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Sample records for high-affinity camp phosphodiesterase

  1. PdeH, a High-Affinity cAMP Phosphodiesterase, Is a Key Regulator of Asexual and Pathogenic Differentiation in Magnaporthe oryzae

    PubMed Central

    Ramanujam, Ravikrishna; Naqvi, Naweed I.

    2010-01-01

    Cyclic AMP-dependent pathways mediate the communication between external stimuli and the intracellular signaling machinery, thereby influencing important aspects of cellular growth, morphogenesis and differentiation. Crucial to proper function and robustness of these signaling cascades is the strict regulation and maintenance of intracellular levels of cAMP through a fine balance between biosynthesis (by adenylate cyclases) and hydrolysis (by cAMP phosphodiesterases). We functionally characterized gene-deletion mutants of a high-affinity (PdeH) and a low-affinity (PdeL) cAMP phosphodiesterase in order to gain insights into the spatial and temporal regulation of cAMP signaling in the rice-blast fungus Magnaporthe oryzae. In contrast to the expendable PdeL function, the PdeH activity was found to be a key regulator of asexual and pathogenic development in M. oryzae. Loss of PdeH led to increased accumulation of intracellular cAMP during vegetative and infectious growth. Furthermore, the pdeHΔ showed enhanced conidiation (2–3 fold), precocious appressorial development, loss of surface dependency during pathogenesis, and highly reduced in planta growth and host colonization. A pdeHΔ pdeLΔ mutant showed reduced conidiation, exhibited dramatically increased (∼10 fold) cAMP levels relative to the wild type, and was completely defective in virulence. Exogenous addition of 8-Br-cAMP to the wild type simulated the pdeHΔ defects in conidiation as well as in planta growth and development. While a fully functional GFP-PdeH was cytosolic but associated dynamically with the plasma membrane and vesicular compartments, the GFP-PdeL localized predominantly to the nucleus. Based on data from cAMP measurements and Real-Time RTPCR, we uncover a PdeH-dependent biphasic regulation of cAMP levels during early and late stages of appressorial development in M. oryzae. We propose that PdeH-mediated sustenance and dynamic regulation of cAMP signaling during M. oryzae development is

  2. Regulation of Adrenal Steroidogenesis by the High-affinity Phosphodiesterase 8 Family

    PubMed Central

    Tsai, L-C. L.; Beavo, J. A.

    2014-01-01

    The main function of cyclic AMP phosphodiesterases (PDEs) is to degrade cAMP, a ubiquitous second messenger. Therefore, PDEs can function as prime regulators of cAMP/PKA-dependent processes such as steroidogenesis. Until recently, the roles of the PDE8 family have been largely unexplored, presumably due to the lack of a selective inhibitor. This review focuses on recent reports about the regulatory roles of the PDE8 family in adrenal steroidogenesis, as well as the inhibitory properties and specificity of a new PDE8-selective inhibitor, PF-04957325. We also describe a method of measuring urinary corticosterone levels in vivo as a minimally invasive way of monitoring the stress level in a mouse. PMID:22903278

  3. Phosphodiesterases and subcellular compartmentalized cAMP signaling in the cardiovascular system.

    PubMed

    Stangherlin, Alessandra; Zaccolo, Manuela

    2012-01-01

    Phosphodiesterases are key enzymes in the cAMP signaling cascade. They convert cAMP in its inactive form 5'-AMP and critically regulate the intensity and the duration of cAMP-mediated signals. Multiple isoforms exist that possess different intracellular distributions, different affinities for cAMP, and different catalytic and regulatory properties. This complex repertoire of enzymes provides a multiplicity of ways to modulate cAMP levels, to integrate more signaling pathways, and to respond to the specific needs of the cell within distinct subcellular domains. In this review we summarize key findings on phosphodiesterase compartmentalization in the cardiovascular system.

  4. Modulation of high affinity ATP-dependent cyclic nucleotide transporters by specific and non-specific cyclic nucleotide phosphodiesterase inhibitors.

    PubMed

    Aronsen, Lena; Orvoll, Elin; Lysaa, Roy; Ravna, Aina W; Sager, Georg

    2014-12-15

    Intracellular cyclic nucleotides are eliminated by phosphodiesterases (PDEs) and by ATP Binding cassette transporters such as ABCC4 and ABCC5. PDE5 and ABCC5 have similar affinity for cGMP whereas ABCC5 has much higher affinity for cGMP compared with cAMP. Since the substrate (cGMP) is identical for these two eliminatory processes it is conceivable that various PDE inhibitors also modulate ABCC5-transport. Cyclic GMP is also transported by ABBC4 but the affinity is much lower with a Km 50-100 times higher than for that of ABBCC5. The present study aimed to determine Ki-values for specific or relative specific PDE5 inhibitors (vardenafil, tadalafil, zaprinast and dipyridamole) and the non-specific PDE inhibitors (IBMX, caffeine and theophylline) for ABCC5 and ABCC4 transport. The transport of [(3)H]-cGMP (2 µM) was concentration-dependently inhibited with the following Ki-values: vardenafil (0.62 µM), tadalafil (14.1 µM), zaprinast (0.68 µM) and dipyridamole (1.2 µM), IBMX (10 µM), caffeine (48 µM) and theophylline (69 µM). The Ki-values for the inhibition of the [(3)H]-cAMP (2 µM) transport were: vardenafil (3.4 µM), tadalafil (194 µM), zaprinast (2.8 µM), dipyridamole (5.5 µM), IBMX (16 µM), caffeine (41 µM) and theophylline (85 µM). The specificity for ABCC5 we defined as ratio between Ki-values for inhibition of [(3)H]-cGMP and [(3)H]-cAMP transport. Tadalafil showed the highest specificity (Ki-ratio: 0.073) and caffeine the lowest (Ki-ratio: 1.2). Copyright © 2014 The Authors. Published by Elsevier B.V. All rights reserved.

  5. The High-Affinity Phosphodiesterase BcPde2 Has Impact on Growth, Differentiation and Virulence of the Phytopathogenic Ascomycete Botrytis cinerea

    PubMed Central

    Harren, Karin; Brandhoff, Beate; Knödler, Michael; Tudzynski, Bettina

    2013-01-01

    Components of the cAMP signaling pathway, such as the adenylate cyclase Bac and the protein kinase A (PKA) were shown to affect growth, morphogenesis and differentiation as well as virulence of the phytopathogenic fungus Botrytis cinerea. While loss of Bac caused drastically reduced intracellular cAMP levels, deletion of the PKA resulted in extremely increased cAMP concentrations. To regulate the intracellular level of the second messenger cAMP, a balance between its biosynthesis through adenylate cyclase activity and its hydrolysis by phosphodiesterases (PDEs) is crucial. Here, we report the functional characterization of the two PDEs in the ascomycete B. cinerea, BcPde1 and BcPde2. While deletion of bcpde2 resulted in severely affected vegetative growth, conidiation, germination and virulence, the bcpde1 deletion strain displayed a wild-type-like phenotype. However, the double bcpde1/2 deletion mutant exhibited an even stronger phenotype. Localization studies revealed that BcPde2 accumulates at the plasma membrane, but is also localized in the cytoplasm. BcPde1 was shown to be distributed in the cytoplasm as well, but also accumulates in so far unknown mobile vesicles. Overexpression of bcpde1 in the Δbcpde2 background rescued the deletion phenotype, and in addition an increased transcript level of bcpde1 in the Δbcpde2 strain was observed, indicating redundant functions of both PDEs and an interdependent gene expression. PMID:24265695

  6. Cardiac Hypertrophy Is Inhibited by a Local Pool of cAMP Regulated by Phosphodiesterase 2.

    PubMed

    Zoccarato, Anna; Surdo, Nicoletta C; Aronsen, Jan M; Fields, Laura A; Mancuso, Luisa; Dodoni, Giuliano; Stangherlin, Alessandra; Livie, Craig; Jiang, He; Sin, Yuan Yan; Gesellchen, Frank; Terrin, Anna; Baillie, George S; Nicklin, Stuart A; Graham, Delyth; Szabo-Fresnais, Nicolas; Krall, Judith; Vandeput, Fabrice; Movsesian, Matthew; Furlan, Leonardo; Corsetti, Veronica; Hamilton, Graham; Lefkimmiatis, Konstantinos; Sjaastad, Ivar; Zaccolo, Manuela

    2015-09-25

    Chronic elevation of 3'-5'-cyclic adenosine monophosphate (cAMP) levels has been associated with cardiac remodeling and cardiac hypertrophy. However, enhancement of particular aspects of cAMP/protein kinase A signaling seems to be beneficial for the failing heart. cAMP is a pleiotropic second messenger with the ability to generate multiple functional outcomes in response to different extracellular stimuli with strict fidelity, a feature that relies on the spatial segregation of the cAMP pathway components in signaling microdomains. How individual cAMP microdomains affect cardiac pathophysiology remains largely to be established. The cAMP-degrading enzymes phosphodiesterases (PDEs) play a key role in shaping local changes in cAMP. Here we investigated the effect of specific inhibition of selected PDEs on cardiac myocyte hypertrophic growth. Using pharmacological and genetic manipulation of PDE activity, we found that the rise in cAMP resulting from inhibition of PDE3 and PDE4 induces hypertrophy, whereas increasing cAMP levels via PDE2 inhibition is antihypertrophic. By real-time imaging of cAMP levels in intact myocytes and selective displacement of protein kinase A isoforms, we demonstrate that the antihypertrophic effect of PDE2 inhibition involves the generation of a local pool of cAMP and activation of a protein kinase A type II subset, leading to phosphorylation of the nuclear factor of activated T cells. Different cAMP pools have opposing effects on cardiac myocyte cell size. PDE2 emerges as a novel key regulator of cardiac hypertrophy in vitro and in vivo, and its inhibition may have therapeutic applications. © 2015 American Heart Association, Inc.

  7. Immunological identification of the major platelet low-Km cAMP phosphodiesterase: probable target for anti-thrombotic agents.

    PubMed Central

    Macphee, C H; Harrison, S A; Beavo, J A

    1986-01-01

    Immunoblot and enzyme-activity analyses, using specific immunological probes, indicated that more than 80% of the total low-Km cAMP phosphodiesterase activity present in bovine and human platelets resided in a single phosphodiesterase isozyme. In the presence of protease inhibitors, the platelet enzyme has an apparent subunit size of 110 kDa and appears immunologically and structurally indistinguishable from a recently purified bovine heart isozyme. When protease inhibitors were absent during homogenization and centrifugation, this platelet phosphodiesterase was susceptible to sequential proteolysis forming 80-kDa and 60-kDa peptides. As a previous report on the purification of the platelet low-Km cAMP phosphodiesterase described a 61-kDa protein, our data would suggest that this was a proteolytic fragment. Moreover, in our study a 40-70% increase in catalytic activity was associated with proteolysis. Further similarities between the platelet and heart phosphodiesterases were demonstrated by pharmacological studies that showed identical inhibitor profiles for both enzymes. Several known phosphodiesterase inhibitor compounds that have been found useful in inhibiting platelet aggregation also inhibited the platelet low-Km cAMP phosphodiesterase with potencies very similar to their antithrombotic effects. Cilostamide, Ro 15-2041, milrinone, papaverine, isobutylmethylxanthine, and theophylline inhibited the 110-kDa platelet enzyme with IC50 values of 0.04, 0.13, 0.46, 1.4, 2.6, and 110 microM, respectively. Images PMID:3018742

  8. Effects of repeated treatment with phosphodiesterase-4 inhibitors on cAMP signaling, hippocampal cell proliferation, and behavior in the forced-swim test.

    PubMed

    Xiao, Lan; O'Callaghan, James P; O'Donnell, James M

    2011-08-01

    The effects of repeated treatment with the phosphodiesterase-4 (PDE4) inhibitors rolipram, piclamilast, and 4-(2-(3-(cyclopentyloxy)-4-methoxyphenyl)-2-phenylethyl)pyridine (CDP840), which differ in their interactions with high- and low-affinity binding conformers of the enzyme, were contrasted to those of acute treatment on cAMP signaling, hippocampal cell proliferation, and immobility in the forced-swim test in rats. Repeated treatment with rolipram (1 and 3 mg/kg), piclamilast (0.3 and 1 mg/kg), or CDP840 (10 and 30 mg/kg) for 16 days increased cAMP and phosphorylation of cAMP response element binding protein (pCREB) in hippocampus and prefrontal cortex. In addition, repeated treatment with the PDE4 inhibitors increased proliferation and survival of newborn cells in the hippocampus and produced antidepressant-like effects on behavior, as evidenced by decreased immobility in the forced-swim test. Acute treatment with rolipram (3 mg/kg), piclamilast (1 mg/kg), or CDP840 (30 mg/kg) induced transient increases in cAMP and pCREB in hippocampus and prefrontal cortex, but the dose and time dependence of these effects did not parallel the behavioral effects. Compared with rolipram and piclamilast, repeated treatment with CDP840 exerted lesser effects on neural and behavioral measures, probably because of its weak interaction with the high-affinity binding conformer of PDE4. This suggests the relative importance of the high-affinity binding conformer in the mediation of the long-term effects of PDE4 inhibition on cAMP/pCREB signaling, hippocampal cell proliferation, and antidepressant-like effects on behavior.

  9. Phosphodiesterases in the rat ovary: effect of cAMP in primordial follicles.

    PubMed

    Petersen, Tonny Studsgaard; Stahlhut, Martin; Andersen, Claus Yding

    2015-07-01

    Phosphodiesterases (PDEs) are important regulators of the intracellular cAMP concentration, which is a central second messenger that affects a multitude of intracellular functions. In the ovaries, cAMP exerts diverse functions, including regulation of ovulation and it has been suggested that augmented cAMP levels stimulate primordial follicle growth. The present study examined the gene expression, enzyme activity and immunolocalization of the different cAMP hydrolysing PDEs families in the rat ovary. Further, the effect of PDE4 inhibition on primordial follicle activation in cultured neonatal rat ovaries was also evaluated. We found varied expression of all eight families in the ovary with Pde7b and Pde8a having the highest expression each accounting for more than 20% of the total PDE mRNA. PDE4 accounted for 15-26% of the total PDE activity. Immunoreactive PDE11A was found in the oocytes and PDE2A in the corpora lutea. Incubating neonatal rat ovaries with PDE4 inhibitors did not increase primordial follicle activation or change the expression of the developing follicle markers Gdf9, Amh, Inha, the proliferation marker Mki67 or the primordial follicle marker Tmeff2. In addition, the cAMP analogue 8-bromo-cAMP did not increase AKT1 or FOXO3A phosphorylation associated with follicle activation or increase the expression of Kitlg known to be associated with follicle differentiation but did increase the Tmeff2, Mki67 and Inha expression in a dose-dependent manner. In conclusion, this study shows that both Pde7b and Pde8a are highly expressed in the rodent ovary and that PDE4 inhibition does not cause an increase in primordial follicle activation. © 2015 Society for Reproduction and Fertility.

  10. Resveratrol Ameliorates Aging-Related Metabolic Phenotypes by Inhibiting cAMP Phosphodiesterases

    PubMed Central

    Park, Sung-Jun; Ahmad, Faiyaz; Philp, Andrew; Baar, Keith; Williams, Tishan; Luo, Haibin; Ke, Hengming; Rehmann, Holger; Taussig, Ronald; Brown, Alexandra L.; Kim, Myung K.; Beaven, Michael A.; Burgin, Alex B.; Manganiello, Vincent; Chung, Jay H.

    2012-01-01

    SUMMARY Resveratrol, a polyphenol in red wine, has been reported as a calorie restriction mimetic with potential antiaging and antidiabetogenic properties. It is widely consumed as a nutritional supplement, but its mechanism of action remains a mystery. Here, we report that the metabolic effects of resveratrol result from competitive inhibition of cAMP-degrading phosphodiesterases, leading to elevated cAMP levels. The resulting activation of Epac1, a cAMP effector protein, increases intracellular Ca2+ levels and activates the CamKKβ-AMPK pathway via phospholipase C and the ryanodine receptor Ca2+-release channel. As a consequence, resveratrol increases NAD+ and the activity of Sirt1. Inhibiting PDE4 with rolipram reproduces all of the metabolic benefits of resveratrol, including prevention of diet-induced obesity and an increase in mitochondrial function, physical stamina, and glucose tolerance in mice. Therefore, administration of PDE4 inhibitors may also protect against and ameliorate the symptoms of metabolic diseases associated with aging. PMID:22304913

  11. Characterization of the insulin-sensitive low Km cAMP phosphodiesterase from rat adipose tissue

    SciTech Connect

    Degerman, E.; Belfrage, P.; Manganiello, V.C.

    1986-05-01

    Particulate, but not soluble, low K/sub m/ cAMP phosphodiesterase (PDE) activity of rat adipocytes was increased 50-100% during incubation (10 min) of intact cells with 1-3 nM insulin; activation was less with higher or lower insulin concentrations. Activation was maintained during solubilization with an alkyl polyoxyethylene non-ionic detergent C/sub 13/, E/sub 12/ and NaBr and chromatography on DEAE. Enzyme from DEAE was further purified by chromatography on Sepahadex G-200 and Blue-Sepharose. Activity (with 0.5 ..mu..M (/sup 3/H)cAMP) was rather sensitive to inhibition by p-chloromercuribenzoate (IC/sub 50/, 1 ..mu..M) and less so by 2,2'-dithiobis-(5-nitropyridine) (160 ..mu..M), N-ethylmaleimide (525 ..mu..M) and iodoacetamide (750 ..mu..M). PDE activity was also rather sensitive to inhibition by cilostamide (IC/sub 50/, approx.40 nM) and the cardiotonic drugs CI 930 (450 nM) and milrinone (630 nM) but rather insensitive to RO 20-1724 (190 ..mu..M). Based on effects of these inhibitors, the hormone-sensitive low K/sub m/ particulate cAMP PDE from rat adipocytes seems to be analogous to the insulin-activated particulate PDE from 3T3-L1 adipocytes and the cilostamide-sensitive soluble low K/sub m/ cAMP PDE from bovine liver (designated as III-C), platelets, heart, and other tissues.

  12. The Role of Type 4 Phosphodiesterases in Generating Microdomains of cAMP: Large Scale Stochastic Simulations

    PubMed Central

    Oliveira, Rodrigo F.; Terrin, Anna; Di Benedetto, Giulietta; Cannon, Robert C.; Koh, Wonryull; Kim, MyungSook; Zaccolo, Manuela; Blackwell, Kim T.

    2010-01-01

    Cyclic AMP (cAMP) and its main effector Protein Kinase A (PKA) are critical for several aspects of neuronal function including synaptic plasticity. Specificity of synaptic plasticity requires that cAMP activates PKA in a highly localized manner despite the speed with which cAMP diffuses. Two mechanisms have been proposed to produce localized elevations in cAMP, known as microdomains: impeded diffusion, and high phosphodiesterase (PDE) activity. This paper investigates the mechanism of localized cAMP signaling using a computational model of the biochemical network in the HEK293 cell, which is a subset of pathways involved in PKA-dependent synaptic plasticity. This biochemical network includes cAMP production, PKA activation, and cAMP degradation by PDE activity. The model is implemented in NeuroRD: novel, computationally efficient, stochastic reaction-diffusion software, and is constrained by intracellular cAMP dynamics that were determined experimentally by real-time imaging using an Epac-based FRET sensor (H30). The model reproduces the high concentration cAMP microdomain in the submembrane region, distinct from the lower concentration of cAMP in the cytosol. Simulations further demonstrate that generation of the cAMP microdomain requires a pool of PDE4D anchored in the cytosol and also requires PKA-mediated phosphorylation of PDE4D which increases its activity. The microdomain does not require impeded diffusion of cAMP, confirming that barriers are not required for microdomains. The simulations reported here further demonstrate the utility of the new stochastic reaction-diffusion algorithm for exploring signaling pathways in spatially complex structures such as neurons. PMID:20661441

  13. New fluorescent analogs of cAMP and cGMP available as substrates for cyclic nucleotide phosphodiesterase.

    PubMed

    Hiratsuka, T

    1982-11-25

    The synthesis of fluorescent derivatives of cAMP and cGMP, by reaction with isatoic anhydride in aqueous solution at mild pH and temperature, yielding 2'-O-anthraniloyl derivatives of cyclic nucleotides, is here described. 2'-O-(N-Methylanthraniloyl) derivatives were also synthesized by reaction with N-methylisatoic anhydride. Upon excitation at 330-350 nm, these derivatives exhibited maximum fluorescence emission at 430-445 nm in aqueous solution with quantum yields of 0.11-0.26. Their fluorescence was sensitive to the polarity of solvent; in N,N-dimethylformamide quantum yields of 0.8-0.95. The major differences between the two fluorophores were the longer wavelength of the emission maximum of the N-methylanthraniloyl group and its greater quantum yield. The derivatives were substrates for beef heart cyclic nucleotide phosphodiesterase, 15-24% as effective as the natural substrate cAMP. When combined with thin layer chromatography techniques, two apparent Km values (3-4 microM and 36-76 microM) for the cAMP derivatives and one value (10-18 microM) for the cGMP derivatives were obtained. The results indicate that these 2'-hydroxyl-modified cAMP and cGMP can be useful fluorescent substrate analogs for cyclic nucleotide phosphodiesterase.

  14. Investigation of the alkenyldiarylmethane non-nucleoside reverse transcriptase inhibitors as potential cAMP phosphodiesterase-4B2 inhibitors.

    PubMed

    Cullen, Matthew D; Cheung, York-Fong; Houslay, Miles D; Hartman, Tracy L; Watson, Karen M; Buckheit, Robert W; Pannecouque, Christophe; De Clercq, Erik; Cushman, Mark

    2008-02-15

    The alkenyldiarylmethanes (ADAMs) are currently being investigated as non-nucleoside HIV-1 reverse transcriptase inhibitors (NNRTIs) of potential value in the treatment of HIV infection and AIDS. During the course of these studies, a number of ADAM analogues have been identified that protect HIV-infected cells from the cytopathic effects of the virus by an unknown, HIV-1 RT-independent mechanism. Since the phosphodiesterase 4 family is required for HIV infection, the effect of various ADAMs on the activity of PDE4B2 was investigated in an effort to determine if the ADAMs could possibly be targeting phosphodiesterases. Six compounds representative of the ADAM class were tested for inhibition of cAMP hydrolysis by PDE4B2 enzymatic activity. Four ADAMs were found to be weak inhibitors of PDE4B2 and two of them were inactive. The experimental results are consistent with an antiviral mechanism that does not include inhibition of PDE4 isoforms.

  15. The Glutamic Acid-rich Protein-2 (GARP2) Is a High Affinity Rod Photoreceptor Phosphodiesterase (PDE6)-binding Protein That Modulates Its Catalytic Properties*

    PubMed Central

    Pentia, Dana C.; Hosier, Suzanne; Cote, Rick H.

    2010-01-01

    The glutamic acid-rich protein-2 (GARP2) is a splice variant of the β-subunit of the cGMP-gated ion channel of rod photoreceptors. GARP2 is believed to interact with several membrane-associated phototransduction proteins in rod photoreceptors. In this study, we demonstrated that GARP2 is a high affinity PDE6-binding protein and that PDE6 co-purifies with GARP2 during several stages of chromatographic purification. We found that hydrophobic interaction chromatography succeeds in quantitatively separating GARP2 from the PDE6 holoenzyme. Furthermore, the 17-kDa prenyl-binding protein, abundant in retinal cells, selectively released PDE6 (but not GARP2) from rod outer segment membranes, demonstrating the specificity of the interaction between GARP2 and PDE6. Purified GARP2 was able to suppress 80% of the basal activity of the nonactivated, membrane-bound PDE6 holoenzyme at concentrations equivalent to its endogenous concentration in rod outer segment membranes. However, GARP2 was unable to reverse the transducin activation of PDE6 (in contrast to a previous study) nor did it significantly alter catalysis of the fully activated PDE6 catalytic dimer. The high binding affinity of GARP2 for PDE6 and its ability to regulate PDE6 activity in its dark-adapted state suggest a novel role for GARP2 as a regulator of spontaneous activation of rod PDE6, thereby serving to lower rod photoreceptor “dark noise” and allowing these sensory cells to operate at the single photon detection limit. PMID:16407240

  16. In cardiac myocytes, cAMP elevation triggers the down-regulation of transcripts and promoter activity for cyclic AMP phosphodiesterase-4A10 (PDE4A10).

    PubMed

    McCahill, Angela; Campbell, Lachlan; McSorley, Theresa; Sood, Arvind; Lynch, Martin J; Li, Xiang; Yan, Chen; Baillie, George S; Houslay, Miles D

    2008-11-01

    Transcripts for the PDE4A10 cyclic AMP phosphodiesterase isoform are present in a wide variety of rat tissues including the heart. Sequence comparisons between the putative human and mouse promoters revealed a number of conserved regions including both an Sp1 and a CREB-binding site. The putative mouse PDE4A10 promoter was amplified from genomic DNA and sub-cloned into a luciferase reporter vector for investigation of activity in neonatal cardiac myocytes. Transfection with this construct identified a high level of luciferase expression in neonatal cardiac myocytes. Surprisingly, this activity was down-regulated by elevation of intracellular cAMP through a process involving PKA, but not EPAC, signalling. Such inhibition of the rodent PDE4A10 promoter activity in response to elevated cAMP levels is in contrast to the PDE4 promoters so far described. Site-directed mutagenesis revealed that the Sp1 binding site at promoter position -348 to -336 is responsible for the basal constitutive expression of murine PDE4A10. The conserved CREB-binding motif at position -370 to -363 also contributes to basal promoter activity but does not in itself confer cAMP inhibition upon the PDE4A10 promoter. EMSA analysis confirmed the authenticity of CREB and Sp1 binding sites. The transcriptional start site was identified to be an adenine residue at position -55 in the mouse PDE4A10 promoter. We present evidence that this novel down-regulation of PDE4A10 is mediated by the transcription factor ICER in a PKA dependent manner. The pool of cAMP in cardiac myocytes that down-regulates PDE4A10 is regulated by beta-adrenoceptor coupled adenylyl cyclase activity and via hydrolysis determined predominantly by the action of PDE4 (cAMP phosphodiesterase-4) and not PDE3 (cAMP phosphodiesterase-3). We suggest that increased cAMP may remodel cAMP-mediated signalling events by not only increasing the expression of specific PDE4 cAMP phosphodiesterases but also by down-regulating specific isoforms

  17. Cyclic nucleotide phosphodiesterase-mediated integration of cGMP and cAMP signaling in cells of the cardiovascular system.

    PubMed

    Maurice, Donald H

    2005-05-01

    Numerous pharmacological and physiological agents acting via either cAMP- or cGMP-mediated impact the activities of cells of the cardiovascular system. While most define cAMP and cGMP signaling systems as separate and independent, recent advances in our understanding of cyclic nucleotide signaling, and more specifically, of the roles which cyclic nucleotide phosphodiesterases (PDEs) play in these events, have altered this view. In this short chapter, I will review the data identifying expression of several PDEs in cells of the cardiovascular system. In addition, I will review the data that identify PDEs as enzymes capable of allowing integration between cAMP and cGMP signaling in cells, and propose that cAMP and cGMP signaling systems can represent parallel and interdependent signaling systems. Moreover, I will propose that cGMP-mediated effects on the activities of variants of the Phosphodiesterase 2 (PDE2), PDE3 and PDE5 families may act to coordinate linkage between cAMP and cGMP signaling in these cells.

  18. Neurotrophins elevate cAMP to reach a threshold required to overcome inhibition by MAG through extracellular signal-regulated kinase-dependent inhibition of phosphodiesterase.

    PubMed

    Gao, Ying; Nikulina, Elena; Mellado, Wilfredo; Filbin, Marie T

    2003-12-17

    Inhibitors of regeneration in myelin, such as myelin-associated glycoprotein (MAG), play an important role in preventing regeneration after CNS injury. Elevation of cAMP, either with dibutyryl-cAMP (db-cAMP) or by priming with a variety of neurotrophins, overcomes inhibition by MAG and myelin. However, activation of cAMP is not generally regarded as a signaling pathway for neurotrophins. Here we show that the NGF-like neurotrophins overcome inhibition by MAG by activating tyrosine kinase receptors. We also show that activation of extracellular signal-regulated kinase (Erk) by BDNF is required to overcome inhibition by MAG, and that activated Erk transiently inhibits phosphodiesterase 4 (PDE4), the enzyme that hydrolyzes cAMP. Inhibition of PDE4 then allows cAMP to increase and so initiates the pathway to overcome inhibition. Furthermore, we also show that basal levels of Erk activation and basal cAMP levels contribute to the effects of db-cAMP by pushing the combined levels of cAMP above a threshold required to overcome inhibition. Together, these results not only show how NGF-like neurotrophins can elevate cAMP and overcome inhibition but also point to a novel mechanism of cross talk in neurons from the Erk to the cAMP signaling pathways.

  19. [Effect of red light on activity of cAMP phosphodiesterases in photoperiodically different cereals and vernalized winter wheat].

    PubMed

    Fedenko, E P; Koksharova, T A

    2007-01-01

    Red light illumination of seedlings of photoperiodically different cereals had a different effect on the activity of multiple cyclic adenosine monophosphate phosphodiesterases. The response of all phosphodiesterase forms was reversed in fully vernalized winter wheat Triticum aestivum L.

  20. Intracellular membrane association of the Aplysia cAMP phosphodiesterase long and short forms via different targeting mechanisms.

