqiang; Zhang, Zhenshan; Tian, Yingchuan

    2004-12-01

    The promoter of the pumpkin (Cucurbita moschata) PP2 gene (designated NP) was isolated from the restriction enzyme-digested genomic DNA pool by genome walking and its activity and phloem specificity were examined in transgenic tobacco plants by using GUS as a reporter. Deletion analysis of the promoter revealed that the 473-bp fragment (-465 to + 8 relative to the transcription start site; designated as NPII) exhibited similar activity as the full-length NP promoter and retained its phloem specificity. Furthermore, the sequence from -465 to -171 was shown to contain positive regulatory cis-elements for the promoter activity. An enhanced NP promoter was constructed by duplicating the sequence -465 to -85, and its activity in phloem tissue was shown to be higher than that of the Commelina Yellow Mottle Virus (CoYMV) promoter or a chimeric promoter consisting of the double enhancer sequence from the Cauliflower Mosaic Virus (CaMV) 35S promoter fused upstream to the NPII fragment.

  11. Activity Level-Dependent Synapse-Specific AMPA Receptor Trafficking Regulates Transmission Kinetics

    PubMed Central

    Zhu, J. Julius

    2009-01-01

    Central glutamatergic synapses may express AMPA-sensitive glutamate receptors (AMPA-Rs) with distinct gating properties and exhibit different transmission dynamics, which are important for computing various synaptic inputs received at different populations of synapses. However, how glutamatergic synapses acquire AMPA-Rs with distinct kinetics to influence synaptic integration remains poorly understood. Here I report synapse-specific trafficking of distinct AMPA-Rs in rat cortical layer 4 stellate and layer 5 pyramidal neurons. The analysis indicates that in single layer 4 stellate neurons thalamocortical synapses generate faster synaptic responses than intracortical synapses. Moreover, GluR1-containing AMPA-Rs traffic selectively into intracortical synapses, and this process requires sensory experience-dependent activity and slows down transmission kinetics. GluR4-containing AMPA-Rs traffic more heavily into thalamocortical synapses than intracortical synapses, and this process requires spontaneous synaptic activity and speeds up transmission kinetics. GluR2-containing AMPA-Rs traffic equally into both thalamocortical and intracortical synapses, and this process requires no synaptic activity and resets transmission kinetics. Notably, synaptic trafficking of distinct AMPA-Rs differentially regulates synaptic integration. Thus, synapse-specific AMPA-R trafficking coarsely sets and synaptic activity finely tunes transmission kinetics and integration properties at different synapses in central neurons. PMID:19439609

  12. Self-Specific Stimuli Interact Differently than Non-Self-Specific Stimuli with Eyes-Open Versus Eyes-Closed Spontaneous Activity in Auditory Cortex

    PubMed Central

    Qin, Pengmin; Grimm, Simone; Duncan, Niall W.; Holland, Giles; Guo, Jia shen; Fan, Yan; Weigand, Anne; Baudewig, Juergen; Bajbouj, Malek; Northoff, Georg

    2013-01-01

    Previous studies suggest that there may be a distinct relationship between spontaneous neural activity and subsequent or concurrent self-specific stimulus-induced activity. This study aims to test the impact of spontaneous activity as recorded in an eyes-open (EO) resting state as opposed to eyes-closed (EC) on self-specific versus non-self-specific auditory stimulus-induced activity in fMRI. In our first experiment we used self-specific stimuli comprised of the subject’s own name and non-self-specific stimuli comprised of a friend’s name and an unknown name, presented during EO versus EC baselines in a 3 name condition × 2 baseline design. In Experiment 2 we directly measured spontaneous activity in the absence of stimuli during EO versus EC to confirm a modulatory effect of the two baseline conditions in the regions found to show an interaction effect in Experiment 1. Spontaneous activity during EO was significantly higher than during EC in bilateral auditory cortex and non-self-specific names yielded stronger signal changes relative to EO baseline than to EC. In contrast, there was no difference in response to self-specific names relative to EO baseline than to EC despite the difference between spontaneous activity levels. These results support an impact of spontaneous activity on stimulus-induced activity, moreover an impact that depends on the high-level stimulus characteristic of self-specificity. PMID:23908625

  13. Synthesis of Isomeric Phosphoubiquitin Chains Reveals that Phosphorylation Controls Deubiquitinase Activity and Specificity.

    PubMed

    Huguenin-Dezot, Nicolas; De Cesare, Virginia; Peltier, Julien; Knebel, Axel; Kristaryianto, Yosua Adi; Rogerson, Daniel T; Kulathu, Yogesh; Trost, Matthias; Chin, Jason W

    2016-07-26

    Ubiquitin is post-translationally modified by phosphorylation at several sites, but the consequences of these modifications are largely unknown. Here, we synthesize multi-milligram quantities of ubiquitin phosphorylated at serine 20, serine 57, and serine 65 via genetic code expansion. We use these phosphoubiquitins for the enzymatic assembly of 20 isomeric phosphoubiquitin dimers, with different sites of isopeptide linkage and/or phosphorylation. We discover that phosphorylation of serine 20 on ubiquitin converts UBE3C from a dual-specificity E3 ligase into a ligase that primarily synthesizes K48 chains. We profile the activity of 31 deubiquitinases on the isomeric phosphoubiquitin dimers in 837 reactions, and we discover that phosphorylation at distinct sites in ubiquitin can activate or repress cleavage of a particular linkage by deubiquitinases and that phosphorylation at a single site in ubiquitin can control the specificity of deubiquitinases for distinct ubiquitin linkages. PMID:27425610

  14. Gender specific changes in cortical activation patterns during exposure to artificial gravity

    NASA Astrophysics Data System (ADS)

    Schneider, Stefan; Robinson, Ryan; Smith, Craig; von der Wiesche, Melanie; Goswami, Nandu

    2014-11-01

    Keeping astronauts healthy during long duration spaceflight remains a challenge. Artificial gravity (AG) generated by a short arm human centrifuges (SAHC) is proposed as the next generation of integrated countermeasure devices that will allow human beings to safely spend extended durations in space, although comparatively little is known about any psychological side effects of AG on brain function. 16 participants (8 male and 8 female, GENDER) were exposed to 10 min at a baseline gravitational load (G-Load) of +.03 Gz, then 10 min at +.6 Gz for females and +.8 Gz for males, before being exposed to increasing levels of AG in a stepped manner by increasing the acceleration by +.1 Gz every 3 min until showing signs of pre-syncope. EEG recordings were taken of brain activity during 2 min time periods at each AG level. Analysing the results of the mixed total population of participants by two way ANOVA, a significant effect of centrifugation on alpha and beta activity was found (p<.01). Furthermore results revealed a significant interaction between G-LOAD and GENDER alpha-activity (p<.01), but not for beta-activity. Although the increase in alpha and beta activity with G-LOAD does not reflect a general model of cortical arousal and therefore cannot support previous findings reporting that AG may be a cognitively arousing environment, the gender specific responses identified in this study may have wider implications for EEG and AG research.

  15. Affect-specific activation of shared networks for perception and execution of facial expressions.

    PubMed

    Kircher, Tilo; Pohl, Anna; Krach, Sören; Thimm, Markus; Schulte-Rüther, Martin; Anders, Silke; Mathiak, Klaus

    2013-04-01

    Previous studies have shown overlapping neural activations for observation and execution or imitation of emotional facial expressions. These shared representations have been assumed to provide indirect evidence for a human mirror neuron system, which is suggested to be a prerequisite of action comprehension. We aimed at clarifying whether shared representations in and beyond human mirror areas are specifically activated by affective facial expressions or whether they are activated by facial expressions independent of the emotional meaning. During neuroimaging, participants observed and executed happy and non-emotional facial expressions. Shared representations were revealed for happy facial expressions in the pars opercularis, the precentral gyrus, in the superior temporal gyrus/medial temporal gyrus (MTG), in the pre-supplementary motor area and in the right amygdala. All areas showed less pronounced activation in the non-emotional condition. When directly compared, significant stronger neural responses emerged for happy facial expressions in the pre-supplementary motor area and in the MTG than for non-emotional stimuli. We assume that activation of shared representations depends on the affect and (social) relevance of the facial expression. The pre-supplementary motor area is a core-shared representation-structure supporting observation and execution of affective contagious facial expressions and might have a modulatory role during the preparation of executing happy facial expressions.

  16. Schistosoma mansoni Hemozoin Modulates Alternative Activation of Macrophages via Specific Suppression of Retnla Expression and Secretion

    PubMed Central

    Truscott, Martha; Evans, D. Andrew; Gunn, Matt

    2013-01-01

    The trematode Schistosoma mansoni is one of the etiological agents of schistosomiasis, a key neglected tropical disease responsible for an estimated annual loss of 70 million disability-adjusted life years. Hematophagy represents the primary nutrient acquisition pathway of this parasite, but digestion of hemoglobin also liberates toxic heme. Schistosomes detoxify heme via crystallization into hemozoin, which is subsequently regurgitated into the host's circulation. Here we demonstrate that during experimental schistosomiasis, hemozoin accumulating in the mouse liver is taken up by phagocytes at a time coincident with the development of the egg-induced T-helper 2 (Th2) granulomatous immune response. Furthermore, the uptake of hemozoin also coincides with the hepatic expression of markers of alternative macrophage activation. Alternatively activated macrophages are a key effector cell population associated with protection against schistosomiasis, making hemozoin well placed to play an important immunomodulatory role in this disease. To systematically explore this hypothesis, S. mansoni hemozoin was purified and added to in vitro bone marrow-derived macrophage cultures concurrently exposed to cytokines chosen to reflect the shifting state of macrophage activation in vivo. Macrophages undergoing interleukin-4 (IL-4)-induced alternative activation in the presence of hemozoin developed a phenotype specifically lacking in Retnla, a characteristic alternatively activated macrophage product associated with regulation of Th2 inflammatory responses. As such, in addition to its important detoxification role during hematophagy, we propose that schistosome hemozoin also provides a potent immunomodulatory function in the coevolved network of host-parasite relationships during schistosomiasis. PMID:23090958

  17. How to awaken your nanomachines: Site-specific activation of focal adhesion kinases through ligand interactions.

    PubMed

    Walkiewicz, Katarzyna W; Girault, Jean-Antoine; Arold, Stefan T

    2015-10-01

    The focal adhesion kinase (FAK) and the related protein-tyrosine kinase 2-beta (Pyk2) are highly versatile multidomain scaffolds central to cell adhesion, migration, and survival. Due to their key role in cancer metastasis, understanding and inhibiting their functions are important for the development of targeted therapy. Because FAK and Pyk2 are involved in many different cellular functions, designing drugs with partial and function-specific inhibitory effects would be desirable. Here, we summarise recent progress in understanding the structural mechanism of how the tug-of-war between intramolecular and intermolecular interactions allows these protein 'nanomachines' to become activated in a site-specific manner.

  18. A dynamic model for generating actuator specifications for small arms barrel active stabilization

    NASA Astrophysics Data System (ADS)

    Pathak, Anupam; Brei, Diann; Luntz, Jonathan; Lavigna, Chris

    2006-03-01

    Due to stresses encountered in combat, it is known that soldier marksmanship noticeably decreases regardless of prior training. Active stabilization systems in small arms have potential to address this problem to increase soldier survivability and mission effectiveness. The key to success is proper actuator design, but this is highly dependent on proper specification which is challenging due to the human/weapon interaction. This paper presents a generic analytical dynamic model which is capable of defining the necessary actuation specifications for a wide range of small arms platforms. The model is unique because it captures the human interface--shoulder and arm--that introduces the jitter disturbance in addition to the geometry, inertial properties and active stabilization stiffness of the small arms platform. Because no data to date is available for actual shooter-induced disturbance in field conditions, a method is given using the model to back-solve from measured shooting range variability data the disturbance amplitude information relative to the input source (arm or shoulder). As examples of the applicability of the model to various small arms systems, two different weapon systems were investigated: the M24 sniper weapon and the M16 assault rifle. In both cases, model based simulations provided valuable insight into impact on the actuation specifications (force, displacement, phase, frequency) due to the interplay of the human-weapon-active stabilization interface including the effect of shooter-disturbance frequency, disturbance location (shoulder vs. arm), and system parameters (stiffness, barrel rotation).

  19. How to produce high specific activity tin-117m using alpha particle beam.

    PubMed

    Duchemin, C; Essayan, M; Guertin, A; Haddad, F; Michel, N; Métivier, V

    2016-09-01

    Tin-117m is an interesting radionuclide for both diagnosis and therapy, thanks to the gamma-ray and electron emissions, respectively, resulting from its decay to tin-117g. The high specific activity of tin-117m is required in many medical applications, and it can be obtained using a high energy alpha particle beam and a cadmium target. The experiments performed at the ARRONAX cyclotron (Nantes, France) using an alpha particle beam delivered at 67.4MeV provide a measurement of the excitation function of the Cd-nat(α,x)Sn-117m reaction and the produced contaminants. The Cd-116(α,3n)Sn-117m production cross section has been deduced from these experimental results using natural cadmium. Both production yield and specific activity as a function of the projectile energy have been calculated. These informations help to optimize the irradiation conditions to produce tin-117m with the required specific activity using α particles with a cadmium target.

  20. Sequestration of nanoparticles by an EPS matrix reduces the particle-specific bactericidal activity

    PubMed Central

    Wang, Qian; Kang, Fuxing; Gao, Yanzheng; Mao, Xuewei; Hu, Xiaojie

    2016-01-01

    Most artificial nanomaterials are known to exhibit broad-spectrum bactericidal activity; however, the defence mechanisms that bacteria use based on extracellular polymeric substances (EPS) to detoxify nanoparticles (NPs) are not well known. We ruled out the possibility of ion-specific bactericidal activity by showing the lack of equivalent dissolved zinc and silicon toxicity and determined the particle-specific toxicity of ZnO and SiO2 nanoparticles (ZnONPs/SiO2NPs) through dialysis isolation experiments. Surprisingly, the manipulation of the E. coli EPS (i.e., no EPS manipulation or EPS removal by sonication/centrifugation) showed that their particle-specific bactericidal activity could be antagonized by NP-EPS sequestration. The survival rates of pristine E. coli (no EPS manipulation) reached 65% (ZnONPs, 500 mg L−1) and 79% (SiO2NPs, 500 mg L−1), whereas survival rates following EPS removal by sonication/centrifugation were 11% and 63%, respectively. Transmission electron microscopy (TEM) combined with fluorescence micro-titration analysis and Fourier-transform infrared spectroscopy (FTIR) showed that protein-like substances (N-H and C-N in amide II) and secondary carbonyl groups (C=O) in the carboxylic acids of EPS acted as important binding sites that were involved in NP sequestration. Accordingly, the amount and composition of EPS produced by bacteria have important implications for the bactericidal efficacy and potential environmental effects of NPs. PMID:26856606

  1. Music training leads to the development of timbre-specific gamma band activity.

    PubMed

    Shahin, Antoine J; Roberts, Larry E; Chau, Wilkin; Trainor, Laurel J; Miller, Lee M

    2008-05-15

    Oscillatory gamma band activity (GBA, 30-100 Hz) has been shown to correlate with perceptual and cognitive phenomena including feature binding, template matching, and learning and memory formation. We hypothesized that if GBA reflects highly learned perceptual template matching, we should observe its development in musicians specific to the timbre of their instrument of practice. EEG was recorded in adult professional violinists and amateur pianists as well as in 4- and 5-year-old children studying piano in the Suzuki method before they commenced music lessons and 1 year later. The adult musicians showed robust enhancement of induced (non-time-locked) GBA, specifically to their instrument of practice, with the strongest effect in professional violinists. Consistent with this result, the children receiving piano lessons exhibited increased power of induced GBA for piano tones with 1 year of training, while children not taking lessons showed no effect. In comparison to induced GBA, evoked (time-locked) gamma band activity (30-90 Hz, approximately 80 ms latency) was present only in adult groups. Evoked GBA was more pronounced in musicians than non-musicians, with synchronization equally exhibited for violin and piano tones but enhanced for these tones compared to pure tones. Evoked gamma activity may index the physical properties of a sound and is modulated by acoustical training, while induced GBA may reflect higher perceptual learning and is shaped by specific auditory experiences.

  2. The Amino Acid Specificity for Activation of Phenylalanine Hydroxylase Matches the Specificity for Stabilization of Regulatory Domain Dimers

    PubMed Central

    2016-01-01

    Liver phenylalanine hydroxylase is allosterically activated by phenylalanine. The structural changes that accompany activation have not been identified, but recent studies of the effects of phenylalanine on the isolated regulatory domain of the enzyme support a model in which phenylalanine binding promotes regulatory domain dimerization. Such a model predicts that compounds that stabilize the regulatory domain dimer will also activate the enzyme. Nuclear magnetic resonance spectroscopy and analytical ultracentrifugation were used to determine the ability of different amino acids and phenylalanine analogues to stabilize the regulatory domain dimer. The abilities of these compounds to activate the enzyme were analyzed by measuring their effects on the fluorescence change that accompanies activation and on the activity directly. At concentrations of 10–50 mM, d-phenylalanine, l-methionine, l-norleucine, and (S)-2-amino-3-phenyl-1-propanol were able to activate the enzyme to the same extent as 1 mM l-phenylalanine. Lower levels of activation were seen with l-4-aminophenylalanine, l-leucine, l-isoleucine, and 3-phenylpropionate. The ability of these compounds to stabilize the regulatory domain dimer agreed with their ability to activate the enzyme. These results support a model in which allosteric activation of phenylalanine hydroxylase is linked to dimerization of regulatory domains. PMID:26252467

  3. The Amino Acid Specificity for Activation of Phenylalanine Hydroxylase Matches the Specificity for Stabilization of Regulatory Domain Dimers.

    PubMed

    Zhang, Shengnan; Hinck, Andrew P; Fitzpatrick, Paul F

    2015-08-25

    Liver phenylalanine hydroxylase is allosterically activated by phenylalanine. The structural changes that accompany activation have not been identified, but recent studies of the effects of phenylalanine on the isolated regulatory domain of the enzyme support a model in which phenylalanine binding promotes regulatory domain dimerization. Such a model predicts that compounds that stabilize the regulatory domain dimer will also activate the enzyme. Nuclear magnetic resonance spectroscopy and analytical ultracentrifugation were used to determine the ability of different amino acids and phenylalanine analogues to stabilize the regulatory domain dimer. The abilities of these compounds to activate the enzyme were analyzed by measuring their effects on the fluorescence change that accompanies activation and on the activity directly. At concentrations of 10-50 mM, d-phenylalanine, l-methionine, l-norleucine, and (S)-2-amino-3-phenyl-1-propanol were able to activate the enzyme to the same extent as 1 mM l-phenylalanine. Lower levels of activation were seen with l-4-aminophenylalanine, l-leucine, l-isoleucine, and 3-phenylpropionate. The ability of these compounds to stabilize the regulatory domain dimer agreed with their ability to activate the enzyme. These results support a model in which allosteric activation of phenylalanine hydroxylase is linked to dimerization of regulatory domains.

  4. Specific Determination of α-Amylase Activity in Crude Plant Extracts Containing β-Amylase 1

    PubMed Central

    Doehlert, Douglas C.; Duke, Stanley H.

    1983-01-01

    The specific measurement of α-amylase activity in crude plant extracts is difficult because of the presence of β-amylases which directly interfere with most assay methods. Methods compared in this study include heat treatment at 70°C for 20 min, HgCl2 treatment, and the use of the α-amylase specific substrate starch azure. In comparing alfalfa (Medicago sativa L.), soybeans (Glycine max [L.] Merr.), and malted barley (Hordeum vulgare L.), the starch azure assay was the only satisfactory method for all tissues. While β-amylase can liberate no color alone, over 10 International units per milliliter β-amylase activity has a stimulatory effect on the rate of color release. This stimulation becomes constant (about 4-fold) at β-amylase activities over 1,000 International units per milliliter. Two starch azure procedures were developed to eliminate β-amylase interference: (a) the dilution procedure, the serial dilution of samples until β-amylase levels are below levels that interfere; (b) the β-amylase saturation procedure, addition of exogenous β-amylase to increase endogenous β-amylase activity to saturating levels. Both procedures yield linear calibrations up to 0.3 International units per milliliter. These two procedures produced statistically identical results with most tissues, but not for all tissues. Differences between the two methods with some plant tissues was attributed to inaccuracy with the dilution procedure in tissues high in β-amylase activity or inhibitory effects of the commercial β-amylase. The β-amylase saturation procedure was found to be preferable with most species. The heat treatment was satisfactory only for malted barley, as α-amylases in alfalfa and soybeans are heat labile. Whereas HgCl2 proved to be a potent inhibitor of β-amylase activity at concentrations of 10 to 100 micromolar, these concentrations also partially inhibited α-amylase in barley malt. The reported α-amylase activities in crude enzyme extracts from a number

  5. The efficient production of high specific activity copper-64 using a biomedial cyclotron

    SciTech Connect

    McCarthy, D.W.; Shefer, R.E.; Klinkowstein, R.E.; Bass, L.A.

    1996-05-01

    We have developed a method for the efficient and cost-effective production of high specific activity Cu-64, via the Ni-64(p,n)Cu-64 reaction, using a small biomedical cyclotron. Nickel-64 (95% enriched) has been successfully electroplated on gold disks at thicknesses of {approximately}20-300 {mu}ms and bombarded with protons at beam currents up to {approximately}45 microamps. An automated target has been designed to facilitate the irradiations on a biomedical cyclotron. Techniques have been developed for the rapid and efficient separation of Cu-64 from Ni-64 and other reaction byproducts using ion exchange chromatography. An initial production run using 55 mg of 95% enriched Ni-64 yielded 20 GBq of Cu-64 with specific activity of 4.5 GBq/{mu}g (determined by serial dilution titrations with TETA). In a series of experiments, bombardment of 18.7-23.7 mg of 85% enriched Ni-64 has produced 8.9-18.5 GBq of Cu-64 with specific activity of 4.5 GBq/{mu}g (determined by serial dilution titrations with TETA). In a series of experiments, bombardment of 18.7-23.7 mg of 85% enriched Ni-64 has produced 8.9-18.5 GBq of Cu-64 (133 {plus_minus} 10 MBq/{mu}Ahr) with specific activity of 3.5 GBq-11.5 GBq/{mu}g. The amount and specific activity of the Cu-64 produced is more than adequate for both PET and therapy experiments. The Cu-64 produced in more than adequate for both PET and therapy experiments. The Cu-64 had been used to radiolabel PTSM (pyruvaldehyde bis (N4-methylthiosemicarbazone)-used to quantify blood flow), a monoclonal antibody (1A3) and octreotide. An efficient technique for recycling the costly enriched nickel-64 target material has been developed. Nickel eluted off the separation column is collected, boiled to dryness and redissolved in the electroplating bath. Using this method, 94.2 {plus_minus} 3.2% of the Ni-64 has been recovered. The technique described provides a simple, cost-effective method for the cyclotron production of Cu-64.

  6. Preparation and immunoreactivity of high specific activity indium-111-DTPA labeled monoclonal antibody (MoAb) using ultrapure indium-111

    SciTech Connect

    Zoghbi, S.S.; Neumann, R.D.; Gottschalk, A.

    1986-10-01

    The preparation of high-specific activity /sup 111/In-DTPA-MoAb without increasing the number of DTPA molecules per Ab was investigated. Instant thin layer chromatography was used to assay the relationship between labeling efficiencies and specific activities. With ultrapurified /sup 111/In, the specific activity of the radiolabeled MoAb approached the expected theoretic maximum of 100 muCi/microgram. The bioactivity of such high-specific activity preparation showed no degradation as measured by in vitro cell binding assay.

  7. Intralaminar stimulation of the inferior colliculus facilitates frequency-specific activation in the auditory cortex

    NASA Astrophysics Data System (ADS)

    Allitt, B. J.; Benjaminsen, C.; Morgan, S. J.; Paolini, A. G.

    2013-08-01

    Objective. Auditory midbrain implants (AMI) provide inadequate frequency discrimination for open set speech perception. AMIs that can take advantage of the tonotopic laminar of the midbrain may be able to better deliver frequency specific perception and lead to enhanced performance. Stimulation strategies that best elicit frequency specific activity need to be identified. This research examined the characteristic frequency (CF) relationship between regions of the auditory cortex (AC), in response to stimulated regions of the inferior colliculus (IC), comparing monopolar, and intralaminar bipolar electrical stimulation. Approach. Electrical stimulation using multi-channel micro-electrode arrays in the IC was used to elicit AC responses in anaesthetized male hooded Wistar rats. The rate of activity in AC regions with CFs within 3 kHz (CF-aligned) and unaligned CFs was used to assess the frequency specificity of responses. Main results. Both monopolar and bipolar IC stimulation led to CF-aligned neural activity in the AC. Altering the distance between the stimulation and reference electrodes in the IC led to changes in both threshold and dynamic range, with bipolar stimulation with 400 µm spacing evoking the lowest AC threshold and widest dynamic range. At saturation, bipolar stimulation elicited a significantly higher mean spike count in the AC at CF-aligned areas than at CF-unaligned areas when electrode spacing was 400 µm or less. Bipolar stimulation using electrode spacing of 400 µm or less also elicited a higher rate of elicited activity in the AC in both CF-aligned and CF-unaligned regions than monopolar stimulation. When electrodes were spaced 600 µm apart no benefit over monopolar stimulation was observed. Furthermore, monopolar stimulation of the external cortex of the IC resulted in more localized frequency responses than bipolar stimulation when stimulation and reference sites were 200 µm apart. Significance. These findings have implications for the

  8. Crystal structures of human tissue kallikrein 4: activity modulation by a specific zinc binding site.

    PubMed

    Debela, Mekdes; Magdolen, Viktor; Grimminger, Valerie; Sommerhoff, Christian; Messerschmidt, Albrecht; Huber, Robert; Friedrich, Rainer; Bode, Wolfram; Goettig, Peter

    2006-10-01

    Human tissue kallikrein 4 (hK4) belongs to a 15-member family of closely related serine proteinases. hK4 is predominantly expressed in prostate, activates hK3/PSA, and is up-regulated in prostate and ovarian cancer. We have identified active monomers of recombinant hK4 besides inactive oligomers in solution. hK4 crystallised in the presence of zinc, nickel, and cobalt ions in three crystal forms containing cyclic tetramers and octamers. These structures display a novel metal site between His25 and Glu77 that links the 70-80 loop with the N-terminal segment. Micromolar zinc as present in prostatic fluid inhibits the enzymatic activity of hK4 against fluorogenic substrates. In our measurements, wild-type hK4 exhibited a zinc inhibition constant (IC50) of 16 microM including a permanent residual activity, in contrast to the zinc-independent mutants H25A and E77A. Since the Ile16 N terminus of wild-type hK4 becomes more accessible for acetylating agents in the presence of zinc, we propose that zinc affects the hK4 active site via the salt-bridge formed between the N terminus and Asp194 required for a functional active site. hK4 possesses an unusual 99-loop that creates a groove-like acidic S2 subsite. These findings explain the observed specificity of hK4 for the P1 to P4 substrate residues. Moreover, hK4 shows a negatively charged surface patch, which may represent an exosite for prime-side substrate recognition. PMID:16950394

  9. Non-specific binding sites help to explain mixed inhibition in mushroom tyrosinase activities.

    PubMed

    Hassani, Sorour; Haghbeen, Kamahldin; Fazli, Mostafa

    2016-10-21

    Inhibition and activation studies of tyrosinase could prove beneficial to agricultural, food, cosmetic, and pharmaceutical industries. Although non-competitive and mixed-inhibition are frequent modes observed in kinetics studies on mushroom tyrosinase (MT) activities, the phenomena are left unexplained. In this study, dual effects of phthalic acid (PA) and cinnamic acid (CA) on MT during mono-phenolase activity were demonstrated. PA activated and inhibited MT at concentrations lower and higher than 150 μM, respectively. In contrast, CA inhibited and activated MT at concentrations lower and higher than 5 μM. The mode of inhibition for both effectors was mixed-type. Complex kinetics of MT in the presence of a modulator could partly be ascribed to its mixed-cooperativity. However, to explain mixed-inhibition mode, it is necessary to demonstrate how the ternary complex of substrate/enzyme/effector is formed. Therefore, we looked for possible non-specific binding sites using MT tropolone-bound PDB (2Y9X) in the computational studies. When tropolone was in MTPa (active site), PA and CA occupied different pockets (named MTPb and MTPc, respectively). The close Moldock scores of PA binding posed in MTPb and MTPa suggested that MTPb could be a secondary binding site for PA. Similar results were obtained for CA. Ensuing results from 10 ns molecular dynamics simulations for 2Y9X-effector complexes indicated that the structures were gradually stabilized during simulation. Tunnel analysis by using CAVER Analyst and CHEXVIS resulted in identifying two distinct channels that assumingly participate in exchanging the effectors when the direct channel to MTPa is not accessible.

  10. Rapid direct methods for enumeration of specific, active bacteria in water and biofilms

    NASA Technical Reports Server (NTRS)

    McFeters, G. A.; Pyle, B. H.; Lisle, J. T.; Broadaway, S. C.

    1999-01-01

    Conventional methods for detecting indicator and pathogenic bacteria in water may underestimate the actual population due to sublethal environmental injury, inability of the target bacteria to take up nutrients and other physiological factors which reduce bacterial culturability. Rapid and direct methods are needed to more accurately detect and enumerate active bacteria. Such a methodological advance would provide greater sensitivity in assessing the microbiological safety of water and food. The principle goal of this presentation is to describe novel approaches we have formulated for the rapid and simultaneous detection of bacteria plus the determination of their physiological activity in water and other environmental samples. The present version of our method involves the concentration of organisms by membrane filtration or immunomagnetic separation and combines an intracellular fluorochrome (CTC) for assessment of respiratory activity plus fluorescent-labelled antibody detection of specific bacteria. This approach has also been successfully used to demonstrate spatial and temporal heterogeneities of physiological activities in biofilms when coupled with cryosectioning. Candidate physiological stains include those capable of determining respiratory activity, membrane potential, membrane integrity, growth rate and cellular enzymatic activities. Results obtained thus far indicate that immunomagnetic separation can provide a high degree of sensitivity in the recovery of seeded target bacteria (Escherichia coli O157:H7) in water and hamburger. The captured and stained target bacteria are then enumerated by either conventional fluorescence microscopy or ChemScan(R), a new instrument that is very sensitive and rapid. The ChemScan(R) laser scanning instrument (Chemunex, Paris, France) provides the detection of individual fluorescently labelled bacterial cells using three emission channels in less than 5 min. A high degree of correlation has been demonstrated between

  11. Non-specific binding sites help to explain mixed inhibition in mushroom tyrosinase activities.

    PubMed

    Hassani, Sorour; Haghbeen, Kamahldin; Fazli, Mostafa

    2016-10-21

    Inhibition and activation studies of tyrosinase could prove beneficial to agricultural, food, cosmetic, and pharmaceutical industries. Although non-competitive and mixed-inhibition are frequent modes observed in kinetics studies on mushroom tyrosinase (MT) activities, the phenomena are left unexplained. In this study, dual effects of phthalic acid (PA) and cinnamic acid (CA) on MT during mono-phenolase activity were demonstrated. PA activated and inhibited MT at concentrations lower and higher than 150 μM, respectively. In contrast, CA inhibited and activated MT at concentrations lower and higher than 5 μM. The mode of inhibition for both effectors was mixed-type. Complex kinetics of MT in the presence of a modulator could partly be ascribed to its mixed-cooperativity. However, to explain mixed-inhibition mode, it is necessary to demonstrate how the ternary complex of substrate/enzyme/effector is formed. Therefore, we looked for possible non-specific binding sites using MT tropolone-bound PDB (2Y9X) in the computational studies. When tropolone was in MTPa (active site), PA and CA occupied different pockets (named MTPb and MTPc, respectively). The close Moldock scores of PA binding posed in MTPb and MTPa suggested that MTPb could be a secondary binding site for PA. Similar results were obtained for CA. Ensuing results from 10 ns molecular dynamics simulations for 2Y9X-effector complexes indicated that the structures were gradually stabilized during simulation. Tunnel analysis by using CAVER Analyst and CHEXVIS resulted in identifying two distinct channels that assumingly participate in exchanging the effectors when the direct channel to MTPa is not accessible. PMID:27344491

  12. Anti-rhinovirus-specific activity of the alpha-sympathomimetic oxymetazoline.

    PubMed

    Koelsch, Stephan; Tschaikin, Marion; Sacher, Fritz

    2007-01-01

    Oxymetazoline (CAS 2315-02-8, OMZ, Nasivin) known as the active ingredient in nose drops and sprays demonstrates excellent efficacy in the treatment of rhinitis symptoms that are mainly caused by Rhinovirus infections. To elucidate possible modes of action, the antiviral activity of OMZ was studied in vitro on human pathogenic viruses. No in vitro effects were detected against enveloped RNA viruses, Parainfluenza Virus and Respiratory Syncytial Virus and against Adenovirus, a non-enveloped DNA-virus. In contrast, OMZ showed a specific inhibition of Human Rhinovirus (HRV). Analysis of production of HRV-14 and HRV-39 after treatment of infected HeLa cells using plaque-reduction assay and virus titration showed a strong dose-dependent antiviral activity of OMZ. Additional data demonstrated that OMZ did also directly affect HRV-14 infectivity in a dose-dependent manner. Analysis of a cell-protective effect of OMZ showed that pre-treatment of HeLa cells decreased virus adsorption as well as virus replication. Furthermore, OMZ induced a down-regulation of ICAM-1 expression on Tumor Necrosis Factor-alpha (TNF-alpha)-stimulated HeLa cells and human umbilical vein endothelial cells. Taken together, these results show that OMZ besides its vasoconstrictive action also possesses potent antiviral and anti-inflammatory activities. Therefore, OMZ does not only reduce rhinitis symptoms but additionally offers a causal therapeutic approach.

  13. In vivo bioimaging with tissue-specific transcription factor activated luciferase reporters

    PubMed Central

    Buckley, Suzanne M. K.; Delhove, Juliette M. K. M.; Perocheau, Dany P.; Karda, Rajvinder; Rahim, Ahad A.; Howe, Steven J.; Ward, Natalie J.; Birrell, Mark A.; Belvisi, Maria G.; Arbuthnot, Patrick; Johnson, Mark R.; Waddington, Simon N.; McKay, Tristan R.

    2015-01-01

    The application of transcription factor activated luciferase reporter cassettes in vitro is widespread but potential for in vivo application has not yet been realized. Bioluminescence imaging enables non-invasive tracking of gene expression in transfected tissues of living rodents. However the mature immune response limits luciferase expression when delivered in adulthood. We present a novel approach of tissue-targeted delivery of transcription factor activated luciferase reporter lentiviruses to neonatal rodents as an alternative to the existing technology of generating germline transgenic light producing rodents. At this age, neonates acquire immune tolerance to the conditionally responsive luciferase reporter. This simple and transferrable procedure permits surrogate quantitation of transcription factor activity over the lifetime of the animal. We show principal efficacy by temporally quantifying NFκB activity in the brain, liver and lungs of somatotransgenic reporter mice subjected to lipopolysaccharide (LPS)-induced inflammation. This response is ablated in Tlr4−/− mice or when co-administered with the anti-inflammatory glucocorticoid analogue dexamethasone. Furthermore, we show the malleability of this technology by quantifying NFκB-mediated luciferase expression in outbred rats. Finally, we use somatotransgenic bioimaging to longitudinally quantify LPS- and ActivinA-induced upregulation of liver specific glucocorticoid receptor and Smad2/3 reporter constructs in somatotransgenic mice, respectively. PMID:26138224

  14. A novel enzyme activity involving the demethylation of specific partially methylated oligogalacturonides.

    PubMed Central

    Williams, Martin A K; Benen, Jacques A E

    2002-01-01

    Studies of the enzymic digestion of pectic substrates using different polygalacturonase (PG) preparations have revealed evidence for a previously unreported enzyme activity carried out by a contaminating enzyme in one of the preparations. This observed activity involves the demethylation of specific oligogalacturonides, namely 2-methyltrigalacturonic acid and 2,3-dimethyltetragalacturonic acid. However, no large-scale demethylation of highly methylated polymeric substrates is found, demonstrating that the enzyme responsible is not a conventional pectin methylesterase (PME). Furthermore, it has been shown that a commercial sample of fungal PME from Aspergillus niger demethylates all of the oligogalacturonides present as primary products of endo-PG digestion, in contrast with the activity observed here. On the basis of the known methyl ester distribution of the endo-PG-generated fragments and knowledge of which of these oligogalacturonides are demethylated, it is concluded that the observed activity can be explained by the existence of an exo-acting methylesterase that attacks the non-reducing end of the oligogalacturonide molecules. PMID:12097140

  15. Sin Nombre hantavirus nucleocapsid protein exhibits a metal-dependent DNA-specific endonucleolytic activity.

    PubMed

    Möncke-Buchner, Elisabeth; Szczepek, Michal; Bokelmann, Marcel; Heinemann, Patrick; Raftery, Martin J; Krüger, Detlev H; Reuter, Monika

    2016-09-01

    We demonstrate that the nucleocapsid protein of Sin Nombre hantavirus (SNV-N) has a DNA-specific endonuclease activity. Upon incubation of SNV-N with DNA in the presence of magnesium or manganese, we observed DNA digestion in sequence-unspecific manner. In contrast, RNA was not affected under the same conditions. Moreover, pre-treatment of SNV-N with RNase before DNA cleavage increased the endonucleolytic activity. Structure-based protein fold prediction using known structures from the PDB database revealed that Asp residues in positions 88 and 103 of SNV-N show sequence similarity with the active site of the restriction endonuclease HindIII. Crystal structure of HindIII predicts that residues Asp93 and Asp108 are essential for coordination of the metal ions required for HindIII DNA cleavage. Therefore, we hypothesized that homologous residues in SNV-N, Asp88 and Asp103, may have a similar function. Replacing Asp88 and Asp103 by alanine led to an SNV-N protein almost completely abrogated for endonuclease activity. PMID:27261891

  16. Activation of Plant Plasma Membrane Ca2+-Permeable Channels by Race-Specific Fungal Elicitors.

    PubMed Central

    Gelli, A.; Higgins, V. J.; Blumwald, E.

    1997-01-01

    The response of plant cells to invading pathogens is regulated by fluctuations in cytosolic Ca2+ levels that are mediated by Ca2+-permeable channels located at the plasma membrane of the host cell. The mechanisms by which fungal elicitors can induce Ca2+ uptake by the host cell were examined by the application of conventional patch-clamp techniques. Whole-cell and single-channel experiments on tomato (Lycopersicon esculentum L.) protoplasts revealed a race-specific fungal elicitor-induced activation of a plasma membrane Ca2+-permeable channel. The presence of the fungal elicitor resulted in a greater probability of channel opening. Guanosine 5[prime]-[[beta]-thio]diphosphate, a GDP analog that locks heterotrimeric G-proteins into their inactivated state, abolished the channel activation induced by the fungal elicitor, whereas guanosine 5[prime][[gamma]-thio]triphosphate, a nonhydrolyzable GTP analog that locks heterotrimeric G-proteins into their activated state, produced an effect similar to that observed with the fungal elicitor. Mastoparan, which stimulates GTPase activity, mimicked the effect of GTP[[gamma

  17. Tailoring activated carbons for the development of specific adsorbents of gasoline vapors.

    PubMed

    Vivo-Vilches, J F; Bailón-García, E; Pérez-Cadenas, A F; Carrasco-Marín, F; Maldonado-Hódar, F J

    2013-12-15

    The specific adsorption of oxygenated and aliphatic gasoline components onto activated carbons (ACs) was studied under static and dynamic conditions. Ethanol and n-octane were selected as target molecules. A highly porous activated carbon (CA) was prepared by means of two processes: carbonization and chemical activation of olive stone residues. Different types of oxygenated groups, identified and quantified by TPD and XPS, were generated on the CA surface using an oxidation treatment with ammonium peroxydisulfate and then selectively removed by thermal treatments, as confirmed by TPD results. Chemical and porous transformations were carefully analyzed throughout these processes and related to their VOC removal performance. The analysis of the adsorption process under static conditions and the thermal desorption of VOCs enabled us to determine the total adsorption capacity and regeneration possibilities. Breakthrough curves obtained for the adsorption process carried out under dynamic conditions provided information about the mass transfer zone in each adsorption bed. While n-octane adsorption is mainly determined by the porosity of activated carbons, ethanol adsorption is related to their surface chemistry, and in particular is enhanced by the presence of carboxylic acid groups.

  18. Human thalamus regulates cortical activity via spatially specific and structurally constrained phase-amplitude coupling.

    PubMed

    Malekmohammadi, Mahsa; Elias, W Jeff; Pouratian, Nader

    2015-06-01

    Although the thalamus is believed to regulate and coordinate cortical activity both within and across functional regions, such as motor and visual cortices, direct evidence for such regulation and the mechanism of regulation remains poorly described. Using simultaneous invasive recordings of cortical and thalamic electrophysiological activity in 2 awake and spontaneously behaving human subjects, we provide direct evidence of thalamic regulation of cortical activity through a mechanism of phase-amplitude coupling (PAC), in which the phase of low frequency oscillations regulates the amplitude of higher frequency oscillations. Specifically, we show that cortical PAC between the theta phase and beta amplitude is spatially dependent on and time variant with the magnitude of thalamocortical theta coherence. Moreover, using causality analysis and MR diffusion tractography, we provide evidence that thalamic theta activity drives cortical theta oscillations and PAC across structures and that these thalamocortical relationships are structurally constrained by anatomic pathways. This relationship allows for new evidence of thalamocortical PAC. Given the diffuse connectivity of the thalamus with the cerebral cortex, thalamocortical PAC may play an important role in addressing the binding problem, including both integration and segregation of information within and across cortical areas.

  19. A serendipitous discovery of antifreeze protein-specific activity in C-linked antifreeze glycoprotein analogs.

    PubMed

    Eniade, Adewale; Purushotham, Madhusudhan; Ben, Robert N; Wang, J B; Horwath, Kathleen

    2003-01-01

    Structurally diverse carbon-linked (C-linked) analogs of antifreeze glycoprotein (AFGP) have been prepared via linear or convergent solid phase synthesis. These analogs range in molecular weight from approx 1.5-4.1 KDa and do not possess the beta-D-galactose-1,3-alpha-D-N-acetylgalactosamine carbohydrate moiety or the L-threonine-L-alanine-L-alanine polypeptide backbone native to the AFGP wild-type. Despite these dramatic structural modifications, the 2.7-KDa and 4.1-KDa analogs possess antifreeze protein-specific activity as determined by recrystallization-inhibition (RI) and thermal hysteresis (TH) assays. These analogs are weaker than the wild-type in their activity, but nanoliter osmometry indicates that these compounds are binding to ice and affecting a localized freezing point depression. This is the first example of a C-linked AFGP analog that possesses TH and RI activity and suggests that the rational design and synthesis of chemically and biologically stable AFGP analogs is a feasible and worthwhile endeavor. Given the low degree of TH activity, these compounds may prove useful for the protection of cells during freezing and thawing cycles. PMID:12777711

  20. Identification of three kinds of mutually related composite elements conferring S phase-specific transcriptional activation.

    PubMed

    Taoka, K; Kaya, H; Nakayama, T; Araki, T; Meshi, T; Iwabuchi, M

    1999-06-01

    Conservation of the Oct motif (CGCGGATC) is a remarkable feature of plant histone gene promoters. Many of the Oct motifs are paired with a distinct motif, Hex, TCA or CCAAT-box, constituting the type I element (CCACGTCANCGATCCGCG), type II element (TCACGCGGATC) and type III element (GATCCGCG-N14-ACCAATCA). To clarify the roles of these Oct-containing composite elements (OCEs) in cell cycle-dependent and tissue-specific expression, we performed gain-of-function experiments with transgenic tobacco cell lines and plants harboring a derivative of the 35S core promoter/beta-glucuronidase fusion gene in which three or four copies of an OCE had been placed upstream. Although their activities were slightly different, results showed that each of the three types of OCEs could confer the ability to direct S phase-specific expression on a heterologous promoter. In transgenic plants, the type I and III elements exhibited a similar activity, directing expression in meristematic tissues, whereas the activity of the type II element appeared to be restricted to young cotyledons and maturating guard cells. Mutational analyses demonstrated that the co-operation of Oct with another module (Hex, TCA or CCAAT-box) was absolutely required for both temporal and spatial regulation. Thus, OCEs play a pivotal role in regulation of the expression of plant histone genes.

  1. Cell-specific production and antimicrobial activity of naphthoquinones in roots of lithospermum erythrorhizon

    PubMed

    Brigham; Michaels; Flores

    1999-02-01

    Pigmented naphthoquinone derivatives of shikonin are produced at specific times and in specific cells of Lithospermum erythrorhizon roots. Normal pigment development is limited to root hairs and root border cells in hairy roots grown on "noninducing" medium, whereas induction of additional pigment production by abiotic (CuSO4) or biotic (fungal elicitor) factors increases the amount of total pigment, changes the ratios of derivatives produced, and initiates production of pigment de novo in epidermal cells. When the biological activity of these compounds was tested against soil-borne bacteria and fungi, a wide range of sensitivity was recorded. Acetyl-shikonin and beta-hydroxyisovaleryl-shikonin, the two most abundant derivatives in both Agrobacterium rhizogenes-transformed "hairy-root" cultures and greenhouse-grown plant roots, were the most biologically active of the seven compounds tested. Hyphae of the pathogenic fungi Rhizoctonia solani, Pythium aphanidermatum, and Nectria hematococca induced localized pigment production upon contact with the roots. Challenge by R. solani crude elicitor increased shikonin derivative production 30-fold. We have studied the regulation of this suite of related, differentially produced, differentially active compounds to understand their role(s) in plant defense at the cellular level in the rhizosphere.

  2. What's up? Emotion-specific activation of vertical space during language processing.

    PubMed

    Dudschig, Carolin; de la Vega, Irmgard; Kaup, Barbara

    2015-03-01

    The relationship between language processing and vertical space has been shown for various groups of words including valence words, implicit location words, and words referring to religious concepts. However, it remains unclear whether these are single phenomena or whether there is an underlying common mechanism. Here, we show that the evaluation of word valence interacts with motor responses in the vertical dimension, with positive (negative) evaluations facilitating upward (downward) responses. When valence evaluation was not required, implicit location words (e.g., bird, shoe) influenced motor responses whereas valence words (e.g., kiss, hate) did not. Importantly, a subset of specific emotional valence words that are commonly associated with particular bodily postures (e.g., proud→upright; sad→slouched) did automatically influence motor responses. Together, this suggests that while the vertical spatial dimension is not directly activated by word valence, it is activated when processing words referring to emotional states with stereotypical bodily-postures. These results provide strong evidence that the activation of spatial associations during language processing is experience-specific in nature and cannot be explained with reference to a general mapping between all valence words and space (i.e., all positive and negative words generally relate to spatial processing). These findings support the experiential view of language comprehension, suggesting that the automatic reactivation of bodily experiences is limited to word groups referring to emotions or entities directly associated with spatial experiences (e.g., posture or location in the world). PMID:25454886

  3. Acute stress differentially affects aromatase activity in specific brain nuclei of adult male and female quail.

    PubMed

    Dickens, Molly J; Cornil, Charlotte A; Balthazart, Jacques

    2011-11-01

    The rapid and temporary suppression of reproductive behavior is often assumed to be an important feature of the adaptive acute stress response. However, how this suppression operates at the mechanistic level is poorly understood. The enzyme aromatase converts testosterone to estradiol in the brain to activate reproductive behavior in male Japanese quail (Coturnix japonica). The discovery of rapid and reversible modification of aromatase activity (AA) provides a potential mechanism for fast, stress-induced changes in behavior. We investigated the effects of acute stress on AA in both sexes by measuring enzyme activity in all aromatase-expressing brain nuclei before, during, and after 30 min of acute restraint stress. We show here that acute stress rapidly alters AA in the male and female brain and that these changes are specific to the brain nuclei and sex of the individual. Specifically, acute stress rapidly (5 min) increased AA in the male medial preoptic nucleus, a region controlling male reproductive behavior; in females, a similar increase was also observed, but it appeared delayed (15 min) and had smaller amplitude. In the ventromedial and tuberal hypothalamus, regions associated with female reproductive behavior, stress induced a quick and sustained decrease in AA in females, but in males, only a slight increase (ventromedial) or no change (tuberal) in AA was observed. Effects of acute stress on brain estrogen production, therefore, represent one potential way through which stress affects reproduction.

  4. In vivo efficacy of platelet-delivered, high specific activity factor VIII variants

    PubMed Central

    Greene, Teshell K.; Wang, Cheng; Hirsch, Jessica D.; Zhai, Li; Gewirtz, Jamie; Thornton, Michael A.; Miao, Hongzhi Z.; Pipe, Steven W.; Kaufman, Randal J.; Camire, Rodney M.; Arruda, Valder R.; Kowalska, M. Anna

    2010-01-01

    Ectopically expressed, human B-domainless (hB) factor 8 (F8) in platelets improves hemostasis in hemophilia A mice in several injury models. However, in both a cuticular bleeding model and a cremaster laser arteriole/venule injury model, there were limitations to platelet-derived (p) hBF8 efficacy, including increased clot embolization. We now address whether variants of F8 with enhanced activity, inactivation resistant F8 (IR8) and canine (c) BF8, would improve clotting efficacy. In both transgenic and lentiviral murine model approaches, pIR8 expressed at comparable levels to phBF8, but pcBF8 expressed at only approximately 30%. Both variants were more effective than hBF8 in cuticular bleeding and FeCl3 carotid artery models. However, in the cremaster injury model, only pcBF8 was more effective, markedly decreasing clot embolization. Because inhibitors of F8 are stored in platelet granules and IR8 is not protected by binding to von Willebrand factor, we also tested whether pIR8 was effective in the face of inhibitors and found that pIR8 is protected from the inhibitors. In summary, pF8 variants with high specific activity are more effective in controlling bleeding, but this improved efficacy was inconsistent between bleeding models, perhaps reflecting the underlying mechanism(s) for the increased specific activity of the studied F8 variants. PMID:20852129

  5. Enzymatic activity and substrate specificity of the recombinant tomato β-galactosidase 1.

    PubMed

    Eda, Masahiro; Ishimaru, Megumi; Tada, Toshiji; Sakamoto, Tatsuji; Kotake, Toshihisa; Tsumuraya, Yoichi; Mort, Andrew J; Gross, Kenneth C

    2014-10-15

    The open reading frame of tomato β-galactosidase 1 was expressed in yeast, and the enzymatic properties and substrate specificity were investigated. The enzyme had peak activity at pH 5.0 and 40-50°C. TBG1 was active on β-(1,3)- and β-(1,6)-galactobiose and lactose. TBG1 released galactose from lupin galactan, tomato fruit alkali soluble pectin, arabinogalactan, gum arabic and methyl β-(1,6)-galactohexaoside, but not from labeled β-(1,4)-galactoheptaose. TBG1 was assessed for its ability to degrade three galactosyl-containing cell wall fractions purified from different development and ripening stages of tomato fruit. TBG1 released galactose from all of the fractions from all of the stages tested. TBG1 activity was highest on the hemicellulose fraction at the 10 and 20d after pollination stage. This result is not correlated the with TBG1 expression pattern. TBG1 might act on a small but specific set of polysaccharide containing galactose.

  6. Acetohydroxyacid synthase activity and transcripts profiling reveal tissue-specific regulation of ahas genes in sunflower.

    PubMed

    Ochogavía, Ana C; Breccia, Gabriela; Vega, Tatiana; Felitti, Silvina A; Picardi, Liliana A; Nestares, Graciela

    2014-07-01

    Acetohydroxyacid synthase (AHAS) is the target site of several herbicides and catalyses the first step in the biosynthesis of branched chain amino acid. Three genes coding for AHAS catalytic subunit (ahas1, ahas2 and ahas3) have been reported for sunflower. The aim of this work was to study the expression pattern of ahas genes family and AHAS activity in sunflower (Helianthus annuus L.). Different organs (leaves, hypocotyls, roots, flowers and embryos) were evaluated at several developmental stages. The transcriptional profile was studied through RT-qPCR. The highest expression for ahas1 was shown in leaves, where all the induced and natural gene mutations conferring herbicide resistance were found. The maximal expression of ahas2 and ahas3 occurred in immature flowers and embryos. The highest AHAS activity was found in leaves and immature embryos. Correlation analysis among ahas gene expression and AHAS activity was discussed. Our results show that differences in ahas genes expression are tissue-specific and temporally regulated. Moreover, the conservation of multiple AHAS isoforms in sunflower seems to result from different expression requirements controlled by tissue-specific regulatory mechanisms at different developmental stages. PMID:24908515

  7. Cell-specific activation of the human skeletal alpha-actin by androgens.

    PubMed

    Hong, Mei Hua; Sun, Hong; Jin, Cheng He; Chapman, Mark; Hu, Junlian; Chang, William; Burnett, Kelven; Rosen, Jon; Negro-Vilar, Andres; Miner, Jeffrey N

    2008-03-01

    Although it is evident that androgens increase muscle mass and strength, little is known about the critical molecular targets of androgens in skeletal muscle. In rodents, the skeletal alpha-actin gene is a tissue-specific gene expressed only in the levator ani and other skeletal muscles but not in the prostate or preputial gland, the well-known androgen target tissue. We identified tissue-specific androgen-regulated genes in the skeletal muscle in rats after oral administration of androgens and focused on androgen-dependent up-regulation of the skeletal alpha-actin gene. To investigate the mechanism of action, an in vitro system with various cell lines and a series of deletion mutants of the alpha-actin promoter were used. The human skeletal alpha-actin promoter was activated by androgens in the muscle cell line C2C12 but not in the liver, prostate, or breast cancer cell lines in which exogenous human androgen receptor is expressed. The sequence of the promoter is sufficient for cell-specific androgen response, providing a model for the tissue specificity demonstrated in vivo. Using a series of deletion mutants, the androgen response can be maintained using just the proximal promoter region. The importance of androgen regulation of this small portion of the human skeletal alpha-actin promoter was demonstrated by the correlation between muscle and the alpha-actin promoter activity for an array of selective androgen receptor modulators (SARMs), including an orally active SARM LGD2226. Taken together, the results suggest that the regulation of skeletal alpha-actin by androgens/SARMs may represent an important model system for understanding androgen anabolic action in the muscle.

  8. Sex-specific foraging behavior in response to fishing activities in a threatened seabird

    PubMed Central

    García-Tarrasón, Manuel; Bécares, Juan; Bateman, Santiago; Arcos, José Manuel; Jover, Lluís; Sanpera, Carolina

    2015-01-01

    Some seabird species have learnt to efficiently exploit fishing discards from trawling activities. However, a discard ban has been proposed as necessary in Europe to ensure the sustainability of the seas. It is of crucial importance for the management and conservation purposes to study the potential consequences of a discard ban on the foraging ecology of threatened seabirds. We assessed the influence of fishing activities on the feeding habits of 22 male and 15 female Audouin's gulls (Larus audouinii) from the Ebro Delta (Mediterranean Sea) during the breeding period using GPS loggers together with Stable Isotope Analysis (SIA), which provided new insights into their foraging behavior and trophic ecology, respectively. GPS data revealed different sex-specific foraging patterns between workdays and weekends. Females were highly consistent in that they foraged at sea throughout the week even though discarding stops at weekends. In contrast, males switched from foraging at sea during the week (when discards are produced) to an increased use of rice field habitats at weekends (when fishermen do not work). This sex-specific foraging behavior could be related to specific nutritional requirements associated with previous egg production, an energetically demanding period for females. However, on a broader time scale integrated by the SIA, both sexes showed a high degree of individual specialization in their trophic ecology. The need to obtain detailed information on the dependence and response of seabirds to fishing activities is crucial in conservation sciences. In this regard, sex-specific foraging behavior in relation to fisheries has been overlooked, despite the ecological and conservation implications. For instance, this situation may lead to sex differentiation in bycatch mortality in longlines when trawlers do not operate. Moreover, any new fisheries policy will need to be implemented gradually to facilitate the adaptation of a specialized species to a discard ban

  9. Body side-specific control of motor activity during turning in a walking animal.

    PubMed

    Gruhn, Matthias; Rosenbaum, Philipp; Bockemühl, Till; Büschges, Ansgar

    2016-04-27

    Animals and humans need to move deftly and flexibly to adapt to environmental demands. Despite a large body of work on the neural control of walking in invertebrates and vertebrates alike, the mechanisms underlying the motor flexibility that is needed to adjust the motor behavior remain largely unknown. Here, we investigated optomotor-induced turning and the neuronal mechanisms underlying the differences between the leg movements of the two body sides in the stick insect Carausius morosus. We present data to show that the generation of turning kinematics in an insect are the combined result of descending unilateral commands that change the leg motor output via task-specific modifications in the processing of local sensory feedback as well as modification of the activity of local central pattern generating networks in a body-side-specific way. To our knowledge, this is the first study to demonstrate the specificity of such modifications in a defined motor task.

  10. A multiplexed transcription activator-like effector system for detecting specific DNA sequences.

    PubMed

    Honarmand, Ali; Mayall, Robert; George, Iain; Oberding, Lisa; Dastidar, Himika; Fegan, Jamie; Chaudhuri, Somshukla; Dole, Justin; Feng, Sharon; Hoang, Denny; Moges, Ruth; Osgood, Julie; Remondini, Taylor; van der Meulen, Wm Keith; Wang, Su; Wintersinger, Chris; Zaparoli Zucoloto, Amanda; Chatfield-Reed, Kate; Arcellana-Panlilio, Mayi; Nygren, Anders

    2014-12-19

    Transcription activator-like effectors (TALEs), originating from the Xanthomonas genus of bacteria, bind to specific DNA sequences based on amino acid sequence in the repeat-variable diresidue (RVD) positions of the protein. By altering these RVDs, it has been shown that a TALE protein can be engineered to bind virtually any DNA sequence of interest. The possibility of multiplexing TALEs for the purposes of identifying specific DNA sequences has yet to be explored. Here, we demonstrate a system in which a TALE protein bound to a nitrocellulose strip has been utilized to capture purified DNA, which is then detected using the binding of a second distinct TALE protein conjugated to a protein tag that is then detected by a dot blot. This system provides a signal only when both TALEs bind to their respective sequences, further demonstrating the specificity of the TALE binding.

  11. A multiplexed transcription activator-like effector system for detecting specific DNA sequences.

    PubMed

    Honarmand, Ali; Mayall, Robert; George, Iain; Oberding, Lisa; Dastidar, Himika; Fegan, Jamie; Chaudhuri, Somshukla; Dole, Justin; Feng, Sharon; Hoang, Denny; Moges, Ruth; Osgood, Julie; Remondini, Taylor; van der Meulen, Wm Keith; Wang, Su; Wintersinger, Chris; Zaparoli Zucoloto, Amanda; Chatfield-Reed, Kate; Arcellana-Panlilio, Mayi; Nygren, Anders

    2014-12-19

    Transcription activator-like effectors (TALEs), originating from the Xanthomonas genus of bacteria, bind to specific DNA sequences based on amino acid sequence in the repeat-variable diresidue (RVD) positions of the protein. By altering these RVDs, it has been shown that a TALE protein can be engineered to bind virtually any DNA sequence of interest. The possibility of multiplexing TALEs for the purposes of identifying specific DNA sequences has yet to be explored. Here, we demonstrate a system in which a TALE protein bound to a nitrocellulose strip has been utilized to capture purified DNA, which is then detected using the binding of a second distinct TALE protein conjugated to a protein tag that is then detected by a dot blot. This system provides a signal only when both TALEs bind to their respective sequences, further demonstrating the specificity of the TALE binding. PMID:25524096

  12. Evaluating bacterial activity from cell-specific ribosomal RNA content measured with oligonucleotide probes

    SciTech Connect

    Kemp, P.F.; Lee, S.; LaRoche, J.

    1992-10-01

    We describe a procedure for measuring the cell-specific quantity of ribosomal RNA (rRNA) and DNA in order to evaluate the frequency distribution of activity among cells. The procedure is inherently quantitative, does not require sample incubation and potentially can be taxon-specific. Fluorescently-labelled oligonucleotide probes are hybridized to the complementary 16S rRNA sequences in preserved, intact cells. The resulting cell fluorescence is proportional to cellular rRNA content and can be measured with a microscope-mounted photometer system, by image analysis, or by flow cytometry. Similarly, DNA content is measured as fluorescence of cells stained with the DNA specific fluorochrome DAPI. These are either prepared as separate samples for purposes of enumeration and DNA measurements, or are dual-labelled cells which are also hybridized with oligonucleotide probes.

  13. Evaluating bacterial activity from cell-specific ribosomal RNA content measured with oligonucleotide probes

    SciTech Connect

    Kemp, P.F.; Lee, S.; LaRoche, J.

    1992-01-01

    We describe a procedure for measuring the cell-specific quantity of ribosomal RNA (rRNA) and DNA in order to evaluate the frequency distribution of activity among cells. The procedure is inherently quantitative, does not require sample incubation and potentially can be taxon-specific. Fluorescently-labelled oligonucleotide probes are hybridized to the complementary 16S rRNA sequences in preserved, intact cells. The resulting cell fluorescence is proportional to cellular rRNA content and can be measured with a microscope-mounted photometer system, by image analysis, or by flow cytometry. Similarly, DNA content is measured as fluorescence of cells stained with the DNA specific fluorochrome DAPI. These are either prepared as separate samples for purposes of enumeration and DNA measurements, or are dual-labelled cells which are also hybridized with oligonucleotide probes.

  14. Site-specific fatty chain-modified exenatide analogs with balanced glucoregulatory activity and prolonged in vivo activity.

    PubMed

    Sun, Lidan; Huang, Xun; Han, Jing; Cai, Xingguang; Dai, Yuxuan; Chu, Yingying; Wang, Chuandong; Huang, Wenlong; Qian, Hai

    2016-06-15

    The therapeutic utility of exenatide (Ex-4) is limited due to short plasma half-life of 2.4h and thus numerous approaches have been used to obtain a longer action time. However, such strategies often attend to one thing and lose another. The study aimed to identify a candidate with balanced glucoregulatory activity and prolonged in vivo activity. A series of fatty chain conjugates of Ex-4 were designed and synthesized. First, thirteen cysteine modified peptides (1-13) were prepared. Peptides 1, 10, and 13 showed improved glucagon-like peptide-1 (GLP-1) receptor activate potency and were thus selected for second step modifications to yield conjugates I-1-I-9. All conjugates retained significant GLP-1 receptor activate potency and more importantly exerted enhanced albumin-binding properties and in vitro plasma stability. The protracted antidiabetic effects of the most stable I-3 were further confirmed by both multiple intraperitoneal glucose tolerance test and hypoglycemic efficacies test in vivo. Furthermore, once daily injection of I-3 to streptozotocin (STZ) induced diabetic mice achieved long-term beneficial effects on hemoglobin A1C (HbA1C) lowering and glucose tolerance. Once daily injection of I-3 to diet induced obesity (DIO) mice also achieved favorable effects on food intake, body weight, and blood chemistry. Our results suggested that I-3 was a promising agent deserving further investigation to treat obesity patients with diabetes. PMID:27155328

  15. Polyfunctional Specific Response to Echinococcus Granulosus Associates to the Biological Activity of the Cysts

    PubMed Central

    Petrone, Linda; Vanini, Valentina; Petruccioli, Elisa; Ettorre, Giuseppe Maria; Schininà, Vincenzo; Busi Rizzi, Elisa; Ludovisi, Alessandra; Corpolongo, Angela; Ippolito, Giuseppe; Pozio, Edoardo; Teggi, Antonella; Goletti, Delia

    2015-01-01

    Background Cystic echinococcosis (CE) is a complex disease caused by Echinococcus granulosus (E.granulosus), and its immunophatogenesis is still not clearly defined. A peculiar feature of chronic CE is the coexistence of Th1 and Th2 responses. It has been suggested that Th1 cytokines are related to disease resistance, whereas Th2 cytokines are related to disease susceptibility and chronicity. The aim of this study was to evaluate, by multi-parametric flow cytometry (FACS), the presence of CE specific immune signatures. Methodology/Principal Findings We enrolled 54 subjects with suspected CE; 42 of them had a confirmed diagnosis, whereas 12 were classified as NO-CE. Based on the ultrasonography images, CE patients were further categorized as being in "active stages" (25) and "inactive stages" (17). The ability of CD4+ T-cells to produce IFN-γ, IL-2, TNF-α, Th2 cytokines or IL-10 was assessed by FACS on antigen-specific T-cells after overnight stimulation with Antigen B (AgB) of E.granulosus. Cytokine profiles were evaluated in all the enrolled subjects. The results show that none of the NO-CE subjects had a detectable AgB-specific response. Among the CE patients, the frequency and proportions of AgB-specific CD4+ T-cells producing IL-2+TNF-α+Th2+ or TNF-α+Th2+ were significantly increased in the “active stages” group compared to the “inactive stages” group. Moreover, an increased proportion of the total polyfunctional subsets, as triple-and double-functional CD4 T-cells, was found in CE patients with active disease. The response to the mitogen, used as a control stimulus to evaluate the immune competence status, was characterized by the same cytokine subsets in all the subjects enrolled, independent of CE. Conclusions We demonstrate, for the first time to our knowledge, that polyfunctional T-cell subsets as IL-2+TNF-α+Th2+ triple-positive and TNF-α+Th2+ double-positive specific T-cells associate with cyst biological activity. These results contribute

  16. In Vivo Structure-Activity Relationship Studies Support Allosteric Targeting of a Dual Specificity Phosphatase

    PubMed Central

    Korotchenko, Vasiliy N.; Saydmohammed, Manush; Vollmer, Laura L.; Bakan, Ahmet; Sheetz, Kyle; Debiec, Karl T.; Greene, Kristina A.; Agliori, Christine S.; Bahar, Ivet; Day, Billy W.; Vogt, Andreas; Tsang, Michael

    2014-01-01

    Dual specificity phosphatase 6 (DUSP6) functions as a feedback attenuator of Fibroblast Growth Factor signaling during development. In vitro high throughput chemical screening attempts to discover DUSP6 inhibitors have yielded limited success. Yet, in vivo whole organism screens using zebrafish identified 1 (BCI) as an allosteric inhibitor of DUSP6. Here we designed and synthesized a panel of analogs to define structure-activity relationship (SAR) of DUSP6 inhibition. In vivo, high-content analysis in transgenic zebrafish coupled with cell-based chemical complementation assays identified structural features of the 1 pharmacophore that were essential for biological activity. In vitro assays of DUSP hyperactivation corroborated the results from in vivo and cellular SAR. The results reinforce the notion that DUSPs are druggable through allosteric mechanisms, and illustrate the utility of zebrafish as a model organism for in vivo SAR analyses. PMID:24909879

  17. Biologically active protein fragments containing specific binding regions of serum albumin or related proteins

    NASA Technical Reports Server (NTRS)

    Carter, Daniel C. (Inventor)

    1998-01-01

    In accordance with the present invention, biologically active protein fragments can be constructed which contain only those specific portions of the serum albumin family of proteins such as regions known as subdomains IIA and IIIA which are primarily responsible for the binding properties of the serum albumins. The artificial serums that can be prepared from these biologically active protein fragments are advantageous in that they can be produced much more easily than serums containing the whole albumin, yet still retain all or most of the original binding potential of the full albumin proteins. In addition, since the protein fragment serums of the present invention can be made from non-natural sources using conventional recombinant DNA techniques, they are far safer than serums containing natural albumin because they do not carry the potentially harmful viruses and other contaminants that will be found in the natural substances.

  18. Active route learning in virtual environments: disentangling movement control from intention, instruction specificity, and navigation control.

    PubMed

    von Stülpnagel, Rul; Steffens, Melanie C

    2013-09-01

    Active navigation research examines how physiological and psychological involvement in navigation benefits spatial learning. However, existing conceptualizations of active navigation comprise separable, distinct factors. This research disentangles the contributions of movement control (i.e., self-contained vs. observed movement) as a central factor from learning intention (Experiment 1), instruction specificity and instruction control (Experiment 2), as well as navigation control (Experiment 3) to spatial learning in virtual environments. We tested the effects of these factors on landmark recognition (landmark knowledge), tour-integration and route navigation (route knowledge). Our findings suggest that movement control leads to robust advantages in landmark knowledge as compared to observed movement. Advantages in route knowledge do not depend on learning intention, but on the need to elaborate spatial information. Whenever the necessary level of elaboration is assured for observed movement, too, the development of route knowledge is not inferior to that for self-contained movement.

  19. Removal of damaged proteins during ES cell fate specification requires the proteasome activator PA28

    PubMed Central

    Hernebring, Malin; Fredriksson, Åsa; Liljevald, Maria; Cvijovic, Marija; Norrman, Karin; Wiseman, John; Semb, Henrik; Nyström, Thomas

    2013-01-01

    In embryonic stem cells, removal of oxidatively damaged proteins is triggered upon the first signs of cell fate specification but the underlying mechanism is not known. Here, we report that this phase of differentiation encompasses an unexpected induction of genes encoding the proteasome activator PA28αβ (11S), subunits of the immunoproteasome (20Si), and the 20Si regulator TNFα. This induction is accompanied by assembly of mature PA28-20S(i) proteasomes and elevated proteasome activity. Inhibiting accumulation of PA28α using miRNA counteracted the removal of damaged proteins demonstrating that PA28αβ has a hitherto unidentified role required for resetting the levels of protein damage at the transition from self-renewal to cell differentiation. PMID:23459332

  20. Effects of active vs. passive recovery on repeated rugby-specific exercises.

    PubMed

    Jougla, A; Micallef, J P; Mottet, D

    2010-05-01

    The aim of this study was to determine the effects of active vs. passive recovery on performance of a rugby-specific intermittent test in rugby union players. Seven male rugby players (20.6+/-0.5 yrs; 181.9+/-10.0 cm; 94.5+/-12.8 kg) performed in random order, over two separate sessions, a specific repeated-sprint rugby test, the Narbonne test (6 x 4 consecutive actions: 1, scrummaging; 2, agility sprinting; 3, tackling; 4, straight sprinting) with 30s of passive or active recovery (running at 50% of maximal aerobic speed). The Narbonne tests were completed before (pre-test) and after (post-test) a 30-min rugby match. During the Narbonne test, scrum forces, agility and sprint times, heart rate and rate of perceived exertion were measured. Scrum forces were lower in active (74.9+/-13.4 kg) than in passive recovery (90.4+/-20.9 kg), only during the post-test (p<0.05). Fatigue index (%) (p<0.05) and total sprint time (s) (p<0.01) were significantly greater in active than in passive recovery, both during the pre-test (11.5+/-5.7% vs. 6.7+/-4.5% and 18.1+/-1.3s vs. 16.9+/-0.9s) and the post-test (7.3+/-3.3% vs. 4.3+/-1.5% and 18.3+/-1.6s vs. 16.9+/-1.1s). Consequently, the results indicated that passive recovery enabled better performance during the Narbonne test. However, it is obviously impractical to suggest that players should stand still during and following repeated-sprint bouts: the players have to move to ensure they have taken an optimal position. PMID:19560972

  1. Instrument specific brain activation in sensorimotor and auditory representation in musicians.

    PubMed

    Gebel, B; Braun, Ch; Kaza, E; Altenmüller, E; Lotze, M

    2013-07-01

    Musicians show a remarkable ability to interconnect motor patterns and sensory processing in the somatosensory and auditory domains. Many of these processes are specific for the instrument used. We were interested in the cerebral and cerebellar representations of these instrument-specific changes and therefore applied functional magnetic resonance imaging (fMRI) in two groups of instrumentalists with different instrumental training for comparable periods (approximately 15 years). The first group (trumpet players) uses tight finger and lip interaction; the second (pianists as control group) uses only the extremities for performance. fMRI tasks were balanced for instructions (piano and trumpet notes), sensory feedback (keypad and trumpet), and hand-lip interaction on the trumpet. During fMRI, both groups switched between different devices (trumpet or keypad) and performance was combined with or without auditory feedback. Playing the trumpet without any tone emission or using the mouthpiece showed an instrument training-specific activation increase in trumpet players. This was evident for the posterior-superior cerebellar hemisphere, the dominant primary sensorimotor cortex, and the left Heschl's gyrus. Additionally, trumpet players showed increased activity in the bilateral Heschl's gyrus during actual trumpet playing, although they showed significantly decreased loudness while playing with the mouthpiece in the scanner compared to pianists.

  2. Inhibitory activities of short linear motifs underlie Hox interactome specificity in vivo

    PubMed Central

    Baëza, Manon; Viala, Séverine; Heim, Marjorie; Dard, Amélie; Hudry, Bruno; Duffraisse, Marilyne; Rogulja-Ortmann, Ana; Brun, Christine; Merabet, Samir

    2015-01-01

    Hox proteins are well-established developmental regulators that coordinate cell fate and morphogenesis throughout embryogenesis. In contrast, our knowledge of their specific molecular modes of action is limited to the interaction with few cofactors. Here, we show that Hox proteins are able to interact with a wide range of transcription factors in the live Drosophila embryo. In this context, specificity relies on a versatile usage of conserved short linear motifs (SLiMs), which, surprisingly, often restrains the interaction potential of Hox proteins. This novel buffering activity of SLiMs was observed in different tissues and found in Hox proteins from cnidarian to mouse species. Although these interactions remain to be analysed in the context of endogenous Hox regulatory activities, our observations challenge the traditional role assigned to SLiMs and provide an alternative concept to explain how Hox interactome specificity could be achieved during the embryonic development. DOI: http://dx.doi.org/10.7554/eLife.06034.001 PMID:25869471

  3. The roadmap for estimation of cell-type-specific neuronal activity from non-invasive measurements.

    PubMed

    Uhlirova, Hana; Kılıç, Kıvılcım; Tian, Peifang; Sakadžić, Sava; Gagnon, Louis; Thunemann, Martin; Desjardins, Michèle; Saisan, Payam A; Nizar, Krystal; Yaseen, Mohammad A; Hagler, Donald J; Vandenberghe, Matthieu; Djurovic, Srdjan; Andreassen, Ole A; Silva, Gabriel A; Masliah, Eliezer; Kleinfeld, David; Vinogradov, Sergei; Buxton, Richard B; Einevoll, Gaute T; Boas, David A; Dale, Anders M; Devor, Anna

    2016-10-01

    The computational properties of the human brain arise from an intricate interplay between billions of neurons connected in complex networks. However, our ability to study these networks in healthy human brain is limited by the necessity to use non-invasive technologies. This is in contrast to animal models where a rich, detailed view of cellular-level brain function with cell-type-specific molecular identity has become available due to recent advances in microscopic optical imaging and genetics. Thus, a central challenge facing neuroscience today is leveraging these mechanistic insights from animal studies to accurately draw physiological inferences from non-invasive signals in humans. On the essential path towards this goal is the development of a detailed 'bottom-up' forward model bridging neuronal activity at the level of cell-type-specific populations to non-invasive imaging signals. The general idea is that specific neuronal cell types have identifiable signatures in the way they drive changes in cerebral blood flow, cerebral metabolic rate of O2 (measurable with quantitative functional Magnetic Resonance Imaging), and electrical currents/potentials (measurable with magneto/electroencephalography). This forward model would then provide the 'ground truth' for the development of new tools for tackling the inverse problem-estimation of neuronal activity from multimodal non-invasive imaging data.This article is part of the themed issue 'Interpreting BOLD: a dialogue between cognitive and cellular neuroscience'. PMID:27574309

  4. Involvement of specific calmodulin isoforms in salicylic acid-independent activation of plant disease resistance responses.

    PubMed

    Heo, W D; Lee, S H; Kim, M C; Kim, J C; Chung, W S; Chun, H J; Lee, K J; Park, C Y; Park, H C; Choi, J Y; Cho, M J

    1999-01-19

    The Ca2+ signal is essential for the activation of plant defense responses, but downstream components of the signaling pathway are still poorly defined. Here we demonstrate that specific calmodulin (CaM) isoforms are activated by infection or pathogen-derived elicitors and participate in Ca2+-mediated induction of plant disease resistance responses. Soybean CaM (SCaM)-4 and SCaM-5 genes, which encode for divergent CaM isoforms, were induced within 30 min by a fungal elicitor or pathogen, whereas other SCaM genes encoding highly conserved CaM isoforms did not show such response. This pathogen-triggered induction of these genes specifically depended on the increase of intracellular Ca2+ level. Constitutive expression of SCaM-4 and SCaM-5 in transgenic tobacco plants triggered spontaneous induction of lesions and induces an array of systemic acquired resistance (SAR)-associated genes. Surprisingly, these transgenic plants have normal levels of endogenous salicylic acid (SA). Furthermore, coexpression of nahG gene did not block the induction of SAR-associated genes in these transgenic plants, indicating that SA is not involved in the SAR gene induction mediated by SCaM-4 or SCaM-5. The transgenic plants exhibit enhanced resistance to a wide spectrum of virulent and avirulent pathogens, including bacteria, fungi, and virus. These results suggest that specific CaM isoforms are components of a SA-independent signal transduction chain leading to disease resistance.

  5. Specific activation of the mu opioid receptor (MOR) by endomorphin 1 and endomorphin 2.

    PubMed

    Monory, K; Bourin, M C; Spetea, M; Tömböly, C; Tóth, G; Matthes, H W; Kieffer, B L; Hanoune, J; Borsodi, A

    2000-02-01

    The recently discovered endomorphin 1 (Tyr-Pro-Trp-Phe-NH2) and endomorphin 2 (Tyr-Pro-Phe-Phe-NH2) were investigated with respect to their direct receptor-binding properties, and to their ability to activate G proteins and to inhibit adenylyl cyclase in both cellular and animal models. Both tetrapeptides activated G proteins and inhibited adenylyl cyclase activity in membrane preparations from cells stably expressing the mu opioid receptor, an effect reversed by the mu receptor antagonist CTAP (D-Phe-Cys-Tyr-D-Trp-Arg-Thr-Pen-Thr-NH2), but they had no influence on cells stably expressing the delta opioid receptor. To further establish the selectivity of these peptides for the mu opioid receptor, brain preparations of mice lacking the mu opioid receptor gene were used to study their binding and signalling properties. Endomorphin 2, tritiated by a dehalotritiation method resulting in a specific radioactivity of 1.98 TBq/mmol (53.4 Ci/mmol), labelled the brain membranes of wild-type mice with a Kd value of 1.77 nM and a Bmax of 63.33 fmol/mg protein. In membranes of mice lacking the mu receptor gene, no binding was observed, and both endomorphins failed to stimulate [35S]guanosine-5'-O-(3-thio)triphosphate ([35S]GTPgammaS) binding and to inhibit adenylyl cyclase. These data show that endomorphins are capable of activating G proteins and inhibiting adenylyl cyclase activity, and all these effects are mediated by the mu opioid receptors.

  6. Aberrant Oscillatory Activity during Simple Movement in Task-Specific Focal Hand Dystonia

    PubMed Central

    Hinkley, Leighton B. N.; Dolberg, Rebecca; Honma, Susanne; Findlay, Anne; Byl, Nancy N.; Nagarajan, Srikantan S.

    2012-01-01

    In task-specific focal hand dystonia (tspFHD), the temporal dynamics of cortical activity in the motor system and how these processes are related to impairments in sensory and motor function are poorly understood. Here, we use time-frequency reconstructions of magnetoencephalographic (MEG) data to elaborate the temporal and spatial characteristics of cortical activity during movement. A self-paced finger tapping task during MEG recording was performed by 11 patients with tspFHD and 11 matched healthy controls. In both groups robust changes in beta (12–30 Hz) and high gamma (65–90 Hz) oscillatory activity were identified over sensory and motor cortices during button press. A significant decrease [p < 0.05, 1% False Discovery Rate (FDR) corrected] in high gamma power during movements of the affected hand was identified over ipsilateral sensorimotor cortex in the period prior to (−575 ms) and following (725 ms) button press. Furthermore, an increase (p < 0.05, 1% FDR corrected) in beta power suppression following movement of the affected hand was identified over visual cortex in patients with tspFHD. For movements of the unaffected hand, a significant (p < 0.05, 1% FDR corrected) increase in beta power suppression was identified over secondary somatosensory cortex (S2) in the period following button press in patients with tspFHD. Oscillatory activity within in the tspFHD group was however not correlated with clinical measures. Understanding these aberrant oscillatory dynamics can provide the groundwork for interventions that focus on modulating the timing of this activity. PMID:23226140

  7. Specific marker of feigned memory impairment: The activation of left superior frontal gyrus.

    PubMed

    Chen, Zi-Xiang; Xue, Li; Liang, Chun-Yu; Wang, Li-Li; Mei, Wei; Zhang, Qiang; Zhao, Hu

    2015-11-01

    Faking memory impairment means normal people complain lots of memory problems without organic damage in forensic assessments. Using alternative forced-choice paradigm, containing digital or autobiographical information, previous neuroimaging studies have indicated that faking memory impairment could cause the activation in the prefrontal and parietal regions, and might involve a fronto-parietal-subcortical circuit. However, it is still unclear whether different memory types have influence on faking or not. Since different memory types, such as long-term memory (LTM) and short-term memory (STM), were found supported by different brain areas, we hypothesized that feigned STM or LTM impairment had distinct neural activation mapping. Besides that, some common neural correlates may act as the general characteristic of feigned memory impairment. To verify this hypothesis, the functional magnetic resonance imaging (fMRI) combined with an alternative word forced-choice paradigm were used in this study. A total of 10 right-handed participants, in this study, had to perform both STW and LTM tasks respectively under answering correctly, answering randomly and feigned memory impairment conditions. Our results indicated that the activation of the left superior frontal gyrus and the left medial frontal gyrus was associated with feigned LTM impairment, whereas the left superior frontal gyrus, the left precuneus and the right anterior cingulate cortex (ACC) were highly activated while feigning STM impairment. Furthermore, an overlapping was found in the left superior frontal gyrus, and it suggested that the activity of the left superior frontal gyrus might be acting as a specific marker of feigned memory impairment. PMID:26479324

  8. Stability and activity of an Enterobacter aerogenes-specific bacteriophage under simulated gastro-intestinal conditions.

    PubMed

    Verthé, K; Possemiers, S; Boon, N; Vaneechoutte, M; Verstraete, W

    2004-09-01

    A bacteriophage, designated UZ1 and showing lytic activity against a clinically important strain (BE1) of Enterobacter aerogenes was isolated from hospital sewage. The stability and lytic activity against this strain under simulated gastro-intestinal conditions was evaluated. After addition of bacteriophage UZ1 to a liquid feed at gastric pH 2, the phage was immediately inactivated and could not be recovered. However, by use of an antacid to neutralize stomach acidity, no significant changes in phage titer were observed after 2 h incubation at 37 degrees C. After supplementing pancreatic juice and further incubation for 4 h, the phage titer remained stable. The persistence of UZ1 in a mixed microbial ecosystem that was representative for the large intestine was monitored using an in vitro simulation of the human intestinal microbial ecosystem. A pulse administration of bacteriophage UZ1 at a concentration of 10(5) plaque-forming units (PFU)/ml to reactor 3 (which simulates the ascending colon) showed that, in the absence of the host, bacteriophage UZ1 persisted for 13 days in the simulated colon, while the theoretical washout was calculated at 16 days. To assess its lytic activity in an intestinal microbial ecosystem, a green fluorescent protein (gfp)-labeled E. aerogenes BE1 strain was constructed and gfp-specific primers were designed in order to quantify the host strain using real-time PCR. It was observed that bacteriophage UZ1 was able to replicate and showed lytic activity against E. aerogenes BE1/ gfp in an intestinal microbial ecosystem. Indeed, after 17 h a 2 log unit reduction of E. aerogenes BE1/ gfp was measured as compared with the assay without bacteriophage UZ1, while the phage titer increased by 2 log units at an initial multiplicity of infection of 0.07 PFU/colony-forming unit. This is the first report of an in vitro model to study bacteriophage activity in the complex intestinal microbial community.

  9. Cloning and characterization of a novel Athspr promoter specifically active in vascular tissue.

    PubMed

    Zhang, Liang; Yang, Tao; Li, Xiaoying; Hao, Hongyan; Xu, Shengtao; Cheng, Wei; Sun, Yingli; Wang, Chongying

    2014-05-01

    The vascular system--xylem, phloem and the cambium--is essential for water supply, nutrient transport, and physical support in higher plants. Although it is known that vascular-specific gene expression is regulated by cis-acting regulatory sequences in promoters, it is largely unknown how many regulatory elements exist and what their roles are in promoters. To understand the regulatory elements of vascular-specific promoters and their roles in vascular development, a T-DNA insertion mutant showing delayed growth and diminished resistance to environmental stress was isolated using promoter trap strategy. The novel gene, Arabidopsis thaliana heat shock protein-related (Athspr), was cloned from Arabidopsis ecotype C24. Strong GUS (β-glucuronidase) staining in the original promoter trap line was found in the vascular tissues of all organs in the mutant. The Athspr promoter was cloned and fused with GUS and eGFP (enhanced green fluorescent protein) reporter genes to verify its vascular-specific expression in Arabidopsis. Further histochemical analysis in transgenic plants demonstrated a similar GUS expression pattern in the vascular tissues. In addition, ATHSPR-eGFP driven by Athspr promoter was observed in vascular bundles of the transgenic seedling roots. Finally, comparative analysis with promoter motifs from 37 genes involved in vascular development revealed that Athspr and all other promoters active in vascular tissues contained regulatory elements responding to phytohormones, light, biotic and abiotic stresses, as well as those regulating tissue-specific expression. These results demonstrated that the Athspr promoter has a vascular tissue-specific activity and Athspr may have multiple functions in vascular development and resistance against various stresses. PMID:24675528

  10. Strain-specific antiviral activity of iminosugars against human influenza A viruses

    PubMed Central

    Hussain, S.; Miller, J. L.; Harvey, D. J.; Gu, Y.; Rosenthal, P. B.; Zitzmann, N.; McCauley, J. W.

    2015-01-01

    Objectives Drugs that target host cell processes can be employed to complement drugs that specifically target viruses, and iminosugar compounds that inhibit host α-glucosidases have been reported to show antiviral activity against multiple viruses. Here the effect and mechanism of two iminosugar α-glucosidase inhibitors, N-butyl-deoxynojirimycin (NB-DNJ) and N-nonyl-deoxynojirimycin (NN-DNJ), on human influenza A viruses was examined. Methods The viruses examined were a recently circulating seasonal influenza A(H3N2) virus strain A/Brisbane/10/2007, an older H3N2 strain A/Udorn/307/72, and A/Lviv/N6/2009, a strain representative of the currently circulating pandemic influenza A(H1N1)pdm09 virus. Results The inhibitors had the strongest effect on Brisbane/10 and NN-DNJ was more potent than NB-DNJ. Both compounds showed antiviral activity in cell culture against three human influenza A viruses in a strain-specific manner. Consistent with its action as an α-glucosidase inhibitor, NN-DNJ treatment resulted in an altered glycan processing of influenza haemagglutinin (HA) and neuraminidase (NA), confirmed by MS. NN-DNJ treatment was found to reduce the cell surface expression of the H3 subtype HA. The level of sialidase activity of NA was reduced in infected cells, but the addition of exogenous sialidase to the cells did not complement the NN-DNJ-mediated inhibition of virus replication. Using reassortant viruses, the drug susceptibility profile was determined to correlate with the origin of the HA. Conclusions NN-DNJ inhibits influenza A virus replication in a strain-specific manner that is dependent on the HA. PMID:25223974

  11. Subtle Changes in Motif Positioning Cause Tissue-Specific Effects on Robustness of an Enhancer's Activity

    PubMed Central

    Erceg, Jelena; Saunders, Timothy E.; Girardot, Charles; Devos, Damien P.; Hufnagel, Lars; Furlong, Eileen E. M.

    2014-01-01

    Deciphering the specific contribution of individual motifs within cis-regulatory modules (CRMs) is crucial to understanding how gene expression is regulated and how this process is affected by sequence variation. But despite vast improvements in the ability to identify where transcription factors (TFs) bind throughout the genome, we are limited in our ability to relate information on motif occupancy to function from sequence alone. Here, we engineered 63 synthetic CRMs to systematically assess the relationship between variation in the content and spacing of motifs within CRMs to CRM activity during development using Drosophila transgenic embryos. In over half the cases, very simple elements containing only one or two types of TF binding motifs were capable of driving specific spatio-temporal patterns during development. Different motif organizations provide different degrees of robustness to enhancer activity, ranging from binary on-off responses to more subtle effects including embryo-to-embryo and within-embryo variation. By quantifying the effects of subtle changes in motif organization, we were able to model biophysical rules that explain CRM behavior and may contribute to the spatial positioning of CRM activity in vivo. For the same enhancer, the effects of small differences in motif positions varied in developmentally related tissues, suggesting that gene expression may be more susceptible to sequence variation in one tissue compared to another. This result has important implications for human eQTL studies in which many associated mutations are found in cis-regulatory regions, though the mechanism for how they affect tissue-specific gene expression is often not understood. PMID:24391522

  12. Validity of ActiGraph Child-Specific Equations during Various Physical Activities

    PubMed Central

    Crouter, Scott E.; Horton, Magdalene; Bassett, David R.

    2013-01-01

    PURPOSE The purpose of this study was to examine the validity of seven child-specific ActiGraph prediction equations/cut-points (Crouter vector magnitude 2-regression model (Cvm2RM), Crouter vertical axis 2RM (Cva2RM), Freedson, Treuth, Trost, Puyau and Evenson) for estimating energy expenditure (EE) and time spent in sedentary behaviors, light physical activity (LPA), moderate PA (MPA), and vigorous PA (VPA). METHODS Forty boys and 32 girls (mean±SD; age, 12±0.8 yrs) participated in the study. Participants performed eight structured activities and approximately 2-hrs of free-living activity. Activity data was collected using an ActiGraph GT3X+, positioned on the right hip, and EE (METRMR; activity VO2 divided by resting VO2) was measured using a Cosmed K4b2. ActiGraph prediction equations were compared against the Cosmed for METRMR and time spent in sedentary behaviors, LPA, MPA, VPA, and MVPA. RESULTS For the structured activities, all prediction methods were significantly different from measured METRMR for ≥ 3 activities (P<0.05), however all provided close estimates of METRMR during walking. On average, participants were monitored for 95.0±36.5 minutes during the free-living measurement. The Cvm2RM and Puyau methods were within 0.9 METRMR of measured free-living METRMR (P>0.05); all other methods significantly underestimated measured METRMR (P<0.05). The Cva2RM was within 9.7 minutes of measured time spent in sedentary behaviors, LPA, MPA, and MVPA, which was the best of the methods examined. All prediction equations underestimated VPA by 6.0–13.6 minutes. CONCLUSION Compared to the Cosmed, the Cvm2RM and Puyau methods provided the best estimate of METRMR and the Cva2RM provided the closest estimate of time spent in each intensity category during the free-living measurement. Lastly, all prediction methods had large individual prediction errors. PMID:23439413

  13. Soil Moisture Active Passive (SMAP) Mission Level 4 Carbon (L4_C) Product Specification Document

    NASA Technical Reports Server (NTRS)

    Glassy, Joe; Kimball, John S.; Jones, Lucas; Reichle, Rolf H.; Ardizzone, Joseph V.; Kim, Gi-Kong; Lucchesi, Robert A.; Smith, Edmond B.; Weiss, Barry H.

    2015-01-01

    This is the Product Specification Document (PSD) for Level 4 Surface and Root Zone Soil Moisture (L4_SM) data for the Science Data System (SDS) of the Soil Moisture Active Passive (SMAP) project. The L4_SM data product provides estimates of land surface conditions based on the assimilation of SMAP observations into a customized version of the NASA Goddard Earth Observing System, Version 5 (GEOS-5) land data assimilation system (LDAS). This document applies to any standard L4_SM data product generated by the SMAP Project.

  14. Specific activity to H*(10) conversion coefficients for in situ gamma spectrometry.

    PubMed

    Lemercier, M; Gurriaran, R; Bouisset, P; Cagnat, X

    2008-01-01

    The typical situations found in in situ gamma spectrometry have been simulated by Monte Carlo techniques to obtain the energy spectra of the photon fluence rate existing at 1 m above ground. The main difficulty found with the model is its slow convergence. A method to speed up the calculation has been derived. The results of the model have been thoroughly tested against existing data. In a final stage, the specific activity to H*(10) conversion coefficients have been derived for the typical scenarios encountered in the in situ gamma spectrometry. PMID:17942441

  15. Target design considerations for high specific activity [{sup 11}C]O{sub 2}

    SciTech Connect

    Ferrieri, R.A.; Alexoff, D.L.; Schlyer, D.J.; McDonald, K.; Wolf, A.P.

    1993-12-31

    In the routine preparation of {sup 11}C-labeled compounds through N-[{sup 11}C]-methylation using [{sup 11}C]H{sub 3}I, total masses are always higher than synthesis mass contribution, suggesting that the target system contributes carrier carbon to the final product mass. This conclusion prompted this evaluation of target materials and target design for [{sup 11}C]O{sub 2} production. Ultimately, one is faced with the sprospect of compromising between [{sup 11}C]O{sub 2} specific activity and the amount that can be extracted from the target after a reasonable irradiation time.

  16. Research activities at the Australian Bureau of Meteorology for the regional ionospheric specification and forecasting

    NASA Astrophysics Data System (ADS)

    Bouya, Zahra; Terkildsen, Michael

    2016-07-01

    The Australian Space Forecast Centre (ASFC) provides space weather forecasts to a diverse group of customers. Space Weather Services (SWS) within the Australian Bureau of Meteorology is focussed both on developing tailored products and services for the key customer groups, and supporting ASFC operations. Research in SWS is largely centred on the development of data-driven models using a range of solar-terrestrial data. This paper will cover some data requirements , approaches and recent SWS activities for data driven modelling with a focus on the regional Ionospheric specification and forecasting.

  17. Regulation of the imprinted Dlk1-Dio3 locus by allele-specific enhancer activity.

    PubMed

    Luo, Zhuojuan; Lin, Chengqi; Woodfin, Ashley R; Bartom, Elizabeth T; Gao, Xin; Smith, Edwin R; Shilatifard, Ali

    2016-01-01

    Genomic imprinting is a critical developmental process characteristic of parent of origin-specific gene expression. It is well accepted that differentially DNA-methylated regions (DMRs) and enhancers are two major classes of cis-elements determining parent of origin-specific gene expression, with each recruiting different sets of transcription factors. Previously, we identified the AF4/FMR2 (AFF) family protein AFF3 within the transcription elongation complex SEC-L3. Here, we report that AFF3 can specifically bind both gametic DMRs (gDMRs) and enhancers within imprinted loci in an allele-specific manner. We identify the molecular regulators involved in the recruitment of AFF3 to gDMRs and provide mechanistic insight into the requirement of AFF3 at an enhancer for the expression of an ∼200-kb polycistronic transcript within the imprinted Dlk1-Dio3 locus. Our data suggest that the heterochromatic environment at the gDMR reinforces silencing of its related enhancer by controlling the binding and activity of AFF3 in an allele-specific manner. In summary, this study provides molecular details about the regulation of dosage-critical imprinted gene expression through the regulated binding of the transcription elongation factor AFF3 between a DMR and an enhancer. PMID:26728555

  18. Crosstalk among electrical activity, trophic factors and morphogenetic proteins in the regulation of neurotransmitter phenotype specification.