    PubMed

    Kim, Kun-Hyung; Jun, Yong-Woo; Park, Yongsoo; Lee, Jin-A; Suh, Byung-Chang; Lim, Chae-Seok; Lee, Yong-Seok; Kaang, Bong-Kiun; Jang, Deok-Jin

    2014-09-12

    Phosphodiesterases (PDEs) play key roles in cAMP compartmentalization, which is required for intracellular signaling processes, through specific subcellular targeting. Previously, we showed that the long and short forms of Aplysia PDE4 (ApPDE4), which are localized to the membranes of distinct subcellular organelles, play key roles in 5-hydroxytryptamine-induced synaptic facilitation in Aplysia sensory and motor synapses. However, the molecular mechanism of the isoform-specific distinct membrane targeting was not clear. In this study, we further investigated the molecular mechanism of the membrane targeting of the ApPDE4 long and short forms. We found that the membrane targeting of the long form was mediated by hydrophobic interactions, mainly via 16 amino acids at the N-terminal region, whereas the short form was targeted solely to the plasma membrane, mainly by nonspecific electrostatic interactions between their N termini and the negatively charged lipids such as the phosphatidylinositol polyphosphates PI4P and PI(4,5)P2, which are embedded in the inner leaflet of the plasma membrane. Moreover, oligomerization of the long or short form by interaction of their respective upstream conserved region domains, UCR1 and UCR2, enhanced their plasma membrane targeting. These results suggest that the long and short forms of ApPDE4 are distinctly targeted to intracellular membranes through their direct association with the membranes via hydrophobic and electrostatic interactions, respectively.

  1. Molecular cloning of rat homologues of the Drosophila melanogaster dunce cAMP phosphodiesterase: evidence for a family of genes.

    PubMed Central

    Swinnen, J V; Joseph, D R; Conti, M

    1989-01-01

    To study the structure and function of cyclic nucleotide phosphodiesterases (PDEs) involved in mammalian gametogenesis, a rat testis cDNA library was screened at low stringency with a cDNA clone coding for the Drosophila melanogaster dunce-encoded PDE as a probe. This screening resulted in the isolation of two groups of cDNA clones, differing in their nucleotide sequences (ratPDE1 and ratPDE2). In the rat testis, RNA transcripts corresponding to both groups of clones were expressed predominantly in germ cells. Additional screenings of a Sertoli cell cDNA library with a ratPDE2 clone as a probe led to the isolation of two more groups of clones (rat-PDE3 and ratPDE4). Unlike ratPDE1 and ratPDE2, these clones hybridized to transcripts present predominantly in the Sertoli cell. In the middle of the coding region, all four groups of clones were homologous to each other. The deduced amino acid sequences of part of this region were also homologous to the D. melanogaster dunce PDE and to PDEs from bovine and yeast. These data indicate that a family of genes homologous to the D. melanogaster dunce-encoded PDE is present in the rat and that these genes are differentially expressed in somatic and germ cells of the seminiferous tubule. These findings provide a molecular basis for the observed heterogeneity of cAMP PDEs. Images PMID:2546153

  2. Intracellular Membrane Association of the Aplysia cAMP Phosphodiesterase Long and Short Forms via Different Targeting Mechanisms*

    PubMed Central

    Kim, Kun-Hyung; Jun, Yong-Woo; Park, Yongsoo; Lee, Jin-A; Suh, Byung-Chang; Lim, Chae-Seok; Lee, Yong-Seok; Kaang, Bong-Kiun; Jang, Deok-Jin

    2014-01-01

    Phosphodiesterases (PDEs) play key roles in cAMP compartmentalization, which is required for intracellular signaling processes, through specific subcellular targeting. Previously, we showed that the long and short forms of Aplysia PDE4 (ApPDE4), which are localized to the membranes of distinct subcellular organelles, play key roles in 5-hydroxytryptamine-induced synaptic facilitation in Aplysia sensory and motor synapses. However, the molecular mechanism of the isoform-specific distinct membrane targeting was not clear. In this study, we further investigated the molecular mechanism of the membrane targeting of the ApPDE4 long and short forms. We found that the membrane targeting of the long form was mediated by hydrophobic interactions, mainly via 16 amino acids at the N-terminal region, whereas the short form was targeted solely to the plasma membrane, mainly by nonspecific electrostatic interactions between their N termini and the negatively charged lipids such as the phosphatidylinositol polyphosphates PI4P and PI(4,5)P2, which are embedded in the inner leaflet of the plasma membrane. Moreover, oligomerization of the long or short form by interaction of their respective upstream conserved region domains, UCR1 and UCR2, enhanced their plasma membrane targeting. These results suggest that the long and short forms of ApPDE4 are distinctly targeted to intracellular membranes through their direct association with the membranes via hydrophobic and electrostatic interactions, respectively. PMID:25077971

  3. Evolution of self-organisation in Dictyostelia by adaptation of a non-selective phosphodiesterase and a matrix component for regulated cAMP degradation.

    PubMed

    Kawabe, Yoshinori; Weening, Karin E; Marquay-Markiewicz, Jacques; Schaap, Pauline

    2012-04-01

    Dictyostelium discoideum amoebas coordinate aggregation and morphogenesis by secreting cyclic adenosine monophosphate (cAMP) pulses that propagate as waves through fields of cells and multicellular structures. To retrace how this mechanism for self-organisation evolved, we studied the origin of the cAMP phosphodiesterase PdsA and its inhibitor PdiA, which are essential for cAMP wave propagation. D. discoideum and other species that use cAMP to aggregate reside in group 4 of the four major groups of Dictyostelia. We found that groups 1-3 express a non-specific, low affinity orthologue of PdsA, which gained cAMP selectivity and increased 200-fold in affinity in group 4. A low affinity group 3 PdsA only partially restored aggregation of a D. discoideum pdsA-null mutant, but was more effective at restoring fruiting body morphogenesis. Deletion of a group 2 PdsA gene resulted in disruption of fruiting body morphogenesis, but left aggregation unaffected. Together, these results show that groups 1-3 use a low affinity PdsA for morphogenesis that is neither suited nor required for aggregation. PdiA belongs to a family of matrix proteins that are present in all Dictyostelia and consist mainly of cysteine-rich repeats. However, in its current form with several extensively modified repeats, PdiA is only present in group 4. PdiA is essential for initiating spiral cAMP waves, which, by organising large territories, generate the large fruiting structures that characterise group 4. We conclude that efficient cAMP-mediated aggregation in group 4 evolved by recruitment and adaptation of a non-selective phosphodiesterase and a matrix component into a system for regulated cAMP degradation.

  4. IL-1 binds to high affinity receptors on human osteosarcoma cells and potentiates prostaglandin E2 stimulation of cAMP production

    SciTech Connect

    Rodan, S.B.; Wesolowski, G.; Chin, J.; Limjuco, G.A.; Schmidt, J.A.; Rodan, G.A. )

    1990-08-15

    IL-1 is a potent bone resorbing agent. Its mechanism of action is unknown, but the presence of osteoblasts was shown to be necessary for IL-1 stimulation of bone resorption by isolated osteoclasts. This study examines the presence of IL-1R and IL-1 effects in osteoblastic cells from a clonal human osteosarcoma cell line, Saos-2/B-10. We found that the binding affinity and the number of binding sites increases substantially during the postconfluent stage. Scatchard and curve-fitting analysis revealed one class of high affinity binding sites, with Kd/Ki's of 40 +/- 17 pM (mean +/- SD) for IL-1 alpha (n = 5) and 9 +/- 7 pM for IL-1 beta (n = 5) and 2916 +/- 2438 (n = 6) receptors/cell. Incubation of the cells with 125I-IL-1 alpha (100 pM) at 4 degrees C, followed by incubation at 37 degrees C up to 4 h, revealed internalization of receptor-bound IL-1 alpha. Chemical cross-linking studies showed that the IL-1R in Saos-2/B-10 cells had a molecular mass of approximately 80 kDa. To assess the biologic effect of IL-1 in Saos-2/B-10 cells, we determined PGE2 content and adenylate cyclase activity. Although IL-1 had no effect on PGE2 synthesis, both IL-1 alpha and IL-1 beta enhanced PGE2 stimulation of adenylate cyclase two- to four-fold in a dose-dependent manner. The half-maximal effect for IL-1 alpha was seen at 8 to 10 pM and for IL-1 beta at 0.6 to 1.8 pM. IL-1 did not enhance basal adenylate cyclase or stimulation by parathyroid hormone, isoproterenol, or forskolin. IL-1 enhancement of PGE2-stimulated adenylate cyclase was detected between 1 to 2 h, was maximal at 4 to 5 h, was not prevented by cycloheximide treatment, and was seen in membranes from IL-1 pretreated cells. These data show effects of IL-1 on a human osteoblast-like cell line that are mediated by high affinity receptors. These IL-1 effects could contribute to the biologic action of IL-1 on bone.

  5. 2',3'-cAMP hydrolysis by metal-dependent phosphodiesterases containing DHH, EAL, and HD domains is non-specific: Implications for PDE screening.

    PubMed

    Rao, Feng; Qi, Yaning; Murugan, Elavazhagan; Pasunooti, Swathi; Ji, Qiang

    2010-07-30

    The recent report of 2',3'-cAMP isolated from rat kidney is the first proof of its biological existence, which revived interest in this mysterious molecule. 2',3'-cAMP serves as an extracellular adenosine source, but how it is degraded remains unclear. Here, we report that 2',3'-cAMP can be hydrolyzed by six phosphodiesterases containing three different families of hydrolytic domains, generating invariably 3'-AMP but not 2'-AMP. The catalytic efficiency (k(cat)/K(m)) of each enzyme against 2',3'-cAMP correlates with that against the widely used non-specific substrate bis(p-nitrophenyl)phosphate (bis-pNPP), indicating that 2',3'-cAMP is a previously unknown non-specific substrate for PDEs. Furthermore, we show that the exclusive formation of 3'-AMP is due to the P-O2' bond having lower activation energy and is not the result of steric exclusion at enzyme active site. Our analysis provides mechanistic basis to dissect protein function when 2',3'-cAMP hydrolysis is observed.

  6. Xanthine derivatives: comparison between suppression of tumour necrosis factor-alpha production and inhibition of cAMP phosphodiesterase activity.

    PubMed Central

    Semmler, J; Gebert, U; Eisenhut, T; Moeller, J; Schönharting, M M; Alléra, A; Endres, S

    1993-01-01

    Several in vitro and in vivo studies have demonstrated suppression of tumour necrosis factor-alpha (TNF-alpha) synthesis by pentoxifylline. In the present study we compared the effect of pentoxifylline with that of five other xanthine derivatives. We addressed two questions. First, what is the relative potency of those chemically related compounds in suppressing the lipopolysaccharide (LPS)-induced production of TNF-alpha in human mononuclear cells? Second, does suppression of TNF-alpha production by these xanthine derivatives correlate with their capacity to inhibit 3',5'-cAMP phosphodiesterase (PDE) activity? The experimental drug A 80 2715 [1-(5-hydroxy-5-methylhexyl)-3-methyl-7-propylxanthine] was identified as the most potent agent with an IC50 (concentration exerting 50% suppression of LPS-induced TNF-alpha production) of 41 microM (mean of 13 individuals). The IC50 values of the other substances ranged between 106 microM for HWA 138 and 419 microM for theophylline. The LPS-induced interleukin-1 beta (IL-1 beta) production was not influenced by all substances tested at comparable concentrations. Inhibition of PDE activity was determined in a cell-free system using PDE isolated from bovine heart. All xanthine derivatives dose-dependently inhibited PDE activity. Furthermore, with the exception of theophylline, there was a high degree of correlation between the potency to suppress TNF-alpha production in the cell culture system and the potency to inhibit PDE activity in the cell-free enzymatic assay. This argues for a crucial role of PDE inhibition in the suppression of TNF-alpha synthesis by xanthine derivatives. PMID:8388363

  7. Identification and Characterization of a Novel Phosphodiesterase from the Metagenome of an Indian Coalbed

    PubMed Central

    Singh, Durgesh Narain; Gupta, Ankush; Singh, Vijay Shankar; Mishra, Rajeev; Kateriya, Suneel; Tripathi, Anil Kumar

    2015-01-01

    Phosphoesterases are involved in the degradation of organophosphorus compounds. Although phosphomonoesterases and phosphotriesterases have been studied in detail, studies on phosphodiesterases are rather limited. In our search to find novel phosphodiesterases using metagenomic approach, we cloned a gene encoding a putative phosphodiesterase (PdeM) from the metagenome of the formation water collected from an Indian coal bed. Bioinformatic analysis showed that PdeM sequence possessed the characteristic signature motifs of the class III phosphodiesterases and phylogenetic study of PdeM enabled us to identify three distinct subclasses (A, B, and C) within class III phosphodiesterases, PdeM clustering in new subclass IIIB. Bioinformatic, biochemical and biophysical characterization of PdeM further revealed some of the characteristic features of the phosphodiesterases belonging to newly described subclass IIIB. PdeM is a monomer of 29.3 kDa, which exhibits optimum activity at 25°C and pH 8.5, but low affinity for bis(pNPP) as well as pNPPP. The recombinant PdeM possessed phosphodiesterase, phosphonate-ester hydrolase and nuclease activity. It lacked phosphomonoesterase, phosphotriesterase, and RNAse activities. Overexpression of PdeM in E.coli neither affected catabolite respression nor did the recombinant protein hydrolyzed cAMP in vitro, indicating its inability to hydrolyze cAMP. Although Mn2+ was required for the activity of PdeM, but addition of metals (Mn2+ or Fe3+) did not induce oligomerization. Further increase in concentration of Mn2+ upto 3 mM, increased α-helical content as well as the phosphodiesterase activity. Structural comparison of PdeM with its homologs showed that it lacked critical residues required for dimerization, cAMP hydrolysis, and for the high affinity binding of bis(pNPP). PdeM, thus, is a novel representative of new subclass of class III phosphodiesterases. PMID:25658120

  8. Silymarin Activates c-AMP Phosphodiesterase and Stimulates Insulin Secretion in a Glucose-Dependent Manner in HIT-T15 Cells

    PubMed Central

    Meng, Ran; Mahadevan, Jana; Oseid, Elizabeth; Vallerie, Sara; Robertson, R. Paul

    2016-01-01

    Silymarin (SIL) is a flavonoid extracted from milk thistle seed that has been reported to decrease hyperglycemia in people with type 2 diabetes (T2D). However, it is not known whether SIL has direct secretory effects on β-cells. Using the β-cell line HIT-T15, SIL was shown to decrease intracellular peroxide levels and to augment glucose-stimulated insulin secretion (GSIS). However, the latter was observed using a concentration range of 25–100 µM, which was too low to affect endogenous peroxide levels. The stimulatory effect of SIL dissipated at higher concentrations (100–200 µM), and mild apoptosis was observed. The smaller concentrations of SIL also decreased cAMP phosphodiesterase activity in a Ca2+/calmodulin-dependent manner. The stimulatory effects of SIL on GSIS were inhibited by three different inhibitors of exocytosis, indicating that SIL’s mechanism of stimulating GSIS operated via closing β-cell K-ATP channels, and perhaps more distal sites of action involving calcium influx and G-proteins. We concluded that augmentation of GSIS by SIL can be observed at concentrations that also inhibit cAMP phosphodiesterase without concomitant lowering of intracellular peroxides. PMID:27973458

  9. Hydrogen sulfide-mediated stimulation of mitochondrial electron transport involves inhibition of the mitochondrial phosphodiesterase 2A, elevation of cAMP and activation of protein kinase A.

    PubMed

    Módis, Katalin; Panopoulos, Panagiotis; Coletta, Ciro; Papapetropoulos, Andreas; Szabo, Csaba

    2013-11-01

    Although hydrogen sulfide (H₂S) is generally known as a mitochondrial poison, recent studies show that lower concentrations of H₂S play a physiological role in the stimulation of mitochondrial electron transport and cellular bioenergetics. This effect involves electron donation at Complex II. Other lines of recent studies demonstrated that one of the biological actions of H₂S involves inhibition of cAMP and cGMP phosphodiesterases (PDEs). Given the emerging functional role of the mitochondrial isoform of cAMP PDE (PDE2A) in the regulation of mitochondrial function the current study investigated whether cAMP-dependent mechanisms participate in the stimulatory effect of NaHS on mitochondrial function. In isolated rat liver mitochondria, partial digestion studies localized PDE2A into the mitochondrial matrix. NaHS exerted a concentration-dependent inhibitory effect on recombinant PDE2A enzyme in vitro. Moreover, NaHS induced an elevation of cAMP levels when added to isolated mitochondria and stimulated the mitochondrial electron transport. The latter effect was inhibited by Rp-cAMP, an inhibitor of the cAMP-dependent protein kinase (PKA). The current findings suggest that the direct electron donating effect of NaHS is amplified by an intramitochondrial cAMP system, which may involve the inhibition of PDE2A and subsequent, cAMP-mediated stimulation of PKA.

  10. Fundamental Reaction Pathway and Free Energy Profile for Hydrolysis of Intracellular Second Messenger Adenosine 3',5'-Cyclic Monophosphate (cAMP) Catalyzed by Phosphodiesterase-4

    PubMed Central

    Chen, Xi; Zhao, Xinyun; Xiong, Ying; Liu, Junjun; Zhan, Chang-Guo

    2011-01-01

    As important drug targets for a variety of human diseases, cyclic nucleotide phosphodiesterases (PDEs) are a superfamily of enzymes sharing a similar catalytic site. We have performed pseudobond first-principles quantum mechanical/molecular mechanical-free energy perturbation (QM/MM-FE) and QM/MM-Poisson-Boltzmann surface area (PBSA) calculations to uncover the detailed reaction mechanism for PDE4-catalyzed hydrolysis of adenosine 3',5'-cyclic monophosphate (cAMP). This is the first report on QM/MM reaction-coordinate calculations including the protein environment of any PDE-catalyzed reaction system, demonstrating a unique catalytic reaction mechanism. The QM/MM-FE and QM/MM-PBSA calculations revealed that the PDE4-catalyzed hydrolysis of cAMP consists of two reaction stages: cAMP hydrolysis (stage 1) and bridging hydroxide ion regeneration (stage 2). The stage 1 includes the binding of cAMP in the active site, nucleophilic attack of the bridging hydroxide ion on the phosphorous atom of cAMP, cleavage of O3'-P phosphoesteric bond of cAMP, protonation of the departing O3' atom, and dissociation of hydrolysis product (AMP). The stage 2 includes the binding of solvent water molecules with the metal ions in the active site and regeneration of the bridging hydroxide ion. The dissociation of the hydrolysis product is found to be rate-determining for the enzymatic reaction process. The calculated activation Gibbs free energy of ≥16.0 and reaction free energy of -11.1 kcal/mol are in good agreement with the experimentally derived activation free energy of 16.6 kcal/mol and reaction free energy of -11.5 kcal/mol, suggesting that the catalytic mechanism obtained from this study is reliable and provides a solid base for future rational drug design. PMID:21973014

  11. The Dunce cAMP phosphodiesterase PDE-4 negatively regulates G alpha(s)-dependent and G alpha(s)-independent cAMP pools in the Caenorhabditis elegans synaptic signaling network.

    PubMed

    Charlie, Nicole K; Thomure, Angela M; Schade, Michael A; Miller, Kenneth G

    2006-05-01

    Forward genetic screens for mutations that rescue the paralysis of ric-8 (Synembryn) reduction-of-function mutations frequently reveal mutations that cause hyperactivation of one or more components of the G alpha(s) pathway. Here, we report that one of these mutations strongly reduces the function of the Dunce cAMP phosphodiesterase PDE-4 by disrupting a conserved active site residue. Loss of function and neural overexpression of PDE-4 have profound and opposite effects on locomotion rate, but drug-response assays suggest that loss of PDE-4 function does not affect steady-state acetylcholine release or reception. Our genetic analysis suggests that PDE-4 regulates both G alpha(s)-dependent and G alpha(s)-independent cAMP pools in the neurons controlling locomotion rate. By immunostaining, PDE-4 is strongly expressed throughout the nervous system, where it localizes to small regions at the outside boundaries of synaptic vesicle clusters as well as intersynaptic regions. The synaptic subregions containing PDE-4 are distinct from those containing active zones, as indicated by costaining with an antibody against the long form of UNC-13. This highly focal subsynaptic localization suggests that PDE-4 may exert its effects by spatially regulating intrasynaptic cAMP pools.

  12. The role of the PDE4D cAMP phosphodiesterase in the regulation of glucagon-like peptide-1 release

    PubMed Central

    Ong, WK; Gribble, FM; Reimann, F; Lynch, MJ; Houslay, MD; Baillie, GS; Furman, BL; Pyne, NJ

    2009-01-01

    Background and purpose: Increases in intracellular cyclic AMP (cAMP) augment the release/secretion of glucagon-like peptide-1 (GLP-1). As cAMP is hydrolysed by cAMP phosphodiesterases (PDEs), we determined the role of PDEs and particularly PDE4 in regulating GLP-1 release. Experimental approach: GLP-1 release, PDE expression and activity were investigated using rats and GLUTag cells, a GLP-1-releasing cell line. The effects of rolipram, a selective PDE4 inhibitor both in vivo and in vitro and stably overexpressed catalytically inactive PDE4D5 (D556A-PDE4D5) mutant in vitro on GLP-1 release were investigated. Key results: Rolipram (1.5 mg·kg−1 i.v.) increased plasma GLP-1 concentrations approximately twofold above controls in anaesthetized rats and enhanced glucose-induced GLP-1 release in GLUTag cells (EC50∼1.2 nmol·L−1). PDE4D mRNA transcript and protein were detected in GLUTag cells using RT-PCR with gene-specific primers and Western blotting with a specific PDE4D antibody respectively. Moreover, significant PDE activity was inhibited by rolipram in GLUTag cells. A GLUTag cell clone (C1) stably overexpressing the D556A-PDE4D5 mutant, exhibited elevated intracellular cAMP levels and increased basal and glucose-induced GLP-1 release compared with vector-transfected control cells. A role for intracellular cAMP/PKA in enhancing GLP-1 release in response to overexpression of D556A-PDE4D5 mutant was demonstrated by the finding that the PKA inhibitor H89 reduced both basal and glucose-induced GLP-1 release by 37% and 39%, respectively, from C1 GLUTag cells. Conclusions and implications: PDE4D may play an important role in regulating intracellular cAMP linked to the regulation of GLP-1 release. British Journal of Pharmacology (2009) 157, 633–644; doi:10.1111/j.1476-5381.2009.00194.x; published online 9 April 2009 PMID:19371330

  13. The antitumour agent 5-(3,3-dimethyl-1-triazeno) imidazole-4-carboxamide (DTIC) inhibits rat liver cAMP phosphodiesterase and amplifies hormone effects in hepatocytes and hepatoma cells.

    PubMed Central

    Larsson, P. G.; Haffner, F.; Brłnstad, G. O.; Christoffersen, T.

    1979-01-01

    The antitumour agent 5-(3,3-dimethyl-1-triazeno)imidazole-4-carboxamide (DTIC) was found to inhibit competitively the low-Km cyclic AMP phosphodiesterase activity in an ammonium-sulphate-precipitable fraction of the 2,000g supernatant of rat liver. With substrate concentration at 0.25 microM, I50 was 790 microM for DTIC and 350 microM for theophylline. DTIC at 2 mM more than doubled the cAMP response to glucagon in hepatocytes and to adrenaline in MH1C1 hepatoma cells, indicating that it also exerts its inhibitory effect on the phosphodiesterase in intact cells. The possible contribution of the phosphodiesterase inhibition to the growth-inhibitory and cytotoxic effects of DTIC is discussed. PMID:228692

  14. Important role of phosphodiesterase 3B for the stimulatory action of cAMP on pancreatic beta-cell exocytosis and release of insulin.

    PubMed

    Härndahl, Linda; Jing, Xing-Jun; Ivarsson, Rosita; Degerman, Eva; Ahrén, Bo; Manganiello, Vincent C; Renström, Erik; Holst, Lena Stenson

    2002-10-04

    Cyclic AMP potentiates glucose-stimulated insulin release and mediates the stimulatory effects of hormones such as glucagon-like peptide 1 (GLP-1) on pancreatic beta-cells. By inhibition of cAMP-degrading phosphodiesterase (PDE) and, in particular, selective inhibition of PDE3 activity, stimulatory effects on insulin secretion have been observed. Molecular and functional information on beta-cell PDE3 is, however, scarce. To provide such information, we have studied the specific effects of the PDE3B isoform by adenovirus-mediated overexpression. In rat islets and rat insulinoma cells, approximate 10-fold overexpression of PDE3B was accompanied by a 6-8-fold increase in membrane-associated PDE3B activity. The cAMP concentration was significantly lowered in transduced cells (INS-1(832/13)), and insulin secretion in response to stimulation with high glucose (11.1 mm) was reduced by 40% (islets) and 50% (INS-1). Further, the ability of GLP-1 (100 nm) to augment glucose-stimulated insulin secretion was inhibited by approximately 30% (islets) and 70% (INS-1). Accordingly, when stimulating with cAMP, a substantial decrease (65%) in exocytotic capacity was demonstrated in patch-clamped single beta-cells. In untransduced insulinoma cells, application of the PDE3-selective inhibitor OPC3911 (10 microm) was shown to increase glucose-stimulated insulin release as well as cAMP-enhanced exocytosis. The findings suggest a significant role of PDE3B as an important regulator of insulin secretory processes.

  15. [Effect of vernalization and red light illumination of seedlings of bread wheat (Triticum aestivum L.) on the temperature profile of the cAMP phosphodiesterase activity].

    PubMed

    Fedenko, E P; Koksharova, T A; Agamalova, S R; Beliaeva, E V

    2004-01-01

    Phenotypic manifestations of Vrn (vernalization) and Ppd (photoperiodism) genes responsible for transition of bread wheat Triticum aestivum L. to generative growth (flowering) are mutually related. Since the mechanism of phytochrome-induced photoperiodism involves the enzymes of cyclic adenosine monophosphate metabolism and phosphodiesterase in particular, we tested involvement of phosphodiesterase in the process of winter wheat vernalization and formation of flowering competence in alternate wheat requiring a long day but no vernalization for the transition to flowering. We studied temperature dependence of phosphodiesterase activity in vernalized and unvernalized winter wheat on the one hand and in etiolated and red light illuminated seedlings of alternate wheat on the other hand. Short-term experiments demonstrated that vernalization and red light illumination are similar to long day by the effect on the long-day plants. Both influences induced a pronounced inversion of the temperature profile of phosphodiesterase activity in the 28-45 degrees C range. We propose that phosphodiesterase is involved in vernalization processes and can serve as a sensor of low temperature in winter wheat. Changed temperature profile is a radical control mechanism of phosphodiesterase activity in response to the influences (red light and vernalizing temperatures) responsible for competence of various bread wheat forms for generative growth.

  16. Defining the role of a FYVE domain in the localization and activity of a cAMP phosphodiesterase implicated in osmoregulation in Trypanosoma cruzi

    PubMed Central

    Schoijet, Alejandra C.; Miranda, Kildare; Medeiros, Lia Carolina Soares; de Souza, Wanderley; Flawiá, Mirtha M.; Torres, Héctor N.; Pignataro, Omar P.; Docampo, Roberto; Alonso, Guillermo D.

    2010-01-01

    Summary Intracellular levels of cyclic nucleotide second messengers are regulated predominantly by a large superfamily of phosphodiesterases. Trypanosoma cruzi, the causative agent of Chagas disease, encodes four different phosphodiesterase (PDE) families. One of these PDEs, T. cruzi phosphodiesterase C2 (TcrPDEC2) has been characterized as a FYVE-domain containing protein. Here, we report a novel role for TcrPDEC2 in osmoregulation in T. cruzi and reveal the relevance of its FYVE domain. Our data show that treatment of epimastigotes with TcrPDEC2 inhibitors improves their regulatory volume decrease, whereas cells overexpressing this enzyme are unaffected by the same inhibitors. Consistent with these results, TcrPDEC2 localizes to the contractile vacuole complex, showing strong labeling in the region corresponding to the spongiome. Furthermore, transgenic parasites overexpressing a truncated version of TcrPDEC2 without the FYVE domain show a failure in its targeting to the contractile vacuole complex and a marked decrease in phosphodiesterase activity, supporting the importance of this domain to the localization and activity of TcrPDEC2. Taking together, the results here presented are consistent with the importance of the cyclic AMP signaling pathway in regulatory volume decrease and implicate TcrPDEC2 as a specifically localized phosphodiesterase involved in osmoregulation in T. cruzi. PMID:21166893

  17. Interaction with receptor for activated C-kinase 1 (RACK1) sensitizes the phosphodiesterase PDE4D5 towards hydrolysis of cAMP and activation by protein kinase C

    PubMed Central

    Bird, Rebecca J.; Baillie, George S.; Yarwood, Stephen J.

    2010-01-01

    We have previously identified the PKC (protein kinase C)-anchoring protein RACK1 (receptor for activated C-kinase 1), as a specific binding partner for the cAMP-specific phosphodiesterase PDE4D5, suggesting a potential site for cross-talk between the PKC and cAMP signalling pathways. In the present study we found that elevation of intracellular cAMP, with the β2-adrenoceptor agonist isoproterenol (isoprenaline), led to activation of PDE4 enzymes in the particulate and soluble fractions of HEK (human embryonic kidney)-293 cells. In contrast activation of PDE4D5, with isoproterenol and the PKC activator PMA, was restricted to the particulate fraction, where it interacts with RACK1; however, RACK1 is dispensable for anchoring PDE4D5 to the particulate fraction. Kinetic studies demonstrated that RACK1 alters the conformation of particulate-associated PDE4D5 so that it more readily interacts with its substrate cAMP and with rolipram, a PDE4 inhibitor that specifically targets the active site of the enzyme. Interaction with RACK1 was also essential for PKC-dependent and ERK (extracellular-signal-regulated kinase)-independent phosphorylation (on Ser126), and activation of PDE4D5 in response to PMA and isoproterenol, both of which trigger the recruitment of PKCα to RACK1. Together these results reveal novel signalling cross-talk, whereby RACK1 mediates PKC-dependent activation of PDE4D5 in the particulate fraction of HEK-293 cells in response to elevations in intracellular cAMP. PMID:20819076

  18. Altered phosphodiesterase 3-mediated cAMP hydrolysis contributes to a hypermotile phenotype in obese JCR:LA-cp rat aortic vascular smooth muscle cells: implications for diabetes-associated cardiovascular disease.