    PubMed

    Borodinsky, Laura N; Belgacem, Yesser H

    2016-04-01

    Morphogenetic proteins are responsible for patterning the embryonic nervous system by enabling cell proliferation that will populate all the neural structures and by specifying neural progenitors that imprint different identities in differentiating neurons. The adoption of specific neurotransmitter phenotypes is crucial for the progression of neuronal differentiation, enabling neurons to connect with each other and with target tissues. Preliminary neurotransmitter specification originates from morphogen-driven neural progenitor specification through the combinatorial expression of transcription factors according to morphogen concentration gradients, which progressively restrict the identity that born neurons adopt. However, neurotransmitter phenotype is not immutable, instead trophic factors released from target tissues and environmental stimuli change expression of neurotransmitter-synthesizing enzymes and specific vesicular transporters modifying neuronal neurotransmitter identity. Here we review studies identifying the mechanisms of catecholaminergic, GABAergic, glutamatergic, cholinergic and serotonergic early specification and of the plasticity of these neurotransmitter phenotypes during development and in the adult nervous system. The emergence of spontaneous electrical activity in developing neurons recruits morphogenetic proteins in the process of neurotransmitter phenotype plasticity, which ultimately equips the nervous system and the whole organism with adaptability for optimal performance in a changing environment. PMID:26686293

  19. Regulation of the imprinted Dlk1-Dio3 locus by allele-specific enhancer activity

    PubMed Central

    Luo, Zhuojuan; Lin, Chengqi; Woodfin, Ashley R.; Bartom, Elizabeth T.; Gao, Xin; Smith, Edwin R.; Shilatifard, Ali

    2016-01-01

    Genomic imprinting is a critical developmental process characteristic of parent of origin-specific gene expression. It is well accepted that differentially DNA-methylated regions (DMRs) and enhancers are two major classes of cis-elements determining parent of origin-specific gene expression, with each recruiting different sets of transcription factors. Previously, we identified the AF4/FMR2 (AFF) family protein AFF3 within the transcription elongation complex SEC-L3. Here, we report that AFF3 can specifically bind both gametic DMRs (gDMRs) and enhancers within imprinted loci in an allele-specific manner. We identify the molecular regulators involved in the recruitment of AFF3 to gDMRs and provide mechanistic insight into the requirement of AFF3 at an enhancer for the expression of an ∼200-kb polycistronic transcript within the imprinted Dlk1-Dio3 locus. Our data suggest that the heterochromatic environment at the gDMR reinforces silencing of its related enhancer by controlling the binding and activity of AFF3 in an allele-specific manner. In summary, this study provides molecular details about the regulation of dosage-critical imprinted gene expression through the regulated binding of the transcription elongation factor AFF3 between a DMR and an enhancer. PMID:26728555

  20. TASK DIFFICULTY MODULATES ACTIVITY OF SPECIFIC NEURONAL POPULATIONS IN PRIMARY VISUAL CORTEX

    PubMed Central

    Chen, Yao; Martinez-Conde, Susana; Macknik, Stephen L.; Bereshpolova, Yulia; Swadlow, Harvey A.; Alonso, Jose-Manuel

    2008-01-01

    Spatial attention enhances our ability to detect stimuli at restricted regions of the visual field. This enhancement is thought to depend on the difficulty of the task being performed, but the underlying neuronal mechanisms for this dependency remain largely unknown. Here we demonstrate that task difficulty modulates neuronal firing rate at the earliest stages of cortical visual processing (area V1) in the macaque monkey. These modulations are spatially specific: increasing task difficulty enhances V1 neuronal firing rate at the focus of attention and suppresses it in regions surrounding the focus. Moreover, we show that response enhancement and suppression are mediated by distinct populations of neurons that differ in direction selectivity, spike width, interspike interval distribution and contrast sensitivity. Our results provide strong support for center-surround models of spatial attention and suggest that task difficulty modulates the activity of specific populations of neurons in the primary visual cortex. PMID:18604204

  1. Stage specific activity of synthetic antimalarial endoperoxides, N-89 and N-251, against Plasmodium falciparum.

    PubMed

    Morita, Masayuki; Koyama, Takahiko; Sanai, Hitomi; Sato, Akira; Hiramoto, Akiko; Masuyama, Araki; Nojima, Masatomo; Wataya, Yusuke; Kim, Hye-Sook

    2015-02-01

    We have reported that two endoperoxides, N-89 and N-251, synthesized in 2001, possess potent antimalarial activities. Aiming at their eventual use for curing malaria in humans, we have been investigating various aspects of their antimalarial actions. Here we show that N-89 and N-251 inhibit the growth of Plasmodium falciparum within human erythrocytes in vitro at its lifecycle stage 'trophozoite' specifically. It is known that artemisinin compounds, which are currently used for curing malaria, have other stage-specificities. Therefore, it is likely that the antimalarial mechanism of N-89 and N-251 differs from those of artemisinin compounds. As malaria parasites resistant to artemisinin-based combination therapy are currently emerging in some tropical regions, N-89 and N-251 are candidates for overcoming these new problems. PMID:25449979

  2. Activity and specificity of TRV-mediated gene editing in plants.

    PubMed

    Ali, Zahir; Abul-Faraj, Aala; Piatek, Marek; Mahfouz, Magdy M

    2015-01-01

    Plant trait engineering requires efficient targeted genome-editing technologies. Clustered regularly interspaced palindromic repeats (CRISPRs)/ CRISPR associated (Cas) type II system is used for targeted genome-editing applications across eukaryotic species including plants. Delivery of genome engineering reagents and recovery of mutants remain challenging tasks for in planta applications. Recently, we reported the development of Tobacco rattle virus (TRV)-mediated genome editing in Nicotiana benthamiana. TRV infects the growing points and possesses small genome size; which facilitate cloning, multiplexing, and agroinfections. Here, we report on the persistent activity and specificity of the TRV-mediated CRISPR/Cas9 system for targeted modification of the Nicotiana benthamiana genome. Our data reveal the persistence of the TRV- mediated Cas9 activity for up to 30 d post-agroinefection. Further, our data indicate that TRV-mediated genome editing exhibited no off-target activities at potential off-targets indicating the precision of the system for plant genome engineering. Taken together, our data establish the feasibility and exciting possibilities of using virus-mediated CRISPR/Cas9 for targeted engineering of plant genomes.

  3. SRT1720, a SIRT1 specific activator, protected H2O2-induced senescent endothelium.

    PubMed

    Li, Rui-Lin; Lu, Zhao-Yang; Huang, Jing-Juan; Qi, Jia; Hu, An; Su, Zhi-Xiao; Zhang, Lan; Li, Yue; Shi, Yi-Qin; Hao, Chang-Ning; Duan, Jun-Li

    2016-01-01

    Silent information regulator 1 (SIRT1) plays a critical role in maintaining vascular homeostasis via modulating senescent-related signal pathway, however, the molecular mechanism remains modest clarified. The purpose of this study was to examine whether SIRT1 specific activator SRT1720 would exhibit pro-angiogenic and anti-aging properties in response to hydrogen peroxide (H2O2)-induced endothelial senescence, and determine the underlying mechanisms. We pre-treated senescent human umbilical vein endothelial cells (HUVECs) with SRT1720, senescence-associated beta-galactosidase activity, apoptosis, migration, tube formation, proliferation and angiogenic factors were quantitatively examined. The results revealed that pharmacologic activation of SIRT1 by SRT1720 rescued apoptotic HUVECs and upregulated angiogenic response through reinforcing the protein expressions of angiogenic and survival factors in vitro. Furthermore, we confirmed that the expressions of endothelial nitric oxide synthase (eNOS), vascular endothelial growth factor (VEGF) and phosphoryl-Akt were augmented in SRT1720-treated senescent HUVECs. In conclusion, our data indicated that SRT1720 could protect against endothelial senescence and maintain cell function via Akt/eNOS/VEGF axis. PMID:27508009

  4. SRT1720, a SIRT1 specific activator, protected H2O2-induced senescent endothelium

    PubMed Central

    Li, Rui-Lin; Lu, Zhao-Yang; Huang, Jing-Juan; Qi, Jia; Hu, An; Su, Zhi-Xiao; Zhang, Lan; Li, Yue; Shi, Yi-Qin; Hao, Chang-Ning; Duan, Jun-Li

    2016-01-01

    Silent information regulator 1 (SIRT1) plays a critical role in maintaining vascular homeostasis via modulating senescent-related signal pathway, however, the molecular mechanism remains modest clarified. The purpose of this study was to examine whether SIRT1 specific activator SRT1720 would exhibit pro-angiogenic and anti-aging properties in response to hydrogen peroxide (H2O2)-induced endothelial senescence, and determine the underlying mechanisms. We pre-treated senescent human umbilical vein endothelial cells (HUVECs) with SRT1720, senescence-associated beta-galactosidase activity, apoptosis, migration, tube formation, proliferation and angiogenic factors were quantitatively examined. The results revealed that pharmacologic activation of SIRT1 by SRT1720 rescued apoptotic HUVECs and upregulated angiogenic response through reinforcing the protein expressions of angiogenic and survival factors in vitro. Furthermore, we confirmed that the expressions of endothelial nitric oxide synthase (eNOS), vascular endothelial growth factor (VEGF) and phosphoryl-Akt were augmented in SRT1720-treated senescent HUVECs. In conclusion, our data indicated that SRT1720 could protect against endothelial senescence and maintain cell function via Akt/eNOS/VEGF axis. PMID:27508009

  5. Amplitude and timing of somatosensory cortex activity in Task Specific Focal Hand Dystonia

    PubMed Central

    Dolberg, Rebecca; Hinkley, Leighton B. N.; Honma, Susanne; Zhu, Zhao; Findlay, Anne M.; Byl, Nancy N.; Nagarjan, Srikantan S.

    2011-01-01

    Objective Task-specific focal hand dystonia (tspFHD) is a movement disorder diagnosed in individuals performing repetitive hand behaviors. The extent to which processing anomalies in primary sensory cortex extend to other regions or across the two hemispheres is presently unclear. Methods In response to low/high rate and novel tactile stimuli on the affected and unaffected hands, magnetoencephalography (MEG) was used to elaborate activity timing and amplitude in the primary somatosensory (S1) and secondary somatosensory/parietal ventral (S2/PV) cortices. MEG and clinical performance measures were collected from thirteen patients and matched controls. Results Compared to controls, subjects with tspFHD had increased response amplitude in S2/PV bilaterally in response to high rate and novel stimuli. Subjects with tspFHD also showed increased response latency (low rate, novel) of the affected digits in contralateral S1. For high rate, subjects with tspFHD showed increased response latency in ipsilateral S1 and S2/PV bilaterally. Activation differences correlated with functional sensory deficits (predicting a latency shift in S1), motor speed and muscle strength. Conclusions There are objective differences in the amplitude and timing of activity for both hands across contralateral and ipsilateral somatosensory cortex in patients with tspFHD. Significance Knowledge of cortical processing abnormalities across S1 and S2/PV in dystonia should be applied towards the development of learning based sensorimotor interventions. PMID:21802357

  6. Versatile strategy for controlling the specificity and activity of engineered T cells.

    PubMed

    Ma, Jennifer S Y; Kim, Ji Young; Kazane, Stephanie A; Choi, Sei-Hyun; Yun, Hwa Young; Kim, Min Soo; Rodgers, David T; Pugh, Holly M; Singer, Oded; Sun, Sophie B; Fonslow, Bryan R; Kochenderfer, James N; Wright, Timothy M; Schultz, Peter G; Young, Travis S; Kim, Chan Hyuk; Cao, Yu

    2016-01-26

    The adoptive transfer of autologous T cells engineered to express a chimeric antigen receptor (CAR) has emerged as a promising cancer therapy. Despite impressive clinical efficacy, the general application of current CAR-T--cell therapy is limited by serious treatment-related toxicities. One approach to improve the safety of CAR-T cells involves making their activation and proliferation dependent upon adaptor molecules that mediate formation of the immunological synapse between the target cancer cell and T-cell. Here, we describe the design and synthesis of structurally defined semisynthetic adaptors we refer to as "switch" molecules, in which anti-CD19 and anti-CD22 antibody fragments are site-specifically modified with FITC using genetically encoded noncanonical amino acids. This approach allows the precise control over the geometry and stoichiometry of complex formation between CD19- or CD22-expressing cancer cells and a "universal" anti-FITC-directed CAR-T cell. Optimization of this CAR-switch combination results in potent, dose-dependent in vivo antitumor activity in xenograft models. The advantage of being able to titrate CAR-T-cell in vivo activity was further evidenced by reduced in vivo toxicity and the elimination of persistent B-cell aplasia in immune-competent mice. The ability to control CAR-T cell and cancer cell interactions using intermediate switch molecules may expand the scope of engineered T-cell therapy to solid tumors, as well as indications beyond cancer therapy. PMID:26759368

  7. Chlamydial metabolism revisited: interspecies metabolic variability and developmental stage-specific physiologic activities.

    PubMed

    Omsland, Anders; Sixt, Barbara Susanne; Horn, Matthias; Hackstadt, Ted

    2014-07-01

    Chlamydiae are a group of obligate intracellular bacteria comprising important human and animal pathogens as well as symbionts of ubiquitous protists. They are characterized by a developmental cycle including two main morphologically and physiologically distinct stages, the replicating reticulate body and the infectious nondividing elementary body. In this review, we reconstruct the history of studies that have led to our current perception of chlamydial physiology, focusing on their energy and central carbon metabolism. We then compare the metabolic capabilities of pathogenic and environmental chlamydiae highlighting interspecies variability among the metabolically more flexible environmental strains. We discuss recent findings suggesting that chlamydiae may not live as energy parasites throughout the developmental cycle and that elementary bodies are not metabolically inert but exhibit metabolic activity under appropriate axenic conditions. The observed host-free metabolic activity of elementary bodies may reflect adequate recapitulation of the intracellular environment, but there is evidence that this activity is biologically relevant and required for extracellular survival and maintenance of infectivity. The recent discoveries call for a reconsideration of chlamydial metabolism and future in-depth analyses to better understand how species- and stage-specific differences in chlamydial physiology may affect virulence, tissue tropism, and host adaptation.

  8. Penicillin V acylase from Pectobacterium atrosepticum exhibits high specific activity and unique kinetics.

    PubMed

    Avinash, V S; Ramasamy, Sureshkumar; Suresh, C G; Pundle, Archana

    2015-08-01

    Penicillin V acylases (PVAs, E.C.3.5.11) belong to the Ntn hydrolase super family of enzymes that catalyze the deacylation of the side chain from phenoxymethyl penicillin (penicillin V). Penicillin acylases find use in the pharmaceutical industry for the production of semi-synthetic antibiotics. PVAs employ the N-terminal cysteine residue as catalytic nucleophile and are structurally and evolutionarily related to bile salt hydrolases (BSHs). Here, we report the cloning and characterization of a PVA enzyme from the Gram-negative plant pathogen, Pectobacterium atrosepticum (PaPVA). The enzyme was cloned and expressed in Escherichia coli attaining a very high yield (250 mg/l) and a comparatively high specific activity (430 IU/mg). The enzyme showed marginally better pH and thermo-stability over PVAs characterized from Gram-positive bacteria. The enzyme also showed enhanced activity in presence of organic solvents and detergents. The enzyme kinetics turned out to be significantly different from that of previously reported PVAs, displaying positive cooperativity and substrate inhibition. The presence of bile salts had a modulating effect on PaPVA activity. Sequence analysis and characterization reveal the distinctive nature of these enzymes and underscore the need to study PVAs from Gram-negative bacteria. PMID:25931393

  9. ChIP-seq Accurately Predicts Tissue-Specific Activity of Enhancers

    SciTech Connect

    Visel, Axel; Blow, Matthew J.; Li, Zirong; Zhang, Tao; Akiyama, Jennifer A.; Holt, Amy; Plajzer-Frick, Ingrid; Shoukry, Malak; Wright, Crystal; Chen, Feng; Afzal, Veena; Ren, Bing; Rubin, Edward M.; Pennacchio, Len A.

    2009-02-01

    A major yet unresolved quest in decoding the human genome is the identification of the regulatory sequences that control the spatial and temporal expression of genes. Distant-acting transcriptional enhancers are particularly challenging to uncover since they are scattered amongst the vast non-coding portion of the genome. Evolutionary sequence constraint can facilitate the discovery of enhancers, but fails to predict when and where they are active in vivo. Here, we performed chromatin immunoprecipitation with the enhancer-associated protein p300, followed by massively-parallel sequencing, to map several thousand in vivo binding sites of p300 in mouse embryonic forebrain, midbrain, and limb tissue. We tested 86 of these sequences in a transgenic mouse assay, which in nearly all cases revealed reproducible enhancer activity in those tissues predicted by p300 binding. Our results indicate that in vivo mapping of p300 binding is a highly accurate means for identifying enhancers and their associated activities and suggest that such datasets will be useful to study the role of tissue-specific enhancers in human biology and disease on a genome-wide scale.

  10. AIRE activated tissue specific genes have histone modifications associated with inactive chromatin.

    PubMed

    Org, Tõnis; Rebane, Ana; Kisand, Kai; Laan, Martti; Haljasorg, Uku; Andreson, Reidar; Peterson, Pärt

    2009-12-15

    The Autoimmune Regulator (AIRE) protein is expressed in thymic medullary epithelial cells, where it promotes the ectopic expression of tissue-restricted antigens needed for efficient negative selection of developing thymocytes. Mutations in AIRE cause APECED syndrome, which is characterized by a breakdown of self-tolerance. The molecular mechanism by which AIRE increases the expression of a variety of different genes remains unknown. Here, we studied AIRE-regulated genes using whole genome expression analysis and chromatin immunoprecipitation. We show that AIRE preferentially activates genes that are tissue-specific and characterized by low levels of initial expression in stably transfected HEK293 cell model and mouse thymic medullary epithelial cells. In addition, the AIRE-regulated genes lack active chromatin marks, such as histone H3 trimethylation (H3K4me3) and acetylation (AcH3), on their promoters. We also show that during activation by AIRE, the target genes acquire histone H3 modifications associated with transcription and RNA polymerase II. In conclusion, our data show that AIRE is able to promote ectopic gene expression from chromatin associated with histone modifications characteristic to inactive genes.

  11. Specific hunger- and satiety-induced tuning of guinea pig enteric nerve activity.

    PubMed

    Roosen, Lina; Boesmans, Werend; Dondeyne, Marjan; Depoortere, Inge; Tack, Jan; Vanden Berghe, Pieter

    2012-09-01

    Although hunger and satiety are mainly centrally regulated, there is convincing evidence that also gastrointestinal motor activity and hormone fluctuations significantly contribute to appetite signalling. In this study, we investigated how motility and enteric nerve activity are set by fasting and feeding. By means of video-imaging, we tested whether peristaltic activity differs in ex vivo preparations from fasted and re-fed guinea pigs. Ca(2+) imaging was used to investigate whether the feeding state directly alters neuronal activity, either occurring spontaneously or evoked by (an)orexigenic signalling molecules. We found that pressure-induced (2 cmH(2)O) peristaltic activity occurs at a higher frequency in ileal segments from re-fed animals (re-fed versus fasted, 6.12 ± 0.22 vs. 4.84 ± 0.52 waves min(-1), P = 0.028), even in vitro hours after death. Myenteric neuronal responses were tuned to the feeding status, since neurons in tissues from re-fed animals remained hyper-responsive to high K(+)-evoked depolarization (P < 0.001) and anorexigenic molecules (P < 0.001), while being less responsive to orexigenic ghrelin (P = 0.013). This illustrates that the feeding status remains ‘imprinted' ex vivo. We were able to reproduce this feeding state-related memory in vitro and found humoral feeding state-related factors to be implicated. Although the molecular link with hyperactivity is not entirely elucidated yet, glucose-dependent pathways are clearly involved in tuning neuronal excitability. We conclude that a bistable memory system that tunes neuronal responses to fasting and re-feeding is present in the enteric nervous system, increasing responses to depolarization and anorexigenic molecules in the re-fed state, while decreasing responses to orexigenic ghrelin. Unlike the hypothalamus, where specific cell populations sensitive to either orexigenic or anorexigenic molecules exist, the enteric feeding state-related memory system is present at the functional level

  12. Specific hunger- and satiety-induced tuning of guinea pig enteric nerve activity.

    PubMed

    Roosen, Lina; Boesmans, Werend; Dondeyne, Marjan; Depoortere, Inge; Tack, Jan; Vanden Berghe, Pieter

    2012-09-01

    Although hunger and satiety are mainly centrally regulated, there is convincing evidence that also gastrointestinal motor activity and hormone fluctuations significantly contribute to appetite signalling. In this study, we investigated how motility and enteric nerve activity are set by fasting and feeding. By means of video-imaging, we tested whether peristaltic activity differs in ex vivo preparations from fasted and re-fed guinea pigs. Ca(2+) imaging was used to investigate whether the feeding state directly alters neuronal activity, either occurring spontaneously or evoked by (an)orexigenic signalling molecules. We found that pressure-induced (2 cmH(2)O) peristaltic activity occurs at a higher frequency in ileal segments from re-fed animals (re-fed versus fasted, 6.12 ± 0.22 vs. 4.84 ± 0.52 waves min(-1), P = 0.028), even in vitro hours after death. Myenteric neuronal responses were tuned to the feeding status, since neurons in tissues from re-fed animals remained hyper-responsive to high K(+)-evoked depolarization (P < 0.001) and anorexigenic molecules (P < 0.001), while being less responsive to orexigenic ghrelin (P = 0.013). This illustrates that the feeding status remains ‘imprinted' ex vivo. We were able to reproduce this feeding state-related memory in vitro and found humoral feeding state-related factors to be implicated. Although the molecular link with hyperactivity is not entirely elucidated yet, glucose-dependent pathways are clearly involved in tuning neuronal excitability. We conclude that a bistable memory system that tunes neuronal responses to fasting and re-feeding is present in the enteric nervous system, increasing responses to depolarization and anorexigenic molecules in the re-fed state, while decreasing responses to orexigenic ghrelin. Unlike the hypothalamus, where specific cell populations sensitive to either orexigenic or anorexigenic molecules exist, the enteric feeding state-related memory system is present at the functional level

  13. Domain-specific physical activity and health-related quality of life in university students.

    PubMed

    Pedišić, Zeljko; Rakovac, Marija; Titze, Sylvia; Jurakić, Danijel; Oja, Pekka

    2014-01-01

    Information on the relationship between domain-specific physical activity (PA) and health-related quality of life (HRQoL) in the general population and specific groups is still scarce. The aim of this study was to determine the relationship between PA in work, transport, domestic and leisure-time domains and HRQoL among university students. PA and HRQoL were assessed in a random stratified sample of 1750 university students using the International Physical Activity Questionnaire - long form and 12-item Short Form Health Survey, respectively. The Spearman's rank correlations, adjusted for age, community size, personal monthly budget, body mass index, smoking habits and alcohol intake ranged from -0.11 to 0.18 in female students and -0.29 to 0.19 in male students. Leisure-time, domestic, transport-related PA and total PA were positively related to HRQoL. Inverse correlations with HRQoL were only found for work-related PA in male students. Multiple linear regression analysis showed that only leisure-time PA was related to the Physical Summary Component score (β = 0.08 for females and β = 0.10 for males, P < 0.05). Domain-specific PA levels were not significantly related to the Mental Component Summary score. To get a more comprehensive insight in the relationship between PA and HRQoL, future studies should not only analyse total PA levels but also domain-specific PA levels. The evidence on the positive relationship of leisure-time, transport and domestic PA with HRQoL can potentially be used to support evidence-based promotion of PA in a university setting, and as a hypothesis for future longitudinal studies on such potential causal relationships.

  14. Specific detection of the cleavage activity of mycobacterial enzymes using a quantum dot based DNA nanosensor

    NASA Astrophysics Data System (ADS)

    Jepsen, Morten Leth; Harmsen, Charlotte; Godbole, Adwait Anand; Nagaraja, Valakunja; Knudsen, Birgitta R.; Ho, Yi-Ping

    2015-12-01

    We present a quantum dot based DNA nanosensor specifically targeting the cleavage step in the reaction cycle of the essential DNA-modifying enzyme, mycobacterial topoisomerase I. The design takes advantages of the unique photophysical properties of quantum dots to generate visible fluorescence recovery upon specific cleavage by mycobacterial topoisomerase I. This report, for the first time, demonstrates the possibility to quantify the cleavage activity of the mycobacterial enzyme without the pre-processing sample purification or post-processing signal amplification. The cleavage induced signal response has also proven reliable in biological matrices, such as whole cell extracts prepared from Escherichia coli and human Caco-2 cells. It is expected that the assay may contribute to the clinical diagnostics of bacterial diseases, as well as the evaluation of treatment outcomes.We present a quantum dot based DNA nanosensor specifically targeting the cleavage step in the reaction cycle of the essential DNA-modifying enzyme, mycobacterial topoisomerase I. The design takes advantages of the unique photophysical properties of quantum dots to generate visible fluorescence recovery upon specific cleavage by mycobacterial topoisomerase I. This report, for the first time, demonstrates the possibility to quantify the cleavage activity of the mycobacterial enzyme without the pre-processing sample purification or post-processing signal amplification. The cleavage induced signal response has also proven reliable in biological matrices, such as whole cell extracts prepared from Escherichia coli and human Caco-2 cells. It is expected that the assay may contribute to the clinical diagnostics of bacterial diseases, as well as the evaluation of treatment outcomes. Electronic supplementary information (ESI) available: Characterization of the QD-based DNA Nanosensor. See DOI: 10.1039/c5nr06326d

  15. Episodic specificity induction impacts activity in a core brain network during construction of imagined future experiences.

    PubMed

    Madore, Kevin P; Szpunar, Karl K; Addis, Donna Rose; Schacter, Daniel L

    2016-09-20

    Recent behavioral work suggests that an episodic specificity induction-brief training in recollecting the details of a past experience-enhances performance on subsequent tasks that rely on episodic retrieval, including imagining future experiences, solving open-ended problems, and thinking creatively. Despite these far-reaching behavioral effects, nothing is known about the neural processes impacted by an episodic specificity induction. Related neuroimaging work has linked episodic retrieval with a core network of brain regions that supports imagining future experiences. We tested the hypothesis that key structures in this network are influenced by the specificity induction. Participants received the specificity induction or one of two control inductions and then generated future events and semantic object comparisons during fMRI scanning. After receiving the specificity induction compared with the control, participants exhibited significantly more activity in several core network regions during the construction of imagined events over object comparisons, including the left anterior hippocampus, right inferior parietal lobule, right posterior cingulate cortex, and right ventral precuneus. Induction-related differences in the episodic detail of imagined events significantly modulated induction-related differences in the construction of imagined events in the left anterior hippocampus and right inferior parietal lobule. Resting-state functional connectivity analyses with hippocampal and inferior parietal lobule seed regions and the rest of the brain also revealed significantly stronger core network coupling following the specificity induction compared with the control. These findings provide evidence that an episodic specificity induction selectively targets episodic processes that are commonly linked to key core network regions, including the hippocampus.

  16. Genetic and functional modularity of Hox activities in the specification of limb-innervating motor neurons.

    PubMed

    Lacombe, Julie; Hanley, Olivia; Jung, Heekyung; Philippidou, Polyxeni; Surmeli, Gulsen; Grinstein, Jonathan; Dasen, Jeremy S

    2013-01-01

    A critical step in the assembly of the neural circuits that control tetrapod locomotion is the specification of the lateral motor column (LMC), a diverse motor neuron population targeting limb musculature. Hox6 paralog group genes have been implicated as key determinants of LMC fate at forelimb levels of the spinal cord, through their ability to promote expression of the LMC-restricted genes Foxp1 and Raldh2 and to suppress thoracic fates through exclusion of Hoxc9. The specific roles and mechanisms of Hox6 gene function in LMC neurons, however, are not known. We show that Hox6 genes are critical for diverse facets of LMC identity and define motifs required for their in vivo specificities. Although Hox6 genes are necessary for generating the appropriate number of LMC neurons, they are not absolutely required for the induction of forelimb LMC molecular determinants. In the absence of Hox6 activity, LMC identity appears to be preserved through a diverse array of Hox5-Hox8 paralogs, which are sufficient to reprogram thoracic motor neurons to an LMC fate. In contrast to the apparently permissive Hox inputs to early LMC gene programs, individual Hox genes, such as Hoxc6, have specific roles in promoting motor neuron pool diversity within the LMC. Dissection of motifs required for Hox in vivo specificities reveals that either cross-repressive interactions or cooperativity with Pbx cofactors are sufficient to induce LMC identity, with the N-terminus capable of promoting columnar, but not pool, identity when transferred to a heterologous homeodomain. These results indicate that Hox proteins orchestrate diverse aspects of cell fate specification through both the convergent regulation of gene programs regulated by many paralogs and also more restricted actions encoded through specificity determinants in the N-terminus.

  17. NFκB-inducing kinase inhibits NFκB activity specifically in neurons of the CNS.

    PubMed

    Mao, Xianrong; Phanavanh, Bounleut; Hamdan, Hamdan; Moerman-Herzog, Andréa M; Barger, Steven W

    2016-04-01

    The control of NFκB in CNS neurons appears to differ from that in other cell types. Studies have reported induction of NFκB in neuronal cultures and immunostaining in vivo, but others have consistently detected little or no transcriptional activation by NFκB in brain neurons. To test if neurons lack some component of the signal transduction system for NFκB activation, we transfected cortical neurons with several members of this signaling system along with a luciferase-based NFκB-reporter plasmid; RelA was cotransfected in some conditions. No component of the NFκB pathway was permissive for endogenous NFκB activity, and none stimulated the activity of exogenous RelA. Surprisingly, however, the latter was inhibited by cotransfection of NFκB-inducing kinase (NIK). Fluorescence imaging of RelA indicated that co-expression of NIK sequestered RelA in the cytoplasm, similar to the effect of IκBα. NIK-knockout mice showed elevated expression of an NFκB-reporter construct in neurons in vivo. Cortical neurons cultured from NIK-knockout mice showed elevated expression of an NFκB-reporter transgene. Consistent with data from other cell types, a C-terminal fragment of NIK suppressed RelA activity in astrocytes as well as neurons. Therefore, the inhibitory ability of the NIK C-terminus was unbiased with regard to cell type. However, inhibition of NFκB by full-length NIK is a novel outcome that appears to be specific to CNS neurons. This has implications for unique aspects of transcription in the CNS, perhaps relevant to aspects of development, neuroplasticity, and neuroinflammation. Full-length NIK was found to inhibit (down arrow) transcriptional activation of NFκB in neurons, while it elevated (up arrow) activity in astrocytes. Deletion constructs corresponding to the N-terminus or C-terminus also inhibited NFκB in neurons, while only the C-terminus did so in astrocytes. One possible explanation is that the inhibition in neurons occurs via two different

  18. Specific activity and hazards of granite samples collected from the Eastern Desert of Egypt.

    PubMed

    Arafa, Wafaa

    2004-01-01

    Fifty granitic rock samples were collected from different plutons in the central part of the Eastern Desert of Egypt and were analyzed for specific concentrations of (238)U, (232)Th and (40)K radionuclei. The measurements were carried out using a high performance and stability Nomad Plus spectroscopy system attached to a 1.7 keV (FWHM) HPGe detector. The spectra were analyzed using the direct gamma counting comparison method as well as the traditional absolute efficiency curve method. The highest average value of (238)U concentration (1184 Bq kg(-1)) was observed at EI Misikat region whereas the highest average values of (40)K and (232)Th concentration (2301.8 and 162.5 Bq kg(-1) respectively), were detected at Gabal Homret Waggat area. The radium equivalent activity (Ra(eq)), the absorbed dose rate (D), the external hazard index (H(ex)) and the annual gonadal dose equivalent were also calculated and compared to the international recommended values. Radon exhalation rate from the rock samples were measured using the activated charcoal canister method. The average value of radon exhalation varies from 0.052 to 0.69 Bq m(-2) h(-1) and depends on the specific concentration of uranium.

  19. Allopurinol reduces antigen-specific and polyclonal activation of human T cells

    PubMed Central

    Pérez-Mazliah, Damián; Albareda, María C.; Alvarez, María G.; Lococo, Bruno; Bertocchi, Graciela L.; Petti, Marcos; Viotti, Rodolfo J.; Laucella, Susana A.

    2012-01-01

    Allopurinol is the most popular commercially available xanthine oxidase inhibitor and it is widely used for treatment of symptomatic hyperuricaemia, or gout. Although, several anti-inflammatory actions of allopurinol have been demonstrated in vivo and in vitro, there have been few studies on the action of allopurinol on T cells. In the current study, we have assessed the effect of allopurinol on antigen-specific and mitogen-driven activation and cytokine production in human T cells. Allopurinol markedly decreased the frequency of IFN-γ and IL-2-producing T cells, either after polyclonal or antigen-specific stimulation with Herpes Simplex virus 1, Influenza (Flu) virus, tetanus toxoid and Trypanosoma cruzi-derived antigens. Allopurinol attenuated CD69 upregulation after CD3 and CD28 engagement and significantly reduced the levels of spontaneous and mitogen-induced intracellular reactive oxygen species in T cells. The diminished T cell activation and cytokine production in the presence of allopurinol support a direct action of allopurinol on human T cells, offering a potential pharmacological tool for the management of cell-mediated inflammatory diseases. PMID:23049532

  20. Unnatural amino acids increase activity and specificity of synthetic substrates for human and malarial cathepsin C.

    PubMed

    Poreba, Marcin; Mihelic, Marko; Krai, Priscilla; Rajkovic, Jelena; Krezel, Artur; Pawelczak, Malgorzata; Klemba, Michael; Turk, Dusan; Turk, Boris; Latajka, Rafal; Drag, Marcin

    2014-04-01

    Mammalian cathepsin C is primarily responsible for the removal of N-terminal dipeptides and activation of several serine proteases in inflammatory or immune cells, while its malarial parasite ortholog dipeptidyl aminopeptidase 1 plays a crucial role in catabolizing the hemoglobin of its host erythrocyte. In this report, we describe the systematic substrate specificity analysis of three cathepsin C orthologs from Homo sapiens (human), Bos taurus (bovine) and Plasmodium falciparum (malaria parasite). Here, we present a new approach with a tailored fluorogenic substrate library designed and synthesized to probe the S1 and S2 pocket preferences of these enzymes with both natural and a broad range of unnatural amino acids. Our approach identified very efficiently hydrolyzed substrates containing unnatural amino acids, which resulted in the design of significantly better substrates than those previously known. Additionally, in this study significant differences in terms of the structures of optimal substrates for human and malarial orthologs are important from the therapeutic point of view. These data can be also used for the design of specific inhibitors or activity-based probes.

  1. Organ-specific systemic lupus erythematosus activity during pregnancy is associated with adverse pregnancy outcomes.

    PubMed

    Tedeschi, Sara K; Guan, Hongshu; Fine, Alexander; Costenbader, Karen H; Bermas, Bonnie

    2016-07-01

    Systemic lupus erythematosus (SLE) is a disease of reproductive-age women, and thus questions regarding how disease influences pregnancy outcomes arise. We investigated whether five specific types of SLE activity during the 6 months before conception or during pregnancy (nephritis, cytopenias, skin disease, arthritis, serositis) were associated with adverse pregnancy outcomes. We performed a retrospective cohort study of pregnancy outcomes among women with SLE at the Brigham and Women's Hospital Lupus Center. Adverse pregnancy outcomes included pre-eclampsia, pre-term delivery, elective termination due to SLE, spontaneous miscarriage at weeks 12-20, and stillbirth. SLE and obstetric history, laboratories, and medications were obtained from electronic medical records. Generalized linear mixed models adjusting for potential confounders were used to identify predictors of any adverse pregnancy outcome. Most pregnancies resulted in a live term delivery (76.5 %). After adjustment for Hispanic ethnicity, prior adverse pregnancy outcome and medication use 6 months before conception, nephritis during pregnancy (odds ratio (OR) 3.6, 95 % confidence interval (CI) 1.0-12.8), cytopenias during pregnancy (OR 3.9, 95 % CI 1.3-11.4), and serositis during pregnancy (OR 5.9, 95 % CI 1.0-34.0) were significantly associated with adverse pregnancy outcome. Specific types of SLE disease activity during pregnancy were related to adverse pregnancy outcome. Nephritis, cytopenias, and serositis carried a higher risk of adverse pregnancy outcome, suggesting that these abnormalities should be carefully monitored during pregnancy. PMID:27166627

  2. Deep brain optical measurements of cell type–specific neural activity in behaving mice

    PubMed Central

    Cui, Guohong; Jun, Sang Beom; Jin, Xin; Luo, Guoxiang; Pham, Michael D; Lovinger, David M; Vogel, Steven S; Costa, Rui M

    2014-01-01

    Recent advances in genetically encoded fluorescent sensors enable the monitoring of cellular events from genetically defined groups of neurons in vivo. In this protocol, we describe how to use a time-correlated single-photon counting (tcspc)–based fiber optics system to measure the intensity, emission spectra and lifetime of fluorescent biosensors expressed in deep brain structures in freely moving mice. When combined with cre-dependent selective expression of genetically encoded ca2+ indicators (GecIs), this system can be used to measure the average neural activity from a specific population of cells in mice performing complex behavioral tasks. as an example, we used viral expression of GcaMps in striatal projection neurons (spns) and recorded the fluorescence changes associated with calcium spikes from mice performing a lever-pressing operant task. the whole procedure, consisting of virus injection, behavior training and optical recording, takes 3–4 weeks to complete. With minor adaptations, this protocol can also be applied to recording cellular events from other cell types in deep brain regions, such as dopaminergic neurons in the ventral tegmental area. the simultaneously recorded fluorescence signals and behavior events can be used to explore the relationship between the neural activity of specific brain circuits and behavior. PMID:24784819

  3. Shh and ZRS enhancer colocalisation is specific to the zone of polarising activity.

    PubMed

    Williamson, Iain; Lettice, Laura A; Hill, Robert E; Bickmore, Wendy A

    2016-08-15

    Limb-specific Shh expression is regulated by the (∼1 Mb distant) ZRS enhancer. In the mouse, limb bud-restricted spatiotemporal Shh expression occurs from ∼E10 to E11.5 at the distal posterior margin and is essential for correct autopod formation. Here, we have analysed the higher-order chromatin conformation of Shh in expressing and non-expressing tissues, both by fluorescence in situ hybridisation (FISH) and by chromosome conformation capture (5C). Conventional and super-resolution light microscopy identified significantly elevated frequencies of Shh/ZRS colocalisation only in the Shh-expressing regions of the limb bud, in a conformation consistent with enhancer-promoter loop formation. However, in all tissues and at all developmental stages analysed, Shh-ZRS spatial distances were still consistently shorter than those to a neural enhancer located between Shh and ZRS in the genome. 5C identified a topologically associating domain (TAD) over the Shh/ZRS genomic region and enriched interactions between Shh and ZRS throughout E11.5 embryos. Shh/ZRS colocalisation, therefore, correlates with the spatiotemporal domain of limb bud-specific Shh expression, but close Shh and ZRS proximity in the nucleus occurs regardless of whether the gene or enhancer is active. We suggest that this constrained chromatin configuration optimises the opportunity for the active enhancer to locate and instigate the expression of Shh.

  4. Altered host behaviour and brain serotonergic activity caused by acanthocephalans: evidence for specificity

    PubMed Central

    Tain, Luke; Perrot-Minnot, Marie-Jeanne; Cézilly, Frank

    2006-01-01

    Manipulative parasites can alter the phenotype of intermediate hosts in various ways. However, it is unclear whether such changes are just by-products of infection or adaptive and enhance transmission to the final host. Here, we show that the alteration of serotonergic activity is functionally linked to the alteration of specific behaviour in the amphipod Gammarus pulex infected with acanthocephalan parasites. Pomphorhynchus laevis and, to a lesser extent, Pomphorhynchus tereticollis altered phototactism, but not geotactism, in G. pulex, whereas the reverse was true for Polymorphus minutus. Serotonin (5-hydroxytryptamine, 5-HT) injected to uninfected G. pulex mimicked the altered phototactism, but had no effect on geotactism. Photophilic G. pulex infected with P. laevis or P. tereticollis showed a 40% increase in brain 5-HT immunoreactivity compared to photophobic, uninfected individuals. In contrast, brain 5-HT immunoreactivity did not differ between P. minutus-infected and uninfected G. pulex. Finally, brain 5-HT immunoreactivity differed significantly among P. tereticollis-infected individuals in accordance with their degree of manipulation. Our results demonstrate that altered 5-HT activity is not the mere consequence of infection by acanthocephalans but is specifically linked to the disruption of host photophobic behaviour, whereas the alteration of other behaviours such as geotactism may rely on distinct physiological routes. PMID:17015346

  5. Shh and ZRS enhancer colocalisation is specific to the zone of polarising activity

    PubMed Central

    Williamson, Iain; Lettice, Laura A.; Hill, Robert E.

    2016-01-01

    Limb-specific Shh expression is regulated by the (∼1 Mb distant) ZRS enhancer. In the mouse, limb bud-restricted spatiotemporal Shh expression occurs from ∼E10 to E11.5 at the distal posterior margin and is essential for correct autopod formation. Here, we have analysed the higher-order chromatin conformation of Shh in expressing and non-expressing tissues, both by fluorescence in situ hybridisation (FISH) and by chromosome conformation capture (5C). Conventional and super-resolution light microscopy identified significantly elevated frequencies of Shh/ZRS colocalisation only in the Shh-expressing regions of the limb bud, in a conformation consistent with enhancer-promoter loop formation. However, in all tissues and at all developmental stages analysed, Shh-ZRS spatial distances were still consistently shorter than those to a neural enhancer located between Shh and ZRS in the genome. 5C identified a topologically associating domain (TAD) over the Shh/ZRS genomic region and enriched interactions between Shh and ZRS throughout E11.5 embryos. Shh/ZRS colocalisation, therefore, correlates with the spatiotemporal domain of limb bud-specific Shh expression, but close Shh and ZRS proximity in the nucleus occurs regardless of whether the gene or enhancer is active. We suggest that this constrained chromatin configuration optimises the opportunity for the active enhancer to locate and instigate the expression of Shh. PMID:27402708

  6. Association Between Serum Ceruloplasmin Specific Activity and Risk of Alzheimer's Disease.

    PubMed

    Siotto, Mariacristina; Simonelli, Ilaria; Pasqualetti, Patrizio; Mariani, Stefania; Caprara, Deborah; Bucossi, Serena; Ventriglia, Mariacarla; Molinario, Rossana; Antenucci, Mirca; Rongioletti, Mauro; Rossini, Paolo Maria; Squitti, Rosanna

    2016-01-01

    Meta-analyses demonstrate copper involvement in Alzheimer's disease (AD), and the systemic ceruloplasmin status in relation to copper is an emerging issue. To deepen this matter, we evaluated levels of ceruloplasmin concentration, ceruloplasmin activity, ceruloplasmin specific activity (eCp/iCp), copper, non-ceruloplasmin copper iron, transferrin, the ceruloplasmin/transferrin ratio, and the APOE genotype in a sample of 84 AD patients and 58 healthy volunteers. From the univariate logistic analyses we found that ceruloplasmin concentration, eCp/iCp, copper, transferrin, the ceruloplasmin/transferrin ratio, and the APOE genotype were significantly associated with the probability of AD. In the multivariable logistic regression analysis, we selected the best subset of biological predictors by the forward stepwise procedure. The analysis showed a decrease of the risk of having AD for eCp/iCp (p = 0.001) and an increase of this risk for non-ceruloplasmin copper (p = 0.008), age (p = 0.001), and APOE-ɛ4 allele (p <  0.001). The estimated model showed a good power in discriminating AD patients from healthy controls (area under curve: 88% ; sensitivity: 66% ; specificity 93%). These data strength the breakdown of copper homeostasis and propose eCp/iCp as a reliable marker of ceruloplasmin status. PMID:26836154

  7. Novel Cycloheximide Derivatives Targeting the Moonlighting Protein Mip Exhibit Specific Antimicrobial Activity Against Legionella pneumophila.

    PubMed

    Rasch, Janine; Theuerkorn, Martin; Ünal, Can; Heinsohn, Natascha; Tran, Stefan; Fischer, Gunter; Weiwad, Matthias; Steinert, Michael

    2015-01-01

    Macrophage infectivity potentiator (Mip) and Mip-like proteins are virulence factors in a wide range of pathogens including Legionella pneumophila. These proteins belong to the FK506 binding protein (FKBP) family of peptidyl-prolyl-cis/trans-isomerases (PPIases). In L. pneumophila, the PPIase activity of Mip is required for invasion of macrophages, transmigration through an in vitro lung-epithelial barrier, and full virulence in the guinea pig infection model. Additionally, Mip is a moonlighting protein that binds to collagen IV in the extracellular matrix. Here, we describe the development and synthesis of cycloheximide derivatives with adamantyl moieties as novel FKBP ligands, and analyze their effect on the viability of L. pneumophila and other bacteria. All compounds efficiently inhibited PPIase activity of the prototypic human FKBP12 as well as Mip with IC50-values as low as 180 nM and 1.7 μM, respectively. Five of these derivatives inhibited the growth of L. pneumophila at concentrations of 30-40 μM, but exhibited no effect on other tested bacterial species indicating a specific spectrum of antibacterial activity. The derivatives carrying a 3,5-dimethyladamantan-1-[yl]acetamide substitution (MT_30.32), and a 3-ethyladamantan-1-[yl]acetamide substitution (MT_30.51) had the strongest effects in PPIase- and liquid growth assays. MT_30.32 and MT_30.51 were also inhibitory in macrophage infection studies without being cytotoxic. Accordingly, by applying a combinatorial approach, we were able to generate novel, hybrid inhibitors consisting of cycloheximide and adamantane, two known FKBP inhibitors that interact with different parts of the PPIase domain, respectively. Interestingly, despite the proven Mip-inhibitory activity, the viability of a Mip-deficient strain was affected to the same degree as its wild type. Hence, we also propose that cycloheximide derivatives with adamantyl moieties are potent PPIase inhibitors with multiple targets in L. pneumophila.

  8. Phosphorylation regulates the water channel activity of the seed-specific aquaporin alpha-TIP.

    PubMed Central

    Maurel, C; Kado, R T; Guern, J; Chrispeels, M J

    1995-01-01

    The vacuolar membrane protein alpha-TIP is a seed-specific protein of the Major Intrinsic Protein family. Expression of alpha-TIP in Xenopus oocytes conferred a 4- to 8-fold increase in the osmotic water permeability (Pf) of the oocyte plasma membrane, showing that alpha-TIP forms water channels and is thus a new aquaporin. alpha-TIP has three putative phosphorylation sites on the cytoplasmic side of the membrane (Ser7, Ser23 and Ser99), one of which (Ser7) has been shown to be phosphorylated. We present several lines of evidence that the activity of this aquaporin is regulated by phosphorylation. First, mutation of the putative phosphorylation sites in alpha-TIP (Ser7Ala, Ser23Ala and Ser99Ala) reduced the apparent water transport activity of alpha-TIP in oocytes, suggesting that phosphorylation of alpha-TIP occurs in the oocytes and participates in the control of water channel activity. Second, exposure of oocytes to the cAMP agonists 8-bromoadenosine 3',5'-cyclic monophosphate, forskolin and 3-isobutyl-1-methylxanthine, which stimulate endogenous protein kinase A (PKA), increased the water transport activity of alpha-TIP by 80-100% after 60 min. That the protein can be phosphorylated by PKA was demonstrated by phosphorylating alpha-TIP in isolated oocyte membranes with the bovine PKA catalytic subunit. Third, the integrity of the three sites at positions 7, 23 and 99 was necessary for the cAMP-dependent increase in the Pf of oocytes expressing alpha-TIP, as well as for in vitro phosphorylation of alpha-TIP. These findings demonstrate that the alpha-TIP water channel can be modulated via phosphorylation of Ser7, Ser23 and Ser99.(ABSTRACT TRUNCATED AT 250 WORDS) Images PMID:7542585

  9. Novel Cycloheximide Derivatives Targeting the Moonlighting Protein Mip Exhibit Specific Antimicrobial Activity Against Legionella pneumophila

    PubMed Central

    Rasch, Janine; Theuerkorn, Martin; Ünal, Can; Heinsohn, Natascha; Tran, Stefan; Fischer, Gunter; Weiwad, Matthias; Steinert, Michael

    2015-01-01

    Macrophage infectivity potentiator (Mip) and Mip-like proteins are virulence factors in a wide range of pathogens including Legionella pneumophila. These proteins belong to the FK506 binding protein (FKBP) family of peptidyl-prolyl-cis/trans-isomerases (PPIases). In L. pneumophila, the PPIase activity of Mip is required for invasion of macrophages, transmigration through an in vitro lung–epithelial barrier, and full virulence in the guinea pig infection model. Additionally, Mip is a moonlighting protein that binds to collagen IV in the extracellular matrix. Here, we describe the development and synthesis of cycloheximide derivatives with adamantyl moieties as novel FKBP ligands, and analyze their effect on the viability of L. pneumophila and other bacteria. All compounds efficiently inhibited PPIase activity of the prototypic human FKBP12 as well as Mip with IC50-values as low as 180 nM and 1.7 μM, respectively. Five of these derivatives inhibited the growth of L. pneumophila at concentrations of 30–40 μM, but exhibited no effect on other tested bacterial species indicating a specific spectrum of antibacterial activity. The derivatives carrying a 3,5-dimethyladamantan-1-[yl]acetamide substitution (MT_30.32), and a 3-ethyladamantan-1-[yl]acetamide substitution (MT_30.51) had the strongest effects in PPIase- and liquid growth assays. MT_30.32 and MT_30.51 were also inhibitory in macrophage infection studies without being cytotoxic. Accordingly, by applying a combinatorial approach, we were able to generate novel, hybrid inhibitors consisting of cycloheximide and adamantane, two known FKBP inhibitors that interact with different parts of the PPIase domain, respectively. Interestingly, despite the proven Mip-inhibitory activity, the viability of a Mip-deficient strain was affected to the same degree as its wild type. Hence, we also propose that cycloheximide derivatives with adamantyl moieties are potent PPIase inhibitors with multiple targets in L

  10. Active Components of Fungus Shiraia bambusiscola Can Specifically Induce BGC823 Gastric Cancer Cell Apoptosis

    PubMed Central

    Zhang, Shubing; Qiu, Dewen; Liu, Jingjiang; Li, Zhijian

    2016-01-01

    Objective Gastric cancer is a major health issue worldwide. Using a therapeutic approach, with minor side-effects, is very essential for the treatment of the gastric cancer. Shiraia bambusicola is a parasitic fungus which is widely used in China for curing several diseases with little side-effects. However, the mechanisms are not well understood yet. The aim of this study was to further understand the pharmacological mechanisms of Shiraia bambusicola and investigate whether it can be used for curing gastric cancer. Materials and Methods In this experimental study, we mainly tested the effect of active components extracted from Shiraia bambusicola on BGC823, A549 and HepG2 cells. We used MTT assay to test cell viability. We also analyzed morphologic changes caused by apoptosis using Hoechst 33342 fluorescence staining, as well as cell cycle status and apoptosis ratio using flow-cytometer. In addition, protein expression level was tested by Western-blotting assay. Results BGC-823 cell proliferation was specifically inhibited by active components of Shiraia bambusicola. Meanwhile, these active components could induce BGC-823 cells apoptosis and retard the cell cycle in S/G2 phase. We also determined that two critical protein markers cleaved Poly(ADP-ribose) polymerase-1 (PARP-1) and FLICE-inhibitory protein (FLIP), involved in apoptosis process, were regulated by these active components. Conclusion These data shed light on the treatment of human gastric cancer and conclude that Shiraia bambusicola can be a good therapeutic candidate for treatment of this malignancy. PMID:27540519

  11. Foxi3 Deficiency Compromises Hair Follicle Stem Cell Specification and Activation.

    PubMed

    Shirokova, Vera; Biggs, Leah C; Jussila, Maria; Ohyama, Takahiro; Groves, Andrew K; Mikkola, Marja L

    2016-07-01

    The hair follicle is an ideal system to study stem cell specification and homeostasis due to its well characterized morphogenesis and stereotypic cycles of stem cell activation upon each hair cycle to produce a new hair shaft. The adult hair follicle stem cell niche consists of two distinct populations, the bulge and the more activation-prone secondary hair germ (HG). Hair follicle stem cells are set aside during early stages of morphogenesis. This process is known to depend on the Sox9 transcription factor, but otherwise the establishment of the hair follicle stem cell niche is poorly understood. Here, we show that that mutation of Foxi3, a Forkhead family transcription factor mutated in several hairless dog breeds, compromises stem cell specification. Further, loss of Foxi3 impedes hair follicle downgrowth and progression of the hair cycle. Genome-wide profiling revealed a number of downstream effectors of Foxi3 including transcription factors with a recognized function in hair follicle stem cells such as Lhx2, Runx1, and Nfatc1, suggesting that the Foxi3 mutant phenotype results from simultaneous downregulation of several stem cell signature genes. We show that Foxi3 displays a highly dynamic expression pattern during hair morphogenesis and cycling, and identify Foxi3 as a novel secondary HG marker. Absence of Foxi3 results in poor hair regeneration upon hair plucking, and a sparse fur phenotype in unperturbed mice that exacerbates with age, caused by impaired secondary HG activation leading to progressive depletion of stem cells. Thus, Foxi3 regulates multiple aspects of hair follicle development and homeostasis. Stem Cells 2016;34:1896-1908.