    PubMed

    Netherton, Stuart J; Jimmo, Sandra L; Palmer, Daniel; Tilley, Douglas G; Dunkerley, Heather A; Raymond, Daniel R; Russell, James C; Absher, P Marlene; Sage, E Helene; Vernon, Robert B; Maurice, Donald H

    2002-04-01

    Cardiovascular diseases represent a significant cause of morbidity and mortality in diabetes. Of the many animal models used in the study of non-insulin-dependent (type 2) diabetes, the JCR:LA-cp rat is unique in that it develops insulin resistance in the presence of obesity and manifests both peripheral and coronary vasculopathies. In this animal model, arterial vascular smooth muscle cells (VSMCs) from homozygous obese (cp/cp) rats, but not from age-matched healthy (+/+ or + /cp, collectively defined +/?) littermates, display an " activated" phenotype in vitro and in vivo and have an elevated level of cAMP phosphodiesterase (PDE) activity. In this report, we confirm that cp/cp rat aortic VSMCs have an elevated level of PDE3 activity and show that only particulate PDE3 (PDE3B) activity is elevated. In marked contrast to results obtained in + /? VSMCs, simultaneous activation of adenylyl cyclase and inhibition of PDE3 activity in cp/cp VSMCs synergistically increased cAMP. Although PDE3 inhibition did not potentiate the antimigratory effects of forskolin on +/? VSMCs, PDE3 inhibition did markedly potentiate the forskolin-induced inhibition of migration of cp/cp-derived VSMCs. Although PDE3 activity was elevated in cp/cp rat aortic VSMCs, levels of expression of cytosolic PDE3 (PDE3A) and PDE3B in +/? and cp/cp VSMCs, as well as activation of these enzymes following activation of the cAMP-protein kinase A signaling cascade, were not different. Our data are consistent with an increased role for PDE3 in regulating cAMP-dependent signaling in cp/cp VSMCs and identify PDE3 as a cellular activity potentially responsible for the phenotype of cp/cp VSMCs.

  19. Cardiac Cyclic Nucleotide Phosphodiesterases: Function, Regulation, and Therapeutic Prospects

    PubMed Central

    Knight, W. E.; Yan, C.

    2014-01-01

    The second messengers cAMP and cGMP exist in multiple discrete compartments and regulate a variety of biological processes in the heart. The cyclic nucleotide phosphodiesterases, by catalyzing the hydrolysis of cAMP and cGMP, play crucial roles in controlling the amplitude, duration, and compartmentalization of cyclic nucleotide signaling. Over 60 phosphodiesterase isoforms, grouped into 11 families, have been discovered to date. In the heart, both cAMP- and cGMP-hydrolyzing phosphodiesterases play important roles in physiology and pathology. At least 7 of the 11 phosphodiesterase family members appear to be expressed in the myocardium, and evidence supports phosphodiesterase involvement in regulation of many processes important for normal cardiac function including pacemaking and contractility, as well as many pathological processes including remodeling and myocyte apoptosis. Pharmacological inhibitors for a number of phosphodiesterase families have also been used clinically or preclinically to treat several types of cardiovascular disease. In addition, phosphodiesterase inhibitors are also being considered for treatment of many forms of disease outside the cardiovascular system, raising the possibility of cardiovascular side effects of such agents. This review will discuss the roles of phosphodiesterases in the heart, in terms of expression patterns, regulation, and involvement in physiological and pathological functions. Additionally, the cardiac effects of various phosphodiesterase inhibitors, both potentially beneficial and detrimental, will be discussed. PMID:22951903

  20. Expression of cAMP and cGMP-phosphodiesterase isoenzymes 3, 4, and 5 in the human clitoris: immunohistochemical and molecular biology study.

    PubMed

    Oelke, Matthias; Hedlund, Petter; Albrecht, Knut; Ellinghaus, Peter; Stief, Christian G; Jonas, Udo; Andersson, Karl-Erik; Uckert, Stefan

    2006-05-01

    Only a little research has focused on the evaluation of female sexual function. With sexual stimulation, the clitoris becomes engorged with blood and tumescent. Nevertheless, only little is known about the significance of the cyclic nucleotide-mediated signal transduction in the control of this process. We sought to elucidate the presence of the phosphodiesterase (PDE) isoenzymes 3, 4, and 5 in the human clitoris using immunohistochemical and molecular biology methods. Thin sections of clitoral specimens were incubated with primary antibodies directed against PDE isoenzymes 3, 4, and 5. Next, the sections were incubated with either Texas red or fluorescein isothiocyanate-labeled secondary antibodies, and visualization was done using laser microscopy. The expression of mRNA encoding for various PDE isoenzymes was evaluated using reverse transcriptase polymerase chain reaction. Immunofluorescence indicating the presence of PDE4 (cyclic adenosine monophosphate-PDE) was observed in the nonvascular smooth musculature of the corpus cavernosum clitoris, sinusoidal endothelial and subendothelial layers, and nerve fibers innervating the tissue. Immunoreactivity specific for PDE5 (cyclic guanosine monophosphate-PDE) was limited to the smooth muscle of the clitoral erectile tissue. The fluorescein isothiocyanate reaction indicating the expression of PDE3 (cyclic adenosine monophosphate-PDE) was registered to a certain degree only in the clitoral epidermis. In the reverse transcriptase polymerase chain reaction studies, a predominant expression of mRNA encoding for PDE1A was registered, but only small amounts of mRNA encoding for PDE4 and PDE5 were detected. Our results have demonstrated the presence of cyclic adenosine monophosphate-PDE and cyclic guanosine monophosphate-PDE in the human clitoris and may indicate a regulatory function of these enzymes in the cyclic nucleotide-mediated control of smooth muscle tone.

  1. cAMP-Specific phosphodiesterase-4 enzymes in the cardiovascular system: a molecular toolbox for generating compartmentalized cAMP signaling.

    PubMed

    Houslay, Miles D; Baillie, George S; Maurice, Donald H

    2007-04-13

    Cyclic AMP regulates a vast number of distinct events in all cells. Early studies established that its hydrolysis by cyclic nucleotide phosphodiesterases (PDEs) controlled both the magnitude and the duration of its influence. Recent evidence shows that PDEs also act as coincident detectors linking cyclic-nucleotide- and non-cyclic-nucleotide-based cellular signaling processes and are tethered with great selectively to defined intracellular structures, thereby integrating and spatially restricting their cellular effects in time and space. Although 11 distinct families of PDEs have been defined, and cells invariably express numerous individual PDE enzymes, a large measure of our increased appreciation of the roles of these enzymes in regulating cyclic nucleotide signaling has come from studies on the PDE4 family. Four PDE4 genes encode more than 20 isoforms. Alternative mRNA splicing and the use of different promoters allows cells the possibility of expressing numerous PDE4 enzymes, each with unique amino-terminal-targeting and/or regulatory sequences. Dominant negative and small interfering RNA-mediated knockdown strategies have proven that particular isoforms can uniquely control specific cellular functions. Thus the protein kinase A phosphorylation status of the beta(2) adrenoceptor and, thereby, its ability to switch its signaling to extracellular signal-regulated kinase activation, is uniquely regulated by PDE4D5 in cardiomyocytes. We describe how cardiomyocytes and vascular smooth muscle cells selectively vary both the expression and the catalytic activities of PDE4 isoforms to regulate their various functions and how altered regulation of these processes can influence the development, or resolution, of cardiovascular pathologies, such as heart failure, as well as various vasculopathies.

  2. Receptor-mediated stimulation of lipid signalling pathways in CHO cells elicits the rapid transient induction of the PDE1B isoform of Ca2+/calmodulin-stimulated cAMP phosphodiesterase.

    PubMed

    Spence, S; Rena, G; Sullivan, M; Erdogan, S; Houslay, M D

    1997-01-01

    Chinese hamster ovary cells (CHO cells) do not exhibit any Ca2+/calmodulin-stimulated cAMP phosphodiesterase (PDE1) activity. Challenge of CHO cells with agonists for endogenous P2-purinoceptors, lysophosphatidic acid receptors and thrombin receptors caused a similar rapid transient induction of PDE1 activity in each instance. This was also evident on noradrenaline challenge of a cloned CHO cell line transfected so as to overexpress alpha 1B-adrenoceptors. This novel PDE1 activity appeared within about 15 min of exposure to ligands, rose to a maximum value within 30 min to 1 h and then rapidly decreased. In each case, the expression of novel PDE1 activity was blocked by the transcriptional inhibitor actinomycin D. Challenge with insulin of either native CHO cells or a CHO cell line transfected so as to overexpress the human insulin receptor failed to induce PDE1 activity. Reverse transcriptase-PCR analyses, using degenerate primers able to detect the PDE1C isoform, did not amplify any fragment from RNA preparations of CHO cells expressing PDE1 activity, although they did so from the human thyroid carcinoma FTC133 cell line. Reverse transcriptase-PCR analyses, using degenerate primers able to detect the PDE1A and PDE1B isoforms, successfully amplified a fragment of the predicted size from RNA preparations of both CHO cells expressing PDE1 activity and human Jurkat T-cells. Sequencing of the PCR products, generated using the PDE1A/B primers, yielded a novel sequence which, by analogy with sequences reported for bovine and murine PDE1B forms, suggests that the PDE1 species induced in CHO cells through protein kinase C activation and that expressed in Jurkat T-cells are PDE1B forms.

  3. Two Phosphodiesterase Genes, PDEL and PDEH, Regulate Development and Pathogenicity by Modulating Intracellular Cyclic AMP Levels in Magnaporthe oryzae

    PubMed Central

    Zhang, Haifeng; Liu, Kaiyue; Zhang, Xing; Tang, Wei; Wang, Jiansheng; Guo, Min; Zhao, Qian; Zheng, Xiaobo; Wang, Ping; Zhang, Zhengguang

    2011-01-01

    Cyclic AMP (cAMP) signaling plays an important role in regulating multiple cellular responses, such as growth, morphogenesis, and/or pathogenicity of eukaryotic organisms such as fungi. As a second messenger, cAMP is important in the activation of downstream effector molecules. The balance of intracellular cAMP levels depends on biosynthesis by adenylyl cyclases (ACs) and hydrolysis by cAMP phosphodiesterases (PDEases). The rice blast fungus Magnaporthe oryzae contains a high-affinity (PdeH/Pde2) and a low-affinity (PdeL/Pde1) PDEases, and a previous study showed that PdeH has a major role in asexual differentiation and pathogenicity. Here, we show that PdeL is required for asexual development and conidial morphology, and it also plays a minor role in regulating cAMP signaling. This is in contrast to PdeH whose mutation resulted in major defects in conidial morphology, cell wall integrity, and surface hydrophobicity, as well as a significant reduction in pathogenicity. Consistent with both PdeH and PdeL functioning in cAMP signaling, disruption of PDEH only partially rescued the mutant phenotype of ΔmagB and Δpka1. Further studies suggest that PdeH might function through a feedback mechanism to regulate the expression of pathogenicity factor Mpg1 during surface hydrophobicity and pathogenic development. Moreover, microarray data revealed new insights into the underlying cAMP regulatory mechanisms that may help to identify potential pathogenicity factors for the development of new disease management strategies. PMID:21386978

  4. Cyclic AMP effectors in African trypanosomes revealed by genome-scale RNA interference library screening for resistance to the phosphodiesterase inhibitor CpdA.

    PubMed

    Gould, Matthew K; Bachmaier, Sabine; Ali, Juma A M; Alsford, Sam; Tagoe, Daniel N A; Munday, Jane C; Schnaufer, Achim C; Horn, David; Boshart, Michael; de Koning, Harry P

    2013-10-01

    One of the most promising new targets for trypanocidal drugs to emerge in recent years is the cyclic AMP (cAMP) phosphodiesterase (PDE) activity encoded by TbrPDEB1 and TbrPDEB2. These genes were genetically confirmed as essential, and a high-affinity inhibitor, CpdA, displays potent antitrypanosomal activity. To identify effectors of the elevated cAMP levels resulting from CpdA action and, consequently, potential sites for adaptations giving resistance to PDE inhibitors, resistance to the drug was induced. Selection of mutagenized trypanosomes resulted in resistance to CpdA as well as cross-resistance to membrane-permeable cAMP analogues but not to currently used trypanocidal drugs. Resistance was not due to changes in cAMP levels or in PDEB genes. A second approach, a genome-wide RNA interference (RNAi) library screen, returned four genes giving resistance to CpdA upon knockdown. Validation by independent RNAi strategies confirmed resistance to CpdA and suggested a role for the identified cAMP Response Proteins (CARPs) in cAMP action. CARP1 is unique to kinetoplastid parasites and has predicted cyclic nucleotide binding-like domains, and RNAi repression resulted in >100-fold resistance. CARP2 and CARP4 are hypothetical conserved proteins associated with the eukaryotic flagellar proteome or with flagellar function, with an orthologue of CARP4 implicated in human disease. CARP3 is a hypothetical protein, unique to Trypanosoma. CARP1 to CARP4 likely represent components of a novel cAMP signaling pathway in the parasite. As cAMP metabolism is validated as a drug target in Trypanosoma brucei, cAMP effectors highly divergent from the mammalian host, such as CARP1, lend themselves to further pharmacological development.

  5. Sarcoplasmic reticulum-associated cyclic adenosine 5'-monophosphate phosphodiesterase activity in normal and failing human hearts.

    PubMed Central

    Movsesian, M A; Smith, C J; Krall, J; Bristow, M R; Manganiello, V C

    1991-01-01

    Sarcoplasmic reticulum-associated cAMP phosphodiesterase activity was examined in microsomes prepared from the left ventricular myocardium of eight heart transplant recipients with end-stage idiopathic dilated cardiomyopathy and six unmatched organ donors with normal cardiac function. At cAMP concentrations less than or equal to 1.0 microM, sarcoplasmic reticulum-associated cAMP phosphodiesterase activity was functionally homogeneous. cAMP phosphodiesterase activity was inhibited competitively by cGMP (Ki = 0.031 +/- 0.008 microM) and the cilostamide derivative OPC 3911 (Ki = 0.018 +/- 0.004 microM), but was essentially insensitive to rolipram. Vmax and Km were 781.7 +/- 109.2 nmol/mg per min and 0.188 +/- 0.031 microM, respectively, in microsomes prepared from nonfailing hearts and 793.9 +/- 68.9 nmol/mg per min and 0.150 +/- 0.027 microM in microsomes prepared from failing hearts. Microsomes prepared from nonfailing and failing hearts did not differ with respect to either the ratio of cAMP phosphodiesterase activity to ATP-dependent Ca2+ accumulation activity or the sensitivity of cAMP phosphodiesterase activity to inhibition by OPC 3911. These data suggest that the diminished inotropic efficacy of phosphodiesterase inhibitors in failing human hearts does not result from changes in the level, kinetic properties, or pharmacologic sensitivity of sarcoplasmic reticulum-associated cAMP phosphodiesterase activity. PMID:1647414

  6. Selective Phosphodiesterase 4B Inhibitors: A Review

    PubMed Central

    Azam, Mohammed Afzal; Tripuraneni, Naga Srinivas

    2014-01-01

    Abstract Phosphodiesterase 4B (PDE4B) is a member of the phosphodiesterase family of proteins that plays a critical role in regulating intracellular levels of cyclic adenosine monophosphate (cAMP) by controlling its rate of degradation. It has been demonstrated that this isoform is involved in the orchestra of events which includes inflammation, schizophrenia, cancers, chronic obstructive pulmonary disease, contractility of the myocardium, and psoriatic arthritis. Phosphodiesterase 4B has constituted an interesting target for drug development. In recent years, a number of PDE4B inhibitors have been developed for their use as therapeutic agents. In this review, an up-to-date status of the inhibitors investigated for the inhibition of PDE4B has been given so that this rich source of structural information of presently known PDE4B inhibitors could be helpful in generating a selective and potent inhibitor of PDE4B. PMID:25853062

  7. High-affinity neuropeptide Y receptor antagonists.

    PubMed Central

    Daniels, A J; Matthews, J E; Slepetis, R J; Jansen, M; Viveros, O H; Tadepalli, A; Harrington, W; Heyer, D; Landavazo, A; Leban, J J

    1995-01-01

    Neuropeptide Y (NPY) is one of the most abundant peptide transmitters in the mammalian brain. In the periphery it is costored and coreleased with norepinephrine from sympathetic nerve terminals. However, the physiological functions of this peptide remain unclear because of the absence of specific high-affinity receptor antagonists. Three potent NPY receptor antagonists were synthesized and tested for their biological activity in in vitro, ex vivo, and in vivo functional assays. We describe here the effects of these antagonists inhibiting specific radiolabeled NPY binding at Y1 and Y2 receptors and antagonizing the effects of NPY in human erythroleukemia cell intracellular calcium mobilization perfusion pressure in the isolated rat kidney, and mean arterial blood pressure in anesthetized rats. PMID:7568074

  8. High-affinity neuropeptide Y receptor antagonists.

    PubMed

    Daniels, A J; Matthews, J E; Slepetis, R J; Jansen, M; Viveros, O H; Tadepalli, A; Harrington, W; Heyer, D; Landavazo, A; Leban, J J; Spaltenstein, A

    1995-09-26

    Neuropeptide Y (NPY) is one of the most abundant peptide transmitters in the mammalian brain. In the periphery it is costored and coreleased with norepinephrine from sympathetic nerve terminals. However, the physiological functions of this peptide remain unclear because of the absence of specific high-affinity receptor antagonists. Three potent NPY receptor antagonists were synthesized and tested for their biological activity in in vitro, ex vivo, and in vivo functional assays. We describe here the effects of these antagonists inhibiting specific radiolabeled NPY binding at Y1 and Y2 receptors and antagonizing the effects of NPY in human erythroleukemia cell intracellular calcium mobilization perfusion pressure in the isolated rat kidney, and mean arterial blood pressure in anesthetized rats.

  9. Phosphodiesterases in endocrine physiology and disease.

    PubMed

    Vezzosi, Delphine; Bertherat, Jérôme

    2011-08-01

    The cAMP-protein kinase A pathway plays a central role in the development and physiology of endocrine tissues. cAMP mediates the intracellular effects of numerous peptide hormones. Various cellular and molecular alterations of the cAMP-signaling pathway have been observed in endocrine diseases. Phosphodiesterases (PDEs) are key regulatory enzymes of intracellular cAMP levels. Indeed, PDEs are the only known mechanism for inactivation of cAMP by catalysis to 5'-AMP. It has been suggested that disruption of PDEs could also have a role in the pathogenesis of many endocrine diseases. This review summarizes the most recent advances concerning the role of the PDEs in the physiopathology of endocrine diseases. The potential significance of this knowledge can be easily envisaged by the development of drugs targeting specific PDEs.

  10. Expression cloning of a high-affinity melatonin receptor from Xenopus dermal melanophores.

    PubMed Central

    Ebisawa, T; Karne, S; Lerner, M R; Reppert, S M

    1994-01-01

    Using an expression cloning strategy, a high-affinity melatonin receptor cDNA has been isolated from Xenopus laevis dermal melanophores. Transient expression of the cDNA in COS-7 cells resulted in high-affinity 2-[125I]-iodomelatonin binding (Kd = 6.3 +/- 0.3 x 10(-11) M). In addition, six ligands exhibited a rank order of inhibition of specific 2-[125I]iodomelatonin binding that was identical to that reported for endogenous high-affinity receptors. Functional studies of CHO cells stably expressing the receptor cDNA showed that melatonin acting through the cloned receptor inhibited forskolin-stimulated cAMP accumulation in a dose-dependent manner. Northern blot analysis showed that melatonin receptor transcripts are moderately expressed in Xenopus dermal melanophores. The cDNA encodes a protein of 420 amino acids, which contains seven hydrophobic segments. Structural analysis revealed that the receptor protein is a newly discovered member of the guanine nucleotide binding protein-coupled receptor family. Images PMID:7517042

  11. Phosphodiesterase Inhibitors as Therapeutics for Traumatic Brain Injury

    PubMed Central

    Titus, David J.; Oliva, Anthony A.; Wilson, Nicole M.; Atkins, Coleen M.

    2014-01-01

    Developing therapeutics for traumatic brain injury remains a challenge for all stages of recovery. The pathological features of traumatic brain injury are diverse, and it remains an obstacle to be able to target the wide range of pathologies that vary between traumatic brain injured patients and that evolve during recovery. One promising therapeutic avenue is to target the second messengers cAMP and cGMP with phosphodiesterase inhibitors due to their broad effects within the nervous system. Phosphodiesterase inhibitors have the capability to target different injury mechanisms throughout the time course of recovery after brain injury. Inflammation and neuronal death are early targets of phosphodiesterase inhibitors, and synaptic dysfunction and circuitry remodeling are late potential targets of phosphodiesterase inhibitors. This review will discuss how signaling through cyclic nucleotides contributes to the pathology of traumatic brain injury in the acute and chronic stages of recovery. We will review our current knowledge of the successes and challenges of using phosphodiesterase inhibitors for the treatment of traumatic brain injury and conclude with important considerations in developing phosphodiesterase inhibitors as therapeutics for brain trauma. PMID:25159077

  12. High-affinity K+ uptake in pepper plants.

    PubMed

    Martínez-Cordero, M Angeles; Martínez, Vicente; Rubio, Francisco

    2005-06-01

    High-affinity K+ uptake is an essential process for plant nutrition under K+-limiting conditions. The results presented here demonstrate that pepper (Capsicum annuum) plants grown in the absence of NH4+ and starved of K+ show an NH4+-sensitive high-affinity K+ uptake that allows plant roots to deplete external K+ to values below 1 microM. When plants are grown in the presence of NH4+, high-affinity K+ uptake is not inhibited by NH4+. Although NH4+-grown plants deplete external K+ below 1 microM in the absence of NH4+, when 1 mM NH4+ is present they do not deplete external K+ below 10 microM. A K+ transporter of the HAK family, CaHAK1, is very likely mediating the NH4+-sensitive component of the high-affinity K+ uptake in pepper roots. CaHAK1 is strongly induced in the roots that show the NH4+-sensitive high-affinity K+ uptake and its induction is reduced in K+-starved plants grown in the presence of NH4+. The NH4+-insensitive K+ uptake may be mediated by an AKT1-like K+ channel.

  13. Archaeology Camp.

    ERIC Educational Resources Information Center

    White, John R.

    1984-01-01

    A summer camp for gifted youth (13-16 years old) featured two field archaeology sessions in which students participated in excavation and field trips to nearby archaeological sites along with traditional camp activities. (CL)

  14. Summer Camp.

    ERIC Educational Resources Information Center

    Burns, Maxine; And Others

    Government regulation of children's summer camps, particularly involving health and safety standards, is discussed in a series of brief interviews with camp directors and representatives of camp associations. Transcribed from the National Public Radio weekly broadcast, "Options in Education," the program includes a lengthy montage of…

  15. Astro Camp

    NASA Image and Video Library

    2012-06-12

    Each year, more than 400 Mississippi and out-of-state youths visit Stennis Space Center for weeklong Astro Camp activities. Astro Camp sessions are for children ages 7-12. The focus for 2012 Astro Camp participants was on 'What's Next for NASA! Moon, Mars, Asteroids and Beyond.'

  16. Summer Camp.

    ERIC Educational Resources Information Center

    Pfisterer, Bill

    About 50 participants and 8 supervisors attended the Summer Camp. Visitors were encouraged and parents often came to see what their kids were doing. Before arriving at camp, the students learned how important balancing the supplies was when loading the boats. On the way to camp, students studied the: (1) landmarks so that they could find their way…

  17. Cyclic 3′,5′-Adenosine Monophosphate Phosphodiesterase of Escherichia coli

    PubMed Central

    Nielsen, L. D.; Monard, D.; Rickenberg, H. V.

    1973-01-01

    The cyclic 3′,5′-adenosine monophosphate (c-AMP) phosphodiesterase from Escherichia coli has been partially purified. The enzyme has an apparent molecular weight of 30,000, a Michaelis constant of 0.5 mM c-AMP, and a pH optimum of 7. The partially purified enzyme requires for activity the presence of a reducing compound and of either iron or a protein which seemingly acts as iron carrier. PMID:4355491

  18. Phosphodiesterase 7 Inhibition Preserves Dopaminergic Neurons in Cellular and Rodent Models of Parkinson Disease

    PubMed Central

    Morales-Garcia, Jose A.; Redondo, Miriam; Alonso-Gil, Sandra; Gil, Carmen; Perez, Concepción; Martinez, Ana; Santos, Angel; Perez-Castillo, Ana

    2011-01-01

    Background Phosphodiesterase 7 plays a major role in down-regulation of protein kinase A activity by hydrolyzing cAMP in many cell types. This cyclic nucleotide plays a key role in signal transduction in a wide variety of cellular responses. In the brain, cAMP has been implicated in learning, memory processes and other brain functions. Methodology/Principal Findings Here we show a novel function of phosphodiesterase 7 inhibition on nigrostriatal dopaminergic neuronal death. We found that S14, a heterocyclic small molecule inhibitor of phosphodiesterase 7, conferred significant neuronal protection against different insults both in the human dopaminergic cell line SH-SY5Y and in primary rat mesencephalic cultures. S14 treatment also reduced microglial activation, protected dopaminergic neurons and improved motor function in the lipopolysaccharide rat model of Parkinson disease. Finally, S14 neuroprotective effects were reversed by blocking the cAMP signaling pathways that operate through cAMP-dependent protein kinase A. Conclusions/Significance Our findings demonstrate that phosphodiesterase 7 inhibition can protect dopaminergic neurons against different insults, and they provide support for the therapeutic potential of phosphodiesterase 7 inhibitors in the treatment of neurodegenerative disorders, particularly Parkinson disease. PMID:21390306

  19. Structural origins of high-affinity biotin binding to streptavidin.

    PubMed

    Weber, P C; Ohlendorf, D H; Wendoloski, J J; Salemme, F R

    1989-01-06

    The high affinity of the noncovalent interaction between biotin and streptavidin forms the basis for many diagnostic assays that require the formation of an irreversible and specific linkage between biological macromolecules. Comparison of the refined crystal structures of apo and a streptavidin:biotin complex shows that the high affinity results from several factors. These factors include the formation of multiple hydrogen bonds and van der Waals interactions between biotin and the protein, together with the ordering of surface polypeptide loops that bury the biotin in the protein interior. Structural alterations at the biotin binding site produce quaternary changes in the streptavidin tetramer. These changes apparently propagate through cooperative deformations in the twisted beta sheets that link tetramer subunits.

  20. Selective high affinity polydentate ligands and methods of making such

    DOEpatents

    DeNardo, Sally; DeNardo, Gerald; Balhorn, Rodney

    2010-02-16

    This invention provides novel polydentate selective high affinity ligands (SHALs) that can be used in a variety of applications in a manner analogous to the use of antibodies. SHALs typically comprise a multiplicity of ligands that each bind different region son the target molecule. The ligands are joined directly or through a linker thereby forming a polydentate moiety that typically binds the target molecule with high selectivity and avidity.

  1. Challenge of human Jurkat T-cells with the adenylate cyclase activator forskolin elicits major changes in cAMP phosphodiesterase (PDE) expression by up-regulating PDE3 and inducing PDE4D1 and PDE4D2 splice variants as well as down-regulating a novel PDE4A splice variant.

    PubMed Central

    Erdogan, S; Houslay, M D

    1997-01-01

    The cAMP phosphodiesterase (PDE) 3 and PDE4 isoforms provide the major cAMP-hydrolysing PDE activities in Jurkat T-cells, with additional contributions from the PDE1 and PDE2 isoforms. Challenge of cells with the adenylate cyclase activator forskolin led to a rapid, albeit transient, increase in PDE3 activity occurring over the first 45 min, followed by a sustained increase in PDE3 activity which began after approximately 3 h and continued for at least 24 h. Only this second phase of increase in PDE3 activity was blocked by the transcriptional inhibitor actinomycin D. After approximately 3 h of exposure to forskolin, PDE4 activity had increased, via a process that could be inhibited by actinomycin D, and it remained elevated for at least a 24 h period. Such actions of forskolin were mimicked by cholera toxin and 8-bromo-cAMP. Forskolin increased intracellular cAMP concentrations in a time-dependent fashion and its action was enhanced when PDE induction was blocked with actinomycin D. Reverse transcription (RT)-PCR analysis, using generic primers designed to detect transcripts representing enzymically active products of the four PDE4 genes, identified transcripts for PDE4A and PDE4D but not for PDE4B or PDE4C in untreated Jurkat T-cells. Forskolin treatment did not induce transcripts for either PDE4B or PDE4C; however, it reduced the RT-PCR signal for PDE4A transcripts and markedly enhanced that for PDE4D transcripts. Using RT-PCR primers for PDE4 splice variants, a weak signal for PDE4D1 was evident in control cells whereas, in forskolin-treated cells, clear signals for both PDE4D1 and PDE4D2 were detected. RT-PCR analysis of the PDE4A species indicated that it was not the PDE4A isoform PDE-46 (PDE4A4B). Immunoblotting of control cells for PDE4 forms identified a single PDE4A species of approximately 118 kDa, which migrated distinctly from the PDE4A4B isoform PDE-46, with immunoprecipitation analyses showing that it provided all of the PDE4 activity in control

  2. Camp Chinkapin.

    ERIC Educational Resources Information Center

    Crook County School District, Prineville, OR.

    The Camp Chinkapin program, begun in 1957-58 as a pilot program for the State of Oregon, provides all sixth grade students in Crook County (Oregon) with a 5-day session in a resident camp setting in the early summer. The book serves as an introduction to and workbook for students attending the Crook County Outdoor Classroom at Suttle Lake. The…

  3. Mechanisms Restricting Diffusion of Intracellular cAMP.

    PubMed

    Agarwal, Shailesh R; Clancy, Colleen E; Harvey, Robert D

    2016-01-22

    Although numerous receptors stimulate cAMP production in a wide array of cells, many elicit distinct, highly localized responses, implying that the subcellular distribution of cAMP is not uniform. One often used explanation is that phosphodiesterases, which breakdown cAMP, act as functional barriers limiting diffusion. However, several studies refute the notion that this is sufficient, suggesting that phosphodiesterase-independent movement of cAMP must occur at rates slower than free diffusion. But, until now this has never been demonstrated. Using Raster Image Correlation Spectroscopy (RICS), we measured the diffusion coefficient of a fluorescently-labeled cAMP derivative (φ450-cAMP) as well as other fluorescent molecules in order to investigate the role that molecular size, cell morphology, and buffering by protein kinase A (PKA) play in restricting cAMP mobility in different cell types. Our results demonstrate that cytosolic movement of cAMP is indeed much slower than the rate of free diffusion and that interactions with PKA, especially type II PKA associated with mitochondria, play a significant role. These findings have important implications with respect to cAMP signaling in all cells.