  12. Fast and Specific Assessment of the Halogenating Peroxidase Activity in Leukocyte-enriched Blood Samples.

    PubMed

    Flemmig, Jörg; Schwarz, Pauline; Bäcker, Ingo; Leichsenring, Anna; Lange, Franziska; Arnhold, Jürgen

    2016-01-01

    In this paper a protocol for the quick and standardized enrichment of leukocytes from small whole blood samples is described. This procedure is based on the hypotonic lysis of erythrocytes and can be applied to human samples as well as to blood of non-human origin. The small initial sample volume of about 50 to 100 µl makes this method applicable to recurrent blood sampling from small laboratory animals. Moreover, leukocyte enrichment is achieved within minutes and with low material efforts regarding chemicals and instrumentation, making this method applicable in multiple laboratory environments. Standardized purification of leukocytes is combined with a highly selective staining method to evaluate halogenating peroxidase activity of the heme peroxidases, myeloperoxidase (MPO) and eosinophil peroxidase (EPO), i.e., the formation of hypochlorous and hypobromous acid (HOCl and HOBr). While MPO is strongly expressed in neutrophils, the most abundant immune cell type in human blood as well as in monocytes, the related enzyme EPO is exclusively expressed in eosinophils. The halogenating activity of these enzymes is addressed by using the almost HOCl- and HOBr-specific dye aminophenyl fluorescein (APF) and the primary peroxidase substrate hydrogen peroxide. Upon subsequent flow cytometry analysis all peroxidase-positive cells (neutrophils, monocytes, eosinophils) are distinguishable and their halogenating peroxidase activity can be quantified. Since APF staining may be combined with the application of cell surface markers, this protocol can be extended to specifically address leukocyte sub-fractions. The method is applicable to detect HOCl and HOBr production both in human and in rodent leukocytes. Given the widely and diversely discussed immunological role of these enzymatic products in chronic inflammatory diseases, this protocol may contribute to a better understanding of the immunological relevance of leukocyte-derived heme peroxidases. PMID:27501318

  13. Neuronal precursor-specific activity of a human doublecortin regulatory sequence.

    PubMed

    Karl, Claudia; Couillard-Despres, Sebastien; Prang, Peter; Munding, Matthias; Kilb, Werner; Brigadski, Tanja; Plötz, Sonja; Mages, Wolfgang; Luhmann, Heiko; Winkler, Jürgen; Bogdahn, Ulrich; Aigner, Ludwig

    2005-01-01

    The doublecortin (DCX) gene encodes a 40-kDa microtubule-associated protein specifically expressed in neuronal precursors of the developing and adult CNS. Due to its specific expression pattern, attention was drawn to DCX as a marker for neuronal precursors and neurogenesis, thereby underscoring the importance of its promoter identification and promoter analysis. Here, we analysed the human DCX regulatory sequence and confined it to a 3.5-kb fragment upstream of the ATG start codon. We demonstrate by transient transfection experiments that this fragment is sufficient and specific to drive expression of reporter genes in embryonic and adult neuronal precursors. The activity of this regulatory fragment overlapped with the expression of endogenous DCX and with the young neuronal markers class III beta-tubulin isotype and microtubule-associated protein Map2ab but not with glial or oligodendroglial markers. Electrophysiological data further confirmed the immature neuronal nature of these cells. Deletions within the 3.5-kb region demonstrated the relevance of specific regions containing transcription factor-binding sites. Moreover, application of neurogenesis-related growth factors in the neuronal precursor cultures suggested the lack of direct signalling of these factors on the DCX promoter construct. PMID:15663475

  14. Post-transcription initiation function of the ubiquitous SAGA complex in tissue-specific gene activation

    PubMed Central

    Weake, Vikki M.; Dyer, Jamie O.; Seidel, Christopher; Box, Andrew; Swanson, Selene K.; Peak, Allison; Florens, Laurence; Washburn, Michael P.; Abmayr, Susan M.; Workman, Jerry L.

    2011-01-01

    The Spt–Ada–Gcn5–acetyltransferase (SAGA) complex was discovered from Saccharomyces cerevisiae and has been well characterized as an important transcriptional coactivator that interacts both with sequence-specific transcription factors and the TATA-binding protein TBP. SAGA contains a histone acetyltransferase and a ubiquitin protease. In metazoans, SAGA is essential for development, yet little is known about the function of SAGA in differentiating tissue. We analyzed the composition, interacting proteins, and genomic distribution of SAGA in muscle and neuronal tissue of late stage Drosophila melanogaster embryos. The subunit composition of SAGA was the same in each tissue; however, SAGA was associated with considerably more transcription factors in muscle compared with neurons. Consistent with this finding, SAGA was found to occupy more genes specifically in muscle than in neurons. Strikingly, SAGA occupancy was not limited to enhancers and promoters but primarily colocalized with RNA polymerase II within transcribed sequences. SAGA binding peaks at the site of RNA polymerase pausing at the 5′ end of transcribed sequences. In addition, many tissue-specific SAGA-bound genes required its ubiquitin protease activity for full expression. These data indicate that in metazoans SAGA plays a prominent post-transcription initiation role in tissue-specific gene expression. PMID:21764853

  15. In Vitro Characterization of Chain Depolymerization Activities of SUMO-Specific Proteases.

    PubMed

    Eckhoff, Julia; Dohmen, R Jürgen

    2016-01-01

    SUMO-specific proteases, known as Ulps in baker's yeast and SENPs in humans, have important roles in controlling the dynamics of SUMO-modified proteins. They display distinct modes of action and specificity, in that they may act on the SUMO precursor, mono-sumoylated, and/or polysumoylated proteins, and they might be specific for substrates with certain SUMO paralogs. SUMO chains may be dismantled either by endo or exo mechanisms. Biochemical characterization of a protease usually requires purification of the protein of interest. Developing a purification protocol, however, can be very difficult, and in some cases, isolation of a protease in its pure form may go along with a substantial loss of activity. To characterize the reaction mechanism of Ulps, we have developed an in vitro assay, which makes use of substrates endowed with artificial poly-SUMO chains of defined lengths, and S. cerevisiae Ulp enzymes in crude extract from E. coli. This fast and economic approach should be applicable to SUMO-specific proteases from other species as well. PMID:27631802

  16. Near-infrared spectroscopy based neurofeedback training increases specific motor imagery related cortical activation compared to sham feedback.

    PubMed

    Kober, S E; Wood, G; Kurzmann, J; Friedrich, E V C; Stangl, M; Wippel, T; Väljamäe, A; Neuper, C

    2014-01-01

    In the present study we implemented a real-time feedback system based on multichannel near-infrared spectroscopy (NIRS). Prior studies indicated that NIRS-based neurofeedback can enhance motor imagery related cortical activation. To specify these prior results and to confirm the efficacy of NIRS-based neurofeedback, we examined changes in blood oxygenation level collected in eight training sessions. One group got real feedback about their own brain activity (N=9) and one group saw a playback of another person's feedback recording (N=8). All participants performed motor imagery of a right hand movement. Real neurofeedback induced specific and focused brain activation over left motor areas. This focal brain activation became even more specific over the eight training sessions. In contrast, sham feedback led to diffuse brain activation patterns over the whole cortex. These findings can be useful when training patients with focal brain lesions to increase activity of specific brain areas for rehabilitation purpose.

  17. Variation in energy expenditure among black-legged kittiwakes: Effects of activity-specific metabolic rates and activity budgets

    USGS Publications Warehouse

    Jodice, P.G.R.; Roby, D.D.; Suryan, R.M.; Irons, D.B.; Kaufman, A.M.; Turco, K.R.; Visser, G.H.

    2003-01-01

    We sought to determine the effect of variation in time-activity budgets (TABs) and foraging behavior on energy expenditure rates of parent black-legged kittiwakes (Rissa tridactyla). We quantified TABs using direct observations of radio-tagged adults and simultaneously measured field metabolic rates (FMR) of these same individuals (n = 20) using the doubly labeled water technique. Estimated metabolic rates of kittiwakes attending their brood at the nest or loafing near the colony were similar (ca. 1.3 x basal metabolic rate [BMR]), although loafing during foraging trips was more costly (2.9 x BMR). Metabolic rates during commuting flight (7.3 x BMR) and prey-searching flight (6.2 x BMR) were similar, while metabolic rates during plunge diving were much higher (ca. 47 x BMR). The proportion of the measurement interval spent foraging had a positive effect on FMR (R2 = 0.68), while the combined proportion of time engaged in nest attendance and loafing near the colony had a negative effect on FMR (R2 = 0.72). Thus, more than two-thirds of the variation in kittiwake FMR could be explained by the allocation of time among various activities. The high energetic cost of plunge diving relative to straight flight and searching flight suggests that kittiwakes can optimize their foraging strategy under conditions of low food availability by commuting long distances to feed in areas where gross foraging efficiency is high.

  18. Sex-specific associations of moderate and vigorous physical activity with physical fitness in adolescents.

    PubMed

    Kidokoro, T; Tanaka, H; Naoi, K; Ueno, K; Yanaoka, T; Kashiwabara, K; Miyashita, M

    2016-11-01

    The present study examined the sex-specific associations of moderate and vigorous physical activity (VPA) with physical fitness in 300 Japanese adolescents aged 12-14 years. Participants were asked to wear an accelerometer to evaluate physical activity (PA) levels of various intensities (i.e. moderate PA (MPA), 3-5.9 metabolic equivalents (METs); VPA, ≥6 METs; moderate to vigorous PA (MVPA), ≥3 METs). Eight fitness items were assessed (grip strength, bent-leg sit-up, sit-and-reach, side step, 50 m sprint, standing long jump, handball throw, and distance running) as part of the Japanese standardised fitness test. A fitness composite score was calculated using Japanese fitness norms, and participants were categorised according to their score from category A (most fit) to category E (least fit), with participants in categories D and E defined as having low fitness. It was found that for boys, accumulating more than 80.7 min/day of MVPA may reduce the probability of low fitness (odds ratio (ORs) [95% confidence interval (CI)] = 0.17 [0.06-0.47], p = .001). For girls, accumulating only 8.4 min of VPA could reduce the likelihood of exhibiting low fitness (ORs [95% CI] = 0.23 [0.05-0.89], p = .032). These results reveal that there are sex-specific differences in the relationship between PA and physical fitness in adolescents, suggesting that sex-specific PA recommendation may be needed to improve physical fitness in adolescents.

  19. Specific interaction of tissue-type plasminogen activator (t-PA) with annexin II on the membrane of pancreatic cancer cells activates plasminogen and promotes invasion in vitro

    PubMed Central

    Díaz, V M; Hurtado, M; Thomson, T M; Reventós, J; Paciucci, R

    2004-01-01

    Background: Overexpression of tissue plasminogen activator (t-PA) in pancreatic cancer cells promotes invasion and proliferation in vitro and tumour growth and angiogenesis in vivo. Aims: To understand the mechanisms by which t-PA favours cancer progression, we analysed the surface membrane proteins responsible for binding specifically t-PA and studied the contribution of this interaction to the t-PA promoted invasion of pancreatic cancer cells. Methods: The ability of t-PA to activate plasmin and a fluorogenic plasmin substrate was used to analyse the nature of the binding of active t-PA to cell surfaces. Specific binding was determined in two pancreatic cancer cell lines (SK-PC-1 and PANC-1), and complex formation analysed by co-immunoprecipitation experiments and co-immunolocalisation in tumours. The functional role of the interaction was studied in Matrigel invasion assays. Results: t-PA bound to PANC-1 and SK-PC-1 cells in a specific and saturable manner while maintaining its activity. This binding was competitively inhibited by specific peptides interfering with the interaction of t-PA with annexin II. The t-PA/annexin II interaction on pancreatic cancer cells was also supported by co-immunoprecipitation assays using anti-t-PA antibodies and, reciprocally, with antiannexin II antibodies. In addition, confocal microscopy showed t-PA and annexin II colocalisation in tumour tissues. Finally, disruption of the t-PA/annexin II interaction by a specific hexapeptide significantly decreased the invasive capacity of SK-PC-1 cells in vitro. Conclusion: t-PA specifically binds to annexin II on the extracellular membrane of pancreatic cancer cells where it activates local plasmin production and tumour cell invasion. These findings may be clinically relevant for future therapeutic strategies based on specific drugs that counteract the activity of t-PA or its receptor annexin II, or their interaction at the surface level. PMID:15194650

  20. Active-R filter

    DOEpatents

    Soderstrand, Michael A.

    1976-01-01

    An operational amplifier-type active filter in which the only capacitor in the circuit is the compensating capacitance of the operational amplifiers, the various feedback and coupling elements being essentially solely resistive.

  1. Specific combinations of presenilins and Aph1s affect the substrate specificity and activity of γ-secretase.

    PubMed

    Yonemura, Yoji; Futai, Eugene; Yagishita, Sosuke; Kaether, Christoph; Ishiura, Shoichi

    2016-09-30

    The γ-secretase complex comprises presenilin (PS), nicastrin (NCT), anterior pharynx-defective 1 (Aph1), and presenilin enhancer 2 (Pen2). PS has two homologues, PS1 and PS2. Aph1 has two isoforms, Aph1a and Aph1b, with the former existing as two splice variants Aph1aL and Aph1aS. Each complex consists of one subunit each, resulting in six different γ-secretases. To better understand the functional differences among the γ-secretases, we reconstituted them using a yeast system and compared Notch1-cleavage and amyloid precursor protein (APP)-cleavage activities. Intriguingly, PS2/Aph1b had a clear substrate specificity: APP-Gal4, but not Notch-Gal4, was cleaved. In HEK cell lines expressing defined γ-secretase subunits, we showed that PS1/Aph1b, PS2/Aph1aL, PS2/Aph1aS and PS2/Aph1b γ-secretase produced amyloid β peptide (Aβ) with a higher Aβ42+Aβ43-to-Aβ40 (Aβ42(43)/Aβ40) ratio than the other γ-secretases. In addition, PS2/Aph1aS γ-secretase produced less Notch intracellular domain (NICD) than did the other 5 γ-secretases. Considering that the Aβ42(43)/Aβ40 ratio is relevant in the pathogenesis of Alzheimer's disease (AD), and that inhibition of Notch cleavage causes severe side effect, these results suggest that the PS2/Aph1aS γ-secretase complex is a potential therapeutic target in AD. PMID:27608597

  2. Identification of lipase encoding genes from Antarctic seawater bacteria using degenerate primers: expression of a cold-active lipase with high specific activity.

    PubMed

    Parra, Loreto P; Espina, Giannina; Devia, Javier; Salazar, Oriana; Andrews, Barbara; Asenjo, Juan A

    2015-01-01

    Cold-active enzymes are valuable catalysts showing high activity at low and moderate temperatures and low thermostability. Among cold-active enzymes, lipases offer a great potential in detergent, cosmetic, biofuel and food or feed industries. In this paper we describe the identification of novel lipase coding genes and the expression of a lipase with high activity at low temperatures. The genomic DNA from Antarctic seawater bacteria showing lipolytic activity at 4°C was used to amplify five DNA fragments that partially encode novel lipases using specifically designed COnsensus-DEgenerate Hybrid Oligonucleotide Primers (CODEHOP). All the fragments were found to have a high identity with an α/β-hydrolase domain-containing protein identified by the sequencing of the complete genome of Shewanella frigidimarina NCIMB 400. The complete sequence of one of the lipase-coding gene fragments, lipE13, was obtained by genome walking. Considering that the other fragments had a high identity to the putative lipase from S. frigidimarina NCIMB 400, the complete lipase genes were amplified using oligonucleotide primers designed based on the 5' and 3' regions of the coding sequence of the related protein. This strategy allowed the amplification of 3 lipase-encoding genes of which one was expressed in the periplasm using the Escherichia coli BL21(DE3)/pET-22b(+) expression system. The recombinant protein was obtained with activity toward p-nitrophenyl caproate showing a high specific activity between 15 and 25°C.

  3. Induction of specific cytotoxic activity for bovine herpesvirus-1 by DNA immunization with different adjuvants.

    PubMed

    Langellotti, C A; Pappalardo, J S; Quattrocchi, V; Mongini, C; Zamorano, P

    2011-06-01

    It is well documented that adjuvants improve the immune response generated by traditional viral vaccines; however, less is known about their effects on the immune response elicited by DNA vaccines. In this study, we have investigated the use of adjuvants, and have analyzed the humoral and cellular specific immune responses elicited by DNA vaccines based on the BoHV-1 glycoprotein D (secreted version) in pCIneo vector with and without Montanide ISA25 (O/W), ISA206 VG (SEPPIC) and Cliptox™ (natural microparticles of clinoptilolite). The comparison of the immune response induced in mice by pCIgD formulated with or without adjuvants showed that the immunomodulators affect the total specific humoral and cellular response. The isotypes induced by these adjuvants were of the type Th1/Th2. A significant increase in the mac-3+ and F4/80+ populations of the groups receiving pCIneo with ISA25, ISA206; and an increase in CD4+ populations of the group receiving pCIneo ISA25, in comparison with the pCIneo group was observed. On the other hand, mice vaccinated with pCIgD/ISA25, pCIgD/ISA206, or pCIgD/Cliptox developed a significantly higher specific cytotoxic activity against BoHV-1 than the pCIgD and pCIneo groups. In this report we propose the use of ISA25, ISA206 or Cliptox as adjuvants in a DNA vaccine since they are able to induce not only a specific humoral immune response but also a specific cellular immune response.

  4. Phosphatidylinositol-specific phospholipase C activity of chromaffin granule-binding proteins

    SciTech Connect

    Creutz, C.E.; Dowling, L.G.; Kyger, E.M.; Franson, R.C.

    1985-06-25

    Using (U-/sup 14/C)phosphatidylinositol as substrate, Ca/sup 2 +/-dependent phospholipase C activity was detected in a group of bovine adrenal medullary proteins that bind to chromaffin granule membranes in the presence of Ca/sup 2 +/. The activity was maximal at neutral pH and represented an 80- to 240-fold enrichment of adrenal medullary cytosol phospholipase C activity measured at pH 7.3. The stimulation of activity by Ca/sup 2 +/ was complex; no activity was present in the absence of Ca/sup 2 +/, 25% activation occurred at 1 microM Ca/sup 2 +/, and full activation at 5 mM Ca/sup 2 +/. The enzyme bound to chromaffin granule membranes in the presence of 2 mM Ca/sup 2 +/ but was released at 40 microM Ca/sup 2 +/, suggesting that intrinsic enzyme activity may be regulated by (Ca/sup 2 +/) at 1 microM, but additional activation at higher concentrations of Ca/sup 2 +/ is seen in vitro as a result of Ca/sup 2 +/-dependent binding of the active enzyme to substrate-containing membranes. This enzyme may generate diacylglycerol and phosphorylated inositol to act as intracellular messengers in the vicinity of the chromaffin granule membrane during the process of exocytosis.

  5. Pluripotent stem cells reveal erythroid-specific activities of the GATA1 N-terminus

    PubMed Central

    Byrska-Bishop, Marta; VanDorn, Daniel; Campbell, Amy E.; Betensky, Marisol; Arca, Philip R.; Yao, Yu; Gadue, Paul; Costa, Fernando F.; Nemiroff, Richard L.; Blobel, Gerd A.; French, Deborah L.; Hardison, Ross C.; Weiss, Mitchell J.; Chou, Stella T.

    2015-01-01

    Germline GATA1 mutations that result in the production of an amino-truncated protein termed GATA1s (where s indicates short) cause congenital hypoplastic anemia. In patients with trisomy 21, similar somatic GATA1s-producing mutations promote transient myeloproliferative disease and acute megakaryoblastic leukemia. Here, we demonstrate that induced pluripotent stem cells (iPSCs) from patients with GATA1-truncating mutations exhibit impaired erythroid potential, but enhanced megakaryopoiesis and myelopoiesis, recapitulating the major phenotypes of the associated diseases. Similarly, in developmentally arrested GATA1-deficient murine megakaryocyte-erythroid progenitors derived from murine embryonic stem cells (ESCs), expression of GATA1s promoted megakaryopoiesis, but not erythropoiesis. Transcriptome analysis revealed a selective deficiency in the ability of GATA1s to activate erythroid-specific genes within populations of hematopoietic progenitors. Although its DNA-binding domain was intact, chromatin immunoprecipitation studies showed that GATA1s binding at specific erythroid regulatory regions was impaired, while binding at many nonerythroid sites, including megakaryocytic and myeloid target genes, was normal. Together, these observations indicate that lineage-specific GATA1 cofactor associations are essential for normal chromatin occupancy and provide mechanistic insights into how GATA1s mutations cause human disease. More broadly, our studies underscore the value of ESCs and iPSCs to recapitulate and study disease phenotypes. PMID:25621499

  6. Activity and High-Order Effective Connectivity Alterations in Sanfilippo C Patient-Specific Neuronal Networks

    PubMed Central

    Canals, Isaac; Soriano, Jordi; Orlandi, Javier G.; Torrent, Roger; Richaud-Patin, Yvonne; Jiménez-Delgado, Senda; Merlin, Simone; Follenzi, Antonia; Consiglio, Antonella; Vilageliu, Lluïsa; Grinberg, Daniel; Raya, Angel

    2015-01-01

    Summary Induced pluripotent stem cell (iPSC) technology has been successfully used to recapitulate phenotypic traits of several human diseases in vitro. Patient-specific iPSC-based disease models are also expected to reveal early functional phenotypes, although this remains to be proved. Here, we generated iPSC lines from two patients with Sanfilippo type C syndrome, a lysosomal storage disorder with inheritable progressive neurodegeneration. Mature neurons obtained from patient-specific iPSC lines recapitulated the main known phenotypes of the disease, not present in genetically corrected patient-specific iPSC-derived cultures. Moreover, neuronal networks organized in vitro from mature patient-derived neurons showed early defects in neuronal activity, network-wide degradation, and altered effective connectivity. Our findings establish the importance of iPSC-based technology to identify early functional phenotypes, which can in turn shed light on the pathological mechanisms occurring in Sanfilippo syndrome. This technology also has the potential to provide valuable readouts to screen compounds, which can prevent the onset of neurodegeneration. PMID:26411903

  7. Body side-specific control of motor activity during turning in a walking animal

    PubMed Central

    Gruhn, Matthias; Rosenbaum, Philipp; Bockemühl, Till; Büschges, Ansgar

    2016-01-01

    Animals and humans need to move deftly and flexibly to adapt to environmental demands. Despite a large body of work on the neural control of walking in invertebrates and vertebrates alike, the mechanisms underlying the motor flexibility that is needed to adjust the motor behavior remain largely unknown. Here, we investigated optomotor-induced turning and the neuronal mechanisms underlying the differences between the leg movements of the two body sides in the stick insect Carausius morosus. We present data to show that the generation of turning kinematics in an insect are the combined result of descending unilateral commands that change the leg motor output via task-specific modifications in the processing of local sensory feedback as well as modification of the activity of local central pattern generating networks in a body-side-specific way. To our knowledge, this is the first study to demonstrate the specificity of such modifications in a defined motor task. DOI: http://dx.doi.org/10.7554/eLife.13799.001 PMID:27130731

  8. Activity and High-Order Effective Connectivity Alterations in Sanfilippo C Patient-Specific Neuronal Networks.

    PubMed

    Canals, Isaac; Soriano, Jordi; Orlandi, Javier G; Torrent, Roger; Richaud-Patin, Yvonne; Jiménez-Delgado, Senda; Merlin, Simone; Follenzi, Antonia; Consiglio, Antonella; Vilageliu, Lluïsa; Grinberg, Daniel; Raya, Angel

    2015-10-13

    Induced pluripotent stem cell (iPSC) technology has been successfully used to recapitulate phenotypic traits of several human diseases in vitro. Patient-specific iPSC-based disease models are also expected to reveal early functional phenotypes, although this remains to be proved. Here, we generated iPSC lines from two patients with Sanfilippo type C syndrome, a lysosomal storage disorder with inheritable progressive neurodegeneration. Mature neurons obtained from patient-specific iPSC lines recapitulated the main known phenotypes of the disease, not present in genetically corrected patient-specific iPSC-derived cultures. Moreover, neuronal networks organized in vitro from mature patient-derived neurons showed early defects in neuronal activity, network-wide degradation, and altered effective connectivity. Our findings establish the importance of iPSC-based technology to identify early functional phenotypes, which can in turn shed light on the pathological mechanisms occurring in Sanfilippo syndrome. This technology also has the potential to provide valuable readouts to screen compounds, which can prevent the onset of neurodegeneration.

  9. CpGB DNA activates dermal macrophages and specifically recruits inflammatory monocytes into the skin.

    PubMed

    Mathes, Allison L; Rice, Lisa; Affandi, Alsya J; DiMarzio, Michael; Rifkin, Ian R; Stifano, Giuseppina; Christmann, Romy B; Lafyatis, Robert

    2015-02-01

    Toll-like receptor 9 (TLR9) drives innate immune responses after recognition of foreign or endogenous DNA containing unmethylated CpG motifs. DNA-mediated TLR9 activation is highly implicated in the pathogenesis of several autoimmune skin diseases, yet its contribution to the inflammation seen in these diseases remains unclear. In this study, TLR9 ligand, CpGB DNA, was administered to mice via a subcutaneous osmotic pump with treatment lasting 1 or 4 weeks. Gene expression and immunofluorescence analyses were used to determine chemokine expression and cell recruitment in the skin surrounding the pump outlet. CpGB DNA skin treatment dramatically induced a marked influx of CD11b+ F4/80+ macrophages, increasing over 4 weeks of treatment, and induction of IFNγ and TNFα expression. Chemokines, CCL2, CCL4, CCL5, CXCL9 and CXCL10, were highly induced in CpGB DNA-treated skin, although abrogation of these signalling pathways individually did not alter macrophage accumulation. Flow cytometry analysis showed that TLR9 activation in the skin increased circulating CD11b+ CD115+ Ly6C(hi) inflammatory monocytes following 1 week of CpGB DNA treatment. Additionally, skin-resident CD11b+ cells were found to initially take up subcutaneous CpGB DNA and propagate the subsequent immune response. Using diphtheria toxin-induced monocyte depletion mouse model, gene expression analysis demonstrated that CD11b+ cells are responsible for the CpGB DNA-induced cytokine and chemokine response. Overall, these data demonstrate that chronic TLR9 activation induces a specific inflammatory response, ultimately leading to a striking and selective accumulation of macrophages in the skin. PMID:25425469

  10. Pyridinone derivatives: Specific human immunodeficiency virus type 1 reverse transcriptase inhibitors with antiviral activity

    SciTech Connect

    Goldman, M.E.; Nunberg, J.H.; O'Brien, J.A.; Quintero, J.C.; Schleif, W.A.; Freund, K.F.; Gaul, S.L.; Saari, W.S.; Wai, J.S.; Hoffman, J.M.; Anderson, P.S.; Emini, E.A.; Stern, A.M. ); Hupe, D.J. )

    1991-08-01

    Derivatives of pyridinones were found to inhibit human immunodeficiency virus type 1 (HIV-1) reverse transcriptase (RT) activity and prevent the spread of HIV-1 infection in cell culture without an appreciable effect on other retroviral or cellular polymerases. 3-{l brace}((4,7-Dimethyl-1,3-benzoxazol-2-yl)methyl)amino{r brace}-5-ethyl-6-methylpyridin-2(1H)-one(L-679,639) and 3-{l brace}((4,7-dichloro-1,3-benzoxazol-2-yl)methyl)amino{r brace}-5-ethyl-6-methylpyridin-2(1H)-one (L-697,661), two compounds within this series, had HIV-1 RT IC{sub 50} values in the range of 20-800 nM, depending upon the template-primer used. The most potent inhibition was obtained with rC{center dot}dG, reversible slow-binding noncompetitive inhibition was observed. ({sup 3}H)L-697,639 bound preferentially to enzyme-template-primer complexes. This binding was magnesium-dependent and saturable with a stoichiometry of 1 mol of ({sup 3}H)L-697,639 per mol of RT heterodimer. Synergism between 3{prime}-azido-3{prime}-deoxythymidine or dideoxyinosine and either of these compounds was also demonstrated in cell culture. Based upon their specificity for HIV-1 RT activity, template-primer dependence on potency and ability to displace ({sup 3}H)L-697,639; a tetrahydroimidazo(4,5,1-jk)(1,4)-benzodiazepin-2(1H)-thione derivative R82150 and the dipyridodiazepinone BI-RG-587 appear to inhibit RT activity by the same mechanism as the pyridinones.

  11. Antinociceptive activity of NK1 receptor antagonists: non-specific effects of racemic RP67580.

    PubMed Central

    Rupniak, N. M.; Boyce, S.; Williams, A. R.; Cook, G.; Longmore, J.; Seabrook, G. R.; Caeser, M.; Iversen, S. D.; Hill, R. G.

    1993-01-01

    1. Release of substance P in the dorsal horn is considered a primary event in the perception of pain. The profile of racemic RP67580, a non-peptide antagonist at the NK1 (substance P) receptor, was examined in a range of antinociception tests on rodents. 2. At doses up to 30 mg kg-1, s.c. racemic RP67580 exhibited antinociceptive activity in writhing and formalin paw tests in mice and gerbils. Acetic acid induced writhing and the licking response to formalin were reduced to 40-50% of the level observed in vehicle-treated animals (P < 0.05). However, this agent was not active in mouse tail flick, rat paw pressure or rat and guinea-pig formalin paw tests. 3. Like racemic RP67580, the calcium channel blockers nifedipine (30 mg kg-1, i.p.) and verapamil (10 or 20 mg kg-1, s.c.) inhibited the response to formalin by approximately 60% in gerbils (P < 0.05 compared with vehicle-treated animals). 4. Evidence for calcium channel antagonist activity of RP67580 was obtained in vitro. Racemic RP67580 inhibited calcium entry into depolarized strips of guinea-pig ileum longitudinal muscle myenteric plexus (apparent KB = 587 +/- 115 nM), inhibited [3H]-diltiazem binding to rabbit skeletal membranes (IC50 = 298 nM) and depressed high threshold calcium currents in neurones cultured from rat cortex (10% inhibition at 10 microM). 5. These findings indicate that the acute antinociceptive effects of RP67580 may not be attributable to a specific interaction with NK1 receptors and may be mediated via calcium channel blockade. PMID:8306108

  12. Inhibition of proteolytic activity of poliovirus and rhinovirus 2A proteinases by elastase-specific inhibitors.

    PubMed Central

    Molla, A; Hellen, C U; Wimmer, E

    1993-01-01

    A polyprotein cleavage assay has been developed to assay the proteolytic activities in vitro of the 2A proteinases encoded by poliovirus and human rhinovirus 14, which are representative members of the Enterovirus and Rhinovirus genera of picornaviruses, respectively. The elastase-specific substrate-based inhibitors elastatinal and methoxysuccinyl-Ala-Ala-Pro-Val-chloromethylketone (MPCMK) inhibited both 2A proteinases in vitro. The electrophoretic mobilities of both 2A proteinases were reduced upon incubation with elastatinal, whereas the mobility of a Cys-109-->Ala poliovirus 2Apro mutant was unchanged, an observation suggesting that this inhibitor may have formed a covalent bond with the active-site Cys-109 nucleophile. Iodoacetamide, calpain inhibitor 1, and antipain inhibited poliovirus 2Apro. MPCMK caused a reduction in the yields of the enteroviruses poliovirus type 1 and coxsackievirus A21 and of human rhinovirus 2 in infected HeLa cells but did not affect the growth of encephalomyocarditis virus, a picornavirus of the Cardiovirus genus. MPCMK abrogated the shutoff of host cell protein synthesis that is induced by enterovirus and rhinovirus infection and reduced the synthesis of virus-encoded polypeptides in infected cells. These results indicate that the determinants of substrate recognition by 2A proteinases resemble those of pancreatic and leukocyte elastases. These results may be relevant to the development of broad-range chemotherapeutic agents against entero- and rhinoviruses. Images PMID:8392608

  13. High specific activity enantiomerically enriched juvenile hormones: synthesis and binding assay

    SciTech Connect

    Prestwich, G.D.; Wawrzenczyk, C.

    1985-08-01

    A stereoselective total synthesis of chiral juvenile hormone I is described that allows stoichiometric introduction of two tritium atoms in the final step. Both optical antipodes of the pivotal epoxy alcohol intermediate were prepared in 95% enantiomeric excess by the Sharpless epoxidation of a (Z)-allylic alcohol. Elaboration of the hydroxy-methyl group to a vinyl group followed by selective homogeneous tritiation affords optically active juvenile hormone I analogs at 58 Ci/mmol. Competitive binding of the labeled 10R, 11S and 10S,11R enantiomers with unlabeled enantiomers to the hemolymph binding protein of Manduca sexta larvae was determined by using a dextran-coated charcoal assay. The natural 10R,11S enantiomer has twice the relative binding affinity of the 10S,11R enantiomer. The availability of such high specific activity optically pure hormones will contribute substantially to the search for high-affinity receptors for juvenile hormones in the nuclei of cells. Moreover, the chiral 12-hydroxy-(10R,11S)-epoxy intermediate allows modification of juvenile hormone for solid-phase biochemical and radioimmunochemical work without altering either the biologically important carbomethoxy or epoxy recognition sites.

  14. Region Specific Effects of Maternal Immune Activation on Offspring Neuroimmune Function

    PubMed Central

    Zhou, Heping

    2015-01-01

    Growing evidence suggests that maternal immune activation has a significant impact on the immuno-competence of the offspring. The present study aimed to characterize region-specific effects of maternal immune activation on the offspring’s neuroimmune function. The offspring born to dams treated with saline or lipopolysaccharide (LPS) at gestational day 18 was stimulated with saline or LPS at postnatal day 21, and the mRNA expression of various inflammatory genes in different brain regions of the offspring was analyzed. The offspring born to saline-treated dams exhibited a typical neuroimmune response with elevated levels of cytokines and chemokines following LPS stimulation in all four brain regions examined. In contrast, the offspring born to LPS-treated dams exhibited significantly reduced mRNA induction of cytokines and chemokines following LPS stimulation in the prefrontal cortex but not in the brainstem when compared with pups born to saline-treated dams. Furthermore, the mRNA expression of LPS-induced I-κBζ was significantly attenuated in the prefrontal cortex when compared with pups born to saline-treated dams. These results suggest that maternal LPS may have differential effects on the neuroimmune function in different regions of the offspring brain, and highlight the importance of maternal milieu in the development of neuroimmune function in the offspring. PMID:26229739

  15. High specific activity enantiomerically enriched juvenile hormones: synthesis and binding assay.

    PubMed Central

    Prestwich, G D; Wawrzeńczyk, C

    1985-01-01

    A stereoselective total synthesis of chiral juvenile hormone I is described that allows stoichiometric introduction of two tritium atoms in the final step. Both optical antipodes of the pivotal epoxy alcohol intermediate were prepared in 95% enantiomeric excess by the Sharpless epoxidation of a (Z)-allylic alcohol. Elaboration of the hydroxy-methyl group to a vinyl group followed by selective homogeneous tritiation affords optically active juvenile hormone I analogs at 58 Ci/mmol. Competitive binding of the labeled 10R, 11S and 10S,11R enantiomers with unlabeled enantiomers to the hemolymph binding protein of Manduca sexta larvae was determined by using a dextran-coated charcoal assay. The natural 10R,11S enantiomer has twice the relative binding affinity of the 10S,11R enantiomer. The availability of such high specific activity optically pure hormones will contribute substantially to the search for high-affinity receptors for juvenile hormones in the nuclei of cells. Moreover, the chiral 12-hydroxy-(10R,11S)-epoxy intermediate allows modification of juvenile hormone for solid-phase biochemical and radioimmunochemical work without altering either the biologically important carbomethoxy or epoxy recognition sites. PMID:3860862

  16. Blue Light Activates a Specific Protein Kinase in Higher Plants 1

    PubMed Central

    Reymond, Philippe; Short, Timothy W.; Briggs, Winslow R.

    1992-01-01

    Blue light mediates the phosphorylation of a membrane protein in seedlings from several plant species. When crude microsomal membrane proteins from dark-grown pea (Pisum sativum L.), sunflower (Helianthus annuus L.), zucchini (Cucurbita pepo L.), Arabidopsis (Arabidopsis thaliana L.), or tomato (Lycopersicon esculentum L.) stem segments, or from maize (Zea mays L.), barley (Hordeum vulgare L.), oat (Avena sativa L.), wheat (Triticum aestivum L.), or sorghum (Sorghum bicolor L.) coleoptiles are illuminated and incubated in vitro with [γ-32P]ATP, a protein of apparent molecular mass from 114 to 130 kD is rapidly phosphorylated. Hence, this system is probably ubiquitous in higher plants. Solubilized maize membranes exposed to blue light and added to unirradiated solubilized maize membranes show a higher level of phosphorylation of the light-affected protein than irradiated membrane proteins alone, suggesting that an unirradiated substrate is phosphorylated by a light-activated kinase. This finding is further demonstrated with membrane proteins from two different species, where the phosphorylated proteins are of different sizes and, hence, unambiguously distinguishable on gel electrophoresis. When solubilized membrane proteins from one species are irradiated and added to unirradiated membrane proteins from another species, the unirradiated protein becomes phosphorylated. These experiments indicate that the irradiated fraction can store the light signal for subsequent phosphorylation in the dark. They also support the hypothesis that light activates a specific kinase and that the systems share a close functional homology among different higher plants. Images Figure 1 Figure 2 Figure 5 PMID:16653043

  17. Serotonergic changes in specific areas of rat brain associated with activity--stress gastric lesions.

    PubMed

    Hellhammer, D H; Hingtgen, J N; Wade, S E; Shea, P A; Aprison, M H

    1983-05-01

    To study serotonergic involvement in the development of gastric lesions following activity wheel stress, three groups of rats (gastric lesions, no gastric lesions, and home--cage controls) were killed following exposure to the experimental procedures. The brains were dissected into eight specific areas and subjected to analyses for serotonin (5-HT) and 5-hydroxyindoleacetic acid (5-HIAA) using high performance liquid chromatography with EC detection. Lower levels of 5-HT were found in the midbrain, cortex, and hippocampus of rats with gastric lesions compared to either the no lesion group, subjected to shorter periods of activity--stress, or the home--cage control group. Levels of 5-HT and 5-HIAA were elevated in the pons/medulla oblongata of both the lesion and the no lesion groups compared to the home--cage controls. Corticosterone levels in blood were also significantly elevated in the lesion group. These data on serotonin changes in the CNS suggest a possible role for this neurotransmitter in stress-induced gastric pathology. PMID:6191350

  18. Regulation of protein C inhibitor (PCI) activity by specific oxidized and negatively charged phospholipids.

    PubMed

    Malleier, Julia M; Oskolkova, Olga; Bochkov, Valery; Jerabek, Ingrid; Sokolikova, Barbora; Perkmann, Thomas; Breuss, Johannes; Binder, Bernd R; Geiger, Margarethe

    2007-06-01

    Protein C inhibitor (PCI) is a serpin with affinity for heparin and phosphatidylethanolamine (PE). We analyzed the interaction of PCI with different phospholipids and their oxidized forms. PCI bound to oxidized PE (OxPE), and oxidized and unoxidized phosphatidylserine (PS) immobilized on microtiter plates and in aqueous suspension. Binding to OxPE and PS was competed by heparin, but not by the aminophospholipid-binding protein annexin V or the PCI-binding lipid retinoic acid. PS and OxPE stimulated the inhibition of activated protein C (aPC) by PCI in a Ca(++)-dependent manner, indicating that binding of both, aPC (Ca(++) dependent) and PCI (Ca(++) independent), to phospholipids is necessary. A peptide corresponding to the heparin-binding site of PCI abolished the stimulatory effect of PS on aPC inhibition. No stimulatory effect of phospholipids on aPC inhibition was seen with a PCI mutant lacking the heparin-binding site. A heparin-like effect of phospholipids (OxPE) was not seen with antithrombin III, another heparin-binding serpin, suggesting that it is specific for PCI. PCI and annexin V were found to be endogenously colocalized in atherosclerotic plaques, supporting the hypothesis that exposure of oxidized PE and/or PS may be important for the local regulation of PCI activity in vivo.

  19. Activation of GATA4 gene expression at the early stage of cardiac specification

    NASA Astrophysics Data System (ADS)

    Yilbas, Ayse; Hamilton, Alison; Wang, Yingjian; Mach, Hymn; Lacroix, Natascha; Davis, Darryl; Chen, Jihong; LI, Qiao

    2014-03-01

    Currently, there are no effective treatments to directly repair damaged heart tissue after cardiac injury since existing therapies focus on rescuing or preserving reversibly damaged tissue. Cell-based therapies using cardiomyocytes generated from stem cells present a promising therapeutic approach to directly replace damaged myocardium with new healthy tissue. However, the molecular mechanisms underlying the commitment of stem cells into cardiomyocytes are not fully understood and will be critical to guide this new technology into the clinic. Since GATA4 is a critical regulator of cardiac differentiation, we examined the molecular basis underlying the early activation of GATA4 gene expression during cardiac differentiation of pluripotent stem cells. Our studies demonstrate the direct involvement of histone acetylation and transcriptional coactivator p300 in the regulation of GATA4 gene expression. More importantly, we show that histone acetyltransferase (HAT) activity is important for GATA4 gene expression with the use of curcumin, a HAT inhibitor. In addition, the widely used histone deacetylase inhibitor valproic acid enhances both histone acetylation and cardiac specification.

  20. Specific induction of fibronectin binding activity by hemoglobin in Candida albicans grown in defined media.

    PubMed

    Yan, S; Nègre, E; Cashel, J A; Guo, N; Lyman, C A; Walsh, T J; Roberts, D D

    1996-08-01

    Fibronectin (FN) is a major component of host extracellular matrix that may play an important role in the initiation and dissemination of Candida albicans infections. Expression of FN binding requires growth of C albicans blastoconidia in complex medium, and the regulation of FN receptor expression is poorly understood. We now demonstrate that hemoglobin is a potent and specific inducer of FN receptor expression and describe a defined medium supplemented with hemoglobin that greatly and stably enhances the binding activity of C. albicans for soluble FN. Enhancement of FN binding by hemoglobin in strain 44807 was concentration dependent and was maximal at 0.1% hemoglobin with 20- to 80-fold enhancement. The hemoglobin-induced FN binding to C. albicans was saturable, with a Kd of 2.7 X 10(-8) M. Enhancement required growth of C. albicans in hemoglobin-containing medium, since simply exposing blastoconidia to hemoglobin in a nongrowing status did not enhance binding. Induction was reversible following removal of hemoglobin from the growth medium and not associated with germination. Inorganic or protein-bound iron was not sufficient for the induction, since other iron-containing proteins or inorganic iron salts were inactive. Growth in the simple medium yeast nitrogen base supplemented with hemoglobin increased cell adhesion to immobilized FN and to cultured monolayers of bovine corneal endothelial cells. These data suggest that hemoglobin may be an important regulator of FN binding activity in C. albicans and thus may play a role in its pathogenesis. PMID:8757815

  1. Pdx-1 is required for activation in vivo from a duodenum-specific enhancer.

    PubMed

    Dusing, M R; Florence, E A; Wiginton, D A

    2001-04-27

    The purine metabolic gene adenosine deaminase (ADA) is expressed along a defined spatiotemporal pattern in the developing mammalian small intestine, where high-level expression is limited to the villous epithelium of the duodenum. This activation is observed in rodents as the intestine completes the final maturation resulting in adult crypt-villus structures at 2-3 weeks postpartum. A regulatory module responsible for this pattern of expression has been identified in the second intron of the human ADA gene. Of the multiple duodenal proteins that can interact with this small duodenal enhancer region, the studies contained in this work describe the identification of five of these proteins as the dispersed homeobox protein PDX-1. This transcription factor exhibits a profile of expression in the small intestine similar to that observed for ADA, making it an ideal candidate factor for the duodenum-specific ADA enhancer. Loss of PDX-1 binding, via a PDX-1 mutated enhancer transgenic construction, resulted in complete loss of high-level activation in the duodenum, demonstrating the absolute requirement for this factor in vivo. However, co-transfection experiments suggest that other proteins that bind the enhancer are also required for enhancer function because PDX-1 alone was incapable of significant transactivation.

  2. Tissue Specific Expression of Cre in Rat Tyrosine Hydroxylase and Dopamine Active Transporter-Positive Neurons.

    PubMed

    Liu, Zhenyi; Brown, Andrew; Fisher, Dan; Wu, Yumei; Warren, Joe; Cui, Xiaoxia

    2016-01-01

    The rat is a preferred model system over the mouse for neurological studies, and cell type-specific Cre expression in the rat enables precise ablation of gene function in neurons of interest, which is especially valuable for neurodegenerative disease modeling and optogenetics. Yet, few such Cre rats are available. Here we report the characterization of two Cre rats, tyrosine hydroxylase (TH)-Cre and dopamine active transporter (DAT or Slc6a3)-Cre, by using a combination of immunohistochemistry (IHC) and mRNA fluorescence in situ hybridization (FISH) as well as a fluorescent reporter for Cre activity. We detected Cre expression in expected neurons in both Cre lines. Interestingly, we also found that in Th-Cre rats, but not DAT-Cre rats, Cre is expressed in female germ cells, allowing germline excision of the floxed allele and hence the generation of whole-body knockout rats. In summary, our data demonstrate that targeted integration of Cre cassette lead to faithful recapitulation of expression pattern of the endogenous promoter, and mRNA FISH, in addition to IHC, is an effective method for the analysis of the spatiotemporal gene expression patterns in the rat brain, alleviating the dependence on high quality antibodies that are often not available against rat proteins. The Th-Cre and the DAT-Cre rat lines express Cre in selective subsets of dopaminergic neurons and should be particularly useful for researches on Parkinson's disease.

  3. X-ray structure of human aromatase reveals an androgen-specific active site.

    PubMed

    Ghosh, Debashis; Griswold, Jennifer; Erman, Mary; Pangborn, Walter

    2010-02-28

    Aromatase is a unique cytochrome P450 that catalyzes the removal of the 19-methyl group and aromatization of the A-ring of androgens for the synthesis of estrogens. All human estrogens are synthesized via this enzymatic aromatization pathway. Aromatase inhibitors thus constitute a frontline therapy for estrogen-dependent breast cancer. Despite decades of intense investigation, this enzyme of the endoplasmic reticulum membrane has eluded all structure determination efforts. We have determined the crystal structure of the highly active aromatase purified from human placenta, in complex with its natural substrate androstenedione. The structure shows the binding mode of androstenedione in the catalytically active oxidized high-spin ferric state of the enzyme. Hydrogen bond-forming interactions and tight packing hydrophobic side chains that complement the puckering of the steroid backbone provide the molecular basis for the exclusive androgenic specificity of aromatase. Locations of catalytic residues and water molecules shed new light on the mechanism of the aromatization step. The structure also suggests a membrane integration model indicative of the passage of steroids through the lipid bilayer.