  4. LNP 906, the first high-affinity photoaffinity ligand selective for I1 imidazoline receptors

    PubMed Central

    Dragan, Urosevic; Stephan, Schann; Jean-Daniel, Ehrhardt; Pascal, Bousquet; Hugues, Greney

    2004-01-01

    The hypotensive effect of imidazoline-like drugs, such as clonidine, was attributed both to α2-adrenergic receptors and nonadrenergic imidazoline receptors, which are divided into I1, I2 and I3 subtypes. We have recently synthesized a derivative of (2-(2-chloro-4-iodo-phenylamino)-5-methyl-pyrroline (LNP 911), the first high-affinity and selective ligand for I1 receptors (I1R), with a photoactivable function (LNP 906). This work aims to test whether this derivative retained the binding properties of LNP 911 and bound irreversibly to I1R. Binding studies showed that LNP 906 exhibited nanomolar affinity for I1R and was selective for I1R over I2 receptors and α2-adrenergic receptors (α2Ars). Upon exposure to u.v. light, LNP 906 irreversibly blocked the binding of [125I]-paraiodoclonidine (PIC) to I1R, time- and dose-dependently, on PC12 cell membranes and interacted with I1R in a reversible and competitive manner in the absence of light. Pharmacological studies showed that this blockade was prevented by the concomitant presence of rilmenidine (a well-known I1 agonist), but not by rauwolscine (an α2 antagonist). Finally, LNP 906 clearly antagonized the decrease in forskolin-stimulated cAMP level induced by rilmenidine, but not by melatonin. These results indicate that LNP 906 is the first high-affinity and selective photoaffinity ligand for I1R and that it behaves as an I1R antagonist. PMID:15178642

  5. Alterations of Phosphodiesterases in Adrenocortical Tumors

    PubMed Central

    Hannah-Shmouni, Fady; Faucz, Fabio R.; Stratakis, Constantine A.

    2016-01-01

    Alterations in the cyclic (c)AMP-dependent signaling pathway have been implicated in the majority of benign adrenocortical tumors (ACTs) causing Cushing syndrome (CS). Phosphodiesterases (PDEs) are enzymes that regulate cyclic nucleotide levels, including cyclic adenosine monophosphate (cAMP). Inactivating mutations and other functional variants in PDE11A and PDE8B, two cAMP-binding PDEs, predispose to ACTs. The involvement of these two genes in ACTs was initially revealed by a genome-wide association study in patients with micronodular bilateral adrenocortical hyperplasia. Thereafter, PDE11A or PDE8B genetic variants have been found in other ACTs, including macronodular adrenocortical hyperplasias and cortisol-producing adenomas. In addition, downregulation of PDE11A expression and inactivating variants of the gene have been found in hereditary and sporadic testicular germ cell tumors, as well as in prostatic cancer. PDEs confer an increased risk of ACT formation probably through, primarily, their action on cAMP levels, but other actions might be possible. In this report, we review what is known to date about PDE11A and PDE8B and their involvement in the predisposition to ACTs. PMID:27625633

  6. Isolation and characterization of PDE9A, a novel human cGMP-specific phosphodiesterase.

    PubMed

    Fisher, D A; Smith, J F; Pillar, J S; St Denis, S H; Cheng, J B

    1998-06-19

    We have cloned and characterized the first human isozyme in a new family of cyclic nucleotide phosphodiesterases, PDE9A. By sequence homology in the catalytic domain, PDE9A is almost equidistant from all eight known mammalian PDE families but is most similar to PDE8A (34% amino acid identity) and least like PDE5A (28% amino acid identity). We report the cloning of human cDNA encoding a full-length protein of 593 amino acids, including a 261-amino acid region located near the C terminus that is homologous to the approximately 270-amino acid catalytic domain of other PDEs. PDE9A is expressed in all eight tissues examined as a approximately 2. 0-kilobase mRNA, with highest levels in spleen, small intestine, and brain. The full-length PDE9A was expressed in baculovirus fused to an N-terminal 9-amino acid FLAG tag. Kinetic analysis of the baculovirus-expressed enzyme shows it to be a very high affinity cGMP-specific PDE with a Km of 170 nM for cGMP and 230 microM for cAMP. The Km for cGMP makes PDE9A one of the highest affinity PDEs known. The Vmax for cGMP (4.9 nmol/min/microg recombinant enzyme) is about twice as fast as that of PDE4 for cAMP. The enzyme is about twice as active in vitro in 1-10 mM Mn2+ than in the same concentration of Mg2+ or Ca2+. PDE9A is insensitive (up to 100 microM) to a variety of PDE inhibitors including rolipram, vinpocetine, SKF-94120, dipyridamole, and 3-isobutyl-1-methyl-xanthine but is inhibited (IC50 = 35 microM) by zaprinast, a PDE5 inhibitor. PDE9A lacks a region homologous to the allosteric cGMP-binding regulatory regions found in the cGMP-binding PDEs: PDE2, PDE5, and PDE6.

  7. Assessment of cellular mechanisms contributing to cAMP compartmentalization in pulmonary microvascular endothelial cells.

    PubMed

    Feinstein, Wei P; Zhu, Bing; Leavesley, Silas J; Sayner, Sarah L; Rich, Thomas C

    2012-03-15

    Cyclic AMP signals encode information required to differentially regulate a wide variety of cellular responses; yet it is not well understood how information is encrypted within these signals. An emerging concept is that compartmentalization underlies specificity within the cAMP signaling pathway. This concept is based on a series of observations indicating that cAMP levels are distinct in different regions of the cell. One such observation is that cAMP production at the plasma membrane increases pulmonary microvascular endothelial barrier integrity, whereas cAMP production in the cytosol disrupts barrier integrity. To better understand how cAMP signals might be compartmentalized, we have developed mathematical models in which cellular geometry as well as total adenylyl cyclase and phosphodiesterase activities were constrained to approximate values measured in pulmonary microvascular endothelial cells. These simulations suggest that the subcellular localizations of adenylyl cyclase and phosphodiesterase activities are by themselves insufficient to generate physiologically relevant cAMP gradients. Thus, the assembly of adenylyl cyclase, phosphodiesterase, and protein kinase A onto protein scaffolds is by itself unlikely to ensure signal specificity. Rather, our simulations suggest that reductions in the effective cAMP diffusion coefficient may facilitate the formation of substantial cAMP gradients. We conclude that reductions in the effective rate of cAMP diffusion due to buffers, structural impediments, and local changes in viscosity greatly facilitate the ability of signaling complexes to impart specificity within the cAMP signaling pathway.

  8. In resting COS1 cells a dominant negative approach shows that specific, anchored PDE4 cAMP phosphodiesterase isoforms gate the activation, by basal cyclic AMP production, of AKAP-tethered protein kinase A type II located in the centrosomal region.

    PubMed

    McCahill, Angela; McSorley, Theresa; Huston, Elaine; Hill, Elaine V; Lynch, Martin J; Gall, Irene; Keryer, Guy; Lygren, Birgitte; Tasken, Kjetil; van Heeke, Gino; Houslay, Miles D

    2005-09-01

    We employ a novel, dominant negative approach to identify a key role for certain tethered cyclic AMP specific phosphodiesterase-4 (PDE4) isoforms in regulating cyclic AMP dependent protein kinase A (PKA) sub-populations in resting COS1 cells. A fraction of PKA is clearly active in resting COS1 cells and this activity increases when cells are treated with the selective PDE4 inhibitor, rolipram. Point mutation of a critical, conserved aspartate residue in the catalytic site of long PDE4A4, PDE4B1, PDE4C2 and PDE4D3 isoforms renders them catalytically inactive. Overexpressed in resting COS1 cells, catalytically inactive forms of PDE4C2 and PDE4D3, but not PDE4A4 and PDE4B1, are constitutively PKA phosphorylated while overexpressed active versions of all these isoforms are not. Inactive and active versions of all these isoforms are PKA phosphorylated in cells where protein kinase A is maximally activated with forskolin and IBMX. By contrast, rolipram challenge of COS1 cells selectively triggers the PKA phosphorylation of recombinant, active PDE4D3 and PDE4C2 but not recombinant, active PDE4A4 and PDE4B1. Purified, recombinant PDE4D3 and PDE4A4 show a similar dose-dependency for in vitro phosphorylation by PKA. Disruption of the tethering of PKA type-II to PKA anchor proteins (AKAPs), achieved using the peptide Ht31, prevents inactive forms of PDE4C2 and PDE4D3 being constitutively PKA phosphorylated in resting cells as does siRNA-mediated knockdown of PKA-RII, but not PKA-RI. PDE4C2 and PDE4D3 co-immunoprecipitate from COS1 cell lysates with 250 kDa and 450 kDa AKAPs that tether PKA type-II and not PKA type-I. PKA type-II co-localises with AKAP450 in the centrosomal region of COS1 cells. The perinuclear distribution of recombinant, inactive PDE4D3, but not inactive PDE4A4, overlaps with AKAP450 and PKA type-II. The distribution of PKA phosphorylated inactive PDE4D3 also overlaps with that of AKAP450 in the centrosomal region of COS1 cells. We propose that a novel role

  9. An Effect of Dexamethasone on Adenosine 3′,5′ -Monophosphate Content and Adenosine 3′,5′ -Monophosphate Phosphodiesterase Activity of Cultured Hepatoma Cells

    PubMed Central

    Manganiello, Vincent; Vaughan, Martha

    1972-01-01

    The effect of dexamethasone on adenosine 3′,5′-monophosphate (cAMP) phosphodiesterase activity in cultured HTC hepatoma cells was investigated. Homogenates of these cells contain phosphodiesterase activity with two apparent Michaelis constants for cAMP (2-5 μm and 50 μm). At all substrate concentrations tested, phosphodiesterase activity was decreased 25-40% in cells incubated for 36 hr or more with 1 μm dexamethasone. Acid phosphatase activity in the same cells was not decreased. α-Methyl testosterone, 1 μm, was without effect on phosphodiesterase activity. Incubation for 10 min with epinephrine plus theophylline increased the cAMP content of the HTC cells 3- to 6-fold. In cells incubated for 72 hr with dexamethasone, the basal concentration of cAMP was slightly increased and the increment produced by epinephrine plus theophylline was markedly increased. We believe that in many cells the so-called permissive effects of steroid hormones on cAMP mediated processes may be due to an effect of these hormones on cAMP phosphodiesterase activity similar to that observed in HTC cells incubated with dexamethasone. PMID:4341439

  10. Astro Camp

    NASA Technical Reports Server (NTRS)

    1996-01-01

    Children who attend NASA's summer Astro Camp at Stennis Space Center enjoy a week of fun-filled activities. Campers learn what it feels like to be a couple of inches taller in space and treck through an astronaut obstacle course. They also have the opportunity to build their own model rockets, which are then launched on the last day of each camp. Campers also travel on field trips to places such as the Challenger Learning Center at the Louisiana Arts and Science Center in Baton Rouge. Four weeks of Astro Camp are held each year during the summer-two camps for 8- to 10-year-olds and two for 11- to 13-year olds.

  11. Astro Camp

    NASA Technical Reports Server (NTRS)

    1995-01-01

    Children who attend NASA's summer Astro Camp at Stennis Space Center enjoy a week of fun-filled activities. Campers learn what it is like to be a couple of inches taller in space and go through an astronaut obstacle course. They also learn how to build their own model rockets, which are launched on the last day of each camp. Campers also attend field trips to places such as the Challenger Learning Center at the Louisiana Arts and Science Center in Baton Rouge. Four weeks of Astro Camp are held during the summer each year-two camps for 8- to 10-year-olds and two for 11- to 13-year olds.

  12. Low and high affinity receptors mediate cellular uptake of heparanase

    PubMed Central

    Ben-Zaken, Olga; Shafat, Itay; Gingis-Velitski, Svetlana; Bangio, Haim; Kelson, Idil Kasuto; Alergand, Tal; Amor, Yehudit; Maya, Ruth Ben-Yakar; Vlodavsky, Israel; Ilan, Neta

    2008-01-01

    Heparanase is an endoglycosidase which cleaves heparan sulfate and hence participates in degradation and remodeling of the extracellular matrix. Importantly, heparanase activity correlated with the metastatic potential of tumor-derived cells, attributed to enhanced cell dissemination as a consequence of heparan sulfate cleavage and remodeling of the extracellular matrix barrier. Heparanase has been characterized as a glycoprotein, yet glycan biochemical analysis was not performed to date. Here, we applied the Qproteome™ GlycoArray kit to perform glycan analysis of heparanase, and compared the kit results with the more commonly used biochemical analyses. We employed fibroblasts isolated from patients with I-cell disease (mucolipidosis II), fibroblasts deficient of low density lipoprotein receptor-related protein and fibroblasts lacking mannose 6-phosphate receptor, to explore the role of mannose 6-phosphate in heparanase uptake. Iodinated heparanase has been utilized to calculate binding affinity. We provide evidence for hierarchy of binding to cellular receptors as a function of heparanase concentration. We report the existence of a high affinity, low abundant (i.e., low density lipoprotein receptor-related protein, mannose 6-phosphate receptor), as well as a low affinity, high abundant (i.e., heparan sulfate proteoglycan) receptors that mediate heparanase binding, and suggest that these receptors cooperate to establish high affinity binding sites for heparanase, thus maintaining extracellular retention of the enzyme tightly regulated. PMID:17981072

  13. 01-ERD-111 - The Development of Synthetic High Affinity Ligands

    SciTech Connect

    Perkins, J; Balhorn, R; Cosman, M; Lightstone, F; Zeller, L

    2004-02-05

    The aim of this project was to develop Synthetic High-Affinity Ligands (SHALs), which bind with high affinity and specificity to proteins of interest for national security and cancer therapy applications. The aim of producing synthetic ligands for sensory devices as an alternative to antibody-based detection assays and therapeutic agents is to overcome the drawbacks associated with antibody-based in next-generation sensors and systems. The focus area of the project was the chemical synthesis of the SHALs. The project concentrated on two different protein targets. (a) The C fragment of tetanus and botulinum toxin, potential biowarfare agents. A SHAL for tetanus or botulinum toxin would be incorporated into a sensory device for the toxins. (b) HLA-DR10, a protein found in high abundance on the surface of Non-Hodgkins Lymphoma. A SHAL specific to a tumor marker, labeled with a radionuclide, would enable the targeted delivery of radiation therapy to metastatic disease. The technical approach used to develop a SHAL for each protein target will be described in more detail below. However, in general, the development of a SHAL requires a combination of computational modeling techniques, modern nuclear magnetic resonance spectroscopy (NMR) and synthetic chemistry.

  14. Direct measurement of equilibrium constants for high-affinity hemoglobins.

    PubMed

    Kundu, Suman; Premer, Scott A; Hoy, Julie A; Trent, James T; Hargrove, Mark S

    2003-06-01

    The biological functions of heme proteins are linked to their rate and affinity constants for ligand binding. Kinetic experiments are commonly used to measure equilibrium constants for traditional hemoglobins comprised of pentacoordinate ligand binding sites and simple bimolecular reaction schemes. However, kinetic methods do not always yield reliable equilibrium constants with more complex hemoglobins for which reaction mechanisms are not clearly understood. Furthermore, even where reaction mechanisms are clearly understood, it is very difficult to directly measure equilibrium constants for oxygen and carbon monoxide binding to high-affinity (K(D) < 1 micro M) hemoglobins. This work presents a method for direct measurement of equilibrium constants for high-affinity hemoglobins that utilizes a competition for ligands between the "target" protein and an array of "scavenger" hemoglobins with known affinities. This method is described for oxygen and carbon monoxide binding to two hexacoordinate hemoglobins: rice nonsymbiotic hemoglobin and Synechocystis hemoglobin. Our results demonstrate that although these proteins have different mechanisms for ligand binding, their affinities for oxygen and carbon monoxide are similar. Their large affinity constants for oxygen, 285 and approximately 100 micro M(-1) respectively, indicate that they are not capable of facilitating oxygen transport.

  15. Histamine receptors on adult rat cardiomyocytes: antagonism of alpha/sub 1/-receptor stimulation of cAMP degradation

    SciTech Connect

    Buxton, I.L.O.; Bowen, S.M.

    1986-03-01

    Incubation of intact cardiomyocytes with the histamine antagonist (/sup 3/H)mepyramine results in rapid reversible binding to a single class of high affinity sites (K/sub D/ = 1.2nM; 50,000 sites/myocyte). In membranes from purified myocytes histamine competition of (/sup 3/H)mepyramine binding (K/sub D/ = 300nM) is not altered by GTP (10..mu..M). Competition of (/sup 3/H)mepyramine binding by H-receptor subtype-selective antagonists suggests the presence of a single class of H/sub 1/-receptors. Incubation of intact myocytes with histamine (luM, H/sub 1/ receptor activation) plus norepinephrine (NE 1uM, alpha/sub 1/ + beta/sub 1/ receptor activation) for 3 min leads to significantly more cAMP accumulation (36.5 pmol/10/sup 6/ myocytes) than NE alone (30 pmol/10/sup 6/ myocytes). Histamine alone does not alter basal cAMP = 10.4 pmol/10/sup 6/ myocytes, or beta/sub 1/ stimulation (isoproternol, 1uM) = 39.6 pmol/10/sup 6/ myocytes. Cyclic AMP accumulation with NE plus prazosin 10nM, (alpha/sub 1/ + beta/sub 1/ + alpha/sub 1/ blockade) is indistinguishable from NE + histamine, (alpha/sub 1/ + beta/sub 1/ + H/sub 1/) stimulation. Histamine competition for (/sup 3/H)prazosin binding suggests that histamine does not block alpha/sub 1/ receptors on the myocyte. These data suggest that H/sub 1/ receptor activation leads to antagonism of the alpha/sub 1/ receptor mediated activation of cAMP phosphodiesterase the authors have recently described.

  16. HIGH AFFINITY, DSRNA BINDING BY DISCONNECTED INTERACTING PROTEIN 1†

    PubMed Central

    Catanese, Daniel J.; Matthews, Kathleen S.

    2010-01-01

    Disconnected Interacting Protein 1 (DIP1) appears from sequence analysis and preliminary binding studies to be a member of the dsRNA-binding protein family. Of interest, DIP1 was shown previously to interact with and influence multiple proteins involved in transcription regulation in Drosophila melanogaster. We show here that the longest isoform of this protein, DIP1-c, exhibits a 500-fold preference for dsRNA over dsDNA of similar nucleotide sequence. Further, DIP1-c demonstrated very high affinity for a subset of dsRNA ligands, with binding in the picomolar range for VA1 RNA and miR-iab-4 precursor stem-loop, a potential physiological RNA target involved in regulating expression of its protein partner, Ultrabithorax. PMID:20643095

  17. High-affinity ammonium transporters and nitrogen sensing in mycorrhizas.

    PubMed

    Javelle, Arnaud; André, Bruno; Marini, Anne Marie; Chalot, Michel

    2003-02-01

    Most terrestrial plants live in mutualistic symbiosis with root-infecting mycorrhizal fungi. This association requires a molecular dialogue between the two partners. However, the nature of the chemical signals that induce hyphal differentiation are not well characterized and the mechanisms for signal reception are still unknown. In addition to its role in ammonium scavenging, the Mep2 protein from Saccharomyces cerevisiae has been proposed to act as an ammonium sensor that is essential for pseudohyphal differentiation in response to ammonium limitation. We propose that the high-affinity ammonium transporters from mycorrhizal fungi act in a similar manner to sense the environment and induce, via as-yet-unidentified signal transduction cascades, the switch in the mode of fungal growth observed during the formation of mycorrhiza.

  18. Complex high affinity interactions occur between MHCI and superantigens

    NASA Technical Reports Server (NTRS)

    Chapes, S. K.; Herpich, A. R.; Spooner, B. S. (Principal Investigator)

    1998-01-01

    Staphylococcal enterotoxins A and C1 (SEA or SEC1) bound to major histocompatibility-I (MHCI) molecules with high affinity (binding constants ranging from 1.1 microM to 79 nM). SEA and SEC1 directly bound MHCI molecules that had been captured by monoclonal antibodies specific for H-2Kk, H-2Dk, or both. In addition, MHCI-specific antibodies inhibited the binding of SEC1 to LM929 cells and SEA competitively inhibited SEC1 binding; indicating that the superantigens bound to MHCI on the cell surface. The affinity and number of superantigen binding sites differed depending on whether MHCI was expressed in the membrane of LM929 cells or whether it was captured. These data support the hypothesis that MHCI molecules can serve as superantigen receptors.

  19. [Hemophilia camps.

    PubMed

    Juárez-Sierra, Julieta; Del Pilar Torres-Arreola, Laura; Marín-Palomares, Teresa; Dueñas-González, María Teresa; Monteros-Rincón, Martha Patricia; Osorio-Guzmán, Maricela

    2013-01-01

    We reported the experience of hemophilia camps which was accomplished with patients from hospitals of the Instituto Mexicano del Seguro Social. The aim was to prepare the families and patients regarding the disease treatment, in order to promote the self sufficiency and to know the impact of the program on the course of the disease. Surveys were applied about treatment items and personal opinions were collected. The results of the national hemophilia camp were: group of 56 patients, average 14 years, 2 % women, 51 % severe hemophilia and 43 % had hemophilic brothers. Benefits: patients increased their knowledge about earlier bleeding identification and the self-infusion method; they became aware on their responsibility in self care, timely treatment and duties at home. Hemophilia camps with patients are an option for attitude change before disease complications. Social network creation and the increase in self-sufficiency are other benefits.

  20. Organized Camping's Honorable Tradition.

    ERIC Educational Resources Information Center

    Miranda, Wilma

    1990-01-01

    Profiles three twentieth-century outdoor camping leaders. Describes early camping programs as "experimental intentional communities" teaching personal and social empowerment. Portia Mansfield's Wyonegonic Camps emphasized cooperation and artistic freedom. Joshua Lieberman's Pioneer Youth Camp taught workers' children social…

  1. Organized Camping's Honorable Tradition.

    ERIC Educational Resources Information Center

    Miranda, Wilma

    1990-01-01

    Profiles three twentieth-century outdoor camping leaders. Describes early camping programs as "experimental intentional communities" teaching personal and social empowerment. Portia Mansfield's Wyonegonic Camps emphasized cooperation and artistic freedom. Joshua Lieberman's Pioneer Youth Camp taught workers' children social…

  2. Camp Minden

    EPA Pesticide Factsheets

    Camp Minden is a Superfund Site located near the City of Minden, Louisiana. In October 2012, one of the storage bunkers exploded. In October 2014, the EPA signed a Settlement Agreement and selected a method to dispose of the remaining explosives.

  3. Discovery of PF-06928215 as a high affinity inhibitor of cGAS enabled by a novel fluorescence polarization assay

    PubMed Central

    Brault, Amy; Vincent, Fabien; Weng, Shawn; Wang, Hong; Dumlao, Darren; Aulabaugh, Ann; Aivazian, Dikran; Castro, Dana; Chen, Ming; Culp, Jeffrey; Dower, Ken; Gardner, Joseph; Hawrylik, Steven; Golenbock, Douglas; Hepworth, David; Horn, Mark; Jones, Lyn; Jones, Peter; Latz, Eicke; Li, Jing; Lin, Lih-Ling; Lin, Wen; Lin, David; Lovering, Frank; Niljanskul, Nootaree; Nistler, Ryan; Pierce, Betsy; Plotnikova, Olga; Schmitt, Daniel; Shanker, Suman; Smith, James; Snyder, William; Subashi, Timothy; Trujillo, John; Tyminski, Edyta; Wang, Guoxing; Wong, Jimson; Lefker, Bruce; Dakin, Leslie; Leach, Karen

    2017-01-01

    Cyclic GMP-AMP synthase (cGAS) initiates the innate immune system in response to cytosolic dsDNA. After binding and activation from dsDNA, cGAS uses ATP and GTP to synthesize 2′, 3′ -cGAMP (cGAMP), a cyclic dinucleotide second messenger with mixed 2′-5′ and 3′-5′ phosphodiester bonds. Inappropriate stimulation of cGAS has been implicated in autoimmune disease such as systemic lupus erythematosus, thus inhibition of cGAS may be of therapeutic benefit in some diseases; however, the size and polarity of the cGAS active site makes it a challenging target for the development of conventional substrate-competitive inhibitors. We report here the development of a high affinity (KD = 200 nM) inhibitor from a low affinity fragment hit with supporting biochemical and structural data showing these molecules bind to the cGAS active site. We also report a new high throughput cGAS fluorescence polarization (FP)-based assay to enable the rapid identification and optimization of cGAS inhibitors. This FP assay uses Cy5-labelled cGAMP in combination with a novel high affinity monoclonal antibody that specifically recognizes cGAMP with no cross reactivity to cAMP, cGMP, ATP, or GTP. Given its role in the innate immune response, cGAS is a promising therapeutic target for autoinflammatory disease. Our results demonstrate its druggability, provide a high affinity tool compound, and establish a high throughput assay for the identification of next generation cGAS inhibitors. PMID:28934246

  4. Discovery of PF-06928215 as a high affinity inhibitor of cGAS enabled by a novel fluorescence polarization assay.

    PubMed

    Hall, Justin; Brault, Amy; Vincent, Fabien; Weng, Shawn; Wang, Hong; Dumlao, Darren; Aulabaugh, Ann; Aivazian, Dikran; Castro, Dana; Chen, Ming; Culp, Jeffrey; Dower, Ken; Gardner, Joseph; Hawrylik, Steven; Golenbock, Douglas; Hepworth, David; Horn, Mark; Jones, Lyn; Jones, Peter; Latz, Eicke; Li, Jing; Lin, Lih-Ling; Lin, Wen; Lin, David; Lovering, Frank; Niljanskul, Nootaree; Nistler, Ryan; Pierce, Betsy; Plotnikova, Olga; Schmitt, Daniel; Shanker, Suman; Smith, James; Snyder, William; Subashi, Timothy; Trujillo, John; Tyminski, Edyta; Wang, Guoxing; Wong, Jimson; Lefker, Bruce; Dakin, Leslie; Leach, Karen

    2017-01-01

    Cyclic GMP-AMP synthase (cGAS) initiates the innate immune system in response to cytosolic dsDNA. After binding and activation from dsDNA, cGAS uses ATP and GTP to synthesize 2', 3' -cGAMP (cGAMP), a cyclic dinucleotide second messenger with mixed 2'-5' and 3'-5' phosphodiester bonds. Inappropriate stimulation of cGAS has been implicated in autoimmune disease such as systemic lupus erythematosus, thus inhibition of cGAS may be of therapeutic benefit in some diseases; however, the size and polarity of the cGAS active site makes it a challenging target for the development of conventional substrate-competitive inhibitors. We report here the development of a high affinity (KD = 200 nM) inhibitor from a low affinity fragment hit with supporting biochemical and structural data showing these molecules bind to the cGAS active site. We also report a new high throughput cGAS fluorescence polarization (FP)-based assay to enable the rapid identification and optimization of cGAS inhibitors. This FP assay uses Cy5-labelled cGAMP in combination with a novel high affinity monoclonal antibody that specifically recognizes cGAMP with no cross reactivity to cAMP, cGMP, ATP, or GTP. Given its role in the innate immune response, cGAS is a promising therapeutic target for autoinflammatory disease. Our results demonstrate its druggability, provide a high affinity tool compound, and establish a high throughput assay for the identification of next generation cGAS inhibitors.

  5. [Interaction of adenosin-3',5'-cyclosulfate with adenosine-3'5'-cyclophosphate dependent protein kinase and phosphodiesterase].

    PubMed

    Severin, E S; Tkachuk, V A; Guliaev, N N

    1976-02-01

    Interaction of adenosine-3',5'-cyclosulphate (cAMS) cAMP analogue, having sulphur atom instead of phosphorus in a six-term cyclic system with pig brain proteinkinase and rabbit skeletal muscle phosphodiesterase is studied. The affinity of proteinkinase to cAMS was found to be in 25000 times lower than the affinity of cAMP, the affinity of cAMS to the active site of phosphodiesterase being high enough. It is suggested that in the regulatory subunit of proteinkinase positive kationic group participates in nucleotide binding by interacting with negative oxygen atom of six-term cyclophosphate system. There is no such a group in the active site of phospodiesterase, because the absence of negative charge in case of cAMS only slightly affects the constant of cAMS binding by phosphodiesterase.

  6. Jen1p: A High Affinity Selenite Transporter in Yeast

    PubMed Central

    McDermott, Joseph R.; Rosen, Barry P.

    2010-01-01

    Selenium is a micronutrient in most eukaryotes, including humans, which is well known for having an extremely thin border between beneficial and toxic concentrations. Soluble tetravalent selenite is the predominant environmental form and also the form that is applied in the treatment of human diseases. To acquire this nutrient from low environmental concentrations as well as to avoid toxicity, a well-controlled transport system is required. Here we report that Jen1p, a proton-coupled monocarboxylate transporter in S. cerevisiae, catalyzes high-affinity uptake of selenite. Disruption of JEN1 resulted in selenite resistance, and overexpression resulted in selenite hypersensitivity. Transport assay showed that overexpression of Jen1p enables selenite accumulation in yeast compared with a JEN1 knock out strain, indicating the Jen1p transporter facilitates selenite accumulation inside cells. Selenite uptake by Jen1p had a Km of 0.91 mM, which is comparable to the Km for lactate. Jen1p transported selenite in a proton-dependent manner which resembles the transport mechanism for lactate. In addition, selenite and lactate can inhibit the transport of each other competitively. Therefore, we postulate selenite is a molecular mimic of monocarboxylates which allows selenite to be transported by Jen1p. PMID:20861301

  7. Jen1p: a high affinity selenite transporter in yeast.

    PubMed

    McDermott, Joseph R; Rosen, Barry P; Liu, Zijuan

    2010-11-15

    Selenium is a micronutrient in most eukaryotes, including humans, which is well known for having an extremely thin border between beneficial and toxic concentrations. Soluble tetravalent selenite is the predominant environmental form and also the form that is applied in the treatment of human diseases. To acquire this nutrient from low environmental concentrations as well as to avoid toxicity, a well-controlled transport system is required. Here we report that Jen1p, a proton-coupled monocarboxylate transporter in S. cerevisiae, catalyzes high-affinity uptake of selenite. Disruption of JEN1 resulted in selenite resistance, and overexpression resulted in selenite hypersensitivity. Transport assay showed that overexpression of Jen1p enables selenite accumulation in yeast compared with a JEN1 knock out strain, indicating the Jen1p transporter facilitates selenite accumulation inside cells. Selenite uptake by Jen1p had a Km of 0.91 mM, which is comparable to the Km for lactate. Jen1p transported selenite in a proton-dependent manner which resembles the transport mechanism for lactate. In addition, selenite and lactate can inhibit the transport of each other competitively. Therefore, we postulate selenite is a molecular mimic of monocarboxylates which allows selenite to be transported by Jen1p.

  8. Phosphatidylserine Reversibly Binds Cu2+ with Extremely High Affinity

    PubMed Central

    Monson, Christopher F.; Cong, Xiao; Robison, Aaron; Pace, Hudson P.; Liu, Chunming; Poyton, Matthew F.; Cremer, Paul S.

    2012-01-01

    Phosphatidylserine (PS) embedded within supported lipid bilayers (SLBs) was found to bind Cu2+ from solution with extraordinarily high affinity. In fact, the equilibrium dissociation constant was in the femtomolar range. The resulting complex formed in a 1:2 Cu2+ to PS ratio and quenches a broad spectrum of lipid-bound fluorophores in a reversible and pH-dependent fashion. At acidic pH values, the fluorophores were almost completely unquenched, while at basic pH values significant quenching (85–90%) was observed. The pH at which the transition occurred was dependent on the PS concentration and ranged from approximately pH 5 to 8. The quenching kinetics was slow at low Cu2+ concentrations and basic values pH (up to several hours), while the unquenching reaction was orders of magnitude more rapid upon lowering the pH. This was consistent with diffusion limited complex formation at basic pH, but rapid dissociation under acidic conditions. The tight binding of Cu2+ to PS may have physiological consequences under certain circumstances. PMID:22548290

  9. Identification, characterization and subcellular localization of TcPDE1, a novel cAMP-specific phosphodiesterase from Trypanosoma cruzi.

    PubMed Central

    D'Angelo, Maximiliano A; Sanguineti, Santiago; Reece, Jeffrey M; Birnbaumer, Lutz; Torres, Héctor N; Flawiá, Mirtha M

    2004-01-01

    Compartmentalization of cAMP phosphodiesterases plays a key role in the regulation of cAMP signalling in mammals. In the present paper, we report the characterization and subcellular localization of TcPDE1, the first cAMP-specific phosphodiesterase to be identified from Trypanosoma cruzi. TcPDE1 is part of a small gene family and encodes a 929-amino-acid protein that can complement a heat-shock-sensitive yeast mutant deficient in phospho-diesterase genes. Recombinant TcPDE1 strongly associates with membranes and cannot be released with NaCl or sodium cholate, suggesting that it is an integral membrane protein. This enzyme is specific for cAMP and its activity is not affected by cGMP, Ca2+, calmodulin or fenotiazinic inhibitors. TcPDE1 is sensitive to the phosphodiesterase inhibitor dipyridamole but is resistant to 3-isobutyl-1-methylxanthine, theophylline, rolipram and zaprinast. Papaverine, erythro-9-(2-hydroxy-3-nonyl)-adenine hydrochloride, and vinpocetine are poor inhibitors of this enzyme. Confocal laser scanning of T. cruzi epimastigotes showed that TcPDE1 is associated with the plasma membrane and concentrated in the flagellum of the parasite. The association of TcPDE1 with this organelle was confirmed by subcellular fractionation and cell-disruption treatments. The localization of this enzyme is a unique feature that distinguishes it from all the trypanosomatid phosphodiesterases described so far and indicates that compartmentalization of cAMP phosphodiesterases could also be important in these parasites. PMID:14556647

  10. High-affinity binding of fibronectin to cultured Kupffer cells

    SciTech Connect

    Cardarelli, P.M.; Blumenstock, F.A.; McKeown-Longo, P.J.; Saba, T.M.; Mazurkiewicz, J.E.; Dias, J.A. )

    1990-11-01

    Hepatic Kupffer cells are a major component of the reticuloendothelial or macrophage system. They were the first phagocytic cell type whose phagocytosis was shown to be influenced by plasma fibronectin, a dimeric opsonic glycoprotein. In the current study, the binding of soluble radioiodinated fibronectin purified from rat serum to isolated rat hepatic Kupffer cells was investigated using a cultured Kupffer cell monolayer technique. Binding was specific, since unlabeled purified fibronectin competed in a dose-dependent manner with the 125I-fibronectin for binding to the Kupffer cells. Addition of gelatin enhanced the binding of 125I-fibronectin to Kupffer cells. The phagocytosis of gelatinized-coated red cells by Kupffer cells was increased either by preopsonizing the target particles with purified fibronectin or by the addition of purified fibronectin to the culture medium. In contrast, exposure of the Kupffer cells to medium containing purified fibronectin followed by wash-removal of the fibronectin did not increase the uptake of gelatin-coated red blood cells, even though fibronectin was detected on the surface of the Kupffer cells by immunofluorescence. Trypsinized monolayers expressed decreased capacity to bind 125I-fibronectin as well as fibronectin-coated sheep erythrocytes. The binding of 125I-fibronectin-gelatin complexes was inhibited by excess unlabeled fibronectin. We calculated that specific high-affinity (Kd = 7.46 x 10(-9) M) binding sites for fibronectin exist on Kupffer cells. There are approximately 2,800-3,500 binding sites or putative fibronectin receptors per Kupffer cell. These sites appear to mediate the enhanced phagocytosis of gelatin-coated particles opsonized by fibronectin.

  11. Phosphodiesterase 3 inhibitors suppress oocyte maturation and consequent pregnancy without affecting ovulation and cyclicity in rodents.

    PubMed Central

    Wiersma, A; Hirsch, B; Tsafriri, A; Hanssen, R G; Van de Kant, M; Kloosterboer, H J; Conti, M; Hsueh, A J

    1998-01-01

    During each reproductive cycle, a preovulatory surge of gonadotropins induces meiotic maturation of the oocyte in the preovulatory follicle followed by ovulation. Although gonadotropins stimulate cAMP production in somatic cells of the follicle, a decrease in intra-oocyte cAMP levels is required for resumption of meiosis in oocytes. Based on the observed compartmentalization of the cAMP-degrading enzyme, phosphodiesterase, in follicular somatic and germ cells, inhibitors of phosphodiesterase 3 were used to block meiosis in ovulating oocytes in rodents. By this strategy, we demonstrated that fertilization and pregnancy could be prevented without disturbing follicle rupture and normal estrous cyclicity. In contrast to conventional contraceptive pills that disrupt ovarian steroidogenesis and reproductive cycles, the present strategy achieves effective contraception by selective blockage of oocyte maturation and development without alterations in ovulation and reproductive cyclicity. PMID:9691090

  12. Cloning and functional characterization of the high-affinity K+ transporter HAK1 of pepper.

    PubMed

    Martínez-Cordero, M Angeles; Martínez, Vicente; Rubio, Francisco

    2004-10-01

    High-affinity K+ uptake in plants plays a crucial role in K+ nutrition and different systems have been postulated to contribute to the high-affinity K+ uptake. The results presented here with pepper (Capsicum annum) demonstrate that a HAK1-type transporter greatly contributes to the high-affinity K+ uptake observed in roots. Pepper plants starved of K+ for 3 d showed high-affinity K+ uptake (Km of 6 microM K+) that was very sensitive to NH and their roots expressed a high-affinity K+ transporter, CaHAK1, which clusters in group I of the KT/HAK/KUP family of transporters. When expressed in yeast ( Saccharomyces cerevisiae ), CaHAK1 mediated high-affinity K+ and Rb+ uptake with Km values of 3.3 and 1.9 microM, respectively. Rb+ uptake was competitively inhibited by micromolar concentrations of NH and Cs+, and by millimolar concentrations of Na+.

  13. Cyclic nucleotide specific phosphodiesterases of Leishmania major

    PubMed Central

    Johner, Andrea; Kunz, Stefan; Linder, Markus; Shakur, Yasmin; Seebeck, Thomas

    2006-01-01

    Background Leishmania represent a complex of important human pathogens that belong to the systematic order of the kinetoplastida. They are transmitted between their human and mammalian hosts by different bloodsucking sandfly vectors. In their hosts, the Leishmania undergo several differentiation steps, and their coordination and optimization crucially depend on numerous interactions between the parasites and the physiological environment presented by the fly and human hosts. Little is still known about the signalling networks involved in these functions. In an attempt to better understand the role of cyclic nucleotide signalling in Leishmania differentiation and host-parasite interaction, we here present an initial study on the cyclic nucleotide-specific phosphodiesterases of Leishmania major. Results This paper presents the identification of three class I cyclic-nucleotide-specific phosphodiesterases (PDEs) from L. major, PDEs whose catalytic domains exhibit considerable sequence conservation with, among other, all eleven human PDE families. In contrast to other protozoa such as Dictyostelium, or fungi such as Saccharomyces cerevisiae, Candida ssp or Neurospora, no genes for class II PDEs were found in the Leishmania genomes. LmjPDEA contains a class I catalytic domain at the C-terminus of the polypeptide, with no other discernible functional domains elsewhere. LmjPDEB1 and LmjPDEB2 are coded for by closely related, tandemly linked genes on chromosome 15. Both PDEs contain two GAF domains in their N-terminal region, and their almost identical catalytic domains are located at the C-terminus of the polypeptide. LmjPDEA, LmjPDEB1 and LmjPDEB2 were further characterized by functional complementation in a PDE-deficient S. cerevisiae strain. All three enzymes conferred complementation, demonstrating that all three can hydrolyze cAMP. Recombinant LmjPDEB1 and LmjPDEB2 were shown to be cAMP-specific, with Km values in the low micromolar range. Several PDE inhibitors were

  14. High Affinity Binding of Indium and Ruthenium Ions by Gastrins

    PubMed Central

    Baldwin, Graham S.; George, Graham N.; Pushie, M. Jake

    2015-01-01

    The peptide hormone gastrin binds two ferric ions with high affinity, and iron binding is essential for the biological activity of non-amidated forms of the hormone. Since gastrins act as growth factors in gastrointestinal cancers, and as peptides labelled with Ga and In isotopes are increasingly used for cancer diagnosis, the ability of gastrins to bind other metal ions was investigated systematically by absorption spectroscopy. The coordination structures of the complexes were characterized by extended X-ray absorption fine structure (EXAFS) spectroscopy. Changes in the absorption of gastrin in the presence of increasing concentrations of Ga3+ were fitted by a 2 site model with dissociation constants (Kd) of 3.3 x 10−7 and 1.1 x 10−6 M. Although the absorption of gastrin did not change upon the addition of In3+ ions, the changes in absorbance on Fe3+ ion binding in the presence of indium ions were fitted by a 2 site model with Kd values for In3+ of 6.5 x 10−15 and 1.7 x 10−7 M. Similar results were obtained with Ru3+ ions, although the Kd values for Ru3+ of 2.6 x 10−13 and 1.2 x 10−5 M were slightly larger than observed for In3+. The structures determined by EXAFS all had metal:gastrin stoichiometries of 2:1 but, while the metal ions in the Fe, Ga and In complexes were bridged by a carboxylate and an oxygen with a metal-metal separation of 3.0–3.3 Å, the Ru complex clearly demonstrated a short range Ru—Ru separation, which was significantly shorter, at 2.4 Å, indicative of a metal-metal bond. We conclude that gastrin selectively binds two In3+ or Ru3+ ions, and that the affinity of the first site for In3+ or Ru3+ ions is higher than for ferric ions. Some of the metal ion-gastrin complexes may be useful for cancer diagnosis and therapy. PMID:26457677

  15. High Affinity Binding of Indium and Ruthenium Ions by Gastrins.

    PubMed

    Baldwin, Graham S; George, Graham N; Pushie, M Jake

    2015-01-01

    The peptide hormone gastrin binds two ferric ions with high affinity, and iron binding is essential for the biological activity of non-amidated forms of the hormone. Since gastrins act as growth factors in gastrointestinal cancers, and as peptides labelled with Ga and In isotopes are increasingly used for cancer diagnosis, the ability of gastrins to bind other metal ions was investigated systematically by absorption spectroscopy. The coordination structures of the complexes were characterized by extended X-ray absorption fine structure (EXAFS) spectroscopy. Changes in the absorption of gastrin in the presence of increasing concentrations of Ga3+ were fitted by a 2 site model with dissociation constants (Kd) of 3.3 x 10-7 and 1.1 x 10-6 M. Although the absorption of gastrin did not change upon the addition of In3+ ions, the changes in absorbance on Fe3+ ion binding in the presence of indium ions were fitted by a 2 site model with Kd values for In3+ of 6.5 x 10-15 and 1.7 x 10-7 M. Similar results were obtained with Ru3+ ions, although the Kd values for Ru3+ of 2.6 x 10-13 and 1.2 x 10-5 M were slightly larger than observed for In3+. The structures determined by EXAFS all had metal:gastrin stoichiometries of 2:1 but, while the metal ions in the Fe, Ga and In complexes were bridged by a carboxylate and an oxygen with a metal-metal separation of 3.0-3.3 Å, the Ru complex clearly demonstrated a short range Ru-Ru separation, which was significantly shorter, at 2.4 Å, indicative of a metal-metal bond. We conclude that gastrin selectively binds two In3+ or Ru3+ ions, and that the affinity of the first site for In3+ or Ru3+ ions is higher than for ferric ions. Some of the metal ion-gastrin complexes may be useful for cancer diagnosis and therapy.

  16. Marketing Your Day Camp.

    ERIC Educational Resources Information Center

    Coleman, George

    1997-01-01

    Marketing strategies for day camps include encouraging camp staff to get involved in organizations involving children, families, and communities; holding camp fairs; offering the use of camp facilities to outside groups; hosting sport leagues and local youth outings; planning community fairs; and otherwise involving the camp in the community. (LP)

  17. Clinical and Molecular Genetics of the Phosphodiesterases (PDEs)

    PubMed Central

    Azevedo, Monalisa F.; Faucz, Fabio R.; Bimpaki, Eirini; Horvath, Anelia; Levy, Isaac; de Alexandre, Rodrigo B.; Ahmad, Faiyaz; Manganiello, Vincent

    2014-01-01

    Cyclic nucleotide phosphodiesterases (PDEs) are enzymes that have the unique function of terminating cyclic nucleotide signaling by catalyzing the hydrolysis of cAMP and GMP. They are critical regulators of the intracellular concentrations of cAMP and cGMP as well as of their signaling pathways and downstream biological effects. PDEs have been exploited pharmacologically for more than half a century, and some of the most successful drugs worldwide today affect PDE function. Recently, mutations in PDE genes have been identified as causative of certain human genetic diseases; even more recently, functional variants of PDE genes have been suggested to play a potential role in predisposition to tumors and/or cancer, especially in cAMP-sensitive tissues. Mouse models have been developed that point to wide developmental effects of PDEs from heart function to reproduction, to tumors, and beyond. This review brings together knowledge from a variety of disciplines (biochemistry and pharmacology, oncology, endocrinology, and reproductive sciences) with emphasis on recent research on PDEs, how PDEs affect cAMP and cGMP signaling in health and disease, and what pharmacological exploitations of PDEs may be useful in modulating cyclic nucleotide signaling in a way that prevents or treats certain human diseases. PMID:24311737

  18. Activated G-protein releases cGMP from high affinity binding sites on PDE from toad rod outer segments (ROS)

    SciTech Connect

    Yuen, P.S.T.; Walseth, T.F.; Panter, S.S.; Sundby, S.R.; Graeff, R.M.; Goldberg, N.D.

    1987-05-01

    cGMP binding proteins in toad ROS were identified by direct photoaffinity labeling (PAL) with /sup 32/P-cGMP and quantified by retention of complexes on nitrocellulose filters. By PAL, high affinity sites were present on the ..cap alpha.. and ..beta.. subunits of the cGMP-specific phosphodiesterase (PDE) which have MW/sub app/ of 94 and 90 kDa. A doublet was deduced from its photolabeling properties to represent PDE/sub ..gamma../ photocrosslinked with PDE/sub ..cap alpha../ or PDE/sub ..beta../, respectively. cGMP prebound to these high affinity sites was released by light-activated G-protein or its ..cap alpha.. subunit complexed with GTP..gamma..S; this inhibition of cGMP binding to PDE did not result from decreased cGMP availability due to enhanced hydrolysis. A low affinity cGMP binding component identified by PAL is tightly associated with ROS membranes. Apparent ATP/light-dependent stimulation of cGMP binding was shown to result from light activated cGMP hydrolysis in conjunction with ATP-promoted conversion of GMP to GDP/GTP and increased GDP/GTP binding. These findings coincide with a model for light-related regulation of cGMP binding and metabolism predicted from intact and cellfree kinetic measurements: in the dark state the cGMP hydrolic rate is constrained by the availability of cGMP because of its binding to high affinity sites on PDE. Light activated G-protein releases cGMP from these sites and allows for its redistribution to lower affinity sites represented by PDE catalytic site(s) and possible cGMP-dependent membrane cation channels.

  19. High-affinity binding of (/sup 3/H)acetylcholine to muscarinic cholinergic receptors

    SciTech Connect

    Kellar, K.J.; Martino, A.M.; Hall, D.P. Jr.; Schwartz, R.D.; Taylor, R.L.

    1985-06-01

    High-affinity binding of (/sup 3/H)acetylcholine to muscarinic cholinergic sites in rat CNS and peripheral tissues was measured in the presence of cytisin, which occupies nicotinic cholinergic receptors. The muscarinic sites were characterized with regard to binding kinetics, pharmacology, anatomical distribution, and regulation by guanyl nucleotides. These binding sites have characteristics of high-affinity muscarinic cholinergic receptors with a Kd of approximately 30 nM. Most of the muscarinic agonist and antagonist drugs tested have high affinity for the (/sup 3/H)acetylcholine binding site, but pirenzepine, an antagonist which is selective for M-1 receptors, has relatively low affinity. The ratio of high-affinity (/sup 3/H)acetylcholine binding sites to total muscarinic binding sites labeled by (/sup 3/H)quinuclidinyl benzilate varies from 9 to 90% in different tissues, with the highest ratios in the pons, medulla, and heart atrium. In the presence of guanyl nucleotides, (/sup 3/H) acetylcholine binding is decreased, but the extent of decrease varies from 40 to 90% in different tissues, with the largest decreases being found in the pons, medulla, cerebellum, and heart atrium. The results indicate that (/sup 3/H)acetylcholine binds to high-affinity M-1 and M-2 muscarinic receptors, and they suggest that most M-2 sites have high affinity for acetylcholine but that only a small fraction of M-1 sites have such high affinity.

  20. A Computational Modeling and Simulation Approach to Investigate Mechanisms of Subcellular cAMP Compartmentation

    PubMed Central

    Yang, Pei-Chi; Boras, Britton W.; Jeng, Mao-Tsuen; Lewis, Timothy J.; McCulloch, Andrew D.; Harvey, Robert D.; Clancy, Colleen E.

    2016-01-01

    Subcellular compartmentation of the ubiquitous second messenger cAMP has been widely proposed as a mechanism to explain unique receptor-dependent functional responses. How exactly compartmentation is achieved, however, has remained a mystery for more than 40 years. In this study, we developed computational and mathematical models to represent a subcellular sarcomeric space in a cardiac myocyte with varying detail. We then used these models to predict the contributions of various mechanisms that establish subcellular cAMP microdomains. We used the models to test the hypothesis that phosphodiesterases act as functional barriers to diffusion, creating discrete cAMP signaling domains. We also used the models to predict the effect of a range of experimentally measured diffusion rates on cAMP compartmentation. Finally, we modeled the anatomical structures in a cardiac myocyte diad, to predict the effects of anatomical diffusion barriers on cAMP compartmentation. When we incorporated experimentally informed model parameters to reconstruct an in silico subcellular sarcomeric space with spatially distinct cAMP production sites linked to caveloar domains, the models predict that under realistic conditions phosphodiesterases alone were insufficient to generate significant cAMP gradients. This prediction persisted even when combined with slow cAMP diffusion. When we additionally considered the effects of anatomic barriers to diffusion that are expected in the cardiac myocyte dyadic space, cAMP compartmentation did occur, but only when diffusion was slow. Our model simulations suggest that additional mechanisms likely contribute to cAMP gradients occurring in submicroscopic domains. The difference between the physiological and pathological effects resulting from the production of cAMP may be a function of appropriate compartmentation of cAMP signaling. Therefore, understanding the contribution of factors that are responsible for coordinating the spatial and temporal

  1. Astro Camp Plus

    NASA Technical Reports Server (NTRS)

    2006-01-01

    Stennis Space Center's new Astro Camp Plus camp kicked off June 19 for teens ages 13-15. The new camp delves more deeply into the science, math and technology concepts introduced in the center's popular Astro Camp series. Campers including Jasmyne White (left) and Dana Yingst, both of Slidell, La., learn how NASA uses 'podcasting' to broadcast video, and made their own podcasts.

  2. Victory Junction Gang Camp

    ERIC Educational Resources Information Center

    Shell, Ryan

    2007-01-01

    This article describes the Victory Junction Gang Camp, a not-for-profit, NASCAR-themed camp for children with chronic medical conditions that serves 24 different disease groups. The mission of the camp is to give children life-changing camping experiences that are exciting, fun, and empowering in a safe and medically sound environment. While doing…

  3. Astro Camp Plus

    NASA Image and Video Library

    2006-06-19

    Stennis Space Center's new Astro Camp Plus camp kicked off June 19 for teens ages 13-15. The new camp delves more deeply into the science, math and technology concepts introduced in the center's popular Astro Camp series. Campers including Jasmyne White (left) and Dana Yingst, both of Slidell, La., learn how NASA uses 'podcasting' to broadcast video, and made their own podcasts.

  4. Camp through the Decades.

    ERIC Educational Resources Information Center

    Nicodemus, Teresa

    2003-01-01

    Camp pioneers relate how camping has grown to become more diverse, environmentally aware, safe, and conscious of its responsibility to promote healthy development of children. Changing trends in clothing, transportation, and food preparation at camp are described. The joys, discoveries, and teachable moments that camp offers children have endured.…

  5. Astro Camp Plus

    NASA Technical Reports Server (NTRS)

    2006-01-01

    Stennis Space Center's new Astro Camp Plus camp kicked off June 19 for teens ages 13-15. The new camp delves more deeply into the science, math and technology concepts introduced in the center's popular Astro Camp series. Campers including Jasmyne White (left) and Dana Yingst, both of Slidell, La., learn how NASA uses 'podcasting' to broadcast video, and made their own podcasts.

  6. [Thermostable extracellular cyclic nucleotide phosphodiesterase from Physarum polycephalum plasmodium].

    PubMed

    Nezvetskiĭ, A R; Orlova, T G; Beĭlina, S I; Orlov, N Ia

    2006-01-01

    The cyclic nucleotide phosphodiesterase secreted by Physarum polycephalum plasmodium into extracellular medium has been partially purified by DEAE cellulose chromatography, ultrafiltration, and HPLC. The results obtained by gel filtration, HPLC, electrophoresis, and isoelectric focusing suggest that, the native enzyme in solution is a monomer with a molecular mass of about 90 kDa and pI in the range 3.6 - 4.0. The Km values were estimated to be about 0.9 mM and 7.7 mM, respectively, and Vm for both substrates were similar (up to several thousand micromoles of cAMP hydrolyzed/hour per mg of enzyme). The partially purified enzyme was shown to be extremely stable. It did not lose the activity after heat treatment at 100 degrees C during 30 min. The enzyme was active in the presence of 1% SDS, but it was fully inactivated under the same conditions in the presence of beta-mercaptoethanol. The properties of the phosphodiesterase from Physarum polycephalum are discussed.

  7. Role of phosphodiesterase-4 on ethanol elicited locomotion and narcosis.

    PubMed

    Baliño, Pablo; Ledesma, Juan Carlos; Aragon, Carlos M G

    2016-02-01

    The cAMP signaling pathway has emerged as an important modulator of the pharmacological effects of ethanol. In this respect, the cAMP-dependent protein kinase has been shown to play an important role in the modulation of several ethanol-induced behavioral actions. Cellular levels of cAMP are maintained by the activity of adenylyl cyclases and phosphodiesterases. In the present work we have focused on ascertaining the role of PDE4 in mediating the neurobehavioral effects of ethanol. For this purpose, we have used the selective PDE4 inhibitor Ro 20-1724. This compound has been proven to enhance cellular cAMP response by PDE4 blockade and can be administered systemically. Swiss mice were injected intraperitoneally (i.p.) with Ro 20-1724 (0-5 mg/kg; i.p.) at different time intervals before ethanol (0-4 g/kg; i.p.) administration. Immediately after the ethanol injection, locomotor activity, loss of righting reflex, PKA footprint and enzymatic activity were assessed. Pretreatment with Ro 20-1724 increased ethanol-induced locomotor stimulation in a dose-dependent manner. Doses that increased locomotor stimulation did not modify basal locomotion or the suppression of motor activity produced by high doses of this alcohol. Ro 20-1724 did not alter the locomotor activation produced by amphetamine or cocaine. The time of loss of righting reflex evoked by ethanol was increased after pretreatment with Ro 20-1724. This effect was selective for the narcotic effects of ethanol since Ro 20-1724 did not affect pentobarbital-induced narcotic effects. Moreover, Ro 20-1724 administration increased the PKA footprint and enzymatic activity response elicited by ethanol. These data provide further evidence of the key role of the cAMP signaling pathway in the central effects of ethanol.

  8. Intracellular tortuosity underlies slow cAMP diffusion in adult ventricular myocytes

    PubMed Central

    Richards, Mark; Lomas, Oliver; Jalink, Kees; Ford, Kerrie L.; Vaughan-Jones, Richard D.; Lefkimmiatis, Konstantinos; Swietach, Pawel

    2016-01-01

    Aims 3′,5′-Cyclic adenosine monophosphate (cAMP) signals in the heart are often confined to concentration microdomains shaped by cAMP diffusion and enzymatic degradation. While the importance of phosphodiesterases (degradative enzymes) in sculpting cAMP microdomains is well established in cardiomyocytes, less is known about cAMP diffusivity (DcAMP) and factors affecting it. Many earlier studies have reported fast diffusivity, which argues against sharply defined microdomains. Methods and results [cAMP] dynamics in the cytoplasm of adult rat ventricular myocytes were imaged using a fourth generation genetically encoded FRET-based sensor. The [cAMP]-response to the addition and removal of isoproterenol (β-adrenoceptor agonist) quantified the rates of cAMP synthesis and degradation. To obtain a read out of DcAMP, a stable [cAMP] gradient was generated using a microfluidic device which delivered agonist to one half of the myocyte only. After accounting for phosphodiesterase activity, DcAMP was calculated to be 32 µm2/s; an order of magnitude lower than in water. Diffusivity was independent of the amount of cAMP produced. Saturating cAMP-binding sites with the analogue 6-Bnz-cAMP did not accelerate DcAMP, arguing against a role of buffering in restricting cAMP mobility. cAMP diffused at a comparable rate to chemically unrelated but similar sized molecules, arguing for a common physical cause of restricted diffusivity. Lower mitochondrial density and order in neonatal cardiac myocytes allowed for faster diffusion, demonstrating the importance of mitochondria as physical barriers to cAMP mobility. Conclusion In adult cardiac myocytes, tortuosity due to physical barriers, notably mitochondria, restricts cAMP diffusion to levels that are more compatible with microdomain signalling. PMID:27089919

  9. Intracellular tortuosity underlies slow cAMP diffusion in adult ventricular myocytes.

    PubMed

    Richards, Mark; Lomas, Oliver; Jalink, Kees; Ford, Kerrie L; Vaughan-Jones, Richard D; Lefkimmiatis, Konstantinos; Swietach, Pawel

    2016-06-01

    3',5'-Cyclic adenosine monophosphate (cAMP) signals in the heart are often confined to concentration microdomains shaped by cAMP diffusion and enzymatic degradation. While the importance of phosphodiesterases (degradative enzymes) in sculpting cAMP microdomains is well established in cardiomyocytes, less is known about cAMP diffusivity (DcAMP) and factors affecting it. Many earlier studies have reported fast diffusivity, which argues against sharply defined microdomains. [cAMP] dynamics in the cytoplasm of adult rat ventricular myocytes were imaged using a fourth generation genetically encoded FRET-based sensor. The [cAMP]-response to the addition and removal of isoproterenol (β-adrenoceptor agonist) quantified the rates of cAMP synthesis and degradation. To obtain a read out of DcAMP, a stable [cAMP] gradient was generated using a microfluidic device which delivered agonist to one half of the myocyte only. After accounting for phosphodiesterase activity, DcAMP was calculated to be 32 µm(2)/s; an order of magnitude lower than in water. Diffusivity was independent of the amount of cAMP produced. Saturating cAMP-binding sites with the analogue 6-Bnz-cAMP did not accelerate DcAMP, arguing against a role of buffering in restricting cAMP mobility. cAMP diffused at a comparable rate to chemically unrelated but similar sized molecules, arguing for a common physical cause of restricted diffusivity. Lower mitochondrial density and order in neonatal cardiac myocytes allowed for faster diffusion, demonstrating the importance of mitochondria as physical barriers to cAMP mobility. In adult cardiac myocytes, tortuosity due to physical barriers, notably mitochondria, restricts cAMP diffusion to levels that are more compatible with microdomain signalling. © The Author 2016. Published by Oxford University Press on behalf of the European Society of Cardiology.