  4. Ciguatoxins activate specific cold pain pathways to elicit burning pain from cooling

    PubMed Central

    Vetter, Irina; Touska, Filip; Hess, Andreas; Hinsbey, Rachel; Sattler, Simon; Lampert, Angelika; Sergejeva, Marina; Sharov, Anastasia; Collins, Lindon S; Eberhardt, Mirjam; Engel, Matthias; Cabot, Peter J; Wood, John N; Vlachová, Viktorie; Reeh, Peter W; Lewis, Richard J; Zimmermann, Katharina

    2012-01-01

    Ciguatoxins are sodium channel activator toxins that cause ciguatera, the most common form of ichthyosarcotoxism, which presents with peripheral sensory disturbances, including the pathognomonic symptom of cold allodynia which is characterized by intense stabbing and burning pain in response to mild cooling. We show that intraplantar injection of P-CTX-1 elicits cold allodynia in mice by targeting specific unmyelinated and myelinated primary sensory neurons. These include both tetrodotoxin-resistant, TRPA1-expressing peptidergic C-fibres and tetrodotoxin-sensitive A-fibres. P-CTX-1 does not directly open heterologously expressed TRPA1, but when co-expressed with Nav channels, sodium channel activation by P-CTX-1 is sufficient to drive TRPA1-dependent calcium influx that is responsible for the development of cold allodynia, as evidenced by a large reduction of excitatory effect of P-CTX-1 on TRPA1-deficient nociceptive C-fibres and of ciguatoxin-induced cold allodynia in TRPA1-null mutant mice. Functional MRI studies revealed that ciguatoxin-induced cold allodynia enhanced the BOLD (Blood Oxygenation Level Dependent) signal, an effect that was blunted in TRPA1-deficient mice, confirming an important role for TRPA1 in the pathogenesis of cold allodynia. PMID:22850668

  5. Tissue Specific Expression of Cre in Rat Tyrosine Hydroxylase and Dopamine Active Transporter-Positive Neurons

    PubMed Central

    Liu, Zhenyi; Brown, Andrew; Fisher, Dan; Wu, Yumei; Warren, Joe; Cui, Xiaoxia

    2016-01-01

    The rat is a preferred model system over the mouse for neurological studies, and cell type-specific Cre expression in the rat enables precise ablation of gene function in neurons of interest, which is especially valuable for neurodegenerative disease modeling and optogenetics. Yet, few such Cre rats are available. Here we report the characterization of two Cre rats, tyrosine hydroxylase (TH)-Cre and dopamine active transporter (DAT or Slc6a3)-Cre, by using a combination of immunohistochemistry (IHC) and mRNA fluorescence in situ hybridization (FISH) as well as a fluorescent reporter for Cre activity. We detected Cre expression in expected neurons in both Cre lines. Interestingly, we also found that in Th-Cre rats, but not DAT-Cre rats, Cre is expressed in female germ cells, allowing germline excision of the floxed allele and hence the generation of whole-body knockout rats. In summary, our data demonstrate that targeted integration of Cre cassette lead to faithful recapitulation of expression pattern of the endogenous promoter, and mRNA FISH, in addition to IHC, is an effective method for the analysis of the spatiotemporal gene expression patterns in the rat brain, alleviating the dependence on high quality antibodies that are often not available against rat proteins. The Th-Cre and the DAT-Cre rat lines express Cre in selective subsets of dopaminergic neurons and should be particularly useful for researches on Parkinson’s disease. PMID:26886559

  6. Cytotoxic T-lymphocyte activity specific for hemagglutinin (H) protein of canine distemper virus in dogs.

    PubMed

    Hirama, Kyoko; Togashi, Ken-ichi; Wakasa, Chiaki; Yoneda, Misako; Nishi, Toshiya; Endo, Yasuyuki; Miura, Ryuichi; Tsukiyama-Kohara, Kyoko; Kai, Chieko

    2003-01-01

    Cytotoxic T-lymphocyte (CTL) responses to hemagglutinin (H) protein of canine distemper virus (CDV) were evaluated in dogs using the replication-deficient adenovirus protein expression system. Skin fibroblasts were isolated from two dogs and were infected with recombinant adenovirus bearing the CDV-H gene (Ade-CDVH). CTL assay was performed using fibroblasts expressing CDV-H protein as target cells and peripheral blood lymphocytes (PBL) collected from the same dogs one week after immunization of CDV as effector cells. Specific cytotoxic activity was observed against autologous but not heterologous fibroblasts expressing CDV-H protein. These results indicate that the CTL epitope(s) were localized in the H protein. PMID:12576714

  7. Specification of haematopoietic stem cell fate via modulation of mitochondrial activity

    PubMed Central

    Vannini, Nicola; Girotra, Mukul; Naveiras, Olaia; Nikitin, Gennady; Campos, Vasco; Giger, Sonja; Roch, Aline; Auwerx, Johan; Lutolf, Matthias P.

    2016-01-01

    Haematopoietic stem cells (HSCs) differ from their committed progeny by relying primarily on anaerobic glycolysis rather than mitochondrial oxidative phosphorylation for energy production. However, whether this change in the metabolic program is the cause or the consequence of the unique function of HSCs remains unknown. Here we show that enforced modulation of energy metabolism impacts HSC self-renewal. Lowering the mitochondrial activity of HSCs by chemically uncoupling the electron transport chain drives self-renewal under culture conditions that normally induce rapid differentiation. We demonstrate that this metabolic specification of HSC fate occurs through the reversible decrease of mitochondrial mass by autophagy. Our data thus reveal a causal relationship between mitochondrial metabolism and fate choice of HSCs and also provide a valuable tool to expand HSCs outside of their native bone marrow niches. PMID:27731316

  8. Purification and characterization of a galactose-specific lectin with mitogenic activity from pinto beans.

    PubMed

    Wong, Jack H; Wong, Clarence C T; Ng, T B

    2006-05-01

    A galactose-specific dimeric lectin from pinto beans was purified using a procedure that involved affinity chromatography on Affi-gel blue gel, anion exchange chromatography on Q-Sepharose, fast protein liquid chromatography (FPLC)-ion exchange chromatography on Mono S, and FPLC-gel filtration on Superdex 200. The molecular mass of this homodimeric lectin was 62 kDa and that of each of its subunits was 31 kDa. The hemagglutinating activity of pinto bean lectin was stable within the pH range of 3-12 and the temperature range of 0-70 degrees C. By using the [3H-methyl]-thymidine incorporation assay, it was shown that the lectin had the ability to evoke a mitogenic response from murine splenocytes but it did not inhibit proliferation of L1210 leukemia cells. The pinto bean lectin inhibited HIV-1 reverse transcriptase with an IC50 of 3 microM. PMID:16600511

  9. Signaling, Regulation, and Specificity of the Type II p21-activated Kinases*

    PubMed Central

    Ha, Byung Hak; Morse, Elizabeth M.; Turk, Benjamin E.; Boggon, Titus J.

    2015-01-01

    The p21-activated kinases (PAKs) are a family of six serine/threonine kinases that act as key effectors of RHO family GTPases in mammalian cells. PAKs are subdivided into two groups: type I PAKs (PAK1, PAK2, and PAK3) and type II PAKs (PAK4, PAK5, and PAK6). Although these groups are involved in common signaling pathways, recent work indicates that the two groups have distinct modes of regulation and have both unique and common substrates. Here, we review recent insights into the molecular level details that govern regulation of type II PAK signaling. We also consider mechanisms by which signal transduction is regulated at the level of substrate specificity. Finally, we discuss the implications of these studies for clinical targeting of these kinases. PMID:25855792

  10. Disposition and transportation of surplus radioactive low specific activity nitric acid. Volume 1, Environmental Assessment

    SciTech Connect

    1995-05-01

    DOE is deactivating the PUREX plant at Hanford; this will involve the disposition of about 692,000 liters (183,000 gallons) of surplus nitric acid contaminated with low levels of U and other radionuclides. The nitric acid, designated as low specific activity, is stored in 4 storage tanks at PUREX. Five principal alternatives were evaluated: transfer for reuse (sale to BNF plc), no action, continued storage in Hanford upgraded or new facility, consolidation of DOE surplus acid, and processing the LSA nitric acid as waste. The transfer to BNF plc is the preferred alternative. From the analysis, it is concluded that the proposed disposition and transportation of the acid does not constitute a major federal action significantly affecting the quality of the human environment within the meaning of NEPA; therefore an environmental impact statement is not required.

  11. MECHANISMS IN ENDOCRINOLOGY: Tissue-specific activation of cortisol in Cushing's syndrome.

    PubMed

    Morgan, Stuart A; Hassan-Smith, Zaki K; Lavery, Gareth G

    2016-08-01

    Glucocorticoids are widely prescribed for their anti-inflammatory properties, but have 'Cushingoid' side effects including visceral obesity, muscle myopathy, hypertension, insulin resistance, type 2 diabetes mellitus, osteoporosis, and hepatic steatosis. These features are replicated in patients with much rarer endogenous glucocorticoid (GC) excess (Cushing's syndrome), which has devastating consequences if left untreated. Current medical therapeutic options that reverse the tissue-specific consequences of hypercortisolism are limited. In this article, we review the current evidence that local GC metabolism via the enzyme 11β-hydroxysteroid dehydrogenase type 1 (11β-HSD1) plays a central role in mediating the adverse metabolic complications associated with circulatory GC excess - challenging our current view that simple delivery of active GCs from the circulation represents the most important mode of GC action. Furthermore, we explore the potential for targeting this enzyme as a novel therapeutic strategy for the treatment of both endogenous and exogenous Cushing's syndrome.

  12. Structure-Specific nuclease activities of Artemis and the Artemis: DNA-PKcs complex

    PubMed Central

    Chang, Howard H.Y.; Lieber, Michael R.

    2016-01-01

    Artemis is a vertebrate nuclease with both endo- and exonuclease activities that acts on a wide range of nucleic acid substrates. It is the main nuclease in the non-homologous DNA end-joining pathway (NHEJ). Not only is Artemis important for the repair of DNA double-strand breaks (DSBs) in NHEJ, it is essential in opening the DNA hairpin intermediates that are formed during V(D)J recombination. Thus, humans with Artemis deficiencies do not have T- or B-lymphocytes and are diagnosed with severe combined immunodeficiency (SCID). While Artemis is the only vertebrate nuclease capable of opening DNA hairpins, it has also been found to act on other DNA substrates that share common structural features. Here, we discuss the key structural features that all Artemis DNA substrates have in common, thus providing a basis for understanding how this structure-specific nuclease recognizes its DNA targets. PMID:27198222

  13. Mitochondrial Complex 1 Activity Measured by Spectrophotometry Is Reduced across All Brain Regions in Ageing and More Specifically in Neurodegeneration

    PubMed Central

    Chakrabarti, Lisa

    2016-01-01

    Mitochondrial function, in particular complex 1 of the electron transport chain (ETC), has been shown to decrease during normal ageing and in neurodegenerative disease. However, there is some debate concerning which area of the brain has the greatest complex 1 activity. It is important to identify the pattern of activity in order to be able to gauge the effect of age or disease related changes. We determined complex 1 activity spectrophotometrically in the cortex, brainstem and cerebellum of middle aged mice (70–71 weeks), a cerebellar ataxic neurodegeneration model (pcd5J) and young wild type controls. We share our updated protocol on the measurements of complex1 activity and find that mitochondrial fractions isolated from frozen tissues can be measured for robust activity. We show that complex 1 activity is clearly highest in the cortex when compared with brainstem and cerebellum (p<0.003). Cerebellum and brainstem mitochondria exhibit similar levels of complex 1 activity in wild type brains. In the aged brain we see similar levels of complex 1 activity in all three-brain regions. The specific activity of complex 1 measured in the aged cortex is significantly decreased when compared with controls (p<0.0001). Both the cerebellum and brainstem mitochondria also show significantly reduced activity with ageing (p<0.05). The mouse model of ataxia predictably has a lower complex 1 activity in the cerebellum, and although reductions are measured in the cortex and brain stem, the remaining activity is higher than in the aged brains. We present clear evidence that complex 1 activity decreases across the brain with age and much more specifically in the cerebellum of the pcd5j mouse. Mitochondrial impairment can be a region specific phenomenon in disease, but in ageing appears to affect the entire brain, abolishing the pattern of higher activity in cortical regions. PMID:27333203

  14. [Specific features of fear conditioning expression in active and passive rabbits].

    PubMed

    2013-11-01

    The active and passive rabbits selected previously on the basis of their behavior in open field and light-dark test, were subjected to fear conditioning using pairing light (4 s) with footshocks (10 Hz, 0.5 s). Heart rate and respiration rate were measured during the classical fear conditioning. Heart rate and respiration rate decreased in response to light before footshock in case of passive-defensive reaction. There were no heart rate and respiration rate reduction in the course of the active defensive reaction. In active rabbits, as compared to passive ones, the frequency of active locomotors reactions and heart rate were higher, the decrease of respiration rate to light was observed at later stages of training, and the detected bradycardia was not stable. Thus, based upon vegetative characteristics, the active rabbits had lower level of fear than passive ones. The active or passive behavioral strategies of animals were preserved during fear conditioning.

  15. Standardized methods for the production of high specific-activity zirconium-89.

    PubMed

    Holland, Jason P; Sheh, Yiauchung; Lewis, Jason S

    2009-10-01

    Zirconium-89 is an attractive metallo-radionuclide for use in immuno-PET due to favorable decay characteristics. Standardized methods for the routine production and isolation of high-purity and high-specific-activity (89)Zr using a small cyclotron are reported. Optimized cyclotron conditions reveal high average yields of 1.52+/-0.11 mCi/muA.h at a proton beam energy of 15 MeV and current of 15 muA using a solid, commercially available (89)Y-foil target (0.1 mm, 100% natural abundance). (89)Zr was isolated in high radionuclidic and radiochemical purity (>99.99%) as [(89)Zr]Zr-oxalate by using a solid-phase hydroxamate resin with >99.5% recovery of the radioactivity. The effective specific-activity of (89)Zr was found to be in the range 5.28-13.43 mCi/microg (470-1195 Ci/mmol) of zirconium. New methods for the facile production of [(89)Zr]Zr-chloride are reported. Radiolabeling studies using the trihydroxamate ligand desferrioxamine B (DFO) gave 100% radiochemical yields in <15 min at room temperature, and in vitro stability measurements confirmed that [(89)Zr]Zr-DFO is stable with respect to ligand dissociation in human serum for >7 days. Small-animal positron emission tomography (PET) imaging studies have demonstrated that free (89)Zr(IV) ions administered as [(89)Zr]Zr-chloride accumulate in the liver, whilst [(89)Zr]Zr-DFO is excreted rapidly via the kidneys within <20 min. These results have important implication for the analysis of immuno-PET imaging of (89)Zr-labeled monoclonal antibodies. The detailed methods described can be easily translated to other radiochemistry facilities and will facilitate the use of (89)Zr in both basic science and clinical investigations.

  16. Is Neural Activity Detected by ERP-Based Brain-Computer Interfaces Task Specific?

    PubMed Central

    Wenzel, Markus A.; Almeida, Inês; Blankertz, Benjamin

    2016-01-01

    Objective Brain-computer interfaces (BCIs) that are based on event-related potentials (ERPs) can estimate to which stimulus a user pays particular attention. In typical BCIs, the user silently counts the selected stimulus (which is repeatedly presented among other stimuli) in order to focus the attention. The stimulus of interest is then inferred from the electroencephalogram (EEG). Detecting attention allocation implicitly could be also beneficial for human-computer interaction (HCI), because it would allow software to adapt to the user’s interest. However, a counting task would be inappropriate for the envisaged implicit application in HCI. Therefore, the question was addressed if the detectable neural activity is specific for silent counting, or if it can be evoked also by other tasks that direct the attention to certain stimuli. Approach Thirteen people performed a silent counting, an arithmetic and a memory task. The tasks required the subjects to pay particular attention to target stimuli of a random color. The stimulus presentation was the same in all three tasks, which allowed a direct comparison of the experimental conditions. Results Classifiers that were trained to detect the targets in one task, according to patterns present in the EEG signal, could detect targets in all other tasks (irrespective of some task-related differences in the EEG). Significance The neural activity detected by the classifiers is not strictly task specific but can be generalized over tasks and is presumably a result of the attention allocation or of the augmented workload. The results may hold promise for the transfer of classification algorithms from BCI research to implicit relevance detection in HCI. PMID:27792781

  17. Melanoma-associated antigen synthesized in vitro for active specific immunotherapy.

    PubMed

    Hayashibe, K

    1992-11-01

    The immunogenicity of the antigen molecule is a prerequisite for active specific immunotherapy for melanoma. Since most of the melanoma-associated antigens recognized by the murine immune system are known to be not immunogenic in man, a detection and analysis system for melanoma-associated antigens is required to reflect in vivo immune responses in patients with melanoma. One of the promising approaches, an attempt to develop human monoclonal antibodies from B lymphocytes of patients with melanoma, has met with limited success due to the difficulties of producing large amounts of antibodies and using them in immunochemical assays, because most of them belong to the IgM class and have low affinity. Our approach is to utilize the screening of a cDNA expression library constructed from mRNA extracted from cultured melanoma cells with antibodies from patients with melanoma. The cloned cDNA, designated as D-1, had 1029 bp and showed no significant homology with viral and mammalian sequences stored in GENETYX. cDNA D-1 hybridized to a 2.0 kb mRNA species from 3 different cell lines of human melanoma, neuroblastoma, erythroleukemia, B lymphoid, and T lymphoid cells, but not from a renal carcinoma cell line, normal peripheral lymphocytes, or normal fibroblasts. The in vivo expression and distribution of mRNA related to cDNA D-1 has been examined in tissue specimens by in situ hybridization and shown to be rather restricted on melanoma cells. The polypeptide antigen encoded by cDNA D-1 may be a valuable immunogen for implementing active specific immunotherapy in patients with melanoma.

  18. Specific binding, internalization, and degradation of human neutrophil activating factor by human polymorphonuclear leukocytes

    SciTech Connect

    Besemer, J.; Hujber, A.; Kuhn, B. )

    1989-10-15

    The interaction of {sup 125}I-labeled recombinant human neutrophil activating factor (NAF) with polymorphonuclear leukocytes (PMN) was studied by means of a radioreceptor assay. The binding was characterized by a rapid transition (t1/2 less than or equal to 1 min) from a pH 3-sensitive state at 4{degree}C to pH 3 resistance at 37{degree}C. This was not caused by internalization of NAF since pH 3-resistant bound iodinated NAF could still be exchanged by an excess of nonlabeled NAF, i.e. was dissociable. Internalized iodinated NAF was processed into trichloroacetic acid-soluble forms. Scatchard transformation of binding isotherms at 4 and 37{degree}C led to nonlinear curves, a finding which is consistent with the expression of two receptor populations, one with high (KD = 11-35 pM) and the other with lower affinity (KD = 640-830 pM) at 4 degrees C. Numbers of the low affinity binding sites were approximately 34,000, and those with high affinity were 5,200/PMN when estimated at 4 degrees C. Binding of iodinated NAF to PMN was specific since it could be competed by an excess of nonlabeled NAF but not by two other activators of PMN function, formylmethionyl-leucyl-phenylalanine or human recombinant granulocyte-macrophage colony-stimulating factor. In addition to human PMN, NAF also bound specifically to two human monocytic cell lines; however, only the low affinity binding site could be detected on these cells.

  19. Tissue-Specific Activation of a Single Gustatory Receptor Produces Opposing Behavioral Responses in Drosophila

    PubMed Central

    Joseph, Ryan M.; Heberlein, Ulrike

    2012-01-01

    Understanding sensory systems that perceive environmental inputs and neural circuits that select appropriate motor outputs is essential for studying how organisms modulate behavior and make decisions necessary for survival. Drosophila melanogaster oviposition is one such important behavior, in which females evaluate their environment and choose to lay eggs on substrates they may find aversive in other contexts. We employed neurogenetic techniques to characterize neurons that influence the choice between repulsive positional and attractive egg-laying responses toward the bitter-tasting compound lobeline. Surprisingly, we found that neurons expressing Gr66a, a gustatory receptor normally involved in avoidance behaviors, receive input for both attractive and aversive preferences. We hypothesized that these opposing responses may result from activation of distinct Gr66a-expressing neurons. Using tissue-specific rescue experiments, we found that Gr66a-expressing neurons on the legs mediate positional aversion. In contrast, pharyngeal taste cells mediate the egg-laying attraction to lobeline, as determined by analysis of mosaic flies in which subsets of Gr66a neurons were silenced. Finally, inactivating mushroom body neurons disrupted both aversive and attractive responses, suggesting that this brain structure is a candidate integration center for decision-making during Drosophila oviposition. We thus define sensory and central neurons critical to the process by which flies decide where to lay an egg. Furthermore, our findings provide insights into the complex nature of gustatory perception in Drosophila. We show that tissue-specific activation of bitter-sensing Gr66a neurons provides one mechanism by which the gustatory system differentially encodes aversive and attractive responses, allowing the female fly to modulate her behavior in a context-dependent manner. PMID:22798487

  20. Neuron-specific enolase as a novel biomarker reflecting tuberculosis activity and treatment response

    PubMed Central

    Nam, Sung-Jin; Jeong, Jee-Yeong; Jang, Tae-Won; Jung, Mann-Hong; Chun, Bong-Kwon; Cha, Hee-Jae; Oak, Chul-Ho

    2016-01-01

    Background/Aims: It is not clear which tests are indicative of the activity and severity of tuberculosis (TB). This study aimed to investigate the predictive value of neuron-specific enolase (NSE) and to determine the origin of NSE in TB patients. Methods: A single-center retrospective analysis was conducted on newly diagnosed TB patients between January and December 2010. Patients were categorized into one of two disease groups (focal segmental or extensive) based on chest X-ray. Pre- and post-treatment NSE concentrations were evaluated. To determine the origin of serum NSE concentration, NSE staining was compared with macrophage-specific CD68 staining in lung tissues and with a tissue microarray using immunohistochemistry and immunofluorescence. Results: A total of 60 newly diagnosed TB patients were analyzed. In TB patients, NSE serum concentration was significantly increased and NSE level decreased after treatment (p < 0.001). In proportion to serum high-sensitivity C-reactive protein concentration, the mean serum concentration of NSE in the extensive group (25.12 ng/mL) was significantly higher than that in the focal segmental group (20.23 ng/mL, p = 0.04). Immunohistochemical staining revealed a large number of macrophages that stained positively for both NSE and CD68 in TB tissues. In addition, NSE signals mostly co-localized with CD68 signals in the tissue microarray of TB patients. Conclusions: Our results suggest that NSE may be a practical parameter that can be used to monitor TB activity and treatment response. Elevated serum NSE level originates, at least in part, from macrophages in granulomatous lesions. PMID:27271274

  1. Ceruloplasmin ferroxidase activity stimulates cellular iron uptake by a trivalent cation-specific transport mechanism

    NASA Technical Reports Server (NTRS)

    Attieh, Z. K.; Mukhopadhyay, C. K.; Seshadri, V.; Tripoulas, N. A.; Fox, P. L.

    1999-01-01

    The balance required to maintain appropriate cellular and tissue iron levels has led to the evolution of multiple mechanisms to precisely regulate iron uptake from transferrin and low molecular weight iron chelates. A role for ceruloplasmin (Cp) in vertebrate iron metabolism is suggested by its potent ferroxidase activity catalyzing conversion of Fe2+ to Fe3+, by identification of yeast copper oxidases homologous to Cp that facilitate high affinity iron uptake, and by studies of "aceruloplasminemic" patients who have extensive iron deposits in multiple tissues. We have recently shown that Cp increases iron uptake by cultured HepG2 cells. In this report, we investigated the mechanism by which Cp stimulates cellular iron uptake. Cp stimulated the rate of non-transferrin 55Fe uptake by iron-deficient K562 cells by 2-3-fold, using a transferrin receptor-independent pathway. Induction of Cp-stimulated iron uptake by iron deficiency was blocked by actinomycin D and cycloheximide, consistent with a transcriptionally induced or regulated transporter. Cp-stimulated iron uptake was completely blocked by unlabeled Fe3+ and by other trivalent cations including Al3+, Ga3+, and Cr3+, but not by divalent cations. These results indicate that Cp utilizes a trivalent cation-specific transporter. Cp ferroxidase activity was required for iron uptake as shown by the ineffectiveness of two ferroxidase-deficient Cp preparations, copper-deficient Cp and thiomolybdate-treated Cp. We propose a model in which iron reduction and subsequent re-oxidation by Cp are essential for an iron uptake pathway with high ion specificity.

  2. Breadth of Scientific Activities and Network Station Specifications in the IGS

    NASA Technical Reports Server (NTRS)

    Moore, A. W.; Springer, T. A.; Reigber, Ch.

    1999-01-01

    This presentation provides a brief overview of the scientific activities of the International GPS Service (IGS). This was an approved activity of the International Association of Geodesy (IAG) with official start of service on 1 Jan 1994. The mission of the IGS is "To provide a service to support geodetic and geophysical research activities, through GPS data and data products." The presentation explains the concept of the IGS working group, and pilot projects, and reviews the current working groups and pilot projects.

  3. Active hydrocarbon (methane) seepage at the Alboran Sea mud volcanoes indicated by specific lipid biomarkers.

    NASA Astrophysics Data System (ADS)

    Lopez-Rodriguez, C.; Stadnitskaia, A.; De Lange, G. J.; Martínez-Ruiz, F.; Comas, M.; Sinninghe Damsté, J. S.

    2012-04-01

    Mud volcanoes (MVs) and pockmark fields are known to occur in the Alboran Basin (Westernmost Mediterranean). These MVs occur above a major sedimentary depocenter that includes up to 7 km thick early Miocene to Holocene sequences. MVs located on the top of diapiric structures that originated from undercompacted Miocene clays and olistostromes. Here we provide results from geochemical data-analyses of four gravity cores acquired in the Northern Mud Volcano Field (north of the 36°N): i.e. Perejil, Kalinin and Schneiderś Heart mud expulsion structures. Extruded materials include different types of mud breccias. Specific lipid biomarkers (n-alkanes, hopanes, irregular isoprenoid hydrocarbons and Dialkyl Glycerol Diethers (DGDs) were analysed by gas chromatography (GC) and gas chromatography mass spectrometry (GC-MS). Determination of Glycerol Dialkyl Glycerol Tetraethers (GDGTs) by high performance liquid chromatography-spectrometry (HPLC-MS), and analysis of biomarker δ13C values were performed in selected samples. Lipid biomarker analysis from the three MVs revealed similar n-alkane distributions in all mud breccia intervals, showing significant hydrocarbon-derived signals and the presence of thermally immature organic-matter admixture. This suggests that similar strata fed these MVs. The hemipelagic drapes reveal comparable n-alkane distributions, suggesting that significant upward diffusion of fluids occurs. Distributions of GDGTs are generally accepted as usefull biomarkers to locate the anaerobic oxidation of methane (AOM) in marine sediments. However, our GDGT profiles only reflect the marine thaumarchaeotal signature. There seems to be no archaea producing specific GDGTs involved in AOM in the recovered interval. Evidence of recent activity (i.e., methane gas-bubbling and chemosynthetic fauna at the Perejil MV) and the presence of specific lipid biomarker related with methanotropic archaea (Irregular Isoprenoids and DGDs), however, suggest the existence of

  4. Carbon-14 Specific Activity Model Validation for Biota in Wetland Environments

    SciTech Connect

    Yankovich, T.L.; Sharp, K.J.; Benz, M.L.; Carr, J.; Killey, R.W.D.

    2008-01-15

    In many cases, contaminants, such as radionuclides, can show highly localized spatial distributions in natural systems. Therefore, a key question for environmental assessment and monitoring becomes, how can these localized distributions of contaminants in the environment lead to organism exposure, and ultimately, the potential for effects to receptor biota? To address this question, an important first step is to conduct field surveys at sites of interest to map out the spatial distribution and extent of contaminants in areas that are being occupied and utilized by resident receptor biota. Work can then be conducted to establish predictive relationships between contaminant concentrations in biota tissues and those in environmental media with which biota interact, to gain an understanding of how representative ambient contaminant concentrations are of biota exposure. The objectives of this study were: - To conduct a field survey in a wetland ecosystem to characterize the spatial distribution of carbon- 14 ({sup 14}C), a radionuclide with dynamics in natural systems that can be described using a specific activity model; and - To determine whether {sup 14}C concentrations in environmental media reflect those measured in tissues of resident flora and fauna. A detailed field campaign was carried out in summer 2001 to characterize the spatial distribution and areal coverage of {sup 14}C in Duke Swamp, a wetland ecosystem on Atomic Energy of Canada Limited (AECL)'s Chalk River Laboratories (CRL) site that receives {sup 14}C through releases from an up-gradient Waste Management Area (WMA), primarily through groundwater influx. Sampling of surface vegetation (dominantly comprised of Sphagnum moss) was conducted at a total of 69 locations, with complementary sampling of air, soil, fungi, aerial insects, ground-dwelling insects, amphibians, small mammals and snakes being carried out at a subset of five locations with varying {sup 14}C concentrations. Concentrations of {sup 14

  5. Post-activation Potentiation in Propulsive Force after Specific Swimming Strength Training.

    PubMed

    Barbosa, A C; Barroso, R; Andries, O

    2016-04-01

    We investigated whether a conditioning activity (8×12.5 m with 2.5 min-interval using both hand paddles and parachute) induced post-activation potentiation in swimming propulsive force and whether a swimmer's force level affected a post-activation potentiation response. 8 competitive swimmers (5 males and 3 females, age: 18.4±1.3 years; IPS=796±56) performed a 10 s maximum tethered swimming test 8 and 4 min before (the highest value was considered as PRE), and 2.5 and 6.5 min after (POST1 and POST2, respectively) the conditioning activity. Rate of force development was not affected, but peak force in POST1 (p=0.02) and impulse in both POST1 (p=0.007) and POST2 (p=0.004) were reduced. Possibly the conditioning activity induced greater fatigue than post-activation potentiation benefits. For instance, the number of repetitions might have been excessive, and rest intervals between the conditioning activity and POST1 and POST2 were possibly too short. There were positive correlations between PRE peak force and changes in peak force and rate of force development. Although conditioning activity was detrimental, positive correlations suggest that weaker swimmers experience a deterioration of performance more than the stronger ones. This conditioning activity is not recommended for swimmers with the current competitive level before a competitive event. PMID:26667922

  6. Male-specific tetraene and triene hydrocarbons ofCarpophilus hemipterus: Structure and pheromonal activity.

    PubMed

    Bartelt, R J; Weisleder, D; Dowd, P F; Plattner, R D

    1992-03-01

    Males ofCarpophilus hemipterus (L.), the dried-fruit beetle, (Coleoptera: Nitidulidae) were found to emit nine all-E tetraene and one all-E triene hydrocarbons in addition to two pheromonally active tetraenes that had been reported previously. The previously known compounds are (2E,4E,6E,8E)-3,5,7-trimethyl-2,4,6,8-decatetraene(1) and (2E,4E,6E,8E)-3, 5,7-trimethyl-2,4,6,8-undecatetraene(2). The new tetraenes were all related to structure1 by having one additional carbon at either one or two of the following four locations: at carbon 1 of the chain, at carbon 10 of the chain, at the 5-alkyl branch, or at the 7-alkyl branch. (Structure 2 also fits within this pattern.) The triene inC. hemipterus is (2E,4E,6E)-5-ethyl-3-methyl-2, 4,6-nonatriene. Also identified from volatile collections from the beetles were the 2Z and 4Z isomers of1. All structures were proven by synthesis, with NMR and mass spectral data for the compounds provided. Two of the newly discovered compounds, (2E,4E,6E,8E)-7-ethyl-3,5-dimethyl-2,4,6,8-decatetraene and (2E,4E,6E,8E)-7-ethyl-3,5-dimethyl-2,4,6,8-undecatetraene, were quite active in the wind-tunnel bioassay, but others, such as (2E,4E,6E,8E)-5-ethyl-3,7-dimethyl-2,4,6,8-decatetraene and (2E,4E,6E,8E)-4,6,8-trimethyl-2,4,6,8-undecatetraene were not. Structureactivity relationships are explored among the natural compounds and additional, synthetic analogs, which were never detected from the beetles. Some of these analogs, such as (2E,4E,6E,8E)-3,5-dimethyl-7-propyl-2,4,6,8-undecatetraene, were quite active in the bioassay. The biosynthesis of the beetle-derived compounds is discussed. A single biosynthetic scheme that lacks complete enzyme specificity at four specific steps could account for the entire series of compounds found in the beetles and their relative proportions. The definition of "pheromone" is discussed in relation to these hydrocarbons. PMID:24254944

  7. Phosphate oxygen isotope ratio proxy for specific microbial activity in marine sediments (Peru Margin)

    NASA Astrophysics Data System (ADS)

    Liang, Y.; Blake, R. E.

    2005-12-01

    Oxygen (O) isotope ratios of biogenic apatites have been widely used as paleotemperature and environmental geochemical proxies. With improved knowledge of the phosphate O isotope effects of different P cycling pathways, the δ18O value of inorganic phosphate (δ18OP) has been proposed as a useful proxy and tracer of biological reactions and P cycling in natural environments[1,2,3,4]. Being the only way of removing P from oceanic water, sedimentary P burial is one of the most important processes during biogeochemical cycling of P. The high concentrations of organic matter and pronounced microbial activity at ODP Site 1230 along the Peru Margin result in unusually high interstitial water phosphate concentrations, which provides a unique opportunity to use δ18OP to investigate inorganic phosphate (Pi) regeneration and P cycling pathways in marine sediments. The isotopic measurements of both dissolved inorganic phosphate (DIP) and bulk sediment Pi show that DIP δ18OP values are affected by three different processes, which are all induced by specific microbial activities present in the sediments. In sediments at ~ 65 to 120 mbsf, porewater DIP is derived from dissolved organophosphorus compounds (DOP) through enzymatic degradation pathways, evidenced by both DIP δ18OP values and interstitial water chemistry. Measured porewater DIP δ18OP values also suggest that 4 to 8% of interstitial water DIP reflects regeneration of Pi from Porg by microbially-synthesized enzymes. Throughout the sediment column and especially at ~ 120 to 150 mbsf, DIP is released from the sediments by microbially-induced reductive dissolution of Fe-oxides, which contributes to the overall high DIP concentrations at Site 1230. The third and dominant process controlling measured DIP δ18OP values is microbial turnover of regenerated Pi. The presence of high microbial activities in organic-rich Site 1230 sediments promotes the remobilization of P and affects marine P cycling by potentially enhancing

  8. Ganglioside GD2-specific trifunctional surrogate antibody Surek demonstrates therapeutic activity in a mouse melanoma model

    PubMed Central

    2012-01-01

    Background Trifunctional bispecific antibodies (trAb) are a special class of bispecific molecules recruiting and activating T cells and accessory immune cells simultaneously at the targeted tumor. The new trAb Ektomab that targets the melanoma-associated ganglioside antigen GD2 and the signaling molecule human CD3 (hCD3) on T cells demonstrated potent T-cell activation and tumor cell destruction in vitro. However, the relatively low affinity for the GD2 antigen raised the question of its therapeutic capability. To further evaluate its efficacy in vivo it was necessary to establish a mouse model. Methods We generated the surrogate trAb Surek, which possesses the identical anti-GD2 binding arm as Ektomab, but targets mouse CD3 (mCD3) instead of hCD3, and evaluated its chemical and functional quality as a therapeutic antibody homologue. The therapeutic and immunizing potential of Surek was investigated using B78-D14, a B16 melanoma transfected with GD2 and GD3 synthases and showing strong GD2 surface expression. The induction of tumor-associated and autoreactive antibodies was evaluated. Results Despite its low affinity of approximately 107 M-1 for GD2, Surek exerted efficient tumor cell destruction in vitro at an EC50 of 70ng/ml [0.47nM]. Furthermore, Surek showed strong therapeutic efficacy in a dose-dependent manner and is superior to the parental GD2 mono-specific antibody, while the use of a control trAb with irrelevant target specificity had no effect. The therapeutic activity of Surek was strictly dependent on CD4+ and CD8+ T cells, and cured mice developed a long-term memory response against a second challenge even with GD2-negative B16 melanoma cells. Moreover, tumor protection was associated with humoral immune responses dominated by IgG2a and IgG3 tumor-reactive antibodies indicating a Th1-biased immune response. Autoreactive antibodies against the GD2 target antigen were not induced. Conclusion Our data suggest that Surek revealed strong tumor elimination

  9. Enrichment of specific electro-active microorganisms and enhancement of methane production by adding granular activated carbon in anaerobic reactors.

    PubMed

    Lee, Jung-Yeol; Lee, Sang-Hoon; Park, Hee-Deung

    2016-04-01

    Direct interspecies electron transfer (DIET) via conductive materials can provide significant benefits to anaerobic methane formation in terms of production amount and rate. Although granular activated carbon (GAC) demonstrated its applicability in facilitating DIET in methanogenesis, DIET in continuous flow anaerobic reactors has not been verified. Here, evidences of DIET via GAC were explored. The reactor supplemented with GAC showed 1.8-fold higher methane production rate than that without GAC (35.7 versus 20.1±7.1mL-CH4/d). Around 34% of methane formation was attributed to the biomass attached to GAC. Pyrosequencing of 16S rRNA gene demonstrated the enrichment of exoelectrogens (e.g. Geobacter) and hydrogenotrophic methanogens (e.g. Methanospirillum and Methanolinea) from the biomass attached to GAC. Furthermore, anodic and cathodic currents generation was observed in an electrochemical cell containing GAC biomass. Taken together, GAC supplementation created an environment for enriching the microorganisms involved in DIET, which increased the methane production rate. PMID:26836607

  10. A specific method for measurement of equine active myeloperoxidase in biological samples and in in vitro tests.

    PubMed

    Franck, Thierry; Kohnen, S; Deby-Dupont, G; Grulke, S; Deby, C; Serteyn, D

    2006-07-01

    An original method called SIEFED (specific immunological extraction followed by enzymatic detection) was developed for the specific detection of the activity of equine myeloperoxidase (MPO). The method consists of the extraction of MPO from aqueous solutions by immobilized anti-MPO antibodies followed by washing (to eliminate proteins and interfering molecules) and measurement of MPO activity using a detection system containing a fluorogenic substrate, hydrogen peroxide, and nitrite as reaction enhancer. The SIEFED technique was applied to study active MPO in horse biological fluids and the effects of 2 polyphenolic molecules, curcumin and resveratrol, on MPO activity. The detection limit of the SIEFED was 0.23 mU/ml. The SIEFED exhibited good precision with intra-assay and interassay coefficients of variation below 10% and 20%, respectively, for MPO activities ranging from 0.25 to 6.4 mU/ml. The activity of MPO was generally higher than 1 mU/ml in the fluids collected from horses with inflammatory diseases. Curcumin and resveratrol exerted a dose-dependent inhibition on MPO activity and, as they were removed before the enzymatic detection of MPO, the results suggest a direct drug-nzyme interaction or an enzyme structure modification by the drug. The SIEFED is a new tool that would be useful for specific detection of active MPO in complex media and for selection of MPO activity modulators.

  11. Approximated maximum adsorption of His-tagged enzyme/mutants on Ni2+-NTA for comparison of specific activities.

    PubMed

    Li, Yuanli; Long, Gaobo; Yang, Xiaolan; Hu, Xiaolei; Feng, Yiran; Tan, Deng; Xie, Yanling; Pu, Jun; Liao, Fei

    2015-03-01

    By approximating maximum activities of six-histidine (6His)-tagged enzyme/mutants adsorbed on Ni2+-NTA-magnetic-submicron-particle (Ni2+-NTA-MSP), a facile approach was tested for comparing enzyme specific activities in cell lysates. On a fixed quantity of Ni2+-NTA-MSP, the activity of an adsorbed 6His-tagged enzyme/mutant was measured via spectrophotometry; the activity after saturation adsorption (Vs) was predicted from response curve with quantities of total proteins from the same lysate as the predictor; Vs was equivalent of specific activity for comparison. This approach required abundance of a 6His-tagged enzyme/mutant over 3% among total proteins in lysate, an accurate series of quantities of total proteins from the same lysate, the largest activity generated by enzyme occupying over 85% binding sites on Ni2+-NTA-MSP and the minimum activity as absorbance change rates of 0.003 min(-1) for analysis. The prediction of Vs tolerated errors in concentrations of total proteins in lysates and was effective to 6His-tagged alkaline phosphatase and its 6His-tagged mutant in lysates. Notably, of those two 6His-tagged enzymes, Vs was effectively approximated with just one optimized quantity of lysates. Hence, this approach with Ni2+-NTA-MSP worked for comparison of specific activities of 6His-tagged enzyme/mutants in lysates when they had sufficient abundance among proteins and activities of adsorbed enzymes were measurable.

  12. Assessing General and Specific Attitudes in Human Learning Behavior: An Activity Perspective and a Multilevel Modeling Approach

    ERIC Educational Resources Information Center

    Sun, Jun; Willson, Victor L.

    2008-01-01

    This article proposes a multilevel modeling approach to study the general and specific attitudes formed in human learning behavior. Based on the premises of activity theory, it conceptualizes the unit of analysis for attitude measurement as a scalable and evolving activity system rather than a single action. Measurement issues related to this…

  13. Responses of absolute and specific soil enzyme activities to long term additions of organic and mineral fertilizer.

    PubMed

    Zhang, Xinyu; Dong, Wenyi; Dai, Xiaoqin; Schaeffer, Sean; Yang, Fengting; Radosevich, Mark; Xu, Lili; Liu, Xiyu; Sun, Xiaomin

    2015-12-01

    Long-term phosphorus (P) and nitrogen (N) applications may seriously affect soil microbial activity. A long-term field fertilizer application trial was established on reddish paddy soils in the subtropical region of southern China in 1998. We assessed the effects of swine manure and seven different rates or ratios of NPK fertilizer treatments on (1) the absolute and specific enzyme activities per unit of soil organic carbon (SOC) or microbial biomass carbon (MBC) involved in C, N, and P transformations and (2) their relationships with soil environmental factors and soil microbial community structures. The results showed that manure applications led to increases in the absolute and specific activities of soil β-1,4-glucosidase(βG), β-1,4-N-acetylglucosaminidase (NAG), and leucine aminopeptidase (LAP). The absolute and specific acid phosphatase (AP) activities decreased as mineral P fertilizer application rates and ratios increased. Redundancy analysis (RDA) showed that there were negative correlations between absolute and specific AP activities, pH, and total P contents, while there were positive correlations between soil absolute and specific βG, NAG, and LAP enzyme activities, and SOC and total N contents. RDA showed that the contents of actinomycete and Gram-positive bacterium PLFA biomarkers are more closely related to the absolute and specific enzyme activities than the other PLFA biomarkers (P<0.01). Our results suggest that both the absolute and specific enzyme activities could be used as sensitive soil quality indicators that provide useful linkages with the microbial community structures and environmental factors. To maintain microbial activity and to minimize environmental impacts, P should be applied as a combination of inorganic and organic forms, and total P fertilizer application rates to subtropical paddy soils should not exceed 44 kg P ha(-1) year(-1). PMID:26196069

  14. Responses of absolute and specific soil enzyme activities to long term additions of organic and mineral fertilizer.

    PubMed

    Zhang, Xinyu; Dong, Wenyi; Dai, Xiaoqin; Schaeffer, Sean; Yang, Fengting; Radosevich, Mark; Xu, Lili; Liu, Xiyu; Sun, Xiaomin

    2015-12-01

    Long-term phosphorus (P) and nitrogen (N) applications may seriously affect soil microbial activity. A long-term field fertilizer application trial was established on reddish paddy soils in the subtropical region of southern China in 1998. We assessed the effects of swine manure and seven different rates or ratios of NPK fertilizer treatments on (1) the absolute and specific enzyme activities per unit of soil organic carbon (SOC) or microbial biomass carbon (MBC) involved in C, N, and P transformations and (2) their relationships with soil environmental factors and soil microbial community structures. The results showed that manure applications led to increases in the absolute and specific activities of soil β-1,4-glucosidase(βG), β-1,4-N-acetylglucosaminidase (NAG), and leucine aminopeptidase (LAP). The absolute and specific acid phosphatase (AP) activities decreased as mineral P fertilizer application rates and ratios increased. Redundancy analysis (RDA) showed that there were negative correlations between absolute and specific AP activities, pH, and total P contents, while there were positive correlations between soil absolute and specific βG, NAG, and LAP enzyme activities, and SOC and total N contents. RDA showed that the contents of actinomycete and Gram-positive bacterium PLFA biomarkers are more closely related to the absolute and specific enzyme activities than the other PLFA biomarkers (P<0.01). Our results suggest that both the absolute and specific enzyme activities could be used as sensitive soil quality indicators that provide useful linkages with the microbial community structures and environmental factors. To maintain microbial activity and to minimize environmental impacts, P should be applied as a combination of inorganic and organic forms, and total P fertilizer application rates to subtropical paddy soils should not exceed 44 kg P ha(-1) year(-1).

  15. FOXA1 potentiates lineage-specific enhancer activation through modulating TET1 expression and function

    PubMed Central

    Yang, Yeqing A.; Zhao, Jonathan C.; Fong, Ka-wing; Kim, Jung; Li, Shangze; Song, Chunxiao; Song, Bing; Zheng, Bin; He, Chuan; Yu, Jindan

    2016-01-01

    Forkhead box A1 (FOXA1) is an FKHD family protein that plays pioneering roles in lineage-specific enhancer activation and gene transcription. Through genome-wide location analyses, here we show that FOXA1 expression and occupancy are, in turn, required for the maintenance of these epigenetic signatures, namely DNA hypomethylation and histone 3 lysine 4 methylation. Mechanistically, this involves TET1, a 5-methylcytosine dioxygenase. We found that FOXA1 induces TET1 expression via direct binding to its cis-regulatory elements. Further, FOXA1 physically interacts with the TET1 protein through its CXXC domain. TET1 thus co-occupies FOXA1-dependent enhancers and mediates local DNA demethylation and concomitant histone 3 lysine 4 methylation, further potentiating FOXA1 recruitment. Consequently, FOXA1 binding events are markedly reduced following TET1 depletion. Together, our results suggest that FOXA1 is not only able to recognize but also remodel the epigenetic signatures at lineage-specific enhancers, which is mediated, at least in part, by a feed-forward regulatory loop between FOXA1 and TET1. PMID:27257062

  16. Cancer-Specific Synthetic Lethality between ATR and CHK1 Kinase Activities

    PubMed Central

    Sanjiv, Kumar; Hagenkort, Anna; Calderón-Montaño, José Manuel; Koolmeister, Tobias; Reaper, Philip M.; Mortusewicz, Oliver; Jacques, Sylvain A.; Kuiper, Raoul V.; Schultz, Niklas; Scobie, Martin; Charlton, Peter A.; Pollard, John R.; Berglund, Ulrika Warpman; Altun, Mikael; Helleday, Thomas

    2015-01-01

    Summary ATR and CHK1 maintain cancer cell survival under replication stress and inhibitors of both kinases are currently undergoing clinical trials. As ATR activity is increased after CHK1 inhibition, we hypothesized that this may indicate an increased reliance on ATR for survival. Indeed, we observe that replication stress induced by the CHK1 inhibitor AZD7762 results in replication catastrophe and apoptosis, when combined with the ATR inhibitor VE-821 specifically in cancer cells. Combined treatment with ATR and CHK1 inhibitors leads to replication fork arrest, ssDNA accumulation, replication collapse, and synergistic cell death in cancer cells in vitro and in vivo. Inhibition of CDK reversed replication stress and synthetic lethality, demonstrating that regulation of origin firing by ATR and CHK1 explains the synthetic lethality. In conclusion, this study exemplifies cancer-specific synthetic lethality between two proteins in the same pathway and raises the prospect of combining ATR and CHK1 inhibitors as promising cancer therapy. PMID:26748709

  17. A Catalytic DNA Activated by a Specific Strain of Bacterial Pathogen

    PubMed Central

    Shen, Zhifa; Wu, Zaisheng; Chang, Dingran; Zhang, Wenqing; Tram, Kha; Lee, Christine; Kim, Peter; Salena, Bruno J.

    2015-01-01

    Abstract Pathogenic strains of bacteria are known to cause various infectious diseases and there is a growing demand for molecular probes that can selectively recognize them. Here we report a special DNAzyme (catalytic DNA), RFD‐CD1, that shows exquisite specificity for a pathogenic strain of Clostridium difficile (C. difficile). RFD‐CD1 was derived by an in vitro selection approach where a random‐sequence DNA library was allowed to react with an unpurified molecular mixture derived from this strain of C. difficle, coupled with a subtractive selection strategy to eliminate cross‐reactivities to unintended C. difficile strains and other bacteria species. RFD‐CD1 is activated by a truncated version of TcdC, a transcription factor, that is unique to the targeted strain of C. difficle. Our study demonstrates for the first time that in vitro selection offers an effective approach for deriving functional nucleic acid probes that are capable of achieving strain‐specific recognition of bacterial pathogens. PMID:26676768

  18. Cancer-Specific Synthetic Lethality between ATR and CHK1 Kinase Activities.

    PubMed

    Sanjiv, Kumar; Hagenkort, Anna; Calderón-Montaño, José Manuel; Koolmeister, Tobias; Reaper, Philip M; Mortusewicz, Oliver; Jacques, Sylvain A; Kuiper, Raoul V; Schultz, Niklas; Scobie, Martin; Charlton, Peter A; Pollard, John R; Berglund, Ulrika Warpman; Altun, Mikael; Helleday, Thomas

    2016-01-12

    ATR and CHK1 maintain cancer cell survival under replication stress and inhibitors of both kinases are currently undergoing clinical trials. As ATR activity is increased after CHK1 inhibition, we hypothesized that this may indicate an increased reliance on ATR for survival. Indeed, we observe that replication stress induced by the CHK1 inhibitor AZD7762 results in replication catastrophe and apoptosis, when combined with the ATR inhibitor VE-821 specifically in cancer cells. Combined treatment with ATR and CHK1 inhibitors leads to replication fork arrest, ssDNA accumulation, replication collapse, and synergistic cell death in cancer cells in vitro and in vivo. Inhibition of CDK reversed replication stress and synthetic lethality, demonstrating that regulation of origin firing by ATR and CHK1 explains the synthetic lethality. In conclusion, this study exemplifies cancer-specific synthetic lethality between two proteins in the same pathway and raises the prospect of combining ATR and CHK1 inhibitors as promising cancer therapy. PMID:26748709

  19. Evolutionary Selection on Barrier Activity: Bar1 Is an Aspartyl Protease with Novel Substrate Specificity

    PubMed Central

    Jones, Stephen K.; Clarke, Starlynn C.; Craik, Charles S.