  10. Chromosome localizations of genes for five cAMP-specific phosphodiesterases in man and mouse

    SciTech Connect

    Milatovich, A.; Francke, U. ); Bolger, G.; Michaeli, T. )

    1994-03-01

    Cyclic nucleotides are important second messengers that mediate a number of cellular responses to external signals. Cyclic nucleotide phosphodiesterases play a role in signal transduction by regulating the cellular concentrations of these messengers. Here, the authors have applied Southern analyses of somatic cell hybrid lines and of recombinant inbred (RI) mouse strains as well as fluorescence chromosomal in situ hybridization (FISH) to chromosomally localize five cAMP-specific nucleotide phosphodiesterase genes in human and mouse. Genes DPDE1, DPDE2, DPDE3, and DPDE4 that share sequence homology with the Drosophila dunce gene were assigned to human chromosomes 19 (DPDE1 and DPDE2), ga12 (DPDE3), and 1p31 (DPDE4) and to mouse chromosomes 8, 9, 13, and 4, respectively. The high-affinity cAMP-specific phosphodiesterase gene (HCP1) was mapped to human chromosome 8q13-q22. Since these genes are potential candidates for involvement in psychiatric or behavioral disorders, knowledge of their chromosomal localizations will facilitate the discovery of their association with disease genes as they are being mapped by linkage studies.

  11. ElaC encodes a novel binuclear zinc phosphodiesterase.

    PubMed

    Vogel, Andreas; Schilling, Oliver; Niecke, Manfred; Bettmer, Jorg; Meyer-Klaucke, Wolfram

    2002-08-09

    ElaC is a widespread gene found in eubacteria, archaebacteria, and mammals with a highly conserved sequence. Two human ElaC variants were recently associated with cancer (Tavtigian, S. V., Simard, J., Teng, D. H., Abtin, V., Baumgard, M., Beck, A., Camp, N. J., Carillo, A. R., Chen, Y., Dayananth, P., Desrochers, M., Dumont, M., Farnham, J. M., Frank, D., Frye, C., Ghaffari, S., Gupte, J. S., Hu, R., Iliev, D., Janecki, T., Kort, E. N., Laity, K. E., Leavitt, A., Leblanc, G., McArthur-Morrison, J., Pederson, A., Penn, B., Peterson, K. T., Reid, J. E., Richards, S., Schroeder, M., Smith, R., Snyder, S. C., Swedlund, B., Swensen, J., Thomas, A., Tranchant, M., Woodland, A. M., Labrie, F., Skolnick, M. H., Neuhausen, S., Rommens, J., and Cannon-Albright, L. A. (2001) Nat. Genet. 27, 172-180; Yanaihara, N., Kohno, T., Takakura, S., Takei, K., Otsuka, A., Sunaga, N., Takahashi, M., Yamazaki, M., Tashiro, H., Fukuzumi, Y., Fujimori, Y., Hagiwara, K., Tanaka, T., and Yokota, J. (2001) Genomics 72, 169-179). Analysis of the primary sequence indicates homology to an arylsulfatase and predicts a metallo-beta-lactamase fold. At present, no ElaC gene product has been investigated. We cloned the Escherichia coli ElaC gene and purified the recombinant gene product. An enzymatic analysis showed that ElaC does not encode an arylsulfatase but rather encodes a phosphodiesterase that hydrolyzes bis(p-nitrophenyl)phosphate with a k(cat) of 59 s(-1) and K' of 4 mm. Kinetic analysis of the dimeric enzyme revealed positive cooperativity for the substrate bis(p-nitrophenyl)phosphate with a Hill coefficient of 1.6, whereas hydrolysis of the substrate thymidine-5'-p-nitrophenyl phosphate followed Michaelis-Menten kinetics. Furthermore, the enzyme is capable of binding two zinc or two iron ions. However, it displays phosphodiesterase activity only in the zinc form. The metal environment characterized by zinc K-edge x-ray absorption spectroscopy was modeled with two histidine residues, one

  12. Compartmentation of cAMP signalling in cardiomyocytes in health and disease.

    PubMed

    Perera, R K; Nikolaev, V O

    2013-04-01

    3',5'-cyclic adenosine monophosphate (cAMP) is a ubiquitous second messenger critically involved in the regulation of heart function. It has been shown to act in discrete subcellular signalling compartments formed by differentially localized receptors, phosphodiesterases and protein kinases. Cardiac diseases such as hypertrophy or heart failure are associated with structural and functional remodelling of these microdomains which leads to changes in cAMP compartmentation. In this review, we will discuss recent key findings which provided new insights into cAMP compartmentation in cardiomyocytes with a particular focus on its alterations in heart disease.

  13. Phosphodiesterase Inhibition to Target the Synaptic Dysfunction in Alzheimer's Disease

    NASA Astrophysics Data System (ADS)

    Bales, Kelly R.; Plath, Niels; Svenstrup, Niels; Menniti, Frank S.

    Alzheimer's Disease (AD) is a disease of synaptic dysfunction that ultimately proceeds to neuronal death. There is a wealth of evidence that indicates the final common mediator of this neurotoxic process is the formation and actions on synaptotoxic b-amyloid (Aβ). The premise in this review is that synaptic dysfunction may also be an initiating factor in for AD and promote synaptotoxic Aβ formation. This latter hypothesis is consistent with the fact that the most common risk factors for AD, apolipoprotein E (ApoE) allele status, age, education, and fitness, encompass suboptimal synaptic function. Thus, the synaptic dysfunction in AD may be both cause and effect, and remediating synaptic dysfunction in AD may have acute effects on the symptoms present at the initiation of therapy and also slow disease progression. The cyclic nucleotide (cAMP and cGMP) signaling systems are intimately involved in the regulation of synaptic homeostasis. The phosphodiesterases (PDEs) are a superfamily of enzymes that critically regulate spatial and temporal aspects of cyclic nucleotide signaling through metabolic inactivation of cAMP and cGMP. Thus, targeting the PDEs to promote improved synaptic function, or 'synaptic resilience', may be an effective and facile approach to new symptomatic and disease modifying therapies for AD. There continues to be a significant drug discovery effort aimed at discovering PDE inhibitors to treat a variety of neuropsychiatric disorders. Here we review the current status of those efforts as they relate to potential new therapies for AD.

  14. Identification and functional study of phosphodiesterases in rat urinary bladder.

    PubMed

    Qiu, Y; Kraft, P; Craig, E C; Liu, X; Haynes-Johnson, D

    2001-12-01

    Abstract Cyclic nucleotides are important secondary messengers involved in modulating the contractility of various smooth muscles. Phosphodiesterases (PDE) play important roles in this process by modulating the levels of cyclic nucleotides and their duration of action. This study was designed to identify and characterize the PDE isoenzymes in rat urinary bladder and to evaluate their roles in regulating bladder smooth muscle tone. The involvement of cAMP and cGMP pathways in this process was also assessed. The studies were carried out with tissues from male and female rats and no significant sex-related difference was found in the results. Utilizing the unique pharmacological properties of different isoenzymes, PDE1, 2, 3, 4, and 5 were identified in rat bladder. Organ bath experiments showed that forskolin was most potent in relaxing pre-contracted rat bladder strips while sodium nitroprusside was moderately effective, suggesting the relaxation was mainly mediated by the cAMP pathway and that the cGMP pathway is moderately involved. For PDE inhibitors, the non-specific inhibitor papaverine was most effective in relaxing pre-contracted bladder strips. Among isoenzyme-selective inhibitors, vinpocetine, EHNA, and sildenafil induced more relaxation than milrinone and rolipram.

  15. Cadmium inhibits the induction of high-affinity nitrate uptake in maize (Zea mays L.) roots.

    PubMed

    Rizzardo, Cecilia; Tomasi, Nicola; Monte, Rossella; Varanini, Zeno; Nocito, Fabio F; Cesco, Stefano; Pinton, Roberto

    2012-12-01

    Cadmium (Cd) detoxification involves glutathione and phytochelatins biosynthesis: the higher need of nitrogen should require increased nitrate (NO(3)(-)) uptake and metabolism. We investigated inducible high-affinity NO(3)(-) uptake across the plasma membrane (PM) in maize seedlings roots upon short exposure (10 min to 24 h) to low Cd concentrations (0, 1 or 10 μM): the activity and gene transcript abundance of high-affinity NO(3)(-) transporters, NO(3)(-) reductases and PM H(+)-ATPases were analyzed. Exposure to 1 mM NO(3)(-) led to a peak in high-affinity (0.2 mM) NO(3)(-) uptake rate (induction), which was markedly lowered in Cd-treated roots. Plasma membrane H(+)-ATPase activity was also strongly limited, while internal NO(3)(-) accumulation and NO(3)(-) reductase activity in extracts of Cd treated roots were only slightly lowered. Kinetics of high- and low-affinity NO(3)(-) uptake showed that Cd rapidly (10 min) blocked the inducible high-affinity transport system; the constitutive high-affinity transport system appeared not vulnerable to Cd and the low-affinity transport system appeared to be less affected and only after a prolonged exposure (12 h). Cd-treatment also modified transcript levels of genes encoding high-affinity NO(3)(-) transporters (ZmNTR2.1, ZmNRT2.2), PM H(+)-ATPases (ZmMHA3, ZmMHA4) and NO(3)(-) reductases (ZmNR1, ZmNADH:NR). Despite an expectable increase in NO(3)(-) demand, a negative effect of Cd on NO(3)(-) nutrition is reported. Cd effect results in alterations at the physiological and transcriptional levels of NO(3)(-) uptake from the external solution and it is particularly severe on the inducible high-affinity anion transport system. Furthermore, Cd would limit the capacity of the plant to respond to changes in NO(3) (-) availability.

  16. Increased social interaction in mice deficient of the striatal medium spiny neuron-specific phosphodiesterase 10A2.

    PubMed

    Sano, Hiromi; Nagai, Yumiko; Miyakawa, Tsuyoshi; Shigemoto, Ryuichi; Yokoi, Mineto

    2008-04-01

    Cyclic nucleotide phosphodiesterase 10A (PDE10A) is a member of phosphodiesterase families that degrade cAMP and/or cGMP in distinct intracellular sites. PDE10A has a dual activity on hydrolysis of both cAMP and cGMP, and is prominently expressed in the striatum and the testis. Previous studies suggested that PDE10A is involved in regulation of locomotor activity and potentially related to psychosis, but concrete physiological roles of PDE10A remains elusive yet. In this study, we genetically inactivated PDE10A2, a prominent isoform of PDE10A in the brain, in mice, and demonstrate that PDE10A2 deficiency results in increased social interaction without any major influence on different other behaviors, along with increased levels of striatal cAMP. We also demonstrate that PDE10A2 is selectively distributed in medium spiny neurons, but not interneurons, of the striatal complex. Thus, our results establish a physiological role for PDE10A2 in regulating cAMP pathway and social interaction, and suggest that cAMP signaling cascade in striatal medium spiny neurons might be involved in regulating social interaction behavior in mice.

  17. Engineering of a red-light-activated human cAMP/cGMP-specific phosphodiesterase.

    PubMed

    Gasser, Carlos; Taiber, Sandra; Yeh, Chen-Min; Wittig, Charlotte Helene; Hegemann, Peter; Ryu, Soojin; Wunder, Frank; Möglich, Andreas

    2014-06-17

    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.

  18. [Cyclic nucleotide phosphodiesterase IV expression, activity and targeting in cells of cardiovascular system].

    PubMed

    Yan, Jun; Zhu, Hai-Bo

    2007-06-01

    Cyclic nucleotide second messages (cAMP and cGMP) play a central role in signal transduction and regulation of physiologic responses. The only way to inactivate them is to degrade them through the action of phosphodiesterases (PDEs). Recent advances show that PDE4, a cAMP specific phosphodiesterase, has specific functions in regulating the activities of the cardiovascular system. PDE4 is expressed in the cells of cardiovascular systems including cardiomyocytes, vascular smooth muscle cells, and vascular endothelial cells. The expression level of PDE4 is shown to be downregulated in the failure hearts, while it is upregulated in hypertrophied hearts. And PDE4 deficiency in mice is associated with a cardiac phenotype comprised of a progressive, age-related cardiomyopathy, accelerated heart failure after myocardial infarction and exercise-induced arrhythmias. Local levels of cAMP regulate the precise opening of the ryanodine receptor complex (RyR2) which releases calcium at the start of a heartbeat. Loss or inhibition of PDE4 activity increases calcium flow through RyR2, and causes leakiness and heart failure in mice. These finding may show us a new target for treating cardiovascular diseases.

  19. Michelle: Growing Through Camping

    ERIC Educational Resources Information Center

    Ouellet, Annette M.

    1977-01-01

    A mother, whose physically handicapped 7-year-old daughter has prosthetic feet, describes how her child adjusted well first to a summer day camp and then to a week long camping program run by the Girl Scouts. (GW)

  20. Michelle: Growing Through Camping

    ERIC Educational Resources Information Center

    Ouellet, Annette M.

    1977-01-01

    A mother, whose physically handicapped 7-year-old daughter has prosthetic feet, describes how her child adjusted well first to a summer day camp and then to a week long camping program run by the Girl Scouts. (GW)

  1. A Summer Camp Assessment.

    ERIC Educational Resources Information Center

    Pratte, Janice L.; DiNardi, Salvatore R.

    1979-01-01

    Reported are the results of a project assessing the impact of a revised Massachusetts sanitary code on 500 summer camps for children. The study compared camp compliances with the proposed regulations to the level of compliance with existing regulations. (BT)

  2. Ustilago maydis phosphodiesterases play a role in the dimorphic switch and in pathogenicity.

    PubMed

    Agarwal, Charu; Aulakh, Kavita B; Edelen, Kaly; Cooper, Michael; Wallen, R Margaret; Adams, Seth; Schultz, David J; Perlin, Michael H

    2013-05-01

    Components of the cAMP (cyclic AMP) signalling cascades are conserved from fungi to humans, and are particularly important for fungal dimorphism and pathogenicity. Previous work has described two phosphodiesterases, UmPde1 and UmPde2, in Ustilago maydis which show strong phosphodiesterase activity. We further characterized the biological function(s) of these phosphodiesterases in U. maydis. Specifically, we examined their possible role(s) in regulation of the cAMP-dependent protein kinase A (PKA) pathway and their roles in filamentous growth and pathogenicity. We found that UmPde1, which shares 35 % similarity with Cryptococcus neoformans Pde1, also displays functional homology with this enzyme. UmPde1 complements the capsule-formation defect of C. neoformans strains deleted for Pde1. In U. maydis, the cell morphology of the umpde1 deletion mutant resembled the multiple budding phenotypes seen with the ubc1 mutant, which lacks the regulatory subunit of PKA. Interestingly, on low-ammonium medium, umpde2 deletion strains showed a reduction in filamentation that was comparable to that of ubc1 deletion strains; however, umpde1 deletion strains showed normal filamentation on low-ammonium medium. Furthermore, both the ubc1 deletion strain in which the PKA pathway was constitutively active and the umpde1 deletion strains were significantly reduced in pathogenicity, while the umpde2 deletion strains showed a trend for reduced pathogenicity compared with wild-type strains. These data support a role for the phosphodiesterases UmPde1 and UmPde2 in regulating the U. maydis cAMP-dependent PKA pathway through modulation of cAMP levels, thus affecting dimorphic growth and pathogenicity.

  3. GHB receptor targets in the CNS: focus on high-affinity binding sites.

    PubMed

    Bay, Tina; Eghorn, Laura F; Klein, Anders B; Wellendorph, Petrine

    2014-01-15

    γ-Hydroxybutyric acid (GHB) is an endogenous compound in the mammalian brain with both low- and high-affinity receptor targets. GHB is used clinically in the treatment of symptoms of narcolepsy and alcoholism, but also illicitly abused as the recreational drug Fantasy. Major pharmacological effects of exogenous GHB are mediated by GABA subtype B (GABAB) receptors that bind GHB with low affinity. The existence of GHB high-affinity binding sites has been known for more than three decades, but the uncovering of their molecular identity has only recently begun. This has been prompted by the generation of molecular tools to selectively study high-affinity sites. These include both genetically modified GABAB knock-out mice and engineered selective GHB ligands. Recently, certain GABA subtype A (GABAA) receptor subtypes emerged as high-affinity GHB binding sites and potential physiological mediators of GHB effects. In this research update, a description of the various reported receptors for GHB is provided, including GABAB receptors, certain GABAA receptor subtypes and other reported GHB receptors. The main focus will thus be on the high-affinity binding targets for GHB and their potential functional roles in the mammalian brain.

  4. Atrazine Acts as an Endocrine Disrupter by Inhibiting cAMP-specific Phosphodiesterase-4

    PubMed Central

    Kucka, Marek; Pogrmic-Majkic, Kristina; Fa, Svetlana; Stojilkovic, Stanko S.; Kovacevic, Radmila

    2014-01-01

    Atrazine, one of the most commonly used herbicides worldwide, acts as an endocrine disruptor, but the mechanism of its action has not been characterized. In this study, we show that atrazine rapidly increases cAMP levels in cultured rat pituitary and testicular Leydig cells in a concentration-dependent manner, but less effectively than 3-isobutyl-1-methylxanthine, a competitive non-specific inhibitor of phosphodiesterases (PDEs). In forskolin (an activator of adenylyl cyclase)- and probenecid (an inhibitor of cyclic nucleotide transporters)-treated cells, but not in 3-isobutyl-1-methylxanthine-treated cells, atrazine further increased cAMP levels, indicating that inhibition of PDEs accounts for accumulation of cAMP. In contrast to cAMP, atrazine did not alter cGMP levels, further indicating that it inhibits cAMP-specific PDEs. Atrazine-induced changes in cAMP levels were sufficient to stimulate prolactin release in pituitary cells and androgen production in Leydig cells, indicating that it acts as an endocrine disrupter both in cells that secrete by exocytosis of prestored hormones and in cells that secrete by de novo hormone synthesis. Rolipram abolished the stimulatory effect of atrazine on cAMP release in both cell types, suggesting that it acts as an inhibitor of PDE4s, isoforms whose mRNA transcripts dominate in pituitary and Leydig cells together with mRNA for PDE8A. In contrast, immortalized lacto-somatotrophs showed low expression of these mRNA transcripts and several fold higher cAMP levels compared to normal pituitary cells, and atrazine was unable to further increase cAMP levels. These results indicate that atrazine acts as a general endocrine disrupter by inhibiting cAMP-specific PDE4s. PMID:23022511

  5. Camp is a Celebration.

    ERIC Educational Resources Information Center

    North Dakota Farmers Union, Jamestown. Dept. of Youth Activities.

    A camping workbook provides materials for use in discussion of 3 important aspects of farm living: rural power, communication, and conservation. It is intended that this material be used in class sessions held during a junior youth camp. A sample of the youth camp schedule reveals 3 forty-five minute time periods during the day designated as class…

  6. Specific Inhibition of Phosphodiesterase-4B Results in Anxiolysis and Facilitates Memory Acquisition.

    PubMed

    McGirr, Alexander; Lipina, Tatiana V; Mun, Ho-Suk; Georgiou, John; Al-Amri, Ahmed H; Ng, Enoch; Zhai, Dongxu; Elliott, Christina; Cameron, Ryan T; Mullins, Jonathan G L; Liu, Fang; Baillie, George S; Clapcote, Steven J; Roder, John C

    2016-03-01

    Cognitive dysfunction is a core feature of dementia and a prominent feature in psychiatric disease. As non-redundant regulators of intracellular cAMP gradients, phosphodiesterases (PDE) mediate fundamental aspects of brain function relevant to learning, memory, and higher cognitive functions. Phosphodiesterase-4B (PDE4B) is an important phosphodiesterase in the hippocampal formation, is a major Disrupted in Schizophrenia 1 (DISC1) binding partner and is itself a risk gene for psychiatric illness. To define the effects of specific inhibition of the PDE4B subtype, we generated mice with a catalytic domain mutant form of PDE4B (Y358C) that has decreased ability to hydrolyze cAMP. Structural modeling predictions of decreased function and impaired binding with DISC1 were confirmed in cell assays. Phenotypic characterization of the PDE4B(Y358C) mice revealed facilitated phosphorylation of CREB, decreased binding to DISC1, and upregulation of DISC1 and β-Arrestin in hippocampus and amygdala. In behavioral assays, PDE4B(Y358C) mice displayed decreased anxiety and increased exploration, as well as cognitive enhancement across several tests of learning and memory, consistent with synaptic changes including enhanced long-term potentiation and impaired depotentiation ex vivo. PDE4B(Y358C) mice also demonstrated enhanced neurogenesis. Contextual fear memory, though intact at 24 h, was decreased at 7 days in PDE4B(Y358C) mice, an effect replicated pharmacologically with a non-selective PDE4 inhibitor, implicating cAMP signaling by PDE4B in a very late phase of consolidation. No effect of the PDE4B(Y358C) mutation was observed in the prepulse inhibition and forced swim tests. Our data establish specific inhibition of PDE4B as a promising therapeutic approach for disorders of cognition and anxiety, and a putative target for pathological fear memory.

  7. Involvement of type 4 cAMP-phosphodiesterase in the myogenic differentiation of L6 cells.

    PubMed

    Naro, F; Sette, C; Vicini, E; De Arcangelis, V; Grange, M; Conti, M; Lagarde, M; Molinaro, M; Adamo, S; Némoz, G

    1999-12-01

    Myogenic cell differentiation is induced by Arg(8)-vasopressin, whereas high cAMP levels and protein kinase A (PKA) activity inhibit myogenesis. We investigated the role of type 4 phosphodiesterase (PDE4) during L6-C5 myoblast differentiation. Selective PDE4 inhibition resulted in suppression of differentiation induced by vasopressin. PDE4 inhibition prevented vasopressin-induced nuclear translocation of the muscle-specific transcription factor myogenin without affecting its overall expression level. The effects of PDE4 inhibition could be attributed to an increase of cAMP levels and PKA activity. RNase protection, reverse transcriptase PCR, immunoprecipitation, Western blot, and enzyme activity assays demonstrated that the PDE4D3 isoform is the major PDE4 expressed in L6-C5 myoblasts and myotubes, accounting for 75% of total cAMP-hydrolyzing activity. Vasopressin cell stimulation caused a biphasic increase of PDE4 activity, which peaked at 2 and 15 min and remained elevated for 48 h. In the continuous presence of vasopressin, cAMP levels and PKA activity were lowered. PDE4D3 overexpression increased spontaneous and vasopressin-dependent differentiation of L6-C5 cells. These results show that PDE4D3 plays a key role in the control of cAMP levels and differentiation of L6-C5 cells. Through the modulation of PDE4 activity, vasopressin inhibits the cAMP signal transduction pathway, which regulates myogenesis possibly by controlling the subcellular localization of myogenin.

  8. Involvement of Type 4 cAMP-Phosphodiesterase in the Myogenic Differentiation of L6 Cells

    PubMed Central

    Naro, Fabio; Sette, Claudio; Vicini, Elena; De Arcangelis, Vania; Grange, Muriel; Conti, Marco; Lagarde, Michel; Molinaro, Mario; Adamo, Sergio; Némoz, Georges

    1999-01-01

    Myogenic cell differentiation is induced by Arg8-vasopressin, whereas high cAMP levels and protein kinase A (PKA) activity inhibit myogenesis. We investigated the role of type 4 phosphodiesterase (PDE4) during L6-C5 myoblast differentiation. Selective PDE4 inhibition resulted in suppression of differentiation induced by vasopressin. PDE4 inhibition prevented vasopressin-induced nuclear translocation of the muscle-specific transcription factor myogenin without affecting its overall expression level. The effects of PDE4 inhibition could be attributed to an increase of cAMP levels and PKA activity. RNase protection, reverse transcriptase PCR, immunoprecipitation, Western blot, and enzyme activity assays demonstrated that the PDE4D3 isoform is the major PDE4 expressed in L6-C5 myoblasts and myotubes, accounting for 75% of total cAMP-hydrolyzing activity. Vasopressin cell stimulation caused a biphasic increase of PDE4 activity, which peaked at 2 and 15 min and remained elevated for 48 h. In the continuous presence of vasopressin, cAMP levels and PKA activity were lowered. PDE4D3 overexpression increased spontaneous and vasopressin-dependent differentiation of L6-C5 cells. These results show that PDE4D3 plays a key role in the control of cAMP levels and differentiation of L6-C5 cells. Through the modulation of PDE4 activity, vasopressin inhibits the cAMP signal transduction pathway, which regulates myogenesis possibly by controlling the subcellular localization of myogenin. PMID:10588663

  9. Creating healthy camp experiences.

    PubMed

    Walton, Edward A; Tothy, Alison S

    2011-04-01

    The American Academy of Pediatrics has created recommendations for health appraisal and preparation of young people before participation in day or resident camps and to guide health and safety practices for children at camp. These recommendations are intended for parents, primary health care providers, and camp administration and health center staff. Although camps have diverse environments, there are general guidelines that apply to all situations and specific recommendations that are appropriate under special conditions. This policy statement has been reviewed and is supported by the American Camp Association.

  10. Exploring high-affinity binding properties of octamer peptides by principal component analysis of tetramer peptides.

    PubMed

    Kume, Akiko; Kawai, Shun; Kato, Ryuji; Iwata, Shinmei; Shimizu, Kazunori; Honda, Hiroyuki

    2017-02-01

    To investigate the binding properties of a peptide sequence, we conducted principal component analysis (PCA) of the physicochemical features of a tetramer peptide library comprised of 512 peptides, and the variables were reduced to two principal components. We selected IL-2 and IgG as model proteins and the binding affinity to these proteins was assayed using the 512 peptides mentioned above. PCA of binding affinity data showed that 16 and 18 variables were suitable for localizing IL-2 and IgG high-affinity binding peptides, respectively, into a restricted region of the PCA plot. We then investigated whether the binding affinity of octamer peptide libraries could be predicted using the identified region in the tetramer PCA. The results show that octamer high-affinity binding peptides were also concentrated in the tetramer high-affinity binding region of both IL-2 and IgG. The average fluorescence intensity of high-affinity binding peptides was 3.3- and 2.1-fold higher than that of low-affinity binding peptides for IL-2 and IgG, respectively. We conclude that PCA may be used to identify octamer peptides with high- or low-affinity binding properties from data from a tetramer peptide library. Copyright © 2016 The Society for Biotechnology, Japan. Published by Elsevier B.V. All rights reserved.

  11. SORPTION OF LEAD ON A HIGH AFFINITY OXIDE: MACROSCOPIC AND MICROSCOPIC STUDIES

    EPA Science Inventory

    Sorption of lead (Pb) was investigated on an innovative metal oxide compound using macroscopic and microscopic techniques. The objective of this study was to elucidate the sorption mechanism of Pb on the high-affinity engineered oxide with time at pH 6 employing batch methods an...

  12. SORPTION OF LEAD ON A HIGH AFFINITY OXIDE: MACROSCOPIC AND MICROSCOPIC STUDIES (ABSTRACT)

    EPA Science Inventory

    Sorption of lead (Pb) was investigated on an innovative metal oxide compound using macroscopic and microscopic techniques. The objective of this study was to elucidate the sorption mechanism of Pb on the high-affinity engineered oxide with time at pH 6 employing batch methods an...

  13. High Affinity Iron Permease is Required for Virulence of Rhizopus oryzae

    USDA-ARS?s Scientific Manuscript database

    Rhizopus oryzae is the most common cause of mucormycosis. Clinical and animal model data clearly demonstrate that the presence of elevated available serum iron predisposes the host to develop mucormycosis. The high affinity iron permease gene (rFTR1) is required for R. oryzae iron transport in iro...

  14. SORPTION OF LEAD ON A HIGH AFFINITY OXIDE: MACROSCOPIC AND MICROSCOPIC STUDIES

    EPA Science Inventory

    Sorption of lead (Pb) was investigated on an innovative metal oxide compound using macroscopic and microscopic techniques. The objective of this study was to elucidate the sorption mechanism of Pb on the high-affinity engineered oxide with time at pH 6 employing batch methods an...

  15. SORPTION OF LEAD ON A HIGH AFFINITY OXIDE: MACROSCOPIC AND MICROSCOPIC STUDIES (ABSTRACT)

    EPA Science Inventory

    Sorption of lead (Pb) was investigated on an innovative metal oxide compound using macroscopic and microscopic techniques. The objective of this study was to elucidate the sorption mechanism of Pb on the high-affinity engineered oxide with time at pH 6 employing batch methods an...

  16. Lesbian camp: An unearthing.

    PubMed

    Nielsen, Elly-Jean

    2016-01-01

    Camp-a sensibility, a style, and a form of artistic self-expression-is an elusive concept said to be in the eye of the beholder. To refute Susan Sontag's ( 1966 ) claims that camp is apolitical and not especially homosexual, a number of recent scholarly works have been geared toward revealing camp's fundamental gayness. With the odd footnote aside, lesbian camp has been collapsed into the category of gay male camp, if not eclipsed entirely. Despite the negligible efforts made to legitimize lesbian camp, there are numerous salient cultural examples one might draw on to illustrate, typify, and substantiate a lesbian camp sensibility. I lay the ground work for this scholarly exercise by outlining various definitions and critiques of camp, and by discussing its history and application to queer theory. Then, to unveil lesbian camp, three non-mutually exclusive categories are discussed: classic, erotic, and radical. By gathering various strands of inquiry, and various textual examples (e.g., photography, artistic performances, and literary tropes), this article attempts to reach a more inclusive and nuanced understanding of lesbian camp.

  17. Free radical modification of high-affinity GABA transport in the central nervous system

    SciTech Connect

    Debler, E.A.

    1985-01-01

    Presynaptic nerve terminals of DNS neurons can be characterized as dynamic self-contained subunits of the overall neuron. These neuron subunits together with the adjacent post-synaptic terminals constitute the functional unit of neurotransmission. One major function of the presynaptic nerve terminal in neurotransmission is its role in the release and subsequent reuptake of neurotransmitters. Neurochemical and morphological analysis of this high-affinity uptake process, for the amino acid neurotransmitter gamma-aminobutyric acid (GABA), revealed that free radical modification (lipid peroxidation) of presynaptic nerve terminal membrane structures resulted in a decrease in high-affinity (/sup 14/C)GABA uptake. Depolarized nerve terminals exposed to horseradish peroxidase (HRP)-generated superoxide radicals (.O/sub 2//sup -/) exhibited an alteration of synaptic vesicle membrane structure and a reduction of high-affinity (/sup 14/C)GABA uptake. In the kinetic analysis of high affinity (/sup 14/C)GABA uptake, hypoxanthine/xanthine oxidase (HPX/XOD)-treated synaptosomes exhibited a significant reduction in V/sub max/ but no significant alteration of K/sub m/. This HPX/XOD treatment also resulted in significant structural alterations of both the synaptic vesicle membrane and the synaptosomal plasma membrane. Structural and functional correlates appear to exist between synaptic vesicle and plasma membrane systems and high-affinity (/sup 14/C)GABA uptake. Together, the selectivity of the .O/sub 2//sup -/ toxicity and that of the high-K/sup +//Na/sup +/-free effect support the idea of two separate and different amino acid uptake systems in CNS tissue.