    2015-01-01

    ABSTRACT Peptide-based pheromones are used throughout the fungal kingdom for coordinating sexual responses between mating partners. Here, we address the properties and function of Bar1, an aspartyl protease that acts as a “barrier” and antagonist to pheromone signaling in multiple species. Candida albicans Bar1 was purified and shown to exhibit preferential cleavage of native α pheromone over pheromones from related fungal species. This result establishes that protease substrate specificity coevolved along with changes in its pheromone target. Pheromone cleavage by Bar1 occurred between residues Thr-5 and Asn-6 in the middle of the tridecapeptide sequence. Surprisingly, proteolytic activity was independent of the amino acid residues present at the scissile bond and instead relied on residues at the C terminus of α pheromone. Unlike most aspartyl proteases, Bar1 also exhibited a near-neutral pH optimum and was resistant to the class-wide inhibitor pepstatin A. In addition, genetic analysis was performed on C. albicans BAR1 and demonstrated that the protease not only regulates endogenous pheromone signaling but also can limit interspecies pheromone signaling. We discuss these findings and propose that the unusual substrate specificity of Bar1 is a consequence of its coevolution with the α pheromone receptor Ste2 for their shared peptide target. PMID:26604258

  20. A novel and essential mechanism determining specificity and activity of protein phosphatase 2A (PP2A) in vivo.

    PubMed

    Fellner, Thomas; Lackner, Daniel H; Hombauer, Hans; Piribauer, Patrick; Mudrak, Ingrid; Zaragoza, Katrin; Juno, Claudia; Ogris, Egon

    2003-09-01

    Protein phosphatase 2A (PP2A) is an essential intracellular serine/threonine phosphatase containing a catalytic subunit that possesses the potential to dephosphorylate promiscuously tyrosine-phosphorylated substrates in vitro. How PP2A acquires its intracellular specificity and activity for serine/threonine-phosphorylated substrates is unknown. Here we report a novel and phylogenetically conserved mechanism to generate active phospho-serine/threonine-specific PP2A in vivo. Phosphotyrosyl phosphatase activator (PTPA), a protein of so far unknown intracellular function, is required for the biogenesis of active and specific PP2A. Deletion of the yeast PTPA homologs generated a PP2A catalytic subunit with a conformation different from the wild-type enzyme, as indicated by its altered substrate specificity, reduced protein stability, and metal dependence. Complementation and RNA-interference experiments showed that PTPA fulfills an essential function conserved from yeast to man.

  1. Comparison of measured and calculated concrete and rebar specific activity during decommissioning of the Dalhousie SLOWPOKE-2 reactor.

    PubMed

    Smith, Philip G; Everall, Dave; Ariani, Imelda; Tsang, Kwok

    2013-07-01

    Following the defuelling and dismantling of the Dalhousie University SLOWPOKE-2 Reactor (DUSR) in 2011, the reactor pool concrete and rebar were sampled to support the unconditional free release of the material such that the facility could be classified for unrestricted use. A detailed MCNP5 model of the critical core assembly was simulated to calculate the thermal, intermediate and fast neutron flux profile below the reactor pool floor. The neutron fluxes were used to calculate the specific activity of significant radionuclides in the concrete and rebar. The calculated specific activity and consequently the calculated neutron fluxes were validated at a number of sample locations. The calculated concrete and rebar specific activity were found to be in good agreement with the measured specific activity at the sample locations. The unrestricted use of the facility was granted through the approval of the licence to abandon the facility in August 2011.

  2. Comparison of measured and calculated concrete and rebar specific activity during decommissioning of the Dalhousie SLOWPOKE-2 reactor.

    PubMed

    Smith, Philip G; Everall, Dave; Ariani, Imelda; Tsang, Kwok

    2013-07-01

    Following the defuelling and dismantling of the Dalhousie University SLOWPOKE-2 Reactor (DUSR) in 2011, the reactor pool concrete and rebar were sampled to support the unconditional free release of the material such that the facility could be classified for unrestricted use. A detailed MCNP5 model of the critical core assembly was simulated to calculate the thermal, intermediate and fast neutron flux profile below the reactor pool floor. The neutron fluxes were used to calculate the specific activity of significant radionuclides in the concrete and rebar. The calculated specific activity and consequently the calculated neutron fluxes were validated at a number of sample locations. The calculated concrete and rebar specific activity were found to be in good agreement with the measured specific activity at the sample locations. The unrestricted use of the facility was granted through the approval of the licence to abandon the facility in August 2011. PMID:23658212

  3. 15 CFR 922.105 - Prohibited or otherwise regulated activities-Unit-specific.

    Code of Federal Regulations, 2013 CFR

    2013-01-01

    ... and Foreign Trade (Continued) NATIONAL OCEANIC AND ATMOSPHERIC ADMINISTRATION, DEPARTMENT OF COMMERCE...) through (15) of § 922.103 do not apply to scientific exploration or research activities conducted by or... Wildlife Refuge: (i) Vessels engaged in scientific exploration or research activities conducted by or...

  4. 15 CFR 922.105 - Prohibited or otherwise regulated activities-Unit-specific.

    Code of Federal Regulations, 2014 CFR

    2014-01-01

    ... and Foreign Trade (Continued) NATIONAL OCEANIC AND ATMOSPHERIC ADMINISTRATION, DEPARTMENT OF COMMERCE...) through (15) of § 922.103 do not apply to scientific exploration or research activities conducted by or... Wildlife Refuge: (i) Vessels engaged in scientific exploration or research activities conducted by or...

  5. Functionally Specific Oscillatory Activity Correlates between Visual and Auditory Cortex in the Blind

    ERIC Educational Resources Information Center

    Schepers, Inga M.; Hipp, Joerg F.; Schneider, Till R.; Roder, Brigitte; Engel, Andreas K.

    2012-01-01

    Many studies have shown that the visual cortex of blind humans is activated in non-visual tasks. However, the electrophysiological signals underlying this cross-modal plasticity are largely unknown. Here, we characterize the neuronal population activity in the visual and auditory cortex of congenitally blind humans and sighted controls in a…

  6. Site-specific incorporation of redox active amino acids into proteins

    DOEpatents

    Alfonta, Lital; Schultz, Peter G.; Zhang, Zhiwen

    2009-02-24

    Compositions and methods of producing components of protein biosynthetic machinery that include orthogonal tRNAs, orthogonal aminoacyl-tRNA synthetases, and orthogonal pairs of tRNAs/synthetases, which incorporate redox active amino acids into proteins are provided. Methods for identifying these orthogonal pairs are also provided along with methods of producing proteins with redox active amino acids using these orthogonal pairs.

  7. Site-specific incorporation of redox active amino acids into proteins

    DOEpatents

    Alfonta; Lital , Schultz; Peter G. , Zhang; Zhiwen

    2010-10-12

    Compositions and methods of producing components of protein biosynthetic machinery that include orthogonal tRNAs, orthogonal aminoacyl-tRNA synthetases, and orthogonal pairs of tRNAs/synthetases, which incorporate redox active amino acids into proteins are provided. Methods for identifying these orthogonal pairs are also provided along with methods of producing proteins with redox active amino acids using these orthogonal pairs.

  8. DEVELOPMENTAL REGULATION OF PROTEIN KINASE B ACTIVATION IS ISOFORM SPECIFIC IN SKELETAL MUSCLE OF NEONATAL PIGS

    Technology Transfer Automated Retrieval System (TEKTRAN)

    The postprandial activation of the insulin signaling pathway that leads to translation initiation is enhanced in skeletal muscle of the neonate and decreases with development in parallel with the developmental decline in muscle protein synthesis. Our previous study showed that the activity of protei...

  9. Site-specific incorporation of redox active amino acids into proteins

    SciTech Connect

    Alfonta, Lital; Schultz, Peter G.; Zhang, Zhiwen

    2012-02-14

    Compositions and methods of producing components of protein biosynthetic machinery that include orthogonal tRNAs, orthogonal aminoacyl-tRNA synthetases, and orthogonal pairs of tRNAs/synthetases, which incorporate redox active amino acids into proteins are provided. Methods for identifying these orthogonal pairs are also provided along with methods of producing proteins with redox active amino acids using these orthogonal pairs.

  10. Site-specific incorporation of redox active amino acids into proteins

    SciTech Connect

    Alfonta, Lital; Schultz, Peter G.; Zhang, Zhiwen

    2011-08-30

    Compositions and methods of producing components of protein biosynthetic machinery that include orthogonal tRNAs, orthogonal aminoacyl-tRNA synthetases, and orthogonal pairs of tRNAs/synthetases, which incorporate redox active amino acids into proteins are provided. Methods for identifying these orthogonal pairs are also provided along with methods of producing proteins with redox active amino acids using these orthogonal pairs.

  11. Oocyte-specific deletion of Pten causes premature activation of the primordial follicle pool.

    PubMed

    Reddy, Pradeep; Liu, Lian; Adhikari, Deepak; Jagarlamudi, Krishna; Rajareddy, Singareddy; Shen, Yan; Du, Chun; Tang, Wenli; Hämäläinen, Tuula; Peng, Stanford L; Lan, Zi-Jian; Cooney, Austin J; Huhtaniemi, Ilpo; Liu, Kui

    2008-02-01

    In the mammalian ovary, progressive activation of primordial follicles from the dormant pool serves as the source of fertilizable ova. Menopause, or the end of female reproductive life, occurs when the primordial follicle pool is exhausted. However, the molecular mechanisms underlying follicle activation are poorly understood. We provide genetic evidence that in mice lacking PTEN (phosphatase and tensin homolog deleted on chromosome 10) in oocytes, a major negative regulator of phosphatidylinositol 3-kinase (PI3K), the entire primordial follicle pool becomes activated. Subsequently, all primordial follicles become depleted in early adulthood, causing premature ovarian failure (POF). Our results show that the mammalian oocyte serves as the headquarters of programming of follicle activation and that the oocyte PTEN-PI3K pathway governs follicle activation through control of initiation of oocyte growth.

  12. Context-dependent activation of reduced autobiographical memory specificity as an avoidant coping style.

    PubMed

    Debeer, Elise; Raes, Filip; Williams, J Mark G; Hermans, Dirk

    2011-12-01

    According to the affect-regulation hypothesis (Williams et al., 2007), reduced autobiographical memory specificity (rAMS) or overgeneral memory (OGM) might be considered a cognitive avoidance strategy; that is, people learn to avoid the emotionally painful consequences associated with the retrieval of specific negative memories. Based on this hypothesis, one would predict significant negative associations between AMS and avoidant coping. However, studies investigating this prediction have led to equivocal results. In the present study we tested a possible explanation for these contradictory findings. It was hypothesized that rAMS (in part) reflects an avoidant coping strategy, which might only become apparent under certain conditions, that is, conditions that signal the possibility of 'danger.' To test this hypothesis, we assessed AMS and behavioral avoidance but experimentally manipulated the instructions. In the neutral condition, two parallel versions of the Autobiographical Memory Test (AMT) were presented under neutral instructions. In the threat condition, the first AMT was presented under neutral instructions, while the second AMT was presented under 'threat instructions.' Results showed no significant correlations between avoidance and OGM under neutral conditions but significant and markedly stronger correlations under threat conditions, with more avoidance being associated with fewer specific and more categoric memories. In addition, high avoiders showed a stronger reduction in AMS in the threat condition as compared with the neutral condition, while low avoiders showed no such difference between conditions. The data confirm that OGM can be considered as part of a broader avoidant coping style. However, more importantly, they show that, at least in nonclinical individuals, the activation of this coping style may depend on the context.

  13. A novel carotenoid cleavage activity involved in the biosynthesis of Citrus fruit-specific apocarotenoid pigments.

    PubMed

    Rodrigo, María J; Alquézar, Berta; Alós, Enriqueta; Medina, Víctor; Carmona, Lourdes; Bruno, Mark; Al-Babili, Salim; Zacarías, Lorenzo

    2013-11-01

    Citrus is the first tree crop in terms of fruit production. The colour of Citrus fruit is one of the main quality attributes, caused by the accumulation of carotenoids and their derivative C30 apocarotenoids, mainly β-citraurin (3-hydroxy-β-apo-8'-carotenal), which provide an attractive orange-reddish tint to the peel of oranges and Mandarins. Though carotenoid biosynthesis and its regulation have been extensively studied in Citrus fruits, little is known about the formation of C30 apocarotenoids. The aim of this study was to the identify carotenoid cleavage enzyme(s) [CCD(s)] involved in the peel-specific C30 apocarotenoids. In silico data mining revealed a new family of five CCD4-type genes in Citrus. One gene of this family, CCD4b1, was expressed in reproductive and vegetative tissues of different Citrus species in a pattern correlating with the accumulation of C30 apocarotenoids. Moreover, developmental processes and treatments which alter Citrus fruit peel pigmentation led to changes of β-citraurin content and CCD4b1 transcript levels. These results point to the involvement of CCD4b1 in β-citraurin formation and indicate that the accumulation of this compound is determined by the availability of the presumed precursors zeaxanthin and β-cryptoxanthin. Functional analysis of CCD4b1 by in vitro assays unequivocally demonstrated the asymmetric cleavage activity at the 7',8' double bond in zeaxanthin and β-cryptoxanthin, confirming its role in C30 apocarotenoid biosynthesis. Thus, a novel plant carotenoid cleavage activity targeting the 7',8' double bond of cyclic C40 carotenoids has been identified. These results suggest that the presented enzyme is responsible for the biosynthesis of C30 apocarotenoids in Citrus which are key pigments in fruit coloration.

  14. Transferrin Binding to Peripheral Blood Lymphocytes Activated by Phytohemagglutinin Involves a Specific Receptor

    PubMed Central

    Galbraith, Robert M.; Werner, Phillip; Arnaud, Philippe; Galbraith, Gillian M. P.

    1980-01-01

    Immunohistological studies have indicated that membrane sites binding transferrin are present upon activated human peripheral blood lymphocytes. In this study, we have investigated transferrin uptake in human lymphocytes exposed to phytohemagglutinin (PHA), by quantitative radiobinding and immunofluorescence in parallel. In stimulated lymphocytes, binding was maximal after a 30-min incubation, being greatest at 37°C, and greater at 22°C than at 4°C. Although some shedding and endocytosis of transferrin occurred at 22° and 37°C, these factors, and resulting synthesis of new sites, did not affect measurement of binding which was found to be saturable, reversible, and specific for transferrin (Ka 0.5-2.5 × 108 M−1). Binding was greater after a 48-h exposure to PHA than after 24 h, and was maximal at 66 h. Sequential Scatchard analysis revealed no significant elevation in affinity of interaction. However, although the total number of receptors increased, the proportion of cells in which binding of ligand was detected immunohistologically increased in parallel, and after appropriate correction, the cellular density of receptors remained relatively constant throughout (60,000-80,000 sites/cell). Increments in binding during the culture period were thus due predominantly to expansion of a population of cells bearing receptors. Similar differences in binding were apparent upon comparison of cells cultured in different doses of PHA, and in unstimulated cells binding was negligible. Transferrin receptors appear, therefore, to be readily detectable only upon lymphocytes that have been activated. Images PMID:6253523

  15. A novel carotenoid cleavage activity involved in the biosynthesis of Citrus fruit-specific apocarotenoid pigments

    PubMed Central

    Rodrigo, María J.; Alquézar, Berta; Al-Babili, Salim

    2013-01-01

    Citrus is the first tree crop in terms of fruit production. The colour of Citrus fruit is one of the main quality attributes, caused by the accumulation of carotenoids and their derivative C30 apocarotenoids, mainly β-citraurin (3-hydroxy-β-apo-8′-carotenal), which provide an attractive orange-reddish tint to the peel of oranges and mandarins. Though carotenoid biosynthesis and its regulation have been extensively studied in Citrus fruits, little is known about the formation of C30 apocarotenoids. The aim of this study was to the identify carotenoid cleavage enzyme(s) [CCD(s)] involved in the peel-specific C30 apocarotenoids. In silico data mining revealed a new family of five CCD4-type genes in Citrus. One gene of this family, CCD4b1, was expressed in reproductive and vegetative tissues of different Citrus species in a pattern correlating with the accumulation of C30 apocarotenoids. Moreover, developmental processes and treatments which alter Citrus fruit peel pigmentation led to changes of β-citraurin content and CCD4b1 transcript levels. These results point to the involvement of CCD4b1 in β-citraurin formation and indicate that the accumulation of this compound is determined by the availability of the presumed precursors zeaxanthin and β-cryptoxanthin. Functional analysis of CCD4b1 by in vitro assays unequivocally demonstrated the asymmetric cleavage activity at the 7′,8′ double bond in zeaxanthin and β-cryptoxanthin, confirming its role in C30 apocarotenoid biosynthesis. Thus, a novel plant carotenoid cleavage activity targeting the 7′,8′ double bond of cyclic C40 carotenoids has been identified. These results suggest that the presented enzyme is responsible for the biosynthesis of C30 apocarotenoids in Citrus which are key pigments in fruit coloration. PMID:24006419

  16. Human Serum-Specific Activation of Alternative Sigma Factors, the Stress Responders in Aggregatibacter actinomycetemcomitans

    PubMed Central

    Tang-Siegel, Gaoyan; Bumgarner, Roger; Ruiz, Teresa; Kittichotirat, Weerayuth; Chen, Weizhen; Chen, Casey

    2016-01-01

    Aggregatibacter actinomycetemcomitans, a known pathogen causing periodontal disease and infective endocarditis, is a survivor in the periodontal pocket and blood stream; both environments contain serum as a nutrient source. To screen for unknown virulence factors associated with this microorganism, A. actinomycetemcomitans was grown in serum-based media to simulate its in vivo environment. Different strains of A. actinomycetemcomitans showed distinct growth phenotypes only in the presence of human serum, and they were grouped into high- and low-responder groups. High-responders comprised mainly serotype c strains, and showed an unusual growth phenomenon, featuring a second, rapid increase in turbidity after 9-h incubation that reached a final optical density 2- to 7-fold higher than low-responders. Upon further investigation, the second increase in turbidity was not caused by cell multiplication, but by cell death. Whole transcriptomic analysis via RNA-seq identified 35 genes that were up-regulated by human serum, but not horse serum, in high-responders but not in low-responders, including prominently an alternative sigma factor rpoE (σE). A lacZ reporter construct driven by the 132-bp rpoE promoter sequence of A. actinomycetemcomitans responded dramatically to human serum within 90 min of incubation only when the construct was carried by a high responder strain. The rpoE promoter is 100% identical among high- and low-responder strains. Proteomic investigation showed potential interactions between human serum protein, e.g. apolipoprotein A1 (ApoA1) and A. actinomycetemcomitans. The data clearly indicated a different activation process for rpoE in high- versus low-responder strains. This differential human serum-specific activation of rpoE, a putative extra-cytoplasmic stress responder and global regulator, suggests distinct in vivo adaptations among different strains of A. actinomycetemcomitans. PMID:27490177

  17. Expression of Aspergillus nidulans phy Gene in Nicotiana benthamiana Produces Active Phytase with Broad Specificities

    PubMed Central

    Oh, Tae-Kyun; Oh, Sung; Kim, Seongdae; Park, Jae Sung; Vinod, Nagarajan; Jang, Kyung Min; Kim, Sei Chang; Choi, Chang Won; Ko, Suk-Min; Jeong, Dong Kee; Udayakumar, Rajangam

    2014-01-01

    A full-length phytase gene (phy) of Aspergillus nidulans was amplified from the cDNA library by polymerase chain reaction (PCR), and it was introduced into a bacterial expression vector, pET-28a. The recombinant protein (rPhy-E, 56 kDa) was overexpressed in the insoluble fraction of Escherichia coli culture, purified by Ni-NTA resin under denaturing conditions and injected into rats as an immunogen. To express A. nidulans phytase in a plant, the full-length of phy was cloned into a plant expression binary vector, pPZP212. The resultant construct was tested for its transient expression by Agrobacterium-infiltration into Nicotiana benthamiana leaves. Compared with a control, the agro-infiltrated leaf tissues showed the presence of phy mRNA and its high expression level in N. benthamiana. The recombinant phytase (rPhy-P, 62 kDa) was strongly reacted with the polyclonal antibody against the nonglycosylated rPhy-E. The rPhy-P showed glycosylation, two pH optima (pH 4.5 and pH 5.5), an optimum temperature at 45~55 °C, thermostability and broad substrate specificities. After deglycosylation by peptide-N-glycosidase F (PNGase-F), the rPhy-P significantly lost the phytase activity and retained 1/9 of the original activity after 10 min of incubation at 45 °C. Therefore, the deglycosylation caused a significant reduction in enzyme thermostability. In animal experiments, oral administration of the rPhy-P at 1500 U/kg body weight/day for seven days caused a significant reduction of phosphorus excretion by 16% in rat feces. Besides, the rPhy-P did not result in any toxicological changes and clinical signs. PMID:25192284

  18. Transplant experiments uncover Baltic Sea basin-specific responses in bacterioplankton community composition and metabolic activities.

    PubMed

    Lindh, Markus V; Figueroa, Daniela; Sjöstedt, Johanna; Baltar, Federico; Lundin, Daniel; Andersson, Agneta; Legrand, Catherine; Pinhassi, Jarone

    2015-01-01

    Anthropogenically induced changes in precipitation are projected to generate increased river runoff to semi-enclosed seas, increasing loads of terrestrial dissolved organic matter and decreasing salinity. To determine how bacterial community structure and functioning adjust to such changes, we designed microcosm transplant experiments with Baltic Proper (salinity 7.2) and Bothnian Sea (salinity 3.6) water. Baltic Proper bacteria generally reached higher abundances than Bothnian Sea bacteria in both Baltic Proper and Bothnian Sea water, indicating higher adaptability. Moreover, Baltic Proper bacteria growing in Bothnian Sea water consistently showed highest bacterial production and beta-glucosidase activity. These metabolic responses were accompanied by basin-specific changes in bacterial community structure. For example, Baltic Proper Pseudomonas and Limnobacter populations increased markedly in relative abundance in Bothnian Sea water, indicating a replacement effect. In contrast, Roseobacter and Rheinheimera populations were stable or increased in abundance when challenged by either of the waters, indicating an adjustment effect. Transplants to Bothnian Sea water triggered the initial emergence of particular Burkholderiaceae populations, and transplants to Baltic Proper water triggered Alteromonadaceae populations. Notably, in the subsequent re-transplant experiment, a priming effect resulted in further increases to dominance of these populations. Correlated changes in community composition and metabolic activity were observed only in the transplant experiment and only at relatively high phylogenetic resolution. This suggested an importance of successional progression for interpreting relationships between bacterial community composition and functioning. We infer that priming effects on bacterial community structure by natural episodic events or climate change induced forcing could translate into long-term changes in bacterial ecosystem process rates. PMID

  19. Tumor-Specific Peptide, Selected from a Phage Peptide Library, Enhances Antitumor Activity of Lactaptin

    PubMed Central

    Makartsova, Anna A.; Fomin, Alexandr S.; Nushtaeva, Anna A.; Koval, Olga A.

    2016-01-01

    A recombinant analogue of lactaptin (RL2), a new potential anticancer molecule, induces apoptosis in cultured tumor cells. The tumor suppression efficacy of RL2 was shown against mouse hepatoma-1 cells and MDA-MB-231 human breast adenocarcinoma cells. The RL2-based therapeutic drug lactaptin is distributed evenly throughout the organism, which reduces its antitumor efficacy. In the current study, we obtained a genetic construct that allows production of the recombinant fusion protein T3-RL2, consisting of RL2 and T3 peptide (YTYDPWLIFPAN), in E. coli cells. T3 peptide was selected from a phage peptide library as a result of two screenings: in vitro using MDA-MB-231 cell culture and in vivo using a mouse xenograft model of breast cancer MDA-MB-231. It was shown that the displayed peptide T3 provides binding and internalization of phage particles by MDA-MB-231 cells and their specific accumulation in MDA-MB-231 tumor tissue. In addition, based on the nucleotide sequences coding RL2 and the known tumor-targeting peptide iRGD, we obtained genetic constructs that provide synthesis of fusion proteins RL2-iRGD and RL-iRGD-His. We studied the cytotoxic activity of fusion proteins T3-RL2, RL2-iRGD and RL-iRGD-His in vitro using MDA-MB-231 and MCF-7 human adenocarcinoma cells. The in vitro results showed that the fusion proteins inhibit proliferation of both cell cultures, and their cytotoxic activity is higher than that of RL2. In vivo experiments on the study of the antitumor efficacy of the obtained fusion proteins demonstrated that T3-RL2 protein significantly inhibits MDA-MB-231 tumor growth in a xenograft model compared with RL2, while the antitumor effect of RL2-iRGD and RL-iRGD-His proteins is comparable to the effect of RL2. PMID:27513518

  20. Human Serum-Specific Activation of Alternative Sigma Factors, the Stress Responders in Aggregatibacter actinomycetemcomitans.

    PubMed

    Tang-Siegel, Gaoyan; Bumgarner, Roger; Ruiz, Teresa; Kittichotirat, Weerayuth; Chen, Weizhen; Chen, Casey

    2016-01-01

    Aggregatibacter actinomycetemcomitans, a known pathogen causing periodontal disease and infective endocarditis, is a survivor in the periodontal pocket and blood stream; both environments contain serum as a nutrient source. To screen for unknown virulence factors associated with this microorganism, A. actinomycetemcomitans was grown in serum-based media to simulate its in vivo environment. Different strains of A. actinomycetemcomitans showed distinct growth phenotypes only in the presence of human serum, and they were grouped into high- and low-responder groups. High-responders comprised mainly serotype c strains, and showed an unusual growth phenomenon, featuring a second, rapid increase in turbidity after 9-h incubation that reached a final optical density 2- to 7-fold higher than low-responders. Upon further investigation, the second increase in turbidity was not caused by cell multiplication, but by cell death. Whole transcriptomic analysis via RNA-seq identified 35 genes that were up-regulated by human serum, but not horse serum, in high-responders but not in low-responders, including prominently an alternative sigma factor rpoE (σE). A lacZ reporter construct driven by the 132-bp rpoE promoter sequence of A. actinomycetemcomitans responded dramatically to human serum within 90 min of incubation only when the construct was carried by a high responder strain. The rpoE promoter is 100% identical among high- and low-responder strains. Proteomic investigation showed potential interactions between human serum protein, e.g. apolipoprotein A1 (ApoA1) and A. actinomycetemcomitans. The data clearly indicated a different activation process for rpoE in high- versus low-responder strains. This differential human serum-specific activation of rpoE, a putative extra-cytoplasmic stress responder and global regulator, suggests distinct in vivo adaptations among different strains of A. actinomycetemcomitans. PMID:27490177

  1. Tumor-Specific Peptide, Selected from a Phage Peptide Library, Enhances Antitumor Activity of Lactaptin.

    PubMed

    Nemudraya, Anna A; Makartsova, Anna A; Fomin, Alexandr S; Nushtaeva, Anna A; Koval, Olga A; Richter, Vladimir A; Kuligina, Elena V

    2016-01-01

    A recombinant analogue of lactaptin (RL2), a new potential anticancer molecule, induces apoptosis in cultured tumor cells. The tumor suppression efficacy of RL2 was shown against mouse hepatoma-1 cells and MDA-MB-231 human breast adenocarcinoma cells. The RL2-based therapeutic drug lactaptin is distributed evenly throughout the organism, which reduces its antitumor efficacy. In the current study, we obtained a genetic construct that allows production of the recombinant fusion protein T3-RL2, consisting of RL2 and T3 peptide (YTYDPWLIFPAN), in E. coli cells. T3 peptide was selected from a phage peptide library as a result of two screenings: in vitro using MDA-MB-231 cell culture and in vivo using a mouse xenograft model of breast cancer MDA-MB-231. It was shown that the displayed peptide T3 provides binding and internalization of phage particles by MDA-MB-231 cells and their specific accumulation in MDA-MB-231 tumor tissue. In addition, based on the nucleotide sequences coding RL2 and the known tumor-targeting peptide iRGD, we obtained genetic constructs that provide synthesis of fusion proteins RL2-iRGD and RL-iRGD-His. We studied the cytotoxic activity of fusion proteins T3-RL2, RL2-iRGD and RL-iRGD-His in vitro using MDA-MB-231 and MCF-7 human adenocarcinoma cells. The in vitro results showed that the fusion proteins inhibit proliferation of both cell cultures, and their cytotoxic activity is higher than that of RL2. In vivo experiments on the study of the antitumor efficacy of the obtained fusion proteins demonstrated that T3-RL2 protein significantly inhibits MDA-MB-231 tumor growth in a xenograft model compared with RL2, while the antitumor effect of RL2-iRGD and RL-iRGD-His proteins is comparable to the effect of RL2. PMID:27513518

  2. Pyridinone derivatives: specific human immunodeficiency virus type 1 reverse transcriptase inhibitors with antiviral activity.

    PubMed Central

    Goldman, M E; Nunberg, J H; O'Brien, J A; Quintero, J C; Schleif, W A; Freund, K F; Gaul, S L; Saari, W S; Wai, J S; Hoffman, J M

    1991-01-01

    Derivatives of pyridinones were found to inhibit human immunodeficiency virus type 1 (HIV-1) reverse transcriptase (RT) activity and prevent the spread of HIV-1 infection in cell culture without an appreciable effect on other retroviral or cellular polymerases. 3-[( (4,7-Dimethyl-1,3-benzoxazol-2-yl) methyl]amino ]-5-ethyl-6-methylpyridin-2(1H)-one (L-697,639) and 3-[[ (4,7-dichloro-1,3-benzoxazol-2-yl) methyl]amino]-5-ethyl-6-methylpyridin-2(1H)-one (L-697,661), two compounds within this series, had HIV-1 RT IC50 values in the range of 20-800 nM, depending upon the template-primer used. The most potent inhibition was obtained with rC.dG and dA.dT as template--primers. With rC.dG, reversible slow-binding non-competitive inhibition was observed. [3H]L-697,639 bound preferentially to enzyme-template-primer complexes. This binding was magnesium-dependent and saturable with a stoichiometry of 1 mol of [3H]L-697,639 per mol of RT heterodimer. Displacement of [3H]L-697,639 was seen with phosphonoformate. In human T-lymphoid-cell culture, L-697,639 and L-697,661 inhibited the spread of HIV-1 infection by at least 95% at concentrations of 12-200 nM. Synergism between 3'-azido-3'-deoxythymidine or dideoxyinosine and either of these compounds was also demonstrated in cell culture. Based upon their specificity for HIV-1 RT activity, template-primer dependence on potency and ability to displace [3H]L-697,639; a tetrahydroimidazo [4,5,1-jk] [1,4]-benzodiazepin-2(1H)-thione derivative R82150 and the dipyridodiazepinone BI-RG-587 appear to inhibit RT activity by the same mechanism as the pyridinones. PMID:1713693

  3. Preparation of high specific activity (86)Y using a small biomedical cyclotron.

    PubMed

    Yoo, Jeongsoo; Tang, Lucie; Perkins, Todd A; Rowland, Douglas J; Laforest, Richard; Lewis, Jason S; Welch, Michael J

    2005-11-01

    86Y is an attractive PET radionuclide due to its intermediate half-life. (86)Y was produced via the 86Sr(p,n)86Y nuclear reaction. Enriched SrCO3 or SrO was irradiated with 2-6 microA of beam current for <4 h on a CS-15 cyclotron. It was shown that the SrO target could withstand at least 6 microA of beam current, a significant improvement over a maximum of 2 microA on the SrCO3 target. Average yields of 4.5 mCi/microA.h were achieved with SrO, which represent 71% of the theoretical yield, compared to 2.3 mCi/microA.h with SrCO3. The radioisotopic contaminants were (86m)Y (220%), 87Y (0.27%), (87m)Y (0.43%) and 88Y (0.024%). 86Y was isolated in an electrochemical cell consisting of three Pt electrodes. The solution was electrolyzed at 2000 mA (40 min) using two Pt plate electrodes. A second electrolysis (230 mA for 20 min) was performed using one Pt plate and a Pt wire. On average, 97.1% of the 86Y was recollected on the Pt wire after a second electrolysis. The (86)Y was collected from the Pt wire using 2.8 M HNO3/EtOH (3:1). After evaporation, 86Y was reconstituted in 100 microl of 0.1 M HCl. Target materials were recovered as SrCO3 and then converted to SrO by thermal decomposition at 1150 degrees C. Specific activity of 86Y was determined to be 29+/-19 mCi/microg via titration of 86Y(OAc)3 with DOTA or DTPA. We have established techniques for the routine, economical production of high purity, high specific activity 86Y on a small biomedical cyclotron that are translatable to other institutions.

  4. Production of radiohalogens and [11C]-methane at high specific activity

    NASA Astrophysics Data System (ADS)

    Nye, Jonathon Andrew

    2005-07-01

    The halogens, occupying Group VII of the periodic table, play an important role in the biochemical processes underlying health and disease. A variety of positron emitters covering a broad range of half-lives permit the imaging of the body's physiochemical behavior using PET. Neutron deficient isotopes of the halogen group can be produced by (p,n) reactions from enriched targets with low energy (<13MeV) biomedical cyclotrons. These cyclotrons are distributed relatively evenly throughout the United States at research institutions and commercial distribution sites (i.e., 100+ CTI RDS 11MeV proton cyclotrons). However, these sites concentrate on the core group of positron emitters: 15O, 13N, 11C, and primarily 18F-fluoride. The simplicity of the production process insures their role in the clinical/research environment, labeling H215 O, 13NH3, CH3-compounds and 18F-FDG. Halogens with half-lives longer than 18F have been avoided due to a combination of several factors, such as complexity of the target systems, expense of the enriched substrate, low reaction yields, and extensive post-processing to reclaim the target material. PET research over the last decade has forced a match between drug development and emerging small animal instrumentation, shifting focus to agents labeled with high specific activity 11CH3I and the long-lived radiohalogens, 76Br and 124I. A steady local supply of 18F-fluoride, 11C-methane, 76B-bromide, and 124I-iodide is essential to seize today's research opportunities or for limited distribution outside of our local area. To keep pace, new targetry developments are implemented to reliably produce these isotopes on a batch basis. The research presented details improvements on existing production methods for 18F-fluoride intended for nucleophilic substitution and high specific activity 11C-methane (→CH3I) for the N-methylation of a half-dozen neuroligands. A significant effort is placed on the novel use of low energy cyclotrons for the production

  5. Efficient production of high specific activity 64Cu using a biomedical cyclotron.

    PubMed

    McCarthy, D W; Shefer, R E; Klinkowstein, R E; Bass, L A; Margeneau, W H; Cutler, C S; Anderson, C J; Welch, M J

    1997-01-01

    Copper-64 (T 1/2 = 12.7 h) is an intermediate-lived positron-emitting radionuclide that is a useful radiotracer for positron emission tomography (PET) as well as a promising radiotherapy agent for the treatment for cancer. Currently, copper-64 suitable for biomedical studies is produced in the fast neutron flux trap (irradiation of zinc with fast neutrons) at the Missouri University Research Reactor. Access to the fast neutron flux trap is only possible on a weekly basis, making the availability of this tracer very limited. In order to significantly increase the availability of this intermediate-lived radiotracer, we have investigated and developed a method for the efficient production of high specific activity Cu-64 using a small biomedical cyclotron. It has been suggested that it may be possible to produce Cu-64 on a small biomedical cyclotron utilizing the 64Ni(p,n)64Cu nuclear reaction. We have irradiated both natural nickel and enriched (95% and 98%) Ni-64 plated on gold disks. Nickel has been electroplated successfully at thicknesses of approximately 20-300 mm and bombarded with proton currents of 15-45 microA. A special water-cooled target had been designed to facilitate the irradiations on a biomedical cyclotron up to 60 microA. We have shown that it is possible to separate Cu-64 from Ni-64 and other reaction byproducts rapidly and efficiently by using ion exchange chromatography. Production runs using 19-55 mg of 95% enriched Ni-64 have yielded 150-600 mCi of Cu-64 (2.3-5.0 mCi/microAh) with specific activities of 94-310 mci/microgram Cu. The cyclotron produced Cu-64 had been used to radiolabel PTSM [pyruvaldehyde bis-(N4-methylthiosemicarbazone), used to quantify myocardial, cerebral, renal, and tumor blood flow], MAb 1A3 [monoclonal antibody MAb to colon cancer], and octreotide. A recycling technique for the costly Ni-64 target material has been developed. This technique allows the nickel eluted off the column to be recovered and reused in the

  6. A new easy method for specific measurement of active myeloperoxidase in human biological fluids and tissue extracts.

    PubMed

    Franck, T; Kohnen, S; Boudjeltia, K Zouaoui; Van Antwerpen, P; Bosseloir, A; Niesten, A; Gach, O; Nys, M; Deby-Dupont, G; Serteyn, D

    2009-12-15

    The SIEFED ("Specific Immunological Extraction Followed by Enzymatic Detection") method already developed for the specific detection of the activity of equine myeloperoxidase (MPO) was adapted for the specific measurement of active human MPO in biological fluids or tissue extracts. The method consists of the extraction of MPO from aqueous solutions by immobilized anti-MPO antibodies followed by a washing (to eliminate the extraction medium and the biological fluid with their possible interfering molecules) and the measurement of the activity of MPO with a detection system containing a fluorogenic substrate, H(2)O(2) and nitrite ions as reaction enhancer. The SIEFED was applied to study active MPO in human biological fluids (plasma, bronchoalveolar lavage fluid and supernatant from carotids extracts). The SIEFED for human MPO has a sensitivity limit of 0.080 mU/mL and showed good precision with intra- and inter-assay coefficients of variation below 10 and 20% respectively within a broad range of MPO activities establish from 0.156 to 473 mU/mL. The SIEFED for human MPO will be useful for the specific detection of active MPO in complex fluids and can be complementary to an ELISA to determine an active/total MPO ratio in healthy volunteers and patients especially in case of chronic or acute inflammatory diseases.

  7. A specific CD4 epitope bound by tregalizumab mediates activation of regulatory T cells by a unique signaling pathway.

    PubMed

    Helling, Bianca; König, Martin; Dälken, Benjamin; Engling, Andre; Krömer, Wolfgang; Heim, Katharina; Wallmeier, Holger; Haas, Jürgen; Wildemann, Brigitte; Fritz, Brigitte; Jonuleit, Helmut; Kubach, Jan; Dingermann, Theodor; Radeke, Heinfried H; Osterroth, Frank; Uherek, Christoph; Czeloth, Niklas; Schüttrumpf, Jörg

    2015-04-01

    CD4(+)CD25(+) regulatory T cells (Tregs) represent a specialized subpopulation of T cells, which are essential for maintaining peripheral tolerance and preventing autoimmunity. The immunomodulatory effects of Tregs depend on their activation status. Here we show that, in contrast to conventional anti-CD4 monoclonal antibodies (mAbs), the humanized CD4-specific monoclonal antibody tregalizumab (BT-061) is able to selectively activate the suppressive properties of Tregs in vitro. BT-061 activates Tregs by binding to CD4 and activation of signaling downstream pathways. The specific functionality of BT-061 may be explained by the recognition of a unique, conformational epitope on domain 2 of the CD4 molecule that is not recognized by other anti-CD4 mAbs. We found that, due to this special epitope binding, BT-061 induces a unique phosphorylation of T-cell receptor complex-associated signaling molecules. This is sufficient to activate the function of Tregs without activating effector T cells. Furthermore, BT-061 does not induce the release of pro-inflammatory cytokines. These results demonstrate that BT-061 stimulation via the CD4 receptor is able to induce T-cell receptor-independent activation of Tregs. Selective activation of Tregs via CD4 is a promising approach for the treatment of autoimmune diseases where insufficient Treg activity has been described. Clinical investigation of this new approach is currently ongoing. PMID:25512343

  8. Anti-human cytomegalovirus activity of cytokines produced by CD4+ T-cell clones specifically activated by IE1 peptides in vitro.

    PubMed Central

    Davignon, J L; Castanié, P; Yorke, J A; Gautier, N; Clément, D; Davrinche, C

    1996-01-01

    The control of latent cytomegalovirus (CMV) infections by the immune system is poorly understood. We have previously shown that CD4+ T cells specific for the human CMV major regulatory protein IE1 are frequent in latently infected healthy blood donors. In order to learn about the possible role of these cells, we have developed IE1-specific CD4+ T-cell clones and, in this study, analyzed their epitope specificity and function in vitro. We measured their cytokine production when stimulated with specific IE1 peptides or whole recombinant IE1 protein. Their cytokine profiles, as deduced from gamma interferon (IFN-gamma), tumor necrosis factor alpha (TNF-alpha), and interleukin-4 (IL-4) and IL-6 production, were of the Th0- and Th1-like phenotypes. Supernatants from IE1-specific clones producing IFN-gamma and TNF-alpha were shown to inhibit CMV replication in U373 MG cells. This effect was due, as found by using cytokine-specific neutralizing antibodies, mostly to IFN-gamma, which was secreted at higher levels than TNF-alpha. To better assess the anti-CMV activity of cytokines, recombinant IFN-gamma and TNF-alpha were used and shown to have a synergistic effect on the inhibition of CMV replication and protein expression. Thus, IE1-specific CD4+ T cells display in vitro anti-CMV activity through cytokine secretion and may play a role in the control of in vivo latent infections. PMID:8642638

  9. Organelle-Specific Activity-Based Protein Profiling in Living Cells

    SciTech Connect

    Wiedner, Susan D.; Anderson, Lindsey N.; Sadler, Natalie C.; Chrisler, William B.; Kodali, Vamsi K.; Smith, Richard D.; Wright, Aaron T.

    2014-02-06

    A multimodal acidic organelle targeting activity-based probe was developed for analysis of subcellular native enzymatic activity of cells by fluorescent microscopy and mass spectrometry. A cathepsin reactive warhead was conjugated to an acidotropic amine, and a clickable alkyne for appendage of AlexaFluor 488 or biotin reporter tags. This probe accumulated in punctate vesicles surrounded by LAMP1, a lysosome marker, as observed by Structured Illumination Microscopy (SIM) in J774 mouse macrophage cells. Biotin conjugation, affinity purification, and analysis of in vivo labeled J774 by mass spectrometry showed that the probe was very selective for Cathepsins B and Z, two lysosomal cysteine proteases. Analysis of starvation induced autophagy, which is an increase in cell component catabolism involving lysosomes, showed a large increase in tagged protein number and an increase in cathepsin activity. Organelle targeting activity-based probes and subsequent analysis of resident proteins by mass spectrometry is enabled by tuning the physicochemical properties of the probe.

  10. Lumican Inhibits SNAIL-Induced Melanoma Cell Migration Specifically by Blocking MMP-14 Activity.

    PubMed

    Stasiak, Marta; Boncela, Joanna; Perreau, Corinne; Karamanou, Konstantina; Chatron-Colliet, Aurore; Proult, Isabelle; Przygodzka, Patrycja; Chakravarti, Shukti; Maquart, François-Xavier; Kowalska, M Anna; Wegrowski, Yanusz; Brézillon, Stéphane

    2016-01-01

    Lumican, a small leucine rich proteoglycan, inhibits MMP-14 activity and melanoma cell migration in vitro and in vivo. Snail triggers epithelial-mesenchymal transitions endowing epithelial cells with migratory and invasive properties during tumor progression. The aim of this work was to investigate lumican effects on MMP-14 activity and migration of Snail overexpressing B16F1 (Snail-B16F1) melanoma cells and HT-29 colon adenocarcinoma cells. Lumican inhibits the Snail induced MMP-14 activity in B16F1 but not in HT-29 cells. In Snail-B16F1 cells, lumican inhibits migration, growth, and melanoma primary tumor development. A lumican-based strategy targeting Snail-induced MMP-14 activity might be useful for melanoma treatment. PMID:26930497

  11. Prefrontal set activity predicts rule-specific neural processing during subsequent cognitive performance.

    PubMed

    Sakai, Katsuyuki; Passingham, Richard E

    2006-01-25

    Prefrontal neurons have been shown to represent task rules. Here we show the mechanisms by which the rule-selective activity in the prefrontal cortex influences subsequent cognitive performance based on that rule. Using functional magnetic resonance imaging, we found that the frontopolar cortex interacted with posterior areas differently depending on whether subjects were going to perform a phonological or semantic task. Moreover, we found that the sustained "set" activity in this region predicted the activity that could be recorded in the posterior areas during the performance, as well as the speed of that performance. We argue that the prefrontal set activity does not reflect simple maintenance of the task rules but the process of implementing the rule for subsequent cognitive performance and that this is done through rule-selective interactions with areas involved in execution of the tasks.

  12. Lumican Inhibits SNAIL-Induced Melanoma Cell Migration Specifically by Blocking MMP-14 Activity

    PubMed Central

    Stasiak, Marta; Boncela, Joanna; Perreau, Corinne; Karamanou, Konstantina; Chatron-Colliet, Aurore; Proult, Isabelle; Przygodzka, Patrycja; Chakravarti, Shukti; Maquart, François-Xavier; Kowalska, M. Anna; Wegrowski, Yanusz; Brézillon, Stéphane

    2016-01-01

    Lumican, a small leucine rich proteoglycan, inhibits MMP-14 activity and melanoma cell migration in vitro and in vivo. Snail triggers epithelial-mesenchymal transitions endowing epithelial cells with migratory and invasive properties during tumor progression. The aim of this work was to investigate lumican effects on MMP-14 activity and migration of Snail overexpressing B16F1 (Snail-B16F1) melanoma cells and HT-29 colon adenocarcinoma cells. Lumican inhibits the Snail induced MMP-14 activity in B16F1 but not in HT-29 cells. In Snail-B16F1 cells, lumican inhibits migration, growth, and melanoma primary tumor development. A lumican-based strategy targeting Snail-induced MMP-14 activity might be useful for melanoma treatment. PMID:26930497

  13. Physical activity recommendations for children with specific chronic health conditions: Juvenile idiopathic arthritis, hemophilia, asthma and cystic fibrosis

    PubMed Central

    Philpott, J; Houghton, K; Luke, A

    2010-01-01

    As a group, children with a chronic disease or disability are less active than their healthy peers. There are many reasons for suboptimal physical activity, including biological, psychological and social factors. Furthermore, the lack of specific guidelines for ‘safe’ physical activity participation poses a barrier to increasing activity. Physical activity provides significant general health benefits and may improve disease outcomes. Each child with a chronic illness should be evaluated by an experienced physician for activity counselling and for identifing any contraindications to participation. The present statement reviews the benefits and risks of participation in sport and exercise for children with juvenile arthritis, hemophilia, asthma and cystic fibrosis. Guidelines for participation are included. PMID:21455465

  14. Physical activity recommendations for children with specific chronic health conditions: Juvenile idiopathic arthritis, hemophilia, asthma and cystic fibrosis.

    PubMed

    Philpott, J; Houghton, K; Luke, A

    2010-04-01

    As a group, children with a chronic disease or disability are less active than their healthy peers. There are many reasons for suboptimal physical activity, including biological, psychological and social factors. Furthermore, the lack of specific guidelines for 'safe' physical activity participation poses a barrier to increasing activity. Physical activity provides significant general health benefits and may improve disease outcomes. Each child with a chronic illness should be evaluated by an experienced physician for activity counselling and for identifing any contraindications to participation. The present statement reviews the benefits and risks of participation in sport and exercise for children with juvenile arthritis, hemophilia, asthma and cystic fibrosis. Guidelines for participation are included. PMID:21455465

  15. Physical activity recommendations for children with specific chronic health conditions: juvenile idiopathic arthritis, hemophilia, asthma, and cystic fibrosis.