  18. Two distinct functional high affinity receptors for mouse interleukin-3 (IL-3).

    PubMed Central

    Hara, T; Miyajima, A

    1992-01-01

    The human interleukin-3 receptor (IL-3R) is composed of an IL-3 specific alpha subunit (IL-3R alpha) and a common beta subunit (beta c) that is shared by IL-3, granulocyte/macrophage colony stimulating factor (GM-CSF) and IL-5 receptors. In contrast to the human, the mouse has two distinct but related genes, AIC2A and AIC2B, both of which are homologous to the human beta c gene. AIC2B has proved to encode a common beta subunit between mouse GM-CSF and IL-5 receptors. AIC2A is unique to the mouse and encodes a low affinity IL-3 binding protein. Based on the observation that the AIC2A protein is a component of a high affinity IL-3R, we searched for a cDNA encoding a protein which conferred high affinity IL-3 binding when coexpressed with the AIC2A protein in COS7 cells. We obtained such a cDNA (SUT-1) encoding a mature protein of 70 kDa that has weak homology to the human IL-3R alpha. The SUT-1 protein bound IL-3 with low affinity and formed high affinity receptors not only with the AIC2A protein but also with the AIC2B protein. Both high affinity IL-3Rs expressed on a mouse T cell line, CTLL-2, showed similar IL-3 binding properties and transmitted a growth signal in response to IL-3. Thus, the mouse has two distinct functional high affinity IL-3Rs, providing a molecular explanation for the differences observed between mouse and human IL-3Rs. Images PMID:1582416

  19. Keesler Astro Camp

    NASA Image and Video Library

    2011-06-29

    Young people prepare model rockets during an Astro Camp activity at Keesler Air Force Base in Biloxi. Stennis hosted the camp June 28 - July 1 in support of the White House Military Families Initiative. The camp also marked the beginning of a partnership between Stennis and Keesler to provide NASA education experiences to military children and to train children and youth care-providers. It is hoped that this activity can be expanded to other military bases next summer.

  20. 1978 national camping market survey

    Treesearch

    Wilbur F. LaPage; Gerald L. Cole

    1979-01-01

    This report summarizes the major findings of a 1978 nationwide camping market survey, and compares them with those of similar surveys conducted in 1971 and 1973. It documents recent trends in camping and in the composition of the camping market, and compares camping demand with the available supply of developed campsites. The active camping market in 1978 included 17.5...

  1. Phosphodiesterase inhibitors: history of pharmacology.

    PubMed

    Schudt, Christian; Hatzelmann, Armin; Beume, Rolf; Tenor, Hermann

    2011-01-01

    The first pharmacological investigations of phosphodiesterase (PDE) inhibitors were developed with the clinical efficacies of drugs isolated from coffee, cacao and tea but only later their relevant ingredients were identified as xanthines that act as PDE. With its diuretic, inotropic and bronchodilating clinical efficacy, use of theophylline anticipated the clinical goals, which were later approached with the first-generation of weakly selective PDE inhibitors in the period from 1980 to 1990. Pharmacological and clinical research with these early compounds provided a vast pool of information regarding desired and adverse actions - although most of these new drugs had to be discontinued due to severe adverse effects. The pharmacological models for cardiac, vascular and respiratory indications were analysed for their PDE isoenzyme profiles, and when biochemical and molecular biological approaches expanded our knowledge of the PDE superfamily, the purified isoenzymes that were now available opened the door for more systematic studies of inhibitors and for generation of highly selective isoenzyme-specific drugs. The development of simple screening models and clinically relevant indication models reflecting the growing knowledge about pathomechanisms of disease are summarised here for today's successful application of highly selective PDE3, PDE4 and PDE5 inhibitors. The interplay of serendipitous discoveries, the establishment of intelligent pharmacological models and the knowledge gain by research results with new substances is reviewed. The broad efficacies of new substances in vitro, the enormous biodiversity of the PDE isoenzyme family and the sophisticated biochemical pharmacology enabled Viagra to be the first success story in the field of PDE inhibitor drug development, but probably more success stories will follow.

  2. Epac and the high affinity rolipram binding conformer of PDE4 modulate neurite outgrowth and myelination using an in vitro spinal cord injury model

    PubMed Central

    Boomkamp, S D; McGrath, M A; Houslay, M D; Barnett, S C

    2014-01-01

    Background and Purpose cAMP and pharmacological inhibition of PDE4, which degrades it, are promising therapeutic targets for the treatment of spinal cord injury (SCI). Using our previously described in vitro SCI model, we studied the mechanisms by which cAMP modulators promote neurite outgrowth and myelination using enantiomers of the PDE4-specific inhibitor rolipram and other modulators of downstream signalling effectors. Experimental Approach Rat mixed neural cell myelinating cultures were cut with a scalpel and treated with enantiomers of the PDE4-specific inhibitor rolipram, Epac agonists and PKA antagonists. Neurite outgrowth, density and myelination were assessed by immunocytochemistry and cytokine levels analysed by qPCR. Key Results Inhibition of the high-affinity rolipram-binding state (HARBS), rather than the low-affinity rolipram binding state (LARBS) PDE4 conformer promoted neurite outgrowth and myelination. These effects were mediated through the activation of Epac and not through PKA. Expression of the chemokine CXCL10, known to inhibit myelination, was markedly elevated in astrocytes after Rho inhibition and this was blocked by inhibition of Rho kinase or PDE4. Conclusions and Implications PDE4 inhibitors targeted at the HARBS conformer or Epac agonists may provide promising novel targets for the treatment of SCI. Our study demonstrates the differential mechanisms of action of these compounds, as well as the benefit of a combined pharmacological approach and highlighting potential promising targets for the treatment of SCI. These findings need to be confirmed in vivo. PMID:24467222

  3. Upregulation of high affinity GABAA receptors in cultured rat dorsal root ganglion neurons

    PubMed Central

    Lee, Kwan Yeop; Charbonnet, Marcel; Gold, Michael S.

    2012-01-01

    Despite evidence that high-affinity GABAA receptor subunit mRNA and protein are present in dorsal root ganglia (DRG), low affinity currents dominate those detected in acutely dissociated DRG neurons in vitro. This observation raises the possibility that high affinity receptors are normally trafficked out of the DRG toward central and peripheral terminals. We therefore hypothesized that with time in culture, there would be an increase in high-affinity GABAA currents in DRG neurons. To test this hypothesis, we studied dissociated DRG neurons 2 hrs (acute) and 24 hrs (cultured) after plating with whole cell patch clamp techniques, western blot and qRT-PCR analysis. GABAA current density increases dramatically with time in culture in association with the emergence of two persistent currents with EC50’s of 0.25 ± 0.01 μM and 3.2 ± 0.02 μM for GABA activation. In a subpopulation of neurons, there was also an increase in the potency of GABA activation of the transient current from an EC50 of 78.16 ± 10.1 μM to 9.56 ± 1.3 μM with time in culture. A fraction of the high affinity current was potentiated by δ-subunit agonist 4,5,6,7-tetrahydroisoxazolo[5,4-c]pyridine-3-ol (THIP). δ-subunit immunoreactivity was largely restricted to the cytosolic fraction in acute but the membrane fraction in cultured DRG neurons with no detectable change in δ-subunit mRNA. However, the emergence of a high affinity current blocked by THIP and insensitive to bicuculline was detected in a subpopulation of cultured neurons as well in association with an increase in ρ2 and 3-subunit mRNA in cultured DRG neurons. Our results suggest that high-affinity δ-subunit containing GABAA receptors are normally trafficked out of the DRG where they are targeted to peripheral and central processes. They also highlight that the interpretation of data obtained from cultured DRG neurons should be made with caution. PMID:22366297

  4. Cyclic AMP, Protein Kinase A, and Phosphodiesterases: Proceedings of an International Workshop

    PubMed Central

    Stratakis, C. A.

    2014-01-01

    Cyclic nucleotides cAMP and cGMP are part of almost all major cellular signaling pathways. Phosphodiesterases (PDEs) are enzymes that regulate the intracellular levels of cAMP and cGMP. Protein kinase A or cAMP-dependent protein kinase mediates most cAMP effects in the cell. Over the last 25 years, various components of this group of molecules have been involved in human diseases, both genetic and acquired. Lately, the PDEs attract more attention. The pharmacological exploitation of the PDE’s ability to regulate cGMP and cAMP, and through them, a variety of signaling pathways, has led to a number of new drugs for diverse applications from the treatment of erectile dysfunction to heart failure, asthma, and chronic obstructive pulmonary disease. We present the abstracts (available online) and selected articles from the proceedings of a meeting that took place at the National Institutes of Health (NIH), Bethesda, MD, June 8–10, 2011. PMID:22951901

  5. A tyrosine kinase inhibitor-induced myocardial degeneration in rats through off-target phosphodiesterase inhibition.

    PubMed

    Hu, Wenyue; Hirakawa, Brad; Jessen, Bart; Lee, Michelle; Aguirre, Shirley

    2012-12-01

    PF-04254644 is a selective kinase inhibitor of mesenchymal epithelial transition factor/hepatocyte growth factor receptor with known off-target inhibitory activity against the phosphodiesterase (PDE) family. Rats given repeated oral doses of PF-04254644 developed a mild to moderate myocardial degeneration accompanied by sustained increase in heart rate and contractility. Investigative studies were conducted to delineate the mechanisms of toxicity. Microarray analysis of Sprague-Dawley rat hearts in a 6 day repeat dose study with PF-04254644 or milrinone, a selective PDE3 inhibitor, revealed similar perturbation of the cyclic adenosine monophosphate (c-AMP) pathway. PDE inhibition and activation of c-AMP were further substantiated using PDE3B immunofluorescence staining and through a c-AMP response element reporter gene assay. The intracellular calcium and oxidative stress signaling pathways were more perturbed by treatment with PF-04254644 than milrinone. The rat cardiomyocytes calcium assay found a dose-dependent increase in intracellular calcium with PF-04254644 treatment. These data suggest that cardiotoxicity of PF-04254644 was probably due to activation of c-AMP signaling, and possibly subsequent disruption of intracellular calcium and oxidative stress signaling pathways. The greater response with PF-04254644 as compared with milrinone in gene expression and micro- and ultrastructural changes is probably due to the broader panel of PDEs inhibition. Copyright © 2012 John Wiley & Sons, Ltd.

  6. [Involvement of extracellular cAMP-specific phosphodiesterase in control of motile activity of Physarum polycephalum plasmodium].

    PubMed

    Matveeva, N B; Morozov, M A; Nezvetskiĭ, A R; Orlova, T G; Teplov, V A; Beĭlina, S I

    2010-01-01

    Possible involvement of extracellular cAMP-specific phosphodiesterase in the control of cell motile behavior has been investigated in Physarum polycephalum plasmodium, a multinuclear amoeboid cell with the autooscillatory mode of motility. It was found that the rate of the hydrolysis of 10 mM cAMP by a partially purified preparation of cAMP-specific phosphodiesterase secreted by the plasmodium in the course of migration decreases 20-30 times under the action of 1 mM dithiothreitol. In the presence of 1-5 mM of this strong reducing agent, the onset of the plasmodium spreading and the transition to the stage of migration were delayed in a concentration-dependent manner. In accordance with the morphological pattern of motile behavior, the duration of the maintenance of high frequency autooscillations, which normally precede the increase in the rate of the spreading and appear also in response to the application of attractants at spatially uniform concentrations, strongly increased by the action of dithiothreitol. The results obtained suggest that the autocrine production of cAMP and extracellular cAMP-specific phosphodiesterase is an important constituent of the mechanism controlling the motile behavior of the Physarum polycephalum plasmodium.

  7. The cAMP-specific phosphodiesterase TbPDE2C is an essential enzyme in bloodstream form Trypanosoma brucei

    PubMed Central

    Zoraghi, Roya; Seebeck, Thomas

    2002-01-01

    Chemotherapy of human sleeping sickness, a fatal disease caused by the protozoan parasite Trypanosoma brucei, is in a dismal state, and the identification and characterization of new drug targets is an urgent prerequisite for an improvement of the dramatic situation in the field. Over the last several years, inhibitors of cyclic nucleotide-specific phosphodiesterases have proven to be highly successful drug candidates for an assortment of clinical conditions. Their potential as antiparasitic drugs has not been explored so far. This study reports the characterization of a cAMP-specific phosphodiesterase from T. brucei, TbPDE2C. This enzyme is a class I phosphodiesterase, and it is a member of a small enzyme family in T. brucei, TbPDE2. Inhibitors of this enzyme block the proliferation of bloodstream form trypanosomes in culture. RNA interference experiments demonstrated that the TbPDE2 family, and in particular TbPDE2C, are essential for maintaining intracellular cAMP concentrations within a physiological range. Bloodstream form trypanosomes are exquisitely sensitive to elevated concentrations of intracellular cAMP, and a disruption of TbPDE2C function quickly leads to the disruption of nuclear and cellular cell division, and to cell death. TbPDE2C might represent a novel drug target for the development of new and effective trypanocidal drugs. PMID:11930001

  8. Three Recombinant Engineered Antibodies against Recombinant Tags with High Affinity and Specificity

    PubMed Central

    Zhao, Hongyu; Shen, Ao; Xiang, Yang K.; Corey, David P.

    2016-01-01

    We describe three recombinant engineered antibodies against three recombinant epitope tags, constructed with divalent binding arms to recognize divalent epitopes and so achieve high affinity and specificity. In two versions, an epitope is inserted in tandem into a protein of interest, and a homodimeric antibody is constructed by fusing a high-affinity epitope-binding domain to a human or mouse Fc domain. In a third, a heterodimeric antibody is constructed by fusing two different epitope-binding domains which target two different binding sites in GFP, to polarized Fc fragments. These antibody/epitope pairs have affinities in the low picomolar range and are useful tools for many antibody-based applications. PMID:26943906

  9. The fourth dimension in immunological space: how the struggle for nutrients selects high-affinity lymphocytes.

    PubMed

    Wensveen, Felix M; van Gisbergen, Klaas P J M; Eldering, Eric

    2012-09-01

    Lymphocyte activation via the antigen receptor is associated with radical shifts in metabolism and changes in requirements for nutrients and cytokines. Concomitantly, drastic changes occur in the expression of pro-and anti-apoptotic proteins that alter the sensitivity of lymphocytes to limiting concentrations of key survival factors. Antigen affinity is a primary determinant for the capacity of activated lymphocytes to access these vital resources. The shift in metabolic needs and the variable access to key survival factors is used by the immune system to eliminate activated low-affinity cells and to generate an optimal high-affinity response. In this review, we focus on the control of apoptosis regulators in activated lymphocytes by nutrients, cytokines, and costimulation. We propose that the struggle among individual clones that leads to the formation of high-affinity effector cell populations is in effect an 'invisible' fourth signal required for effective immune responses.

  10. Three Recombinant Engineered Antibodies against Recombinant Tags with High Affinity and Specificity.

    PubMed

    Zhao, Hongyu; Shen, Ao; Xiang, Yang K; Corey, David P

    2016-01-01

    We describe three recombinant engineered antibodies against three recombinant epitope tags, constructed with divalent binding arms to recognize divalent epitopes and so achieve high affinity and specificity. In two versions, an epitope is inserted in tandem into a protein of interest, and a homodimeric antibody is constructed by fusing a high-affinity epitope-binding domain to a human or mouse Fc domain. In a third, a heterodimeric antibody is constructed by fusing two different epitope-binding domains which target two different binding sites in GFP, to polarized Fc fragments. These antibody/epitope pairs have affinities in the low picomolar range and are useful tools for many antibody-based applications.

  11. Selection of short peptides with high affinity for various metal ions: Strategies and applications

    SciTech Connect

    Ataai, M.M.; Whiteford, W.K.

    1995-12-01

    The authors have used a random combinatorial library to generate an immensely diverse population of candidate peptides and have screened the library, using novel strategies, for selection of peptides of displaying high affinity towards a target metal. As the pool of available binding candidates, we have employed a hexapeptide library, containing essentially all the possible combination of sequences for a hexapeptide, expressed on surface of phage. Our experiments with copper demonstrated that our amplification and selection procedure is very effective. Out of 20 clones sequenced, after four rounds of amplification and selection 18 shared the same sequence. The corresponding peptide which contain two histidines was then synthesized and its binding affinity for copper was assessed. The results revealed that the selected peptide has a very high affinity for copper; its affinity is significantly higher than any hexapeptide which we are aware of including six adjacent histidine, (his)6, frequently used as an affinity tag in bioseparation. Selection of short peptides with high affinity and selectivity for arsenic, mercury, and lead is also underway. We envision important application of this work including discovery of highly effective drugs for treating metal toxicity replacing the chelating agents currently in use, and development of inexpensive whole cell adsorbents for toxicity replacing the chelating agents currently in use, and development of inexpensive whole cell adsorbents for the removal of toxic metals from dilute aqueous streams because dead cells or isolated cell wall, covered by the desired metal-binding domain may serve as high affinity and high capacity metal adsorbents. Other potential application will also be discussed in this presentation.

  12. Quantifying high-affinity binding of hydrophobic ligands by isothermal titration calorimetry.

    PubMed

    Krainer, Georg; Broecker, Jana; Vargas, Carolyn; Fanghänel, Jörg; Keller, Sandro

    2012-12-18

    A fast and reliable quantification of the binding thermodynamics of hydrophobic high-affinity ligands employing a new calorimetric competition experiment is described. Although isothermal titration calorimetry is the method of choice for a quantitative characterization of intermolecular interactions in solution, a reliable determination of a dissociation constant (K(D)) is typically limited to the range 100 μM > K(D) > 1 nM. Interactions displaying higher or lower K(D) values can be assessed indirectly, provided that a suitable competing ligand is available whose K(D) falls within the directly accessible affinity window. This established displacement assay, however, requires the high-affinity ligand to be soluble at high concentrations in aqueous buffer and, consequently, poses serious problems in the study of protein binding involving small-molecule ligands dissolved in organic solvents--a familiar case in many drug-discovery projects relying on compound libraries. The calorimetric competition assay introduced here overcomes this limitation, thus allowing for a detailed thermodynamic description of high-affinity receptor-ligand interactions involving poorly water-soluble compounds. Based on a single titration of receptor into a dilute mixture of the two competing ligands, this competition assay provides accurate and precise values for the dissociation constants and binding enthalpies of both high- and moderate-affinity ligands. We discuss the theoretical background underlying the approach, demonstrate its practical application to metal ion chelation and high-affinity protein-inhibitor interactions, and explore its potential and limitations with the aid of simulations and statistical analyses.

  13. High affinity choline uptake: an early index of cholinergic innervation in rat brain.

    PubMed

    Sorimachi, M; Kataoka, K

    1975-08-29

    The uptake of [3H]choline was investigated in nuclei-free homogenates or crude synaptosomal fractions (P2) from rat brain under various stages of development. A comparable sensitivity of uptake to treatment by hyposmotic shock suggested the involvement of synaptosomal populations in choline uptake in immature as well as in adult brains. However, significant changes in the "apparent" Km for the high affinity transport system and quantitative differences in the Na ion requirement for maximal uptake at 0.43 muM choline concentration were found during development; facts which suggested a greater contribution of the low affinity system in the more immature brains. Assuming that the uptake with high and low sensitivity to Na+ reduction reflected that via the high and low affinity system reslectively, we have attempted to obtain real Km values for the high affinity system. These Km values changed less than those measured directly, suggesting that the affinity constant for the high affinity system does not change during development. On these assumptions, the developmental changes of cholinergic synaptogenesis were examined in 5 distinct regions of the brain. It was found that the synaptogenesis begins several days earlier than the increase of choline acetyltransferase (ChAc) level in the frontal cortex, the hippocampus, the superior colliculus and the cerebellum. These regions may be included among the terminal-rich regions according to available evidence related to cholinergic systems. On the other hand, synaptogenesis accompanied the concomitant ChAc increase in the striatum, where the cholinergic interneurons are present. It is concluded that the increase of ChAc in the terminal-rich regions is delayed by the axoplasmic flow; therefore, the earlier index of cholinergic synaptogenesis in these regions is the high affinity uptake activity rather than the enzyme activity.

  14. Amyloid-beta binds catalase with high affinity and inhibits hydrogen peroxide breakdown.

    PubMed Central

    Milton, N G

    1999-01-01

    Amyloid-beta (Abeta) specifically bound purified catalase with high affinity and inhibited catalase breakdown of H(2)O(2). The Abeta-induced catalase inhibition involved formation of the inactive catalase Compound II and was reversible. Catalase<-->Abeta interactions provide rapid functional assays for the cytotoxic domain of Abeta and suggest a mechanism for some of the observed actions of Abeta plus catalase in vitro. PMID:10567208

  15. Selective high-affinity polydentate ligands and methods of making such

    DOEpatents

    DeNardo, Sally; DeNardo, Gerald; Balhorn, Rodney

    2013-09-17

    This invention provides polydentate selective high affinity ligands (SHALs) that can be used in a variety of applications in a manner analogous to the use of antibodies. SHALs typically comprise a multiplicity of ligands that each binds different regions on the target molecule. The ligands are joined directly or through a linker thereby forming a polydentate moiety that typically binds the target molecule with high selectivity and avidity.

  16. The Saccharomyces cerevisiae high affinity phosphate transporter encoded by PHO84 also functions in manganese homeostasis.

    PubMed

    Jensen, Laran T; Ajua-Alemanji, Mispa; Culotta, Valeria Cizewski

    2003-10-24

    In the bakers' yeast Saccharomyces cerevisiae, high affinity manganese uptake and intracellular distribution involve two members of the Nramp family of genes, SMF1 and SMF2. In a search for other genes involved in manganese homeostasis, PHO84 was identified. The PHO84 gene encodes a high affinity inorganic phosphate transporter, and we find that its disruption results in a manganese-resistant phenotype. Resistance to zinc, cobalt, and copper ions was also demonstrated for pho84Delta yeast. When challenged with high concentrations of metals, pho84Delta yeast have reduced metal ion accumulation, suggesting that resistance is due to reduced uptake of metal ions. Pho84p accounted for virtually all the manganese accumulated under metal surplus conditions, demonstrating that this transporter is the major source of excess manganese accumulation. The manganese taken in via Pho84p is indeed biologically active and can not only cause toxicity but can also be incorporated into manganese-requiring enzymes. Pho84p is essential for activating manganese enzymes in smf2Delta mutants that rely on low affinity manganese transport systems. A role for Pho84p in manganese accumulation was also identified in a standard laboratory growth medium when high affinity manganese uptake is active. Under these conditions, cells lacking both Pho84p and the high affinity Smf1p transporter accumulated low levels of manganese, although there was no major effect on activity of manganese-requiring enzymes. We conclude that Pho84p plays a role in manganese homeostasis predominantly under manganese surplus conditions and appears to be functioning as a low affinity metal transporter.

  17. Neurotransmitter/sodium symporter orthologue LeuT has a single high-affinity substrate site.

    PubMed

    Piscitelli, Chayne L; Krishnamurthy, Harini; Gouaux, Eric

    2010-12-23

    Neurotransmitter/sodium symporters (NSSs) couple the uptake of neurotransmitter with one or more sodium ions, removing neurotransmitter from the synaptic cleft. NSSs are essential to the function of chemical synapses, are associated with multiple neurological diseases and disorders, and are the targets of therapeutic and illicit drugs. LeuT, a prokaryotic orthologue of the NSS family, is a model transporter for understanding the relationships between molecular mechanism and atomic structure in a broad range of sodium-dependent and sodium-independent secondary transporters. At present there is a controversy over whether there are one or two high-affinity substrate binding sites in LeuT. The first-reported crystal structure of LeuT, together with subsequent functional and structural studies, provided direct evidence for a single, high-affinity, centrally located substrate-binding site, defined as the S1 site. Recent binding, flux and molecular simulation studies, however, have been interpreted in terms of a model where there are two high-affinity binding sites: the central, S1, site and a second, the S2 site, located within the extracellular vestibule. Furthermore, it was proposed that the S1 and S2 sites are allosterically coupled such that occupancy of the S2 site is required for the cytoplasmic release of substrate from the S1 site. Here we address this controversy by performing direct measurement of substrate binding to wild-type LeuT and to S2 site mutants using isothermal titration calorimetry, equilibrium dialysis and scintillation proximity assays. In addition, we perform uptake experiments to determine whether the proposed allosteric coupling between the putative S2 site and the S1 site manifests itself in the kinetics of substrate flux. We conclude that LeuT harbours a single, centrally located, high-affinity substrate-binding site and that transport is well described by a simple, single-substrate kinetic mechanism.

  18. Internationalize Your Camp Staff.

    ERIC Educational Resources Information Center

    Grier, Linda J.

    1986-01-01

    Provides a rationale for using international applicants for American summer camp positions and summarizes the services of organizations that screen, interview, and orient qualified applicants. Discusses contributions that international staff can make to a camp program with a global perspective and points out staff orientation and other practical…

  19. Camp's "Disneyland" Effect.

    ERIC Educational Resources Information Center

    Renville, Gary

    1999-01-01

    Describes the positive mental, physical, and social growth impacts that the camping experience had on the author, and urges camp program evaluation to plan and implement such changes. Sidebar lists steps of effective evaluation: program goals and objectives, goals of evaluation, implementation of evaluation, data analysis, and findings and…

  20. Camp Nursing: Student Internships.

    ERIC Educational Resources Information Center

    Harwood, Catherine Hoe; Van Hofwegen, Lynn

    2002-01-01

    Camps can meet or supplement their health care delivery needs by using student nurses. Three models for student nurse internships, basic information about nursing education, and tips for negotiating student nurse internships are described. Sidebars present resources for camp health centers, nursing student competence characteristics, types of…

  1. Marketing for Camp Trends.

    ERIC Educational Resources Information Center

    Biddle, Alicia

    1998-01-01

    To effectively market a camp, current trends and issues must be considered: specialty programming, the Americans With Disabilities Act, competing recreational programs, changes in the school year, programming for seniors, and accountability. Camps should have a marketing strategy that includes public relations, a marketing plan, a pricing…

  2. Physical Fitness at Camp.

    ERIC Educational Resources Information Center

    Steen, Thomas B.; And Others

    1990-01-01

    Describes decline in youth fitness, emphasizing role of camping programs in youth fitness education. Describes Michigan camp's fitness program, consisting of daily workouts, fitness education, and record keeping. Describes fitness consultants' role in program. Discusses program's highlights and problems, suggesting changes for future use. Shows…

  3. Camp Nursing: Student Internships.

    ERIC Educational Resources Information Center

    Harwood, Catherine Hoe; Van Hofwegen, Lynn

    2002-01-01

    Camps can meet or supplement their health care delivery needs by using student nurses. Three models for student nurse internships, basic information about nursing education, and tips for negotiating student nurse internships are described. Sidebars present resources for camp health centers, nursing student competence characteristics, types of…

  4. Maximizing Camp Property.

    ERIC Educational Resources Information Center

    Knapp, Clifford

    1987-01-01

    Provides selected 14-item outdoor-environmental education bibliography and 20-item checklist of factors weekend/summer camp directors should consider when pondering entry into the outdoor education market. Covers issues of camp philosophy, staff, facilities additions/alterations, equipment, food service, competition, environmental impact,…

  5. Camp Joy: Embracing Diversity.

    ERIC Educational Resources Information Center

    Krehbiel, Amy

    2001-01-01

    Camp Joy (Ohio) offers a racially integrated program to disadvantaged inner-city foster children. To attract quality minority staff, the camp recruits through former campers, word of mouth, a leader-in-training program, job and internship fairs, and networking with nearby colleges and social agencies. Staff training and the intrinsic rewards of…

  6. Today's Child - Tomorrow's Camp.

    ERIC Educational Resources Information Center

    Ditter, Robert B.

    1988-01-01

    Are camps solely in the business of providing fun, or are they facilitators of crucial life skills? A social worker explores the fun versus character-building debate, concluding that though camp is not group therapy, it contributes to overall personal growth and to the social and emotional development of all children. (JMM)

  7. Orienteering in Camping.

    ERIC Educational Resources Information Center

    Larson, Elston F.

    One of the recent developments in camping is "orienteering", a program using a map and compass. Orienteering can be dovetailed into an overall camping program and used to "point up" the entire program, or it can be confined to a single simple game. The arrangement depends on the situation. The minimum age of the participants should be about 9 or…

  8. Camp Joy: Embracing Diversity.

    ERIC Educational Resources Information Center

    Krehbiel, Amy

    2001-01-01

    Camp Joy (Ohio) offers a racially integrated program to disadvantaged inner-city foster children. To attract quality minority staff, the camp recruits through former campers, word of mouth, a leader-in-training program, job and internship fairs, and networking with nearby colleges and social agencies. Staff training and the intrinsic rewards of…

  9. Friends' Discovery Camp

    ERIC Educational Resources Information Center

    Seymour, Seth

    2008-01-01

    This article features Friends' Discovery Camp, a program that allows children with and without autism spectrum disorder to learn and play together. In Friends' Discovery Camp, campers take part in sensory-rich experiences, ranging from hands-on activities and performing arts to science experiments and stories teaching social skills. Now in its 7th…

  10. Camp's "Disneyland" Effect.

    ERIC Educational Resources Information Center

    Renville, Gary

    1999-01-01

    Describes the positive mental, physical, and social growth impacts that the camping experience had on the author, and urges camp program evaluation to plan and implement such changes. Sidebar lists steps of effective evaluation: program goals and objectives, goals of evaluation, implementation of evaluation, data analysis, and findings and…

  11. Building Communities through Camping.

    ERIC Educational Resources Information Center

    Rubendall, Robert L.