    PubMed

    Philpott, John F; Houghton, Kristin; Luke, Anthony

    2010-05-01

    As a group, children with a chronic disease or disability are less active than their healthy peers. There are many reasons for suboptimal physical activity, including biological, psychological, and social factors. Furthermore, the lack of specific guidelines for 'safe' physical activity participation poses a barrier to increasing activity. Physical activity provides significant general health benefits and may improve disease outcomes. Each child with a chronic illness should be evaluated by an experienced physician for activity counselling and for identifying any contraindications to participation. The present statement reviews the benefits and risks of participation in sport and exercise for children with juvenile arthritis, hemophilia, asthma, and cystic fibrosis. Guidelines for participation are included. PMID:20445355

  16. Engineering of Pseudomonas taiwanensis VLB120 for Constitutive Solvent Tolerance and Increased Specific Styrene Epoxidation Activity

    PubMed Central

    Volmer, Jan; Neumann, Christoph; Schmid, Andreas

    2014-01-01

    The application of whole cells as biocatalysts is often limited by the toxicity of organic solvents, which constitute interesting substrates/products or can be used as a second phase for in situ product removal and as tools to control multistep biocatalysis. Solvent-tolerant bacteria, especially Pseudomonas strains, are proposed as promising hosts to overcome such limitations due to their inherent solvent tolerance mechanisms. However, potential industrial applications suffer from tedious, unproductive adaptation processes, phenotypic variability, and instable solvent-tolerant phenotypes. In this study, genes described to be involved in solvent tolerance were identified in Pseudomonas taiwanensis VLB120, and adaptive solvent tolerance was proven by cultivation in the presence of 1% (vol/vol) toluene. Deletion of ttgV, coding for the specific transcriptional repressor of solvent efflux pump TtgGHI gene expression, led to constitutively solvent-tolerant mutants of P. taiwanensis VLB120 and VLB120ΔC. Interestingly, the increased amount of solvent efflux pumps enhanced not only growth in the presence of toluene and styrene but also the biocatalytic performance in terms of stereospecific styrene epoxidation, although proton-driven solvent efflux is expected to compete with the styrene monooxygenase for metabolic energy. Compared to that of the P. taiwanensis VLB120ΔC parent strain, the maximum specific epoxidation activity of P. taiwanensis VLB120ΔCΔttgV doubled to 67 U/g of cells (dry weight). This study shows that solvent tolerance mechanisms, e.g., the solvent efflux pump TtgGHI, not only allow for growth in the presence of organic compounds but can also be used as tools to improve redox biocatalysis involving organic solvents. PMID:25128338

  17. Anticoagulant activity of a sulfated galactan: serpin-independent effect and specific interaction with factor Xa.

    PubMed

    Glauser, Bianca F; Rezende, Ricardo M; Melo, Fabio R; Pereira, Mariana S; Francischetti, Ivo M B; Monteiro, Robson Q; Rezaie, Alireza R; Mourão, Paulo A S

    2009-12-01

    An algal sulfated galactan has high anticoagulant and antithrombotic activities. Its serpin-dependent anticoagulant action is due to promoting thrombin and factor (F)Xa inhibition by antithrombin and heparin cofactor II. Here, we evaluated the anticoagulant effect of the algal sulfated galactan using serpin-free plasma. In contrast to heparin, the sulfated galactan is still able to prolong coagulation time and delay thrombin and FXa generation in serpin-free plasma. We further investigated this effect using purified blood coagulation proteins, discovering that sulfated galactan inhibits the intrinsic tenase and prothrombinase complexes, which are critical for FXa and thrombin generation, respectively. We also investigated the mechanism by which sulfated galactan promotes FXa inhibition by antithrombin using specific recombinant mutants of the protease. We show that sulfated galactan interacts with the heparin-binding exosite of FXa and Arg-236 and Lys-240 of this site are critical residues for this interaction, as observed for heparin. Thus, sulfated galactan and heparin have similar high-affinity and specificity for interaction with FXa, though they have differences in their chemical structures. Similar to heparin, the ability of sulfated galactan to potentiate FXa inhibition by antithrombin is calcium-dependent. However, in contrast to heparin, this effect is not entirely dependent on the conformation of the gamma-carboxyglutamic acid-rich domain of the protease. In conclusion, sulfated galactan and heparin have some similar effects on blood coagulation, but also differ significantly at the molecular level. This sulfated galactan opens new perspective for the development of antithrombotic drugs.

  18. Anticoagulant activity of a sulfated galactan: serpin-independent effect and specific interaction with factor Xa

    PubMed Central

    Glauser, Bianca F.; Rezende, Ricardo M.; Melo, Fabio R.; Pereira, Mariana S.; Francischetti, Ivo M. B.; Monteiro, Robson Q.; Rezaie, Alireza R.; Mourão, Paulo A.S.

    2009-01-01

    Summary An algal sulfated galactan has high anticoagulant and antithrombotic activities. Its serpin-dependent anticoagulant action is due to promoting thrombin and factor Xa inhibition by antithrombin and heparin cofactor II. Here, we evaluated the anticoagulant effect of the algal sulfated galactan using serpin-free plasma. In contrast to heparin, the sulfated galactan is still able to prolong coagulation time and delay thrombin and factor Xa generation in serpin-free plasma. We further investigated this effect using purified blood coagulation proteins, discovering that sulfated galactan inhibits the intrinsic tenase and prothrombinase complexes, which are critical for factor Xa and thrombin generation, respectively. We also investigated the mechanism by which sulfated galactan promotes factor Xa inhibition by antithrombin using specific recombinant mutants of the protease. We show that sulfated galactan interacts with the heparin-binding exosite of factor Xa and Arg-236 and Lys-240 of this site are critical residues for this interaction, as observed for heparin. Thus, sulfated galactan and heparin have similar high-affinity and specificity for interaction with factor Xa, though they have differences in their chemical structures. Similar to heparin, the ability of sulfated galactan to potentiate factor Xa inhibition by antithrombin is calcium-dependent. However, in contrast to heparin, this effect is not entirely dependent on the conformation of the γ-carboxyglutamic acid-rich domain of the protease. In conclusion, sulfated galactan and heparin have some similar effects on blood coagulation, but also differ significantly at the molecular level. This sulfated galactan opens new perspective for the development of antithrombotic drugs. PMID:19967150

  19. Barriers for recess physical activity: a gender specific qualitative focus group exploration

    PubMed Central

    2014-01-01

    Background Many children, in particular girls, do not reach the recommended amount of daily physical activity. School recess provides an opportunity for both boys and girls to be physically active, but barriers to recess physical activity are not well understood. This study explores gender differences in children’s perceptions of barriers to recess physical activity. Based on the socio-ecological model four types of environmental barriers were distinguished: natural, social, physical and organizational environment. Methods Data were collected through 17 focus groups (at 17 different schools) with in total 111 children (53 boys) from fourth grade, with a mean age of 10.4 years. The focus groups included an open group discussion, go-along group interviews, and a gender segregated post-it note activity. A content analysis of the post-it notes was used to rank the children’s perceived barriers. This was verified by a thematic analysis of transcripts from the open discussions and go-along interviews. Results The most frequently identified barriers for both boys and girls were weather, conflicts, lack of space, lack of play facilities and a newly-found barrier, use of electronic devices. While boys and girls identified the same barriers, there were both inter- and intra-gender differences in the perception of these barriers. Weather was a barrier for all children, apart from the most active boys. Conflicts were perceived as a barrier particularly by those boys who played ballgames. Girls said they would like to have more secluded areas added to the school playground, even in large schoolyards where lack of space was not a barrier. This aligned with girls’ requests for more “hanging-out” facilities, whereas boys primarily wanted activity promoting facilities. Conclusion Based on the results from this study, we recommend promoting recess physical activity through a combination of actions, addressing barriers within the natural, social, physical and

  20. Glycyl-tRNA synthetase specifically binds to the poliovirus IRES to activate translation initiation

    PubMed Central

    Andreev, Dmitri E.; Hirnet, Juliane; Terenin, Ilya M.; Dmitriev, Sergey E.; Niepmann, Michael; Shatsky, Ivan N.

    2012-01-01

    Adaptation to the host cell environment to efficiently take-over the host cell's machinery is crucial in particular for small RNA viruses like picornaviruses that come with only small RNA genomes and replicate exclusively in the cytosol. Their Internal Ribosome Entry Site (IRES) elements are specific RNA structures that facilitate the 5′ end-independent internal initiation of translation both under normal conditions and when the cap-dependent host protein synthesis is shut-down in infected cells. A longstanding issue is which host factors play a major role in this internal initiation. Here, we show that the functionally most important domain V of the poliovirus IRES uses tRNAGly anticodon stem–loop mimicry to recruit glycyl-tRNA synthetase (GARS) to the apical part of domain V, adjacent to the binding site of the key initiation factor eIF4G. The binding of GARS promotes the accommodation of the initiation region of the IRES in the mRNA binding site of the ribosome, thereby greatly enhancing the activity of the IRES at the step of the 48S initiation complex formation. Moonlighting functions of GARS that may be additionally needed for other events of the virus–host cell interaction are discussed. PMID:22373920

  1. Ultrasensitive and specific measurement of protease activity using functionalized photonic crystals.

    PubMed

    Gupta, Bakul; Mai, Kelly; Lowe, Stuart B; Wakefield, Denis; Di Girolamo, Nick; Gaus, Katharina; Reece, Peter J; Gooding, J Justin

    2015-10-01

    Herein is presented a microsensor technology as a diagnostic tool for detecting specific matrix metalloproteinases (MMPs) at very low concentrations. MMP-2 and MMP-9 are detected using label free porous silicon (PSi) photonic crystals that have been made selective for a given MMP by filling the nanopores with synthetic polymeric substrates containing a peptide sequence for that MMP. Proteolytic cleavage of the peptide sequence results in a shift in wavelength of the main peak in the reflectivity spectrum of the PSi device, which is dependent on the amount of MMP present. The ability to detect picogram amounts of MMP-2 and MMP-9 released by primary retinal pigment epithelial (RPE) cells and iris pigment epithelial (IPE) cells stimulated with lipopolysaccharide (LPS) is demonstrated. It was found that both cell types secrete higher amounts of MMP-2 than MMP-9 in their stimulated state, with RPE cells producing higher amounts of MMPs than IPE cells. The microsensor performance was compared to conventional protease detection systems, including gelatin zymography and enzyme linked immunosorbent assay (ELISA). It was found that the PSi microsensors were more sensitive than gelatin zymography; PSi microsensors detected the presence of both MMP-2 and MMP-9 while zymography could only detect MMP-2. The MMP-2 and MMP-9 quantification correlated well with the ELISA. This new method of detecting protease activity shows superior performance to conventional protease assays and has the potential for translation to high-throughput multiplexed analysis.

  2. Simple procedure for the synthesis of high specific activity tritiated (6S)-5-formyltetrahydrofolate

    SciTech Connect

    Moran, R.G.; Colman, P.D.

    1982-05-01

    The 5-position of tetrahydrofolate was found to be unusually reactive with low concentrations of formic acid in the presence of a water-soluble carbodiimide. The product of this reaction has neutral and acid ultraviolet spectra and chromatographic behavior consistent with its identity as 5-formyltetrahydrofolate (leucovoriun). When enzymatically synthesized (6S)-tetrahydrofolate was used as starting material, the product supported the growth of folate-depleted L1210 cells at one-half the concentration required for authentic (6R,S)-leucovorin. This reaction has been used to produce high specific activity (44 Ci/mmol) (/sup 3/H)(6S)-5-formyltetrahydrofolate in high yield. Experiments with (/sup 14/C)formic acid indicate that 1 mol of formate reacted per mol of tetrahydrofolate but that no reaction occurred with a variety of other folate compounds. (6S)-5-Formyltetrahydrofolate, labeled in the formyl group with /sup 14/C, has also been synthesized using this reaction. These easily produced, labeled folates should allow close examination of the transport and utilization of leucovorin and of the mechanism of reversal of methotrexate toxicity by reduced folate cofactors.

  3. Measurement of General and Specific Approaches to Physical Activity Parenting: A Systematic Review

    PubMed Central

    McDonald, Samantha; Cohen, Alysia

    2013-01-01

    Abstract Background Parents play a significant role in shaping youth physical activity (PA). However, interventions targeting PA parenting have been ineffective. Methodological inconsistencies related to the measurement of parental influences may be a contributing factor. The purpose of this article is to review the extant peer-reviewed literature related to the measurement of general and specific parental influences on youth PA. Methods A systematic review of studies measuring constructs of PA parenting was conducted. Computerized searches were completed using PubMed, MEDLINE, Academic Search Premier, SPORTDiscus, and PsycINFO. Reference lists of the identified articles were manually reviewed as well as the authors' personal collections. Articles were selected on the basis of strict inclusion criteria and details regarding the measurement protocols were extracted. A total of 117 articles met the inclusionary criteria. Methodological articles that evaluated the validity and reliability of PA parenting measures (n=10) were reviewed separately from parental influence articles (n=107). Results A significant percentage of studies used measures with indeterminate validity and reliability. A significant percentage of articles did not provide sample items, describe the response format, or report the possible range of scores. No studies were located that evaluated sensitivity to change. Conclusion The reporting of measurement properties and the use of valid and reliable measurement scales need to be improved considerably. PMID:23944923

  4. Isomer-specific biodegradation of nonylphenol in an activated sludge bioreactor and structure-biodegradability relationship.

    PubMed

    Lu, Zhijiang; Reif, Rubén; Gan, Jay

    2015-01-01

    Nonylphenol (NP), one of the priority hazardous substances, is in fact a mixture of numerous isomers. It is inconclusive whether or not biodegradation during wastewater treatment process is isomer-specific, leading to the environmental release of NP in different isomer profiles. In this study, we evaluated the isomer selectivity of 19 NP isomers in a laboratory-scale continuous flow conventional activated sludge bioreactor under various operational conditions. The removal efficiency of NP isomers ranged from 90 to 99%, depending on the operational conditions and isomer structures. Isomer selective biodegradation resulted in the increase of composition of recalcitrant isomers, such as, NP₁₉₃a/b, NP₁₁₀a and NP₁₉₄ in the effluent. Moreover, biodegradability was related to the bulkiness of α-substituents and followed α-dimethyl > α-ethyl-α-methyl > α-methyl-α-n-propyl > α-iso-propyl-α-methyl. Steric effect index, a quantitative descriptor of steric hindrance, was linearly correlated with residues of NP isomers in the effluent (R² = 0.76). Decrease of temperature to 10 °C decreased the overall biodegradability and also enhanced the relative enrichment of recalcitrant isomers. These findings suggest that isomer compositions of NP entering the environment may be different from those in technical mixtures and that isomeric selectivity should be taken into account to better understand the occurrence, fate, and ecological risks of NP.

  5. Specific activity of tissue culture antirabic vaccine Rabivak-Vnukovo-32 with short intramuscular vaccination schedule.

    PubMed

    Selimov, M A; Toigombaeva, V S; Zgurskaya, G N; Kulikova, L G; Kodkind, G Kh

    1988-05-01

    Tissue culture rabies vaccine has been used for subcutaneous immunization of 158 subjects according to official instructions and also for intramuscular immunization of 128 subjects according to a short schedule with booster inoculations. All 286 subjects were either bitten or contaminated with saliva of rabid animals or animals suspected of having rabies. The 1168 serum samples were tested by neutralization test (NT) in mice, by radial haemolysis (RH) and by indirect haemagglutination (IHA). The highest, earliest and longest active post-vaccination immunity was registered after the most intensive subcutaneous vaccination course at a dose of 5 ml for 25 days with 3 booster inoculations. Subcutaneous inoculation of 3 ml vaccine for 12 days (36 ml) failed to produce a satisfactory elevation of antibody titre. After 2 to 4 booster inoculations, however, a satisfactory level of antibody was observed. The tissue culture vaccine was shown to have good prospects for clinical vaccination by intramuscular route. On intramuscular vaccination at 1.5 ml for 9 days with 6 booster inoculations on days 16, 23, 30, 37, 67 and 97 (initial vaccine volume 45 ml) the mean geometric antibody titres (MGT) reached 93, 160, 322 and 165 on days 30, 60, 90 and 112, respectively. The economically efficient and rapid IHA and RH tests were confirmed to be specific and suitable for titration of antirabies antibody.

  6. Screening of bacterial isolates for mannose-specific lectin activity by agglutination of yeasts.

    PubMed Central

    Mirelman, D; Altmann, G; Eshdat, Y

    1980-01-01

    A total of 393 clinical bacterial isolates were tested for their ability to agglutinate yeast cells of either Saccharomyces cerevisiae or Candida albicans. A positive agglutination of yeasts that could be prevented by methyl alpha-D-mannoside was taken as an indication for the possible presence of a mannose-specific lectin (carbohydrate-binding protein) on the surface of the tested bacteria. Agglutination tests on glass slides showed that 38% of all the isolates tested were positive in their capacity to agglutinate yeasts. Among the various strains tested, all isolates of Serratia marcescens, Proteus morganii, and Citrobacter diversus, as well as 94% of Klebsiella pneumoniae, were positive. On the other hand, only 46% of the Escherichia coli, 48% of the salmonellae, 44% of the Citrobacter freundii, and 71% of the Aeromonas hydrophila isolates were positive. A quantitative determination of the lectin activity done by observing the agglutination of yeasts in microtiter plates showed that S. marcescens isolates were the most avid binders to the yeast, whereas Klebsiella and Citrobacter isolates were the weakest. PMID:6989854

  7. Mechanism and Specificity of Pentachloropseudilin-mediated Inhibition of Myosin Motor Activity*

    PubMed Central

    Chinthalapudi, Krishna; Taft, Manuel H.; Martin, René; Heissler, Sarah M.; Preller, Matthias; Hartmann, Falk K.; Brandstaetter, Hemma; Kendrick-Jones, John; Tsiavaliaris, Georgios; Gutzeit, Herwig O.; Fedorov, Roman; Buss, Folma; Knölker, Hans-Joachim; Coluccio, Lynne M.; Manstein, Dietmar J.

    2011-01-01

    Here, we report that the natural compound pentachloropseudilin (PClP) acts as a reversible and allosteric inhibitor of myosin ATPase and motor activity. IC50 values are in the range from 1 to 5 μm for mammalian class-1 myosins and greater than 90 μm for class-2 and class-5 myosins, and no inhibition was observed with class-6 and class-7 myosins. We show that in mammalian cells, PClP selectively inhibits myosin-1c function. To elucidate the structural basis for PClP-induced allosteric coupling and isoform-specific differences in the inhibitory potency of the compound, we used a multifaceted approach combining direct functional, crystallographic, and in silico modeling studies. Our results indicate that allosteric inhibition by PClP is mediated by the combined effects of global changes in protein dynamics and direct communication between the catalytic and allosteric sites via a cascade of small conformational changes along a conserved communication pathway. PMID:21680745

  8. The activity-dependent transcription factor NPAS4 regulates domain-specific inhibition

    PubMed Central

    Bloodgood, Brenda L.; Sharma, Nikhil; Browne, Heidi Adlman; Trepman, Alissa Z.; Greenberg, Michael E.

    2014-01-01

    A heterogeneous population of inhibitory neurons controls the flow of information through a neural circuit1–3. Inhibitory synapses that form on pyramidal neuron dendrites modulate the summation of excitatory synaptic potentials4–6 and prevent the generation of dendritic calcium spikes7,8. Precisely timed somatic inhibition limits both the number of action potentials and the time window during which firing can occur8,9. The activity-dependent transcription factor NPAS4 regulates inhibitory synapse number and function in cell culture10, but how this transcription factor affects the inhibitory inputs that form on distinct domains of a neuron in vivo was unclear. Here we show that in the mouse hippocampus behaviourally driven expression of NPAS4 coordinates the redistribution of inhibitory synapses made onto a CA1 pyramidal neuron, simultaneously increasing inhibitory synapse number on the cell body while decreasing the number of inhibitory synapses on the apical dendrites. This rearrangement of inhibition is mediated in part by the NPAS4 target gene brain derived neurotrophic factor (Bdnf), which specifically regulates somatic, and not dendritic, inhibition. These findings indicate that sensory stimuli, by inducing NPAS4 and its target genes, differentially control spatial features of neuronal inhibition in a way that restricts the output of the neuron while creating a dendritic environment that is permissive for plasticity. PMID:24201284

  9. Adsorption behaviors of some phenolic compounds onto high specific area activated carbon cloth.

    PubMed

    Ayranci, Erol; Duman, Osman

    2005-09-30

    Adsorption of phenol, hydroquinone, m-cresol, p-cresol and p-nitrophenol from aqueous solutions onto high specific area activated carbon cloth has been studied. The effect of ionization on adsorption of these ionizable phenolic compounds was examined by studying the adsorption from acidic, basic and natural pH solutions. Kinetics of adsorption was followed by in situ UV spectroscopy over a period of 90 min. First-order rate law was found to be valid for the kinetics of adsorption processes and the rate constants were determined. The highest rate constants were obtained for the adsorption from solutions at the natural pH. The lowest rate constants were observed in basic solutions. The rate constants decreased in the order p-nitrophenol approximately m-cresol>p-cresol>hydroquinone approximately phenol. Adsorption isotherms were derived at 30 degrees C and the isotherm data were treated according to Langmuir, Freundlich and Tempkin isotherm equations. The goodness of fit of experimental data to these isotherm equations was tested and the parameters of equations were determined. The possible interactions of compounds with the carbon surface were discussed considering the charge of the surface and the possible ionization of compounds at acidic, basic and natural pH conditions. PMID:15941619

  10. Mechanism and specificity of pentachloropseudilin-mediated inhibition of myosin motor activity.

    PubMed

    Chinthalapudi, Krishna; Taft, Manuel H; Martin, René; Heissler, Sarah M; Preller, Matthias; Hartmann, Falk K; Brandstaetter, Hemma; Kendrick-Jones, John; Tsiavaliaris, Georgios; Gutzeit, Herwig O; Fedorov, Roman; Buss, Folma; Knölker, Hans-Joachim; Coluccio, Lynne M; Manstein, Dietmar J

    2011-08-26

    Here, we report that the natural compound pentachloropseudilin (PClP) acts as a reversible and allosteric inhibitor of myosin ATPase and motor activity. IC(50) values are in the range from 1 to 5 μm for mammalian class-1 myosins and greater than 90 μm for class-2 and class-5 myosins, and no inhibition was observed with class-6 and class-7 myosins. We show that in mammalian cells, PClP selectively inhibits myosin-1c function. To elucidate the structural basis for PClP-induced allosteric coupling and isoform-specific differences in the inhibitory potency of the compound, we used a multifaceted approach combining direct functional, crystallographic, and in silico modeling studies. Our results indicate that allosteric inhibition by PClP is mediated by the combined effects of global changes in protein dynamics and direct communication between the catalytic and allosteric sites via a cascade of small conformational changes along a conserved communication pathway.

  11. Lysine Specific Demethylase 1 has Dual Functions as a Major Regulator of Androgen Receptor Transcriptional Activity

    PubMed Central

    Cai, Changmeng; He, Housheng Hansen; Gao, Shuai; Chen, Sen; Yu, Ziyang; Gao, Yanfei; Chen, Shaoyong; Chen, Mei Wei; Zhang, Jesse; Ahmed, Musaddeque; Wang, Yang; Metzger, Eric; Schüle, Roland; Liu, X. Shirley; Brown, Myles; Balk, Steven P.

    2014-01-01

    SUMMARY Lysine Specific Demethylase 1 (LSD1, KDM1A) functions as a transcriptional corepressor through demethylation of histone 3 lysine 4 (H3K4), but has coactivator function on some genes through unclear mechanisms. We show that LSD1, interacting with CoREST, associates with and coactivates androgen receptor (AR) on a large fraction of androgen-stimulated genes. A subset of these AR/LSD1-associated enhancer sites have histone 3 threonine 6 phosphorylation (H3T6ph), and these sites are further enriched for androgen-stimulated genes. Significantly, despite its coactivator activity, LSD1 still mediates H3K4me2 demethylation at these androgen-stimulated enhancers. FOXA1 is also associated with LSD1 at AR regulated enhancer sites, and a FOXA1 interaction with LSD1 enhances binding of both proteins at these sites. These findings show LSD1 functions broadly as a regulator of AR function, that it maintains a transcriptional repression function at AR-regulated enhancers through H3K4 demethylation, and has a distinct AR-linked coactivator function mediated by demethylation of other substrates. PMID:25482560

  12. Active transport, substrate specificity, and methylation of Hg(II) in anaerobic bacteria

    SciTech Connect

    Schasfer, Jeffra; Rocks, Sara; Zheng, Wang; Liang, Liyuan; Gu, Baohua; Morel, Francois M

    2011-01-01

    The formation of methylmercury (MeHg), which is biomagnified in aquatic food chains and poses a risk to human health, is effected by some iron- and sulfate-reducing bacteria (FeRB and SRB) in anaerobic environments. However, very little is known regarding the mechanism of uptake of inorganic Hg by these organisms, in part because of the inherent difficulty in measuring the intracellular Hg concentration. By using the FeRB Geobacter sulfurreducens and the SRB Desulfovibrio desulfuricans ND132 as model organisms, we demonstrate that Hg(II) uptake occurs by active transport. We also establish that Hg(II) uptake by G. sulfurreducens is highly dependent on the characteristics of the thiols that bind Hg(II) in the external medium, with some thiols promoting uptake and methylation and others inhibiting both. The Hg(II) uptake system of D. desulfuricans has a higher affinity than that of G. sulfurreducens and promotes Hg methylation in the presence of stronger complexing thiols. We observed a tight coupling between Hg methylation and MeHg export from the cell, suggesting that these two processes may serve to avoid the build up and toxicity of cellular Hg. Our results bring up the question of whether cellular Hg uptake is specific for Hg(II) or accidental, occurring via some essential metal importer. Our data also point at Hg(II) complexation by thiols as an important factor controlling Hg methylation in anaerobic environments.

  13. Isomer-specific biodegradation of nonylphenol in an activated sludge bioreactor and structure-biodegradability relationship.

    PubMed

    Lu, Zhijiang; Reif, Rubén; Gan, Jay

    2015-01-01

    Nonylphenol (NP), one of the priority hazardous substances, is in fact a mixture of numerous isomers. It is inconclusive whether or not biodegradation during wastewater treatment process is isomer-specific, leading to the environmental release of NP in different isomer profiles. In this study, we evaluated the isomer selectivity of 19 NP isomers in a laboratory-scale continuous flow conventional activated sludge bioreactor under various operational conditions. The removal efficiency of NP isomers ranged from 90 to 99%, depending on the operational conditions and isomer structures. Isomer selective biodegradation resulted in the increase of composition of recalcitrant isomers, such as, NP₁₉₃a/b, NP₁₁₀a and NP₁₉₄ in the effluent. Moreover, biodegradability was related to the bulkiness of α-substituents and followed α-dimethyl > α-ethyl-α-methyl > α-methyl-α-n-propyl > α-iso-propyl-α-methyl. Steric effect index, a quantitative descriptor of steric hindrance, was linearly correlated with residues of NP isomers in the effluent (R² = 0.76). Decrease of temperature to 10 °C decreased the overall biodegradability and also enhanced the relative enrichment of recalcitrant isomers. These findings suggest that isomer compositions of NP entering the environment may be different from those in technical mixtures and that isomeric selectivity should be taken into account to better understand the occurrence, fate, and ecological risks of NP. PMID:25462736

  14. Cilofungin (LY121019), an antifungal agent with specific activity against Candida albicans and Candida tropicalis.

    PubMed Central

    Hall, G S; Myles, C; Pratt, K J; Washington, J A

    1988-01-01

    Cilofungin (LY121019) is an antifungal agent that interferes with beta-glucan synthesis in the cells walls of fungi. The activity of this agent against 256 clinical isolates of yeasts was determined. It was found to be very active in vitro against Candida albicans (MIC for 90% of isolates [MIC90], less than or equal to 0.31 microgram/ml; minimal fungicidal concentration for 90% of isolates [MFC90], less than or equal to 0.31 micrograms/ml) and C. tropicalis (MIC90, less than or equal to 0.31 microgram/ml; MFC90, less than or equal to 0.31 microgram/ml) and moderately active against Torulopsis glabrata (MIC90 and MFC90, less than or equal to 20 micrograms/ml). All C. parapsilosis, Cryptococcus, and Saccharomyces cerevisiae strains were resistant. The activity of cilofungin was affected by medium and inoculum size. Antibiotic medium no. 3 was used as the standard medium. Isolates of C. albicans and C. tropicalis demonstrated a paradoxical effect in Sabouraud dextrose broth and yeast nitrogen base broth in that growth was partially inhibited at MICs equivalent to those in antibiotic medium no. 3, but growth continued, in many instances, throughout all concentrations tested. There was decreased activity of cilofungin with inocula greater than 10(5) CFU/ml. The temperature and duration of incubation did not affect its activity. Images PMID:3058017

  15. Modality specific cerebro-cerebellar activations in verbal working memory: an fMRI study.

    PubMed

    Kirschen, Matthew P; Chen, S H Annabel; Desmond, John E

    2010-01-01

    Verbal working memory (VWM) engages frontal and temporal/parietal circuits subserving the phonological loop, as well as, superior and inferior cerebellar regions which have projections from these neocortical areas. Different cerebro-cerebellar circuits may be engaged for integrating aurally- and visually-presented information for VWM. The present fMRI study investigated load (2, 4, or 6 letters) and modality (auditory and visual) dependent cerebro-cerebellar VWM activation using a Sternberg task. FMRI revealed modality-independent activations in left frontal (BA 6/9/44), insular, cingulate (BA 32), and bilateral inferior parietal/supramarginal (BA 40) regions, as well as in bilateral superior (HVI) and right inferior (HVIII) cerebellar regions. Visual presentation evoked prominent activations in right superior (HVI/CrusI) cerebellum, bilateral occipital (BA19) and left parietal (BA7/40) cortex while auditory presentation showed robust activations predominantly in bilateral temporal regions (BA21/22). In the cerebellum, we noted a visual to auditory emphasis of function progressing from superior to inferior and from lateral to medial regions. These results extend our previous findings of fMRI activation in cerebro-cerebellar networks during VWM, and demonstrate both modality dependent commonalities and differences in activations with increasing memory load.

  16. Exposure and Metabolic Activation Biomarkers of Carcinogenic Tobacco-Specific Nitrosamines.

    PubMed

    Hecht, Stephen S; Stepanov, Irina; Carmella, Steven G

    2016-01-19

    Lung cancer is the leading cause of cancer death in the world, and cigarette smoking is its main cause. Oral cavity cancer is another debilitating and often fatal cancer closely linked to tobacco product use. While great strides have been made in decreasing tobacco use in the United States and some other countries, there are still an estimated 1 billion men and 250 million women in the world who are cigarette smokers and there are hundreds of millions of smokeless tobacco users, all at risk for cancer. Worldwide, lung cancer kills about three people per minute. This Account focuses on metabolites and biomarkers of two powerful tobacco-specific nitrosamine carcinogens, 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone (NNK) and N'-nitrosonornicotine (NNN), considered to be among the main causes of lung cancer and oral cavity cancer in people who use tobacco products. Three properties of NNK and NNN are critical for successful biomarker studies: they are present in all tobacco products, they are tobacco-specific and are not found in any other product, and they are strong carcinogens. NNK and NNN are converted in humans to urinary metabolites that can be quantified by mass spectrometry as biomarkers of exposure to these carcinogens. They are also metabolized to diazonium ions and related electrophiles that react with DNA to form addition products that can be detected and quantified by mass spectrometry. These urinary metabolites and DNA addition products can serve as biomarkers of exposure and metabolic activation, respectively. The biomarkers of exposure, in particular the urinary NNK metabolites 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanol (NNAL) and its glucuronides, have been extensively applied to document tobacco-specific lung carcinogen uptake in smokers and nonsmokers exposed to secondhand tobacco smoke. Highly sensitive mass spectrometric methods have been developed for quantitative analysis of these NNK metabolites as well as metabolites of NNN in human urine

  17. Exposure and Metabolic Activation Biomarkers of Carcinogenic Tobacco-Specific Nitrosamines.

    PubMed

    Hecht, Stephen S; Stepanov, Irina; Carmella, Steven G

    2016-01-19

    Lung cancer is the leading cause of cancer death in the world, and cigarette smoking is its main cause. Oral cavity cancer is another debilitating and often fatal cancer closely linked to tobacco product use. While great strides have been made in decreasing tobacco use in the United States and some other countries, there are still an estimated 1 billion men and 250 million women in the world who are cigarette smokers and there are hundreds of millions of smokeless tobacco users, all at risk for cancer. Worldwide, lung cancer kills about three people per minute. This Account focuses on metabolites and biomarkers of two powerful tobacco-specific nitrosamine carcinogens, 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone (NNK) and N'-nitrosonornicotine (NNN), considered to be among the main causes of lung cancer and oral cavity cancer in people who use tobacco products. Three properties of NNK and NNN are critical for successful biomarker studies: they are present in all tobacco products, they are tobacco-specific and are not found in any other product, and they are strong carcinogens. NNK and NNN are converted in humans to urinary metabolites that can be quantified by mass spectrometry as biomarkers of exposure to these carcinogens. They are also metabolized to diazonium ions and related electrophiles that react with DNA to form addition products that can be detected and quantified by mass spectrometry. These urinary metabolites and DNA addition products can serve as biomarkers of exposure and metabolic activation, respectively. The biomarkers of exposure, in particular the urinary NNK metabolites 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanol (NNAL) and its glucuronides, have been extensively applied to document tobacco-specific lung carcinogen uptake in smokers and nonsmokers exposed to secondhand tobacco smoke. Highly sensitive mass spectrometric methods have been developed for quantitative analysis of these NNK metabolites as well as metabolites of NNN in human urine

  18. Biophysical and mutagenic analysis of Thermoanaerobacter ethanolicus secondary-alcohol dehydrogenase activity and specificity.

    PubMed Central

    Burdette, D S; Secundo, F; Phillips, R S; Dong, J; Scott, R A; Zeikus, J G

    1997-01-01

    The Thermoanaerobacter ethanolicus 39E adhB gene encoding the secondary-alcohol dehydrogenase (secondary ADH) was overexpressed in Escherichia coli at more than 10% of total protein. The recombinant enzyme was purified in high yield (67%) by heat-treatment at 85 degrees C and (NH4)2SO4 precipitation. Site-directed mutants (C37S, H59N, D150N, D150Eand D150C were analysed to test the peptide sequence comparison-based predictions of amino acids responsible for putative catalytic Zn binding. X-ray absorption spectroscopy confirmed the presence of a protein-bound Zn atom with ZnS1(imid)1(N,O)3 co-ordination sphere. Inductively coupled plasma atomic emission spectrometry measured 0.48 Zn atoms per wild-type secondary ADH subunit. The C37S, H59N and D150N mutant enzymes bound only 0.11, 0.13 and 0.33 Zn per subunit respectively,suggesting that these residues are involved in Zn liganding. The D150E and D150C mutants retained 0.47 and 1.2 Zn atoms per subunit, indicating that an anionic side-chain moiety at this position preserves the bound Zn. All five mutant enzymes had activity, suggesting that the T. ethanolicus secondary ADH requires a properly co-ordinated catalytic Zn atom. The His-59 and Asp-150 mutations also altered secondary ADH affinity for propan-2-ol over a 140-fold range, whereas the overall change in affinity for ethanol spanned a range of only 7-fold, supporting the importance of the metal in secondary ADH substrate binding. The lack of significant changes in cofactor affinity as a result of these catalytic Zn ligand mutations suggested that secondary ADH substrate-and cofactor-binding sites are structurally distinct. Altering Gly198 to Asp reduced the enzyme specific activity 2.7-fold, increased the Km(app) for NADP+ 225-fold, and decreased the Km(app) for NAD+ 3-fold, supporting the prediction that the enzyme binds nicotinamide cofactor in a Rossmann fold. Our data indicate therefore that, unlike the liver primary ADH

  19. Inducible Conditional Vascular-Specific Overexpression of Peroxisome Proliferator-Activated Receptor Beta/Delta Leads to Rapid Cardiac Hypertrophy

    PubMed Central

    Wagner, Kay-Dietrich; Vukolic, Ana; Baudouy, Delphine; Michiels, Jean-François

    2016-01-01

    Peroxisome proliferator-activated receptors are nuclear receptors which function as ligand-activated transcription factors. Among them, peroxisome proliferator-activated receptor beta/delta (PPARβ/δ) is highly expressed in the heart and thought to have cardioprotective functions due to its beneficial effects in metabolic syndrome. As we already showed that PPARβ/δ activation resulted in an enhanced cardiac angiogenesis and growth without impairment of heart function, we were interested to determine the effects of a specific activation of PPARβ/δ in the vasculature on cardiac performance under normal and in chronic ischemic heart disease conditions. We analyzed the effects of a specific PPARβ/δ overexpression in endothelial cells on the heart using an inducible conditional vascular-specific mouse model. We demonstrate that vessel-specific overexpression of PPARβ/δ induces rapid cardiac angiogenesis and growth with an increase in cardiomyocyte size. Upon myocardial infarction, vascular overexpression of PPARβ/δ, despite the enhanced cardiac vessel formation, does not protect against chronic ischemic injury. Our results suggest that the proper balance of PPARβ/δ activation in the different cardiac cell types is required to obtain beneficial effects on the outcome in chronic ischemic heart disease. PMID:27057154

  20. Specific capture of the hydrolysate on magnetic beads for sensitive detecting plant vacuolar processing enzyme activity.

    PubMed

    Zhou, Jun; Cheng, Meng; Zeng, Lizhang; Liu, Weipeng; Zhang, Tao; Xing, Da

    2016-05-15

    Conventional plant protease detection always suffers from high background interference caused by the complex coloring metabolites in plant cells. In this study, a bio-modified magnetic beads-based strategy was developed for sensitive and quantitative detection of plant vacuolar processing enzyme (VPE) activity. Cleavage of the peptide substrate (ESENCRK-FITC) after asparagine residue by VPE resulted in the 2-cyano-6-amino-benzothiazole (CABT)-functionalized magnetic beads capture of the severed substrate CRK-FITC via a condensation reaction between CABT and cysteine (Cys). The catalytic activity was subsequently obtained by the confocal microscopy imaging and flow cytometry quantitative analysis. The sensor system integrated advantages of (i) the high efficient enrichment and separation capabilities of magnetic beads and (ii) the catalyst-free properties of the CABT-Cys condensation reaction. It exhibited a linear relationship between the fluorescence signal and the concentration of severed substrate in the range of 10-600 pM. The practical results showed that, compared with normal growth conditions, VPE activity was increased by 2.7-fold (307.2 ± 25.3 μM min(-1)g(-1)) upon cadmium toxicity stress. This platform effectively overcame the coloring metabolites-caused background interference, showing fine applicability for the detection of VPE activity in real samples. The strategy offers great sensitivity and may be further extended to other protease activity detection. PMID:26797250

  1. A small RNA activates CFA synthase by isoform-specific mRNA stabilization

    PubMed Central

    Fröhlich, Kathrin Sophie; Papenfort, Kai; Fekete, Agnes; Vogel, Jörg

    2013-01-01

    Small RNAs use a diversity of well-characterized mechanisms to repress mRNAs, but how they activate gene expression at the mRNA level remains not well understood. The predominant activation mechanism of Hfq-associated small RNAs has been translational control whereby base pairing with the target prevents the formation of an intrinsic inhibitory structure in the mRNA and promotes translation initiation. Here, we report a translation-independent mechanism whereby the small RNA RydC selectively activates the longer of two isoforms of cfa mRNA (encoding cyclopropane fatty acid synthase) in Salmonella enterica. Target activation is achieved through seed pairing of the pseudoknot-exposed, conserved 5′ end of RydC to an upstream region of the cfa mRNA. The seed pairing stabilizes the messenger, likely by interfering directly with RNase E-mediated decay in the 5′ untranslated region. Intriguingly, this mechanism is generic such that the activation is equally achieved by seed pairing of unrelated small RNAs, suggesting that this mechanism may be utilized in the design of RNA-controlled synthetic circuits. Physiologically, RydC is the first small RNA known to regulate membrane stability. PMID:24141880

  2. Insights into Substrate Specificity and Metal Activation of Mammalian Tetrahedral Aspartyl Aminopeptidase

    SciTech Connect

    Chen, Yuanyuan; Farquhar, Erik R.; Chance, Mark R.; Palczewski, Krzysztof; Kiser, Philip D.

    2012-07-11

    Aminopeptidases are key enzymes involved in the regulation of signaling peptide activity. Here, we present a detailed biochemical and structural analysis of an evolutionary highly conserved aspartyl aminopeptidase called DNPEP. We show that this peptidase can cleave multiple physiologically relevant substrates, including angiotensins, and thus may play a key role in regulating neuron function. Using a combination of x-ray crystallography, x-ray absorption spectroscopy, and single particle electron microscopy analysis, we provide the first detailed structural analysis of DNPEP. We show that this enzyme possesses a binuclear zinc-active site in which one of the zinc ions is readily exchangeable with other divalent cations such as manganese, which strongly stimulates the enzymatic activity of the protein. The plasticity of this metal-binding site suggests a mechanism for regulation of DNPEP activity. We also demonstrate that DNPEP assembles into a functionally relevant tetrahedral complex that restricts access of peptide substrates to the active site. These structural data allow rationalization of the enzyme's preference for short peptide substrates with N-terminal acidic residues. This study provides a structural basis for understanding the physiology and bioinorganic chemistry of DNPEP and other M18 family aminopeptidases.

  3. Force-specific activation of Smad1/5 regulates vascular endothelial cell cycle progression in response to disturbed flow.

    PubMed

    Zhou, Jing; Lee, Pei-Ling; Tsai, Chien-Sung; Lee, Chih-I; Yang, Tung-Lin; Chuang, Han-Sheng; Lin, Wei-Wen; Lin, Ting-Er; Lim, Seh Hong; Wei, Shu-Yi; Chen, Yuh-Lien; Chien, Shu; Chiu, Jeng-Jiann

    2012-05-15

    Vascular endothelial cells (ECs) are constantly exposed to blood flow-induced shear stress, but the mechanism of force-specific activation of their signaling to modulate cellular function remains unclear. We have demonstrated that bone morphogenetic protein receptor (BMPR)-specific Smad1/5 can be force-specifically activated by oscillatory shear stress (OSS) in ECs to cause cell cycle progression. Smad1/5 is highly activated in ECs of atherosclerotic lesions in diseased human coronary arteries from patients with end-stage heart failure undergoing heart transplantation and from apolipoprotein E-deficient mice. Application of OSS (0.5 ± 4 dyn/cm(2)) causes the sustained activation of Smad1/5 in ECs through activations of mammalian target of rapamycin and p70S6 kinase, leading to up-regulation of cyclin A and down-regulations of p21(CIP1) and p27(KIP1) and, hence, EC cycle progression. En face examination of rat aortas reveals high levels of phospho-Smad1/5 in ECs of the inner, but not the outer, curvature of aortic arch, nor the straight segment of thoracic aorta [corrected]. Immunohistochemical and en face examinations of the experimentally stenosed abdominal aorta in rats show high levels of phospho-Smad1/5 in ECs at poststenotic sites, where OSS occurs. These OSS activations of EC Smad1/5 in vitro and in vivo are not inhibited by the BMP-specific antagonist Noggin and, hence, are independent of BMP ligand. Transfecting ECs with Smad1/5-specific small interfering RNAs inhibits the OSS-induced EC cycle progression. Our findings demonstrate the force-specificity of the activation of Smad1/5 and its contribution to cell cycle progression in ECs induced by disturbed flow. PMID:22550179

  4. Dynamic ubiquitination of the mitogen-activated protein kinase kinase (MAPKK) Ste7 determines mitogen-activated protein kinase (MAPK) specificity.

    PubMed

    Hurst, Jillian H; Dohlman, Henrik G

    2013-06-28

    Ubiquitination is a post-translational modification that tags proteins for proteasomal degradation. In addition, there is a growing appreciation that ubiquitination can influence protein activity and localization. Ste7 is a prototype MAPKK in yeast that participates in both the pheromone signaling and nutrient deprivation/invasive growth pathways. We have shown previously that Ste7 is ubiquitinated upon pheromone stimulation. Here, we show that the Skp1/Cullin/F-box ubiquitin ligase SCF(Cdc4) and the ubiquitin protease Ubp3 regulate Ste7 ubiquitination and signal specificity. Using purified components, we demonstrate that SCF(Cdc4) ubiquitinates Ste7 directly. Using gene deletion mutants, we show that SCF(Cdc4) and Ubp3 have opposing effects on Ste7 ubiquitination. Although SCF(Cdc4) is necessary for proper activation of the pheromone MAPK Fus3, Ubp3 is needed to limit activation of the invasive growth MAPK Kss1. Finally, we show that Fus3 phosphorylates Ubp3 directly and that phosphorylation of Ubp3 is necessary to limit Kss1 activation. These results reveal a feedback loop wherein one MAPK limits the ubiquitination of an upstream MAPKK and thereby prevents spurious activation of a second competing MAPK. PMID:23645675

  5. Progranulin Deficiency Promotes Circuit-Specific Synaptic Pruning by Microglia via Complement Activation.

    PubMed

    Lui, Hansen; Zhang, Jiasheng; Makinson, Stefanie R; Cahill, Michelle K; Kelley, Kevin W; Huang, Hsin-Yi; Shang, Yulei; Oldham, Michael C; Martens, Lauren Herl; Gao, Fuying; Coppola, Giovanni; Sloan, Steven A; Hsieh, Christine L; Kim, Charles C; Bigio, Eileen H; Weintraub, Sandra; Mesulam, Marek-Marsel; Rademakers, Rosa; Mackenzie, Ian R; Seeley, William W; Karydas, Anna; Miller, Bruce L; Borroni, Barbara; Ghidoni, Roberta; Farese, Robert V; Paz, Jeanne T; Barres, Ben A; Huang, Eric J

    2016-05-01

    Microglia maintain homeostasis in the brain, but whether aberrant microglial activation can cause neurodegeneration remains controversial. Here, we use transcriptome profiling to demonstrate that deficiency in frontotemporal dementia (FTD) gene progranulin (Grn) leads to an age-dependent, progressive upregulation of lysosomal and innate immunity genes, increased complement production, and enhanced synaptic pruning in microglia. During aging, Grn(-/-) mice show profound microglia infiltration and preferential elimination of inhibitory synapses in the ventral thalamus, which lead to hyperexcitability in the thalamocortical circuits and obsessive-compulsive disorder (OCD)-like grooming behaviors. Remarkably, deleting C1qa gene significantly reduces synaptic pruning by Grn(-/-) microglia and mitigates neurodegeneration, behavioral phenotypes, and premature mortality in Grn(-/-) mice. Together, our results uncover a previously unrecognized role of progranulin in suppressing aberrant microglia activation during aging. These results represent an important conceptual advance that complement activation and microglia-mediated synaptic pruning are major drivers, rather than consequences, of neurodegeneration caused by progranulin deficiency.

  6. Progranulin Deficiency Promotes Circuit-Specific Synaptic Pruning by Microglia via Complement Activation.

    PubMed

    Lui, Hansen; Zhang, Jiasheng; Makinson, Stefanie R; Cahill, Michelle K; Kelley, Kevin W; Huang, Hsin-Yi; Shang, Yulei; Oldham, Michael C; Martens, Lauren Herl; Gao, Fuying; Coppola, Giovanni; Sloan, Steven A; Hsieh, Christine L; Kim, Charles C; Bigio, Eileen H; Weintraub, Sandra; Mesulam, Marek-Marsel; Rademakers, Rosa; Mackenzie, Ian R; Seeley, William W; Karydas, Anna; Miller, Bruce L; Borroni, Barbara; Ghidoni, Roberta; Farese, Robert V; Paz, Jeanne T; Barres, Ben A; Huang, Eric J

    2016-05-01

    Microglia maintain homeostasis in the brain, but whether aberrant microglial activation can cause neurodegeneration remains controversial. Here, we use transcriptome profiling to demonstrate that deficiency in frontotemporal dementia (FTD) gene progranulin (Grn) leads to an age-dependent, progressive upregulation of lysosomal and innate immunity genes, increased complement production, and enhanced synaptic pruning in microglia. During aging, Grn(-/-) mice show profound microglia infiltration and preferential elimination of inhibitory synapses in the ventral thalamus, which lead to hyperexcitability in the thalamocortical circuits and obsessive-compulsive disorder (OCD)-like grooming behaviors. Remarkably, deleting C1qa gene significantly reduces synaptic pruning by Grn(-/-) microglia and mitigates neurodegeneration, behavioral phenotypes, and premature mortality in Grn(-/-) mice. Together, our results uncover a previously unrecognized role of progranulin in suppressing aberrant microglia activation during aging. These results represent an important conceptual advance that complement activation and microglia-mediated synaptic pruning are major drivers, rather than consequences, of neurodegeneration caused by progranulin deficiency. PMID:27114033

  7. A Modality-Specific Feedforward Component of Choice-Related Activity in MT.

    PubMed

    Smolyanskaya, Alexandra; Haefner, Ralf M; Lomber, Stephen G; Born, Richard T

    2015-07-01

    The activity of individual sensory neurons can be predictive of an animal's choices. These decision signals arise from network properties dependent on feedforward and feedback inputs; however, the relative contributions of these inputs are poorly understood. We determined the role of feedforward pathways to decision signals in MT by recording neuronal activity while monkeys performed motion and depth tasks. During each session, we reversibly inactivated V2 and V3, which provide feedforward input to MT that conveys more information about depth than motion. We thus monitored the choice-related activity of the same neuron both before and during V2/V3 inactivation. During inactivation, MT neurons became less predictive of decisions for the depth task but not the motion task, indicating that a feedforward pathway that gives rise to tuning preferences also contributes to decision signals. We show that our data are consistent with V2/V3 input conferring structured noise correlations onto the MT population. PMID:26139374

  8. GlnB is specifically required for Azospirillum brasilense NifA activity in Escherichia coli.

    PubMed

    Araújo, Luiza M; Monteiro, Rose A; Souza, Emanuel M; Steffens, M Berenice R; Rigo, Liu U; Pedrosa, Fábio O; Chubatsu, Leda S

    2004-01-01

    The Azospirillum brasilense transcription regulator NifA and the nitrogen-status signaling proteins GlnB, GlnZ and GlnK were expressed in Escherichia coli and analyzed for their ability to activate nif gene expression. When expressed separately, none of the proteins were able to activate nifH promoter expression in any tested conditions; in contrast, nifH expression was observed in cells grown in the absence of ammonium and oxygen and when expressing simultaneously NifA and GlnB proteins, but not when expressing NifA and GlnZ or GlnK. Our results show that the GlnB protein is required for transcription activation by Azospirillum brasilense NifA and it cannot be replaced by GlnZ or GlnK.

  9. Rational design and validation of an anti-protein kinase C active-state specific antibody based on conformational changes

    PubMed Central

    Pena, Darlene Aparecida; Andrade, Victor Piana de; Silva, Gabriela Ávila Fernandes; Neves, José Ivanildo; Oliveira, Paulo Sergio Lopes de; Alves, Maria Julia Manso; Devi, Lakshmi A.; Schechtman, Deborah

    2016-01-01

    Protein kinase C (PKC) plays a regulatory role in key pathways in cancer. However, since phosphorylation is a step for classical PKC (cPKC) maturation and does not correlate with activation, there is a lack of tools to detect active PKC in tissue samples. Here, a structure-based rational approach was used to select a peptide to generate an antibody that distinguishes active from inactive cPKC. A peptide conserved in all cPKCs, C2Cat, was chosen since modeling studies based on a crystal structure of PKCβ showed that it is localized at the interface between the C2 and catalytic domains of cPKCs in an inactive kinase. Anti-C2Cat recognizes active cPKCs at least two-fold better than inactive kinase in ELISA and immunoprecipitation assays, and detects the temporal dynamics of cPKC activation upon receptor or phorbol stimulation. Furthermore, the antibody is able to detect active PKC in human tissue. Higher levels of active cPKC were observed in the more aggressive triple negative breast cancer tumors as compared to the less aggressive estrogen receptor positive tumors. Thus, this antibody represents a reliable, hitherto unavailable and a valuable tool to study PKC activation in cells and tissues. Similar structure-based rational design strategies can be broadly applied to obtain active-state specific antibodies for other signal transduction molecules. PMID:26911897

  10. Rational design and validation of an anti-protein kinase C active-state specific antibody based on conformational changes.

    PubMed

    Pena, Darlene Aparecida; Andrade, Victor Piana de; Silva, Gabriela Ávila Fernandes; Neves, José Ivanildo; Oliveira, Paulo Sergio Lopes de; Alves, Maria Julia Manso; Devi, Lakshmi A; Schechtman, Deborah

    2016-02-25

    Protein kinase C (PKC) plays a regulatory role in key pathways in cancer. However, since phosphorylation is a step for classical PKC (cPKC) maturation and does not correlate with activation, there is a lack of tools to detect active PKC in tissue samples. Here, a structure-based rational approach was used to select a peptide to generate an antibody that distinguishes active from inactive cPKC. A peptide conserved in all cPKCs, C2Cat, was chosen since modeling studies based on a crystal structure of PKCβ showed that it is localized at the interface between the C2 and catalytic domains of cPKCs in an inactive kinase. Anti-C2Cat recognizes active cPKCs at least two-fold better than inactive kinase in ELISA and immunoprecipitation assays, and detects the temporal dynamics of cPKC activation upon receptor or phorbol stimulation. Furthermore, the antibody is able to detect active PKC in human tissue. Higher levels of active cPKC were observed in the more aggressive triple negative breast cancer tumors as compared to the less aggressive estrogen receptor positive tumors. Thus, this antibody represents a reliable, hitherto unavailable and a valuable tool to study PKC activation in cells and tissues. Similar structure-based rational design strategies can be broadly applied to obtain active-state specific antibodies for other signal transduction molecules.

  11. The regulation of glial-specific splicing of Neurexin IV requires HOW and Cdk12 activity.

    PubMed

    Rodrigues, Floriano; Thuma, Leila; Klämbt, Christian

    2012-05-01

    The differentiation of the blood-brain barrier (BBB) is an essential process in the development of a complex nervous system and depends on alternative splicing. In the fly BBB, glial cells establish intensive septate junctions that require the cell-adhesion molecule Neurexin IV. Alternative splicing generates two different Neurexin IV isoforms: Neurexin IV(exon3), which is found in cells that form septate junctions, and Neurexin IV(exon4), which is found in neurons that form no septate junctions. Here, we show that the formation of the BBB depends on the RNA-binding protein HOW (Held out wings), which triggers glial specific splicing of Neurexin IV(exon3). Using a set of splice reporters, we show that one HOW-binding site is needed to include one of the two mutually exclusive exons 3 and 4, whereas binding at the three further motifs is needed to exclude exon 4. The differential splicing is controlled by nuclear access of HOW and can be induced in neurons following expression of nuclear HOW. Using a novel in vivo two-color splicing detector, we then screened for genes required for full HOW activity. This approach identified Cyclin-dependent kinase 12 (Cdk12) and the splicesosomal component Prp40 as major determinants in regulating HOW-dependent splicing of Neurexin IV. Thus, in addition to the control of nuclear localization of HOW, the phosphorylation of the C-terminal domain of the RNA polymerase II by Cdk12 provides an elegant mechanism in regulating timed splicing of newly synthesized mRNA molecules.

  12. Activation of HLA-A2-specific memory B cells in severe combined immunodeficient mice.

    PubMed

    Niguma, T; DeVito, L D; Grailer, A P; Fechner, J H; Sollinger, H W; Burlingham, W J

    1993-05-01

    Peripheral blood leukocytes of all five HLA-A2-sensitized patients produced significant levels of human IgG (> or = 0.25 micrograms/ml) following polyclonal activation in vitro, but PBLs from only one patient (K.H.) who had been transfused recently (< 4 weeks) produced detectable anti-HLA-A2 IgG. PBLs from this in vitro responder and from one in vitro nonresponder (L.G.) were transferred intraperitoneally into SCID mice. A low level (range, 5-40 ng/ml) of human anti-HLA-A2 IgG was detected in the serum of the mice without additional stimulation. This anti-HLA-A2 IgG response was boosted (range, 40-200 ng/ml) when mice received a human skin xenograft or an early challenge with x-irradiated human leukocytes intraperitoneally. Although the anti-HLA antibodies produced were specific for HLA-A2, the boosting of anti-HLA-A2 IgG production did not require the expression of the HLA-A2 protein, since either HLA-A2-negative skin xenografts or HLA-identical x-irradiated PBLs enhanced the production of anti-HLA-A2 IgG. Dose-response of transferred PBLs and kappa:lambda composition of individual mouse anti-HLA-A2 production suggested that low-frequency human memory B-cell clones were stimulated to proliferate and/or triggered to become high Ab secretors by skin graft or PBL boost. PMID:8376189

  13. A specific DNA-binding protein activated by ionizing radiation in normal cells and constitutively present in ataxia telangiectasia cells

    SciTech Connect

    Teale, B.; Lavin, M.F.

    1994-04-01

    Exposure of mammalian cells to ionizing radiation gives rise to a complex series of changes. This response is characterized by the induction of a variety of genes and the activation of pre-existing proteins. We describe here activation of a specific DNA-binding protein by ionizing radiation. The response was dose-dependent and specific for ionizing radiation. The binding factor appears to be normally present in the cytoplasma and responds to radiation by translocation to the nucleus, or is activated within the nucleus by an unknown mechanism. The radiation-induced activation of this protein appears to be mediated through a protein kinase C-associated pathway. A DNA-binding factor recognizing the same binding motif was found to be abnormally distributed in cells from patients with the human genetic disease, ataxiatelangiectasia. The protein was constitutively present in the nucleus and the cytoplasm of ataxia telangiectasia cells and did not respond to radiation. 27 refs., 5 figs.

  14. Highly specific fluorescence detection of T4 polynucleotide kinase activity via photo-induced electron transfer.

    PubMed

    Tao, Mangjuan; Shi, Zhilu; Cheng, Rui; Zhang, Jing; Li, Baoxin; Jin, Yan

    2015-09-15

    Sensitive and reliable study of the activity of polynucleotide kinase (PNK) and its potential inhibitors is of great importance for biochemical interaction related to DNA phosphorylation as well as development of kinase-targeted drug discovery. To achieve facile and reliable detection of PNK activity, we report here a novel fluorescence method for PNK assay based on a combination of exonuclease cleavage reaction and photo-induced electron transfer (PIET) by using T4 PNK as a model target. The fluorescence of 3'-carboxyfluorescein-labeled DNA probe (FDNA) is effectively quenched by deoxyguanosines at the 5' end of its complementary DNA (cDNA) due to an effective PIET between deoxyguanosines and fluorophore. Whereas FDNA/cDNA hybrid is phosphorylated by PNK and then immediately cleaved by lambda exonuclease (λ exo), fluorescence is greatly restored due to the break of PIET. This homogeneous PNK activity assay does not require a complex design by taking advantage of the quenching ability of deoxyguanosines, making the proposed strategy facile and cost-effective. The activity of PNK can be sensitively detected in the range of 0.005 to 10 U mL(-1) with a detection limit of 2.1×10(-3) U mL(-1). Research on inhibition efficiency of different inhibitors demonstrated that it can be explored to evaluate inhibition capacity of inhibitors. The application for detection of PNK activity in complex matrix achieved satisfactory results. Therefore, this PIET strategy opens a promising avenue for studying T4 PNK activity as well as evaluating PNK inhibitors, which is of great importance for discovering kinase-targeted drugs. PMID:26050629

  15. A Specific Component of the Evoked Potential Mirrors Phasic Dopamine Neuron Activity during Conditioning.

    PubMed

    Pan, Wei-Xing; Dudman, Joshua T

    2015-07-22

    Midbrain dopamine (DA) neurons are thought to be a critical node in the circuitry that mediates reward learning. DA neurons receive diverse inputs from regions distributed throughout the neuraxis from frontal neocortex to the mesencephalon. While a great deal is known about changes in the activity of individual DA neurons during learning, much less is known about the functional changes in the microcircuits in which DA neurons are embedded. Here we used local field potentials recorded from the midbrain of behaving mice to show that the midbrain evoked potential (mEP) faithfully reflects the temporal and spatial structure of the phasic response of midbrain neuron populations during conditioning. By comparing the mEP to simultaneously recorded single units, we identified specific components of the mEP that corresponded to phasic DA and non-DA responses to salient stimuli. The DA component of the mEP emerged with the acquisition of a conditioned stimulus, was extinguished following changes in reinforcement contingency, and could be inhibited by pharmacological manipulations that attenuate the phasic responses of DA neurons. In contrast to single-unit recordings, the mEP permitted relatively dense sampling of the midbrain circuit during conditioning and thus could be used to reveal the spatiotemporal structure of multiple intermingled midbrain circuits. Finally, the mEP response was stable for months and thus provides a new approach to study long-term changes in the organization of ventral midbrain microcircuits during learning. Significance statement: Neurons that synthesize and release the neurotransmitter dopamine play a critical role in voluntary reward-seeking behavior. Much of our insight into the function of dopamine neurons comes from recordings of individual cells in behaving animals; however, it is notoriously difficult to record from dopamine neurons due to their sparsity and depth, as well as the presence of intermingled non-dopaminergic neurons. Here we

  16. Active knee joint flexibility and sports activity.

    PubMed

    Hahn, T; Foldspang, A; Vestergaard, E; Ingemann-Hansen, T

    1999-04-01

    The aim of the study was to estimate active knee flexion and active knee extension in athletes and to investigate the potential association of each to different types of sports activity. Active knee extension and active knee flexion was measured in 339 athletes. Active knee extension was significantly higher in women than in men and significantly positively associated with weekly hours of swimming and weekly hours of competitive gymnastics. Active knee flexion was significantly positively associated with participation in basketball, and significantly negatively associated with age and weekly hours of soccer, European team handball and swimming. The results point to sport-specific adaptation of active knee flexion and active knee extension.

  17. Strain- and sex-specific differences in daily flight activity and the circadian clock of Anopheles gambiae mosquitoes.

    PubMed

    Rund, Samuel S C; Lee, Samuel J; Bush, Brian R; Duffield, Giles E

    2012-12-01

    Anopheles gambiae, the primary African malaria vector, is currently speciating into two incipient species, the so-called "molecular forms" M and S. While some geographic areas may contain only one form, in many areas both forms are found coexisting, but reproductively isolated. It appears that spatial segregation of mating swarms may contribute significantly to reproductive isolation as in many locales single-form swarms exist almost exclusively even though they are in close geographic proximity. The mechanism causing this spatial segregation is not well understood. Here, we compare the locomotor flight activity of M and S form male and female An. gambiae mosquitoes with the goal of identifying potential strain-specific temporal differences that could potentially serve as a mating barrier. We use an infrared beam break method to monitor flight activity of individual mosquitoes with a minute-to-minute time resolution under both LD cycle and constant dark conditions. We compare daily total flight activity, activity onset, peak in early nocturnal activity, the build up of dusk-related activity, and the free-running circadian period length. Our investigations revealed strain- and sex-specific differences in total daily activity. In both forms, males commenced nightly flight activity earlier than females, and this corresponded with a significantly shorter circadian period length in males compared to females. We note strain-specific differences in this response to dusk as males of the M form have a pronounced build up in flight activity relative to the S form males prior to complete darkness. This is likely driven by a differential response to the decreasing light intensity at dusk. We hypothesize that this behavioral difference could be a temporal factor contributing to the assembly of single-form swarms. PMID:23068991

  18. Chicken liver glutamate dehydrogenase (GDH) demonstrates a histone H3 specific protease (H3ase) activity in vitro.

    PubMed

    Purohit, Jogeswar S; Tomar, Raghuvir S; Panigrahi, Anil K; Pandey, Shashibhal M; Singh, Divya; Chaturvedi, Madan M

    2013-11-01

    Site-specific proteolysis of the N or C-terminus of histone tails has emerged as a novel form of irreversible post-translational modifications assigned to histones. Though there are many reports describing histone specific proteolysis, there are very few studies on purification of a histone specific protease. Here, we demonstrate a histone H3 specific protease (H3ase) activity in chicken liver nuclear extract. H3ase was purified to homogeneity and identified as glutamate dehydrogenase (GDH) by sequencing. A series of biochemical experiments further confirmed that the H3ase activity was due to GDH. The H3ase clipped histone H3 products were sequenced by N-terminal sequencing and the precise clipping sites of H3ase were mapped. H3ase activity was only specific to chicken liver as it was not demonstrated in other tissues like heart, muscle and brain of chicken. We assign a novel serine like protease activity to GDH which is specific to histone H3. PMID:23856561

  19. Effect of pregnancy-specific β1-glycoprotein on indoleamine-2,3-dioxygenase activity in human monocytes.

    PubMed

    Zamorina, S A; Timganova, V P; Bochkova, M S; Khramtsov, P V; Raev, M B

    2016-07-01

    The role of heterogenic human pregnancy-specific glycoprotein (PSG), obtained by the authors' technology, in the regulation of the indoleamine-2,3-dioxygenase (IDO) activity in female blood monocytes has been studied in vitro. PSG stimulated IDO activity under the conditions of induction of the monocytes by interferon-γ. Upon the induction of cell proliferation by lipopolysaccharides, the stimulating effect was obtained only with 10 μg/mL of PSG. Enhanced IDO activity is probably a factor of peripheral immunological tolerance and antimicrobial protection against intracellular infections in the gestation period. PMID:27595833

  20. Homology modeling studies of yeast Mitogen-Activated Protein Kinases (MAPKS): structural motifs as a basis for specificity.

    PubMed

    Smith, D L; Nilar, S H

    2010-06-01

    Mitogen-activated protein kinases (MAPKs) are key components of cellular signal transduction. It is the objective of this communication to demonstrate that insight into protein-protein interactions in the Common Docking motif of yeast mitogen-activated protein kinases can be obtained based on homology models. Homology models for four yeast MAPKs, FUS3, KSS1, HOG1 and MPK1 were built based on the X-ray structures of active and inactive rat ERK2. The structural motifs required for the basis of specificity were rationalized based on these structures. PMID:19995338

  1. A novel mouse model of intrahepatic cholangiocarcinoma induced by liver-specific Kras activation and Pten deletion

    PubMed Central

    Ikenoue, Tsuneo; Terakado, Yumi; Nakagawa, Hayato; Hikiba, Yohko; Fujii, Tomoaki; Matsubara, Daisuke; Noguchi, Rei; Zhu, Chi; Yamamoto, Keisuke; Kudo, Yotaro; Asaoka, Yoshinari; Yamaguchi, Kiyoshi; Ijichi, Hideaki; Tateishi, Keisuke; Fukushima, Noriyoshi; Maeda, Shin; Koike, Kazuhiko; Furukawa, Yoichi

    2016-01-01

    Intrahepatic cholangiocarcinoma (ICC) is an aggressive malignancy with poor prognosis and its incidence is increasing worldwide. Recently, several types of cells have been considered as the origin of ICC, namely cholangiocytes, liver progenitor cells, and hepatocytes. Here, we have established a novel mouse model of ICC by liver-specific Kras activation and Pten deletion. An activating mutation of Kras in combination with deletion of Pten was introduced in embryonic hepatic bipotential progenitor cells (so-called hepatoblasts) and mature hepatocytes using the Cre-loxP system. As a result, liver-specific Kras activation and homozygous Pten deletion cooperated to induce ICCs exclusively. In contrast, Kras activation in combination with heterozygous Pten deletion induced both ICCs and HCCs, whereas Kras activation alone resulted in HCCs but not ICCs. Furthermore, a cell-lineage visualization system using tamoxifen-inducible Cre-loxP demonstrated that the ICCs did not originate from hepatocytes but from cholangiocytes. Our data suggest that mice carrying liver-specific Kras activation in combination with homozygous Pten deletion should be useful for the investigation of therapeutic strategies for human ICC. PMID:27032374

  2. Tissue-specific changes of glutamine synthetase activity in oats after rhizosphere infestation by Pseudomonas syringae pv. tabaci. Final report

    SciTech Connect

    Knight, T.J.; Temple, S.; Sengupta-Gopalan, C.

    1996-05-15

    Oats (Avena sativa L. lodi) tolerant of rhizosphere infestation by Pseudomonas syringae pv. tabaci when challenged by the pathogen experience tissue-specific alterations of ammonia assimilatory capabilities. Altered ammonia assimilatory potentials between root and leaf tissue result from selective inactivation of glutamine synthetase (GS) by the toxin Tabtoxinine-B-lactam (TBL). Root GS is sensitive and leaf GSs are resistant to TBL inactivation. With prolonged challenge by the pathogen root GS activity decreases but leaf GS specific activity increase. Higher leaf GS activity is due to decreased rates of degradation rather than increased GS synthesis. Higher leaf GS activity and elevated levels of GS polypeptide appear to result from a limited interaction between GS and TBL leading to the accumulation of a less active but more stable GS holoenzyme. Tolerant challenged oats besides surviving rhizosphere infestation, experience enhanced growth. A strong correlation exists between leaf GS activity and whole plant fresh weight, suggesting that tissue-specific changes in ammonia assimilatory capability provides the plant a more efficient mechanism for uptake and utilization of nitrogen.

  3. Science Activities: Specific Problems--Some Solutions. Learning in Science Project. Working Paper No. 48.

    ERIC Educational Resources Information Center

    Tasker, Ross, Ed.; Lambert, John, Ed.

    The Learning in Science Project established that children from a young age construct out of their everyday experiences views which they use to explain their world and that these views are remarkably resistant to replacement by scientifically more useful views. Although science lessons tend to be based upon activities which are designed by teachers…

  4. Timing-specific transfer of adapted muscle activity after walking in an elastic force field.

    PubMed

    Blanchette, Andreanne; Bouyer, Laurent J

    2009-07-01

    Human locomotion results from interactions between feedforward (central commands from voluntary and automatic drive) and feedback (peripheral commands from sensory inputs) mechanisms. Recent studies have shown that locomotion can be adapted when an external force is applied to the lower limb. To better understand the neural control of this adaptation, the present study investigated gait modifications resulting from exposure to a position-dependent force field. Ten subjects walked on a treadmill before, during, and after exposure to a force field generated by elastic tubing that pulled the foot forward and up during swing. Lower limb kinematics and electromyographic (EMG) activity were recorded during each walking period. During force field exposure, peak foot velocity was initially increased by 38%. As subjects adapted, peak foot velocity gradually returned to baseline in activity started earlier (16% before toe off) and remained elevated throughout swing. After force field exposure, foot velocity was initially reduced by 22% and returned to baseline in 9-51 strides. Aftereffects in hamstring EMGs consisted of increased activity around toe off. Contrary to the adapted state, this increase was not maintained during the rest of swing. Together, these results suggest that while the neural control of human locomotion can adapt to force field exposure, the mechanisms underlying this adaptation may vary according to the timing in the gait cycle. Adapted hamstring EMG activity may rely more on feedforward mechanisms around toe off and more on feedback mechanisms during the rest of swing. PMID:19420121

  5. Beyond Rhyme or Reason: ERPs Reveal Task-Specific Activation of Orthography on Spoken Language

    ERIC Educational Resources Information Center

    Pattamadilok, Chotiga; Perre, Laetitia; Ziegler, Johannes C.

    2011-01-01

    Metaphonological tasks, such as rhyme judgment, have been the primary tool for the investigation of the effects of orthographic knowledge on spoken language. However, it has been recently argued that the orthography effect in rhyme judgment does not reflect the automatic activation of orthographic codes but rather stems from sophisticated response…

  6. Liamocin oil from Aureobasidium pullulans has antibacterial activity with specificity for species of Streptococcus

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Liamocin oil from Aureobasidium pullulans NRRL 50380 was tested for antibacterial activity. Liamocins inhibited growth of Streptococcus agalactiae, S. uberis, S. mitis, S. infantarius, and S. mutans, with minimum inhibitory concentrations from 20 'g/ml to 78 'g/ml. Enterococcus faecalis was less sus...

  7. Activity Level as a Dimension of Temperament in Toddlers: Its Relevance for the Organismic Specificity Hypothesis.

    ERIC Educational Resources Information Center

    Gandour, Mary Jane

    1989-01-01

    Findings indicated a significant interaction between activity level and intensity of stimulation in predicting exploratory competence and total exploration among 52 toddlers of 15 months of age who were observed in their homes 6 times over a 4-week period. (RH)

  8. Reduced Specificity of Hippocampal and Posterior Ventrolateral Prefrontal Activity during Relational Retrieval in Normal Aging

    ERIC Educational Resources Information Center

    Giovanello, Kelly S.; Schacter, Daniel L.

    2012-01-01

    Neuroimaging studies of episodic memory in young adults demonstrate greater functional neural activity in ventrolateral pFC and hippocampus during retrieval of relational information as compared with item information. We tested the hypothesis that healthy older adults--individuals who exhibit behavioral declines in relational memory--would show…

  9. Determination of the specific activities of methionine sulfoxide reductase A and B by capillary electrophoresis

    Technology Transfer Automated Retrieval System (TEKTRAN)

    A capillary electrophoresis (CE) method for the determination of methionine sulfoxide reductase A and methionine sulfoxide reductase B activities in mouse liver is described. The method is based on detection of the 4-(dimethylamino)azobenzene-4’-sulfonyl derivative of L-methionine (dabsyl Met), the ...

  10. A Discrete Population of Neurons in the Lateral Amygdala Is Specifically Activated by Contextual Fear Conditioning

    ERIC Educational Resources Information Center

    Wilson, Yvette M.; Murphy, Mark

    2009-01-01

    There is no clear identification of the neurons involved in fear conditioning in the amygdala. To search for these neurons, we have used a genetic approach, the "fos-tau-lacZ" (FTL) mouse, to map functionally activated expression in neurons following contextual fear conditioning. We have identified a discrete population of neurons in the lateral…

  11. A specific mechanism for nonspecific activation in reporter-gene assays.

    PubMed

    Auld, Douglas S; Thorne, Natasha; Nguyen, Dac-Trung; Inglese, James

    2008-08-15

    The importance of bioluminescence in enabling a broad range of high-throughput screening (HTS) assay formats is evidenced by widespread use in industry and academia. Therefore, understanding the mechanisms by which reporter enzyme activity can be modulated by small molecules is critical to the interpretation of HTS data. In this Perspective, we provide evidence for stabilization of luciferase by inhibitors in cell-based luciferase reporter-gene assays resulting in the counterintuitive phenomenon of signal activation. These data were derived from our analysis of luciferase inhibitor compound structures and their prevalence in the Molecular Libraries Small Molecule Repository using 100 HTS experiments available in PubChem. Accordingly, we found an enrichment of luciferase inhibitors in luciferase reporter-gene activation assays but not in assays using other reporters. In addition, for several luciferase inhibitor chemotypes, we measured reporter stabilization and signal activation in cells that paralleled the inhibition determined using purified luciferase to provide further experimental support for these contrasting effects.

  12. Metabolic, anabolic, and mitogenic insulin responses: A tissue-specific perspective for insulin receptor activators

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Insulin acts as the major regulator of the fasting-to-fed metabolic transition by altering substrate metabolism, promoting energy storage, and helping activate protein synthesis. In addition to its glucoregulatory and other metabolic properties, insulin can also act as a growth factor. The metabolic...

  13. Engineering of a red-light–activated human cAMP/cGMP-specific phosphodiesterase

    PubMed Central

    Gasser, Carlos; Taiber, Sandra; Yeh, Chen-Min; Wittig, Charlotte Helene; Hegemann, Peter; Ryu, Soojin; Wunder, Frank; Möglich, Andreas

    2014-01-01

    Sensory photoreceptors elicit vital physiological adaptations in response to incident light. As light-regulated actuators, photoreceptors underpin optogenetics, which denotes the noninvasive, reversible, and spatiotemporally precise perturbation by light of living cells and organisms. Of particular versatility, naturally occurring photoactivated adenylate cyclases promote the synthesis of the second messenger cAMP under blue light. Here, we have engineered a light-activated phosphodiesterase (LAPD) with complementary light sensitivity and catalytic activity by recombining the photosensor module of Deinococcus radiodurans bacterial phytochrome with the effector module of Homo sapiens phosphodiesterase 2A. Upon red-light absorption, LAPD up-regulates hydrolysis of cAMP and cGMP by up to sixfold, whereas far-red light can be used to down-regulate activity. LAPD also mediates light-activated cAMP and cGMP hydrolysis in eukaryotic cell cultures and in zebrafish embryos; crucially, the biliverdin chromophore of LAPD is available endogenously and does not need to be provided exogenously. LAPD thus establishes a new optogenetic modality that permits light control over diverse cAMP/cGMP-mediated physiological processes. Because red light penetrates tissue more deeply than light of shorter wavelengths, LAPD appears particularly attractive for studies in living organisms. PMID:24889611

  14. Exactin: A specific inhibitor of Factor X activation by extrinsic tenase complex from the venom of Hemachatus haemachatus

    PubMed Central

    Girish, Vallerinteavide Mavelli; Kini, R. Manjunatha

    2016-01-01

    Unwanted clots lead to heart attack and stroke that result in a large number of deaths. Currently available anticoagulants have some drawbacks including their non-specific actions. Therefore novel anticoagulants that target specific steps in the coagulation pathway are being sought. Here we describe the identification and characterization of a novel anticoagulant protein from the venom of Hemachatus haemachatus (African Ringhals cobra) that specifically inhibits factor X (FX) activation by the extrinsic tenase complex (ETC) and thus named as exactin. Exactin belongs to the three-finger toxin (3FTx) family, with high sequence identity to neurotoxins and low identity to the well-characterized 3FTx anticoagulants-hemextin and naniproin. It is a mixed-type inhibitor of ETC with the kinetic constants, Ki’ and Ki determined as 30.62 ± 7.73 nM and 153.75 ± 17.96 nM, respectively. Exactin does not bind to the active site of factor VIIa and factor Xa based on its weak inhibition (IC50 ≫ 300 μM) to the amidolytic activities of these proteases. Exactin shows exquisite macromolecular specificity to FX activation as compared to factor IX activation by ETC. Exactin thus displays a distinct mechanism when compared to other anticoagulants targeting ETC, with its selective preference to ETC-FX [ES] complex. PMID:27558950

  15. Exactin: A specific inhibitor of Factor X activation by extrinsic tenase complex from the venom of Hemachatus haemachatus.

    PubMed

    Girish, Vallerinteavide Mavelli; Kini, R Manjunatha

    2016-08-25

    Unwanted clots lead to heart attack and stroke that result in a large number of deaths. Currently available anticoagulants have some drawbacks including their non-specific actions. Therefore novel anticoagulants that target specific steps in the coagulation pathway are being sought. Here we describe the identification and characterization of a novel anticoagulant protein from the venom of Hemachatus haemachatus (African Ringhals cobra) that specifically inhibits factor X (FX) activation by the extrinsic tenase complex (ETC) and thus named as exactin. Exactin belongs to the three-finger toxin (3FTx) family, with high sequence identity to neurotoxins and low identity to the well-characterized 3FTx anticoagulants-hemextin and naniproin. It is a mixed-type inhibitor of ETC with the kinetic constants, Ki' and Ki determined as 30.62 ± 7.73 nM and 153.75 ± 17.96 nM, respectively. Exactin does not bind to the active site of factor VIIa and factor Xa based on its weak inhibition (IC50 ≫ 300 μM) to the amidolytic activities of these proteases. Exactin shows exquisite macromolecular specificity to FX activation as compared to factor IX activation by ETC. Exactin thus displays a distinct mechanism when compared to other anticoagulants targeting ETC, with its selective preference to ETC-FX [ES] complex.

  16. Exactin: A specific inhibitor of Factor X activation by extrinsic tenase complex from the venom of Hemachatus haemachatus.

    PubMed

    Girish, Vallerinteavide Mavelli; Kini, R Manjunatha

    2016-01-01

    Unwanted clots lead to heart attack and stroke that result in a large number of deaths. Currently available anticoagulants have some drawbacks including their non-specific actions. Therefore novel anticoagulants that target specific steps in the coagulation pathway are being sought. Here we describe the identification and characterization of a novel anticoagulant protein from the venom of Hemachatus haemachatus (African Ringhals cobra) that specifically inhibits factor X (FX) activation by the extrinsic tenase complex (ETC) and thus named as exactin. Exactin belongs to the three-finger toxin (3FTx) family, with high sequence identity to neurotoxins and low identity to the well-characterized 3FTx anticoagulants-hemextin and naniproin. It is a mixed-type inhibitor of ETC with the kinetic constants, Ki' and Ki determined as 30.62 ± 7.73 nM and 153.75 ± 17.96 nM, respectively. Exactin does not bind to the active site of factor VIIa and factor Xa based on its weak inhibition (IC50 ≫ 300 μM) to the amidolytic activities of these proteases. Exactin shows exquisite macromolecular specificity to FX activation as compared to factor IX activation by ETC. Exactin thus displays a distinct mechanism when compared to other anticoagulants targeting ETC, with its selective preference to ETC-FX [ES] complex. PMID:27558950

  17. Expression of Overdominance for Specific Activity at the Phosphoglucomutase-2 Locus in the Pacific Oyster, Crassostrea Gigas

    PubMed Central

    Pogson, G. H.

    1991-01-01

    Environmental and genetic components of specific activity variation at the phosphoglucomutase-2 locus in the Pacific oyster, Crassostrea gigas, were examined to assess the direct role played by this polymorphism in a heterozygosity/growth relationship. Both environmental variables studied, season and intertidal position, exerted highly significant effects on phosphoglucomutase specific activity but no interactions occurred between these factors and Pgm-2 genotype. Highly significant differences were also detected between Pgm-2 genotypes. The three most common heterozygotes (Pgm-2(92/100), Pgm-2(96/100) and Pgm-2(100/104)) consistently expressed greater specific activities than the Pgm-2(92/92), Pgm-2(96/96), Pgm-2(100/100) and Pgm-2(104/104) homozygotes. Overall, the specific activities of heterozygotes for the Pgm-2(100) allele exceeded heterozygotes by 24% and 20% in the mantle and adductor muscle tissues, respectively. Heterozygotes formed between the three less frequent Pgm-2(92), Pgm-2(96) and Pgm-2(104) alleles differed sharply from those possessing the Pgm-2(100) allele in being indistinguishable from homozygotes. The possibility of these patterns arising from the undetected presence of an inactive Pgm-2 allele was examined and found to be inconsistent with all of its predicted effects on the specific activity data. Genuine overdominance was shown to be capable of explaining the specific activities of ten structural locus genotypes, allelic frequency distributions in natural populations, and the maintenance of the enzyme polymorphism in a balanced state. The results provide evidence favoring the overdominance explanation for one locus involved in a heterozygosity/growth relationship and suggest that the reported effects of this locus on adult body weight may have been caused by the greater flux capacities of heterozygotes for the Pgm-2(100) allele. PMID:1829426

  18. Inhibition of Plasma Kallikrein by a Highly Specific Active Site Blocking Antibody

    PubMed Central

    Kenniston, Jon A.; Faucette, Ryan R.; Martik, Diana; Comeau, Stephen R.; Lindberg, Allison P.; Kopacz, Kris J.; Conley, Gregory P.; Chen, Jie; Viswanathan, Malini; Kastrapeli, Niksa; Cosic, Janja; Mason, Shauna; DiLeo, Mike; Abendroth, Jan; Kuzmic, Petr; Ladner, Robert C.; Edwards, Thomas E.; TenHoor, Christopher; Adelman, Burt A.; Nixon, Andrew E.; Sexton, Daniel J.

    2014-01-01

    Plasma kallikrein (pKal) proteolytically cleaves high molecular weight kininogen to generate the potent vasodilator and the pro-inflammatory peptide, bradykinin. pKal activity is tightly regulated in healthy individuals by the serpin C1-inhibitor, but individuals with hereditary angioedema (HAE) are deficient in C1-inhibitor and consequently exhibit excessive bradykinin generation that in turn causes debilitating and potentially fatal swelling attacks. To develop a potential therapeutic agent for HAE and other pKal-mediated disorders, we used phage display to discover a fully human IgG1 monoclonal antibody (DX-2930) against pKal. In vitro experiments demonstrated that DX-2930 potently inhibits active pKal (Ki = 0.120 ± 0.005 nm) but does not target either the zymogen (prekallikrein) or any other serine protease tested. These findings are supported by a 2.1-Å resolution crystal structure of pKal complexed to a DX-2930 Fab construct, which establishes that the pKal active site is fully occluded by the antibody. DX-2930 injected subcutaneously into cynomolgus monkeys exhibited a long half-life (t½ ∼12.5 days) and blocked high molecular weight kininogen proteolysis in activated plasma in a dose- and time-dependent manner. Furthermore, subcutaneous DX-2930 reduced carrageenan-induced paw edema in rats. A potent and long acting inhibitor of pKal activity could be an effective treatment option for pKal-mediated diseases, such as HAE. PMID:24970892

  19. Task-specific stability in muscle activation space during unintentional movements.

    PubMed

    Falaki, Ali; Towhidkhah, Farzad; Zhou, Tao; Latash, Mark L

    2014-11-01

    We used robot-generated perturbations applied during position-holding tasks to explore stability of induced unintentional movements in a multidimensional space of muscle activations. Healthy subjects held the handle of a robot against a constant bias force and were instructed not to interfere with hand movements produced by changes in the external force. Transient force changes were applied leading to handle displacement away from the initial position and then back toward the initial position. Intertrial variance in the space of muscle modes (eigenvectors in the muscle activations space) was quantified within two subspaces, corresponding to unchanged handle coordinate and to changes in the handle coordinate. Most variance was confined to the former subspace in each of the three phases of movement, the initial steady state, the intermediate position, and the final steady state. The same result was found when the changes in muscle activation were analyzed between the initial and final steady states. Changes in the dwell time between the perturbation force application and removal led to different final hand locations undershooting the initial position. The magnitude of the undershot scaled with the dwell time, while the structure of variance in the muscle activation space did not depend on the dwell time. We conclude that stability of the hand coordinate is ensured during both intentional and unintentional actions via similar mechanisms. Relative equifinality in the external space after transient perturbations may be associated with varying states in the redundant space of muscle activations. The results fit a hierarchical scheme for the control of voluntary movements with referent configurations and redundant mapping between the levels of the hierarchy. PMID:25092272

  20. TASK-SPECIFIC STABILITY IN MUSCLE ACTIVATION SPACE DURING UNINTENTIONAL MOVEMENTS

    PubMed Central

    Falaki, Ali; Towhidkhah, Farzad; Zhou, Tao; Latash, Mark L.

    2014-01-01

    We used robot-generated perturbations applied during position-holding tasks to explore stability of induced unintentional movements in a multi-dimensional space of muscle activations. Healthy subjects held the handle of a robot against a constant bias force and were instructed not to interfere with hand movements produced by changes in the external force. Transient force changes were applied leading to handle displacement away from the initial position and then back towards the initial position. Inter-trial variance in the space of muscle modes (eigenvectors in the muscle activations space) was quantified within two sub-spaces, corresponding to unchanged handle coordinate and to changes in the handle coordinate. Most variance was confined to the former sub-space in each of the three phases of movement, the initial steady state, the intermediate position, and the final steady state. The same result was found when the changes in muscle activation were analyzed between the initial and final steady states. Changes in the dwell time between the perturbation force application and removal led to different final hand locations undershooting the initial position. The magnitude of the undershot scaled with the dwell time, while the structure of variance in the muscle activation space did not depend on the dwell time. We conclude that stability of the hand coordinate is ensured during both intentional and unintentional actions via similar mechanisms. Relative equifinality in the external space after transient perturbations may be associated with varying states in the redundant space of muscle activations. The results fit a hierarchical scheme for the control of voluntary movements with referent configurations and redundant mapping between the levels of the hierarchy. PMID:25092272

  1. Chain length specificity for activation of cPLA2alpha by C1P: use of the dodecane delivery system to determine lipid-specific effects.

    PubMed

    Wijesinghe, Dayanjan S; Subramanian, Preeti; Lamour, Nadia F; Gentile, Luciana B; Granado, Maria H; Bielawska, Alicja; Szulc, Zdzislaw; Gomez-Munoz, Antonio; Chalfant, Charles E

    2009-10-01

    Previously, our laboratory demonstrated that ceramide-1-phosphate (C1P) specifically activated group IVA cytosolic phospholipase A(2) (cPLA(2)alpha) in vitro. In this study, we investigated the chain length specificity of this interaction. C1P with an acyl-chain of >or=6 carbons efficiently activated cPLA(2)alpha in vitro, whereas C(2)-C1P, was unable to do so. Delivery of C1P to cells via the newly characterized ethanol/dodecane system demonstrated a lipid-specific activation of cPLA(2)alpha, AA release, and PGE(2) synthesis (EC(50) = 400 nM) when compared to structurally similar lipids. C1P delivered as vesicles in water also induced a lipid-specific increase in AA release. Mass spectrometric analysis demonstrated that C1P delivered via ethanol/dodecane induced a 3-fold increase in endogenous C1P with little metabolism to ceramide. C1P was also more efficiently delivered (>3-fold) to internal membranes by ethanol/dodecane as compared to vesiculated C1P. Using this now established delivery method for lipids, C(2)-C1P was shown to be ineffective in the induction of AA release as compared with C(6)-C1P, C(16)-C1P, and C(18:1) C1P. Here, we demonstrate that C1P requires >or=6 carbon acyl-chain to activate cPLA(2)alpha. Thus, published reports on the biological activity of C(2)-C1P are not via eicosanoid synthesis. Furthermore, this study demonstrates that the alcohol/dodecane system can be used to efficiently deliver exogenous phospholipids to cells for the examination of specific biological effects.

  2. Identification of a Male-Specific RNA Binding Protein That Regulates Sex-Specific Splicing of Bmdsx by Increasing RNA Binding Activity of BmPSI▿ §

    PubMed Central

    Suzuki, Masataka G.; Imanishi, Shigeo; Dohmae, Naoshi; Asanuma, Miwako; Matsumoto, Shogo

    2010-01-01

    Bmdsx is a sex-determining gene in the silkworm and is alternatively spliced in males and females. CE1 is a splicing silencer element responsible for the sex-specific splicing of Bmdsx. To identify sex-specific factors implicated in the sex-specific splicing of Bmdsx, we performed RNA affinity chromatography using CE1 RNA as a ligand. We have identified BmIMP, a Bombyx homolog of IGF-II mRNA binding protein (IMP), as a male-specific factor that specifically binds to CE1. The gene encoding BmIMP is localized on the Z chromosome and is male-specifically expressed in various tissues. Antisense inhibition of BmIMP expression increased female-specific splicing of Bmdsx pre-mRNA. Coimmunoprecipitation and glutathione S-transferase (GST) pulldown analyses demonstrated that BmIMP physically interacts with BmPSI, which has been identified as a factor implicated in the sex-specific splicing of Bmdsx, through the KH domains of BmIMP. The functional consequence of this interaction was examined using RNA mobility shift analysis. BmIMP increased BmPSI-CE1 RNA binding activity by decreasing the rate of BmPSI dissociation from CE1 RNA. Truncation analysis of BmIMP suggested that the KH domains are responsible for enhancing BmPSI-CE1 RNA binding activity. These results suggest that BmIMP may enhance the male-specific splicing of Bmdsx pre-mRNA by increasing RNA binding activity of BmPSI. PMID:20956562

  3. Cell-specific activity of the modulator region in the human cytomegalovirus major immediate-early gene

    SciTech Connect

    Lubon, H.K.; Hennighausen, L. ); Ghazal, P.; Reynolds-Kohler, C.; Lockshin, C.; Nelson, J. )

    1989-03-01

    In this paper the authors demonstrate that modular sequences upstream of the enhancer of the major immediate-early promoter of human cytomegalovirus exert a differential effort on the level of transcription in a variety of cells and that this region has the capacity to interact with specific nuclear protein. Depending on the cell type, these modulator sequences increased or decreased transcriptional activation from the IE1 gene promoter-enhancer. The cell lines identified in this report should be useful to study the molecular mechanism of cell-specific transcriptional repression and activation exerted by the major immediate-early promoter upstream region.

  4. Phosphatidylinositol 3-kinase signals activation of p70 S6 kinase in situ through site-specific p70 phosphorylation.

    PubMed Central

    Weng, Q P; Andrabi, K; Klippel, A; Kozlowski, M T; Williams, L T; Avruch, J

    1995-01-01

    The p70 S6 kinase is activated by insulin and mitogens through multisite phosphorylation of the enzyme. One set of activating phosphorylations occurs in a putative autoinhibitory domain in the noncatalytic carboxyl-terminal tail. Deletion of this tail yields a variant (p70 delta CT104) that nevertheless continues to be mitogen regulated. Coexpression with a recombinant constitutively active phosphatidylinositol (PI) 3-kinase (EC 2.7.1.137) gives substantial activation of both full-length p70 and p70 delta CT104 but not Rsk. Activation of p70 delta CT104 by PI 3-kinase and inhibition by wortmannin are each accompanied by parallel and selective changes in the phosphorylation of p70 Thr-252. A Thr or Ser at this site, in subdomain VIII of the catalytic domain just amino-terminal to the APE motif, is necessary for p70 40S kinase activity. The inactive ATP-binding site mutant K123M p70 delta CT104 undergoes phosphorylation of Thr-252 in situ but does not undergo direct phosphorylation by the active PI 3-kinase in vitro. PI 3-kinase provides a signal necessary for the mitogen activation of the p70 S6 kinase, which directs the site-specific phosphorylation of Thr-252 in the p70 catalytic domain, through a distinctive signal transduction pathway. Images Fig. 1 Fig. 2 Fig. 3 PMID:7777579

  5. An M-phase-specific protein kinase of Xenopus oocytes: partial purification and possible mechanism of its periodic activation.

    PubMed

    Labbé, J C; Picard, A; Karsenti, E; Dorée, M

    1988-05-01

    The activity of a Ca2+- and cyclic nucleotide-independent protein kinase(s) which catalyzes hyperphosphorylation of a set of endogenous proteins, including a 95-kDa soluble phosphoprotein, is found to fluctuate in both the meiotic and mitotic cell cycles of Xenopus oocytes and activated eggs. The activity is high in M-phase and hardly detectable in interphase. The activity copurifies with a major histone kinase(s) throughout four purification steps: ammonium sulfate precipitation, DEAE-cellulose chromatography, high-performance liquid chromatography on TSK G3000, and CM-Sepharose chromatography. This suggests that a single enzyme shares activity against endogenous proteins and added histones. Changes in the activity of the M-phase-specific protein kinase(s) as assayed in vitro correlate with changes in the extent of protein phosphorylation in oocytes pulse-labeled with 32P-phosphate by microinjection during meiotic maturation and the early embryonic cell cycle. This suggests that the kinase(s) has a broad specificity and plays a key role in the increased protein phosphorylation which occurs at the transition to M-phase. Microinjection of the maturation-promoting factor (MPF) into immature oocytes triggers, after a 10-min lag period, the activation of the M-phase specific kinase(s), even in the absence of protein synthesis. In contrast MPF microinjection does not induce kinase activation in cycloheximide-treated oocytes arrested after completion of the first meiotic cell cycle or in activated eggs arrested in S-phase by incubation in cycloheximide. This suggests that immature oocytes contain an inactive kinase precursor (prokinase) which is synthesized at each of the following cell cycles. In the absence of MPF addition, the prokinase to kinase transition occurs "spontaneously" after a 2-hr lag period in high-speed supernatants prepared from prophase-arrested oocytes if low-molecular-weight metabolites are eliminated by gel filtration. Addition of ATP, but not of

  6. Rescue of PINK1 Protein Null-specific Mitochondrial Complex IV Deficits by Ginsenoside Re Activation of Nitric Oxide Signaling*

    PubMed Central

    Kim, Kyung-Hee; Song, Karen; Yoon, Seung-Hee; Shehzad, Omer; Kim, Yeong-Shik; Son, Jin H.

    2012-01-01

    PINK1, linked to familial Parkinson's disease, is known to affect mitochondrial function. Here we identified a novel regulatory role of PINK1 in the maintenance of complex IV activity and characterized a novel mechanism by which NO signaling restored complex IV deficiency in PINK1 null dopaminergic neuronal cells. In PINK1 null cells, levels of specific chaperones, including Hsp60, leucine-rich pentatricopeptide repeat-containing (LRPPRC), and Hsp90, were severely decreased. LRPPRC and Hsp90 were found to act upstream of Hsp60 to regulate complex IV activity. Specifically, knockdown of Hsp60 resulted in a decrease in complex IV activity, whereas antagonistic inhibition of Hsp90 by 17-(allylamino) geldanamycin decreased both Hsp60 and complex IV activity. In contrast, overexpression of the PINK1-interacting factor LRPPRC augmented complex IV activity by up-regulating Hsp60. A similar recovery of complex IV activity was also induced by coexpression of Hsp90 and Hsp60. Drug screening identified ginsenoside Re as a compound capable of reversing the deficit in complex IV activity in PINK1 null cells through specific increases of LRPPRC, Hsp90, and Hsp60 levels. The pharmacological effects of ginsenoside Re could be reversed by treatment of the pan-NOS inhibitor l-NG-Nitroarginine Methyl Ester (l-NAME) and could also be reproduced by low-level NO treatment. These results suggest that PINK1 regulates complex IV activity via interactions with upstream regulators of Hsp60, such as LRPPRC and Hsp90. Furthermore, they demonstrate that treatment with ginsenoside Re enhances functioning of the defective PINK1-Hsp90/LRPPRC-Hsp60-complex IV signaling axis in PINK1 null neurons by restoring NO levels, providing potential for new therapeutics targeting mitochondrial dysfunction in Parkinson's disease. PMID:23144451

  7. Rescue of PINK1 protein null-specific mitochondrial complex IV deficits by ginsenoside Re activation of nitric oxide signaling.

    PubMed

    Kim, Kyung-Hee; Song, Karen; Yoon, Seung-Hee; Shehzad, Omer; Kim, Yeong-Shik; Son, Jin H

    2012-12-28

    PINK1, linked to familial Parkinson's disease, is known to affect mitochondrial function. Here we identified a novel regulatory role of PINK1 in the maintenance of complex IV activity and characterized a novel mechanism by which NO signaling restored complex IV deficiency in PINK1 null dopaminergic neuronal cells. In PINK1 null cells, levels of specific chaperones, including Hsp60, leucine-rich pentatricopeptide repeat-containing (LRPPRC), and Hsp90, were severely decreased. LRPPRC and Hsp90 were found to act upstream of Hsp60 to regulate complex IV activity. Specifically, knockdown of Hsp60 resulted in a decrease in complex IV activity, whereas antagonistic inhibition of Hsp90 by 17-(allylamino) geldanamycin decreased both Hsp60 and complex IV activity. In contrast, overexpression of the PINK1-interacting factor LRPPRC augmented complex IV activity by up-regulating Hsp60. A similar recovery of complex IV activity was also induced by coexpression of Hsp90 and Hsp60. Drug screening identified ginsenoside Re as a compound capable of reversing the deficit in complex IV activity in PINK1 null cells through specific increases of LRPPRC, Hsp90, and Hsp60 levels. The pharmacological effects of ginsenoside Re could be reversed by treatment of the pan-NOS inhibitor L-NG-Nitroarginine Methyl Ester (L-NAME) and could also be reproduced by low-level NO treatment. These results suggest that PINK1 regulates complex IV activity via interactions with upstream regulators of Hsp60, such as LRPPRC and Hsp90. Furthermore, they demonstrate that treatment with ginsenoside Re enhances functioning of the defective PINK1-Hsp90/LRPPRC-Hsp60-complex IV signaling axis in PINK1 null neurons by restoring NO levels, providing potential for