    Summer camp is a type of laboratory for community living. Society's needs have changed from providing a positive summer challenge for idle youth to experimenting with the demands and rewards of working in community groups. Decentralized camping emphasizes the team decision-making approach to teach self-reliance and awareness of democratic…

  12. Maximizing Camp Property.

    ERIC Educational Resources Information Center

    Knapp, Clifford

    1987-01-01

    Provides selected 14-item outdoor-environmental education bibliography and 20-item checklist of factors weekend/summer camp directors should consider when pondering entry into the outdoor education market. Covers issues of camp philosophy, staff, facilities additions/alterations, equipment, food service, competition, environmental impact,…

  13. Impact of Therapeutic Camping

    ERIC Educational Resources Information Center

    Shniderman, Craig M.

    1974-01-01

    There has been little interest in, and only slight illumination of, the impact of therapeutic camping for emotionally disturbed children. This study seeks to validate the belief that camping is therapeutic. Subjects were 52 boys, 5 to 11 1/2 years of age. Results support the hypothesis. (Author/HMV)

  14. Reconstitution of high-affinity opioid agonist binding in brain membranes

    SciTech Connect

    Remmers, A.E.; Medzihradsky, F. )

    1991-03-15

    In synaptosomal membranes from rat brain cortex, the {mu} selective agonist ({sup 3}H)dihydromorphine in the absence of sodium, and the nonselective antagonist ({sup 3}H)naltrexone in the presence of sodium, bound to two populations of opioid receptor sites with K{sub d} values of 0.69 and 8.7 nM for dihydromorphine, and 0.34 and 5.5 nM for naltrexone. The addition of 5 {mu}M guanosine 5{prime}-({gamma}-thio)triphosphate (GTP({gamma}S)) strongly reduced high-affinity agonist but not antagonist binding. Exposure of the membranes to high pH reduced the number of GTP({gamma}-{sup 35}S) binding sites by 90% and low K{sub m}, opioid-sensitive GTPase activity by 95%. In these membranes, high-affinity agonist binding was abolished and modulation of residual binding by GTP({gamma}S) was diminished. Alkali treatment of the glioma cell membranes prior to fusion inhibited most of the low K{sub m} GTPase activity and prevented the reconstitution of agonist binding. The results show that high-affinity opioid agonist binding reflects the ligand-occupied receptor - guanine nucleotide binding protein complex.

  15. Structural Basis for High-Affinity Peptide Inhibition of Human Pin1

    PubMed Central

    Zhang, Yan; Daum, Sebastian; Wildemann, Dirk; Zhou, Xiao Zhen; Verdecia, Mark A.; Bowman, Marianne E.; Lücke, Christian; Hunter, Tony; Lu, Kun-Ping; Fischer, Gunter; Noel, Joseph P.

    2009-01-01

    Human Pin1 is a key regulator of cell-cycle progression and plays growth-promoting roles in human cancers. High-affinity inhibitors of Pin1 may provide a unique opportunity for disrupting oncogenic pathways. Here we report two high-resolution X-ray crystal structures of human Pin1 bound to non-natural peptide inhibitors. The structures of the bound high-affinity peptides identify a type-I β-turn conformation for Pin1 prolyl peptide isomerase domain–peptide binding and an extensive molecular interface for high-affinity recognition. Moreover, these structures suggest chemical elements that may further improve the affinity and pharmacological properties of future peptide-based Pin inhibitors. Finally, an intramolecular hydrogen bond observed in both peptide complexes mimics the cyclic conformation of FK506 and rapamycin. Both FK506 and rapamycin are clinically important inhibitors of other peptidyl-prolyl cis-trans isomerases. This comparative discovery suggests that a cyclic peptide polyketide bridge, like that found in FK506 and rapamycin or a similar linkage, may significantly improve the binding affinity of structure-based Pin1 inhibitors. PMID:17518432

  16. Isolation of Anti-Ricin Protective Antibodies Exhibiting High Affinity from Immunized Non-Human Primates

    PubMed Central

    Noy-Porat, Tal; Rosenfeld, Ronit; Ariel, Naomi; Epstein, Eyal; Alcalay, Ron; Zvi, Anat; Kronman, Chanoch; Ordentlich, Arie; Mazor, Ohad

    2016-01-01

    Ricin, derived from the castor bean plant Ricinus communis, is one of the most potent and lethal toxins known, against which there is no available antidote. To date, the use of neutralizing antibodies is the most promising post-exposure treatment for ricin intoxication. The aim of this study was to isolate high affinity anti-ricin antibodies that possess potent toxin-neutralization capabilities. Two non-human primates were immunized with either a ricin-holotoxin- or subunit-based vaccine, to ensure the elicitation of diverse high affinity antibodies. By using a comprehensive set of primers, immune scFv phage-displayed libraries were constructed and panned. A panel of 10 antibodies (five directed against the A subunit of ricin and five against the B subunit) was isolated and reformatted into a full-length chimeric IgG. All of these antibodies were found to neutralize ricin in vitro, and several conferred full protection to ricin-intoxicated mice when given six hours after exposure. Six antibodies were found to possess exceptionally high affinity toward the toxin, with KD values below pM (koff < 1 × 10−7 s−1) that were well correlated with their ability to neutralize ricin. These antibodies, alone or in combination, could be used for the development of a highly-effective therapeutic preparation for post-exposure treatment of ricin intoxication. PMID:26950154

  17. Positron-labeled dopamine agonists for probing the high affinity states of dopamine subtype 2 receptors.

    PubMed

    Hwang, Dah-Ren; Narendran, Raj; Laruelle, Marc

    2005-01-01

    It is well documented that guanidine nucleotide-coupled dopamine subtype 2 receptors (D2) are configured in high and low affinity states for the dopamine agonist in vitro. However, it is still unclear whether these functional states exist in vivo. We hypothesized that positron-labeled D2 agonist and Positron Emission Tomography can be used to probe these functional states noninvasively. Recently, we demonstrated in nonhuman primates that N-[11C]propyl-norapomorphine (NPA), a full D2 agonist, is a suitable tracer for imaging the high affinity states of D2 receptors in vivo. We also developed kinetic modeling method to derive receptor parameters, such as binding potential (BP) and specific uptake ratios (V3''). When coupled with a dopamine releasing drug, amphetamine, NPA was found to be more sensitive than antagonist tracers, such as [11C]raclopride (RAC), to endogenous dopamine concentration changes (by about 42%). This finding suggests that NPA is a superior tracer for reporting endogenous DA concentration. In addition, the difference of the BP or V3'' of NPA and RAC under control and amphetamine challenge conditions could be used to estimate the functional states of D2 receptors in vivo. On the basis of our findings and the assumptions that NPA binds only to the high affinity states and RAC binds equally to both affinity states, we proposed that about 70% of the D2 receptors are configured in the high affinity states in vivo.

  18. Solution assembly of the pseudo-high affinity and intermediate affinity interleukin-2 receptor complexes.

    PubMed Central

    Wu, Z.; Goldstein, B.; Laue, T. M.; Liparoto, S. F.; Nemeth, M. J.; Ciardelli, T. L.

    1999-01-01

    The high affinity interleukin-2 receptor is composed of three cell surface subunits, IL-2Ralpha, IL-2Rbeta, and IL-2Rgamma. Functional forms of the IL-2 receptor exist, however, that enlist only two of the three subunits. On activated T-cells, the alpha- and beta-subunits combine as a preformed heterodimer (the pseudo-high affinity receptor) that serves to capture IL-2. On a subpopulation of natural killer cells, the beta- and gamma-subunits interact in a ligand-dependent manner to form the intermediate affinity receptor site. Previously, we have demonstrated the feasibility of employing coiled-coil molecular recognition for the solution assembly of a heteromeric IL-2 receptor complex. In that study, although the receptor was functional, the coiled-coil complex was a trimer rather than the desired heterodimer. We have now redesigned the hydrophobic heptad sequences of the coiled-coils to generate soluble forms of both the pseudo-high affinity and the intermediate affinity heterodimeric IL-2 receptors. The properties of these complexes were examined and their relevance to the physiological IL-2 receptor mechanism is discussed. PMID:10091650

  19. Single-experiment displacement assay for quantifying high-affinity binding by isothermal titration calorimetry.

    PubMed

    Krainer, Georg; Keller, Sandro

    2015-04-01

    Isothermal titration calorimetry (ITC) is the gold standard for dissecting the thermodynamics of a biomolecular binding process within a single experiment. However, reliable determination of the dissociation constant (KD) from a single titration is typically limited to the range 100 μM>KD>1 nM. Interactions characterized by a lower KD can be assessed indirectly by so-called competition or displacement assays, provided that a suitable competitive ligand is available whose KD falls within the directly accessible window. However, this protocol is limited by the fact that it necessitates at least two titrations to characterize one high-affinity inhibitor, resulting in considerable consumption of both sample material and time. Here, we introduce a fast and efficient ITC displacement assay that allows for the simultaneous characterization of both a high-affinity ligand and a moderate-affinity ligand competing for the same binding site on a receptor within a single experiment. The protocol is based on a titration of the high-affinity ligand into a solution containing the moderate-affinity ligand bound to the receptor present in excess. The resulting biphasic binding isotherm enables accurate and precise determination of KD values and binding enthalpies (ΔH) of both ligands. We discuss the theoretical background underlying the approach, demonstrate its practical application to metal ion chelation, explore its potential and limitations with the aid of simulations and statistical analyses, and elaborate on potential applications to protein-inhibitor interactions.

  20. Isolation of Anti-Ricin Protective Antibodies Exhibiting High Affinity from Immunized Non-Human Primates.

    PubMed

    Noy-Porat, Tal; Rosenfeld, Ronit; Ariel, Naomi; Epstein, Eyal; Alcalay, Ron; Zvi, Anat; Kronman, Chanoch; Ordentlich, Arie; Mazor, Ohad

    2016-03-03

    Ricin, derived from the castor bean plant Ricinus communis, is one of the most potent and lethal toxins known, against which there is no available antidote. To date, the use of neutralizing antibodies is the most promising post-exposure treatment for ricin intoxication. The aim of this study was to isolate high affinity anti-ricin antibodies that possess potent toxin-neutralization capabilities. Two non-human primates were immunized with either a ricin-holotoxin- or subunit-based vaccine, to ensure the elicitation of diverse high affinity antibodies. By using a comprehensive set of primers, immune scFv phage-displayed libraries were constructed and panned. A panel of 10 antibodies (five directed against the A subunit of ricin and five against the B subunit) was isolated and reformatted into a full-length chimeric IgG. All of these antibodies were found to neutralize ricin in vitro, and several conferred full protection to ricin-intoxicated mice when given six hours after exposure. Six antibodies were found to possess exceptionally high affinity toward the toxin, with KD values below pM (k(off )< 1 × 10(-7) s(-1)) that were well correlated with their ability to neutralize ricin. These antibodies, alone or in combination, could be used for the development of a highly-effective therapeutic preparation for post-exposure treatment of ricin intoxication.

  1. High affinity binding of (/sup 3/H)neurotensin of rat uterus

    SciTech Connect

    Pettibone, D.J.; Totaro, J.A.

    1987-11-01

    (/sup 3/H)Neurotensin (NT) was found to bind specifically and with high affinity to crude membranes prepared from rat uterus. Scatchard analysis of saturation binding studies indicated that (/sup 3/H)NT apparently binds to two sites (high affinity Kd 0.5 nM; low affinity Kd 9 nM) with the density of high affinity sites (41 fmoles/mg prot.) being about one-third that of the low affinity sites (100 fmoles/mg prot.). In competition studies, NT and various fragments inhibited (/sup 3/H)NT binding with the following potencies (approximately IC50): NT 8-13 (0.4 nM), NT 1-13 (4 nM), NT 9-13 (130 nM), NT 1-11, NT 1-8 (greater than 100 microM). Quantitatively similar results were obtained using brain tissue. These findings raise the possibility of a role for NT in uterine function.

  2. cGMP Signaling, Phosphodiesterases and Major Depressive Disorder

    PubMed Central

    Reierson, Gillian W; Guo, Shuyu; Mastronardi, Claudio; Licinio, Julio; Wong, Ma-Li

    2011-01-01

    Deficits in neuroplasticity are hypothesized to underlie the pathophysiology of major depressive disorder (MDD): the effectiveness of antidepressants is thought to be related to the normalization of disrupted synaptic transmission and neurogenesis. The cyclic adenosine monophosphate (cAMP) signaling cascade has received considerable attention for its role in neuroplasticity and MDD. However components of a closely related pathway, the cyclic guanosine monophosphate (cGMP) have been studied with much lower intensity, even though this signaling transduction cascade is also expressed in the brain and the activity of this pathway has been implicated in learning and memory processes. Cyclic GMP acts as a second messenger; it amplifies signals received at postsynaptic receptors and activates downstream effector molecules resulting in gene expression changes and neuronal responses. Phosphodiesterase (PDE) enzymes degrade cGMP into 5’GMP and therefore they are involved in the regulation of intracellular levels of cGMP. Here we review a growing body of evidence suggesting that the cGMP signaling cascade warrants further investigation for its involvement in MDD and antidepressant action. PMID:22654729

  3. Cyclic Nucleotide Phosphodiesterases: important signaling modulators and therapeutic targets

    PubMed Central

    Ahmad, Faiyaz; Murata, Taku; Simizu, Kasumi; Degerman, Eva; Maurice, Donald; Manganiello, Vincent

    2014-01-01

    By catalyzing hydrolysis of cAMP and cGMP, cyclic nucleotide phosphodiesterases are critical regulators of their intracellular concentrations and their biological effects. Since these intracellular second messengers control many cellular homeostatic processes, dysregulation of their signals and signaling pathways initiate or modulate pathophysiological pathways related to various disease states, including erectile dysfunction, pulmonary hypertension, acute refractory cardiac failure, intermittent claudication, chronic obstructive pulmonary disease, and psoriasis. Alterations in expression of PDEs and PDE-gene mutations (especially mutations in PDE6, PDE8B, PDE11A and PDE4) have been implicated in various diseases and cancer pathologies. PDEs also play important role in formation and function of multi-molecular signaling/regulatory complexes called signalosomes. At specific intracellular locations, individual PDEs, together with pathway-specific signaling molecules, regulators, and effectors, are incorporated into specific signalosomes, where they facilitate and regulate compartmentalization of cyclic nucleotide signaling pathways and specific cellular functions. Currently, only a limited number of PDE inhibitors (PDE3, PDE4, PDE5 inhibitors) are used in clinical practice. Future paths to novel drug discovery include the crystal structure-based design approach, which has resulted in generation of more effective family-selective inhibitors, as well as burgeoning development of strategies to alter compartmentalized cyclic nucleotide signaling pathways by selectively targeting individual PDEs and their signalosome partners. PMID:25056711

  4. Phosphodiesterase sequence variants may predispose to prostate cancer

    PubMed Central

    de Alexandre, Rodrigo Bertollo; Horvath, Anelia; Szarek, Eva; Manning, Allison D.; Leal, Leticia Ferro; Kardauke, Fabio; Epstein, Jonathan A.; Carraro, Dirce Maria; Soares, Fernando Augusto; Apanasovich, Tatiyana; Stratakis, Constantine A.; Faucz, Fabio Rueda

    2015-01-01

    We hypothesized that mutations that inactivate phosphodiesterase (PDE) activity and lead to increased cyclic AMP (cAMP) and cyclic GMP (cGMP) levels may be associated with prostate cancer (PCa). We sequenced the entire PDE coding sequences in the DNA of 16 biopsy samples from PCa patients. Novel mutations were confirmed in the somatic or germline state by Sanger sequencing. Data were then compared to the 1000 Genome Project. PDE, CREB and pCREB protein expression was also studied in all samples, in both normal and abnormal tissue, by immunofluorescence. We identified 3 previously described PDE sequence variants that were significantly higher in PCa. Four novel sequence variations, one each in the PDE4B, PDE6C, PDE7B and PDE10A genes, respectively, were also found in the PCa samples. Interestingly, PDE10A and PDE4B novel variants that were present in 19% and 6% of the patients, respectively, were found in the tumor tissue only. In patients carrying PDE defects, there was pCREB accumulation (p<0.001), and an increase of the pCREB/CREB ratio (patients 0.97± 0.03; controls 0.52± 0.03; p-value < 0.001) by immunohistochemical analysis. We conclude that PDE sequence variants may play a role in the predisposition and/or progression to PCa at the germline and/or somatic state, respectively. Larger such studies are needed to confirm these findings. PMID:25979379

  5. Pharmacological Validation of Trypanosoma brucei Phosphodiesterases as Novel Drug Targets

    PubMed Central

    de Koning, Harry P.; Gould, Matthew K.; Sterk, Geert Jan; Tenor, Hermann; Kunz, Stefan; Luginbuehl, Edith; Seebeck, Thomas

    2012-01-01

    The development of drugs for neglected infectious diseases often uses parasite-specific enzymes as targets. We here demonstrate that parasite enzymes with highly conserved human homologs may represent a promising reservoir of new potential drug targets. The cyclic nucleotide-specific phosphodiesterases (PDEs) of Trypanosoma brucei, causative agent of the fatal human sleeping sickness, are essential for the parasite. The highly conserved human homologs are well-established drug targets. We here describe what is to our knowledge the first pharmacological validation of trypanosomal PDEs as drug targets. High-throughput screening of a proprietary compound library identified a number of potent hits. One compound, the tetrahydrophthalazinone compound A (Cpd A), was further characterized. It causes a dramatic increase of intracellular cyclic adenosine monophosphate (cAMP). Short-term cell viability is not affected, but cell proliferation is inhibited immediately, and cell death occurs within 3 days. Cpd A prevents cytokinesis, resulting in multinucleated, multiflagellated cells that eventually lyse. These observations pharmacologically validate the highly conserved trypanosomal PDEs as potential drug targets. PMID:22291195

  6. Forskolin increases cAMP and inhibits progesterone induced meiosis reinitiation in Xenopus laevis oocytes.

    PubMed

    Schorderet-Slatkine, S; Baulieu, E E

    1982-10-01

    The diterpene, forskolin, is a potent and reversible inhibitor of progesterone-induced meiosis in Xenopus laevis oocytes (ED50 of inhibition approximately 3 microM). Forskolin alone increases cAMP concentration in oocytes, but, unlike with cholera toxin treatment, there is no lag phase, and reversibility is obtained by washing the cells. Progesterone decreases the forskolin effect on cAMP accumulation, but cAMP concentration remains above the level observed in oocytes treated with progesterone alone. The data corroborate the previously-established antagonistic effect of cAMP on progesterone-induced meiosis. Preliminary experiments in the presence of a phosphodiesterase inhibitor suggest that, as in other biological systems, forskolin is an activator of adenylate cyclase in xenopus laevis oocytes. Contrary to what is observed when forskolin is present in the incubation medium, no effect of the diterpene is recorded after its injection into oocytes, evoking a site of action at the external side of the membrane.

  7. cAMP enhances BMP2-signaling through PKA and MKP1-dependent mechanisms

    SciTech Connect

    Ghayor, Chafik; Ehrbar, Martin; Miguel, Blanca San; Graetz, Klaus W.; Weber, Franz E.

    2009-04-03

    Recent studies suggest that the elevation of intracellular cyclic adenosine monophosphate (cAMP) and the activation of the protein kinase A regulate BMP-induced osteogenesis. However, the precise mechanisms underlying the enhancing effect of cAMP on BMP2 signaling were not completely revealed. In this study we investigated the effect of elevated cAMP level and PKA activation on the BMP2-induced osteoblastic differentiation in pluripotent C2C12 cells. Alkaline phosphatase activity and its mRNA were consistently induced by BMP2 treatment. The pretreatment of C2C12 cells with Forskolin, a cAMP generating agent, dbcAMP, an analogue of cAMP, or IBMX (3-isobutyl 1-methyl xanthine), and a nonspecific inhibitor of phosphodiesterases elicited further activation of alkaline phosphatase. Furthermore, elevated intracellular cAMP level increased BMP2-induced MKP1. On the other hand, BMP2-induced Erk phosphorylation (p44/p42) and cell proliferation were suppressed in the presence of cAMP. Thus, cAMP might enhance BMP2-induced osteoblastic differentiation by a MKP1-Erk-dependent mechanism.

  8. Calmodulin-dependent cyclic nucleotide phosphodiesterase (PDE1).

    PubMed

    Kakkar, R; Raju, R V; Sharma, R K

    1999-07-01

    Ca2+/calmodulin-dependent cyclic nucleotide phosphodiesterase (PDE1) is one of the key enzymes involved in the complex interactions between the cyclic nucleotide and Ca2+ second messenger systems. Currently, three genes encode PDE1, and alternate splicing of these genes gives rise to functionally different isozymes which exhibit distinct catalytic and regulatory properties. Some isozymes have similar kinetic and immunological properties but are differentially regulated by Ca2+ and calmodulin. These isozymes also differ in their mechanism of regulation by phosphorylation. Analysis of various regulatory reactions involving Ca2+ and cyclic adenosine monophosphate (cAMP) has revealed the importance of the time dependence of these reactions during cell activation; however, no measurement is available for the time of occurrence of specific regulatory reactions. cAMP-signalling systems provide a pivotal centre for achieving crosstalk regulation by various signalling pathways. It has been proposed that polypeptide sequences enriched in proline (P), glutamate (E), serine (S) and threonine (T), known as PEST motifs, serve as putative intramolecular signals for rapid proteolytic degradation by calpains. Calpains are Ca(2+)-dependent cysteine proteases that regulate various enzymes, transcription factors and structural proteins through limited proteolysis. Isozyme PDE1A2 has a PEST motif and acts as a substrate for m-calpain. In this paper, we have described PDE1A2 regulation by calpains and its physiological implications. cAMP is an important component of the signal transduction pathway and plays an integral role in various physiological processes such as gene transcription, various neuronal functions, cardiac muscle contraction, vascular relaxation, cell proliferation and a host of other functions. It is important to identify the cellular processes where PDE isoform(s) and cAMP response are altered. This will lead to better understanding of the pathology of disease states

  9. Atrazine acts as an endocrine disrupter by inhibiting cAMP-specific phosphodiesterase-4

    SciTech Connect

    Kucka, Marek; Pogrmic-Majkic, Kristina; Fa, Svetlana; Stojilkovic, Stanko S.; Kovacevic, Radmila

    2012-11-15

    Atrazine, one of the most commonly used herbicides worldwide, acts as an endocrine disruptor, but the mechanism of its action has not been characterized. In this study, we show that atrazine rapidly increases cAMP levels in cultured rat pituitary and testicular Leydig cells in a concentration-dependent manner, but less effectively than 3-isobutyl-1-methylxanthine, a competitive non-specific inhibitor of phosphodiesterases (PDEs). In forskolin (an activator of adenylyl cyclase)- and probenecid (an inhibitor of cyclic nucleotide transporters)-treated cells, but not in 3-isobutyl-1-methylxanthine-treated cells, atrazine further increased cAMP levels, indicating that inhibition of PDEs accounts for accumulation of cAMP. In contrast to cAMP, atrazine did not alter cGMP levels, further indicating that it inhibits cAMP-specific PDEs. Atrazine-induced changes in cAMP levels were sufficient to stimulate prolactin release in pituitary cells and androgen production in Leydig cells, indicating that it acts as an endocrine disrupter both in cells that secrete by exocytosis of prestored hormones and in cells that secrete by de novo hormone synthesis. Rolipram abolished the stimulatory effect of atrazine on cAMP release in both cell types, suggesting that it acts as an inhibitor of PDE4s, isoforms whose mRNA transcripts dominate in pituitary and Leydig cells together with mRNA for PDE8A. In contrast, immortalized lacto-somatotrophs showed low expression of these mRNA transcripts and several fold higher cAMP levels compared to normal pituitary cells, and atrazine was unable to further increase cAMP levels. These results indicate that atrazine acts as a general endocrine disrupter by inhibiting cAMP-specific PDE4s. -- Highlights: ► Atrazine stimulates cAMP accumulation in pituitary and Leydig cells. ► Atrazine also stimulates PRL and androgens secretion. ► Stimulatory effects of atrazine were abolished in cells with IBMX-inhibited PDEs. ► Atrazine specificity toward c

  10. Foreign Language Camps: Camp Waskowitz. Teacher's Guide and Planning Book.

    ERIC Educational Resources Information Center

    Baudin, Phil; And Others

    This guide to running a foreign language camp is intended to cover all aspects of camp administration and program planning. The philosophy of language camps is set forth. The chairperson's responsibilities regarding staff recruitment, staff assignments, and handling finances are outlined. Sample schedules for French, Spanish, and German camps are…

  11. A bimodal modulation of the cAMP pathway is involved in the control of myogenic differentiation in l6 cells.

    PubMed

    Naro, Fabio; De Arcangelis, Vania; Sette, Claudio; Ambrosio, Caterina; Komati, Hiba; Molinaro, Mario; Adamo, Sergio; Nemoz, Georges

    2003-12-05

    We have previously shown that myogenesis induction by Arg8-vasopressin (AVP) in L6 rat myoblasts involves a sustained stimulation of type 4 cAMP-phosphodiesterase. In this model, we observed that a transient cAMP generation occurs in the minutes following AVP addition. Evidence suggests that cAMP generation is due to the prostaglandins produced in response to AVP binding to V1a receptors and subsequent activation of phospholipase A2. The early cAMP increase was effective in activating cAMP-dependent protein kinase (PKA) and increasing phosphorylation of CREB transcription factor. Inhibition of PKA by compound H89 prior to AVP addition led to a significant reduction of expression of the differentiation marker creatine kinase, whereas H89 added 1-5 h after AVP had no significant effect. Furthermore, PKA inhibition 24 h after the beginning of AVP treatment potentiated differentiation. This shows that both an early activation and a later down-regulation of the cAMP pathway are required for AVP induction of myogenesis. Because phosphodiesterase PDE4D3 overexpressed in L6 cells lost its ability to potentiate AVP-induced differentiation when mutated and rendered insensitive to PKA phosphorylation and activation, we hypothesize that the early cAMP increase is required to trigger the down-regulation of cAMP pathway through stimulation of phosphodiesterase.

  12. PC12 cell mutants that possess low- but not high-affinity nerve growth factor receptors neither respond to nor internalize nerve growth factor

    PubMed Central

    1986-01-01

    Four mutant PC12 pheochromocytoma cell lines that are nerve growth factor (NGF)-nonresponsive (PC12nnr) have been selected from chemically mutagenized cultures by a double selection procedure: failure both to grow neurites in the presence of NGF and to survive in NGF-supplemented serum-free medium. The PC12nnr cells were deficient in all additional NGF responses surveyed: abatement of cell proliferation, changes in glycoprotein composition, induction of ornithine decarboxylase, rapid changes in protein phosphorylation, and cell surface ruffling. However, PC12nnr cells closely resembled non-NGF-treated PC12 cells in most properties tested: cell size and shape; division rate; protein, phosphoprotein, and glycoprotein composition; and cell surface morphology. All four PC12nnr lines differed from PC12 cells in three ways in addition to failure of NGF response: PC12nnr cells failed to internalize bound NGF by the normal, saturable, high-affinity mechanism present in PC12 cells. The PC12nnr cells bound NGF but entirely, or nearly entirely, at low-affinity sites only, whereas PC12 cells possess both high- and low-affinity NGF binding sites. The responses to dibutyryl cyclic AMP that were tested appeared to be enhanced or altered in the PC12nnr cells compared to PC12 cells. Internalization of, and responses to, epidermal growth factor were normal in the PC12nnr cells ruling out a generalized defect in hormonal binding, uptake, or response mechanisms. These findings are consistent with a causal association between the presence of high-affinity NGF receptors and of NGF responsiveness and internalization. A possible relationship is also suggested between regulation of cAMP responses and regulation of NGF responses or NGF receptor affinity. PMID:3005338

  13. Better Positioning Those Camp Jobs.

    ERIC Educational Resources Information Center

    Henderson, Karla A.

    1989-01-01

    Discusses summer camps' difficulties in recruiting college students as staff, suggesting camps have "image problem." Describes study of job descriptions to evaluate whether camps offer useful career experiences. Examines frequency and types of job tasks. Examines how camp directors might use job descriptions to recruit more effectively. (TES)

  14. Camp Invention Connects to Classrooms.

    ERIC Educational Resources Information Center

    Krebs, Danute V.; Clark, Barbara D.

    2000-01-01

    This article describes Camp Invention, a national creativity day camp that integrates science, math, social studies, and the arts. The one week camp for children entering grades 2-6 attracts many academically gifted children because of its hands-on curriculum. The camp's curriculum and activities are discussed. (Contains two references.) (CR)

  15. Organized camping: a historical perspective.

    PubMed

    Ramsing, Ron

    2007-10-01

    Organized camping has been part of the fabric of American culture for more than 150 years. Today, organized camps serve more than 11 million youth annually, a significant departure from the first camps that were created to address the challenges of urban living during the Industrial Revolution. This article provides a brief historical perspective of organized camp and its evolution.

  16. Camping Skills. Environmental Education Curriculum.

    ERIC Educational Resources Information Center

    Topeka Public Schools, KS.

    This unit on camping skills is designed for special education students at the high school level. The objective of the unit is to provide students with an adequate camping knowledge and skill development to allow them to participate in camping activities. There is an emphasis on maintaining environmental quality as a part of good camping practices.…

  17. High-affinity maltose/trehalose transport system in the hyperthermophilic archaeon Thermococcus litoralis.

    PubMed

    Xavier, K B; Martins, L O; Peist, R; Kossmann, M; Boos, W; Santos, H

    1996-08-01

    The hyperthermophilic marine archaeon Thermococcus litoralis exhibits high-affinity transport activity for maltose and trehalose at 85 degrees C. The K(m) for maltose transport was 22 nM, and that for trehalose was 17 nM. In cells that had been grown on peptone plus yeast extract, the Vmax for maltose uptake ranged from 3.2 to 7.5 nmol/min/mg of protein in different cell cultures. Cells grown in peptone without yeast extract did not show significant maltose or trehalose uptake. We found that the compound in yeast extract responsible for the induction of the maltose and trehalose transport system was trehalose. [14C]maltose uptake at 100 nM was not significantly inhibited by glucose, sucrose, or maltotriose at a 100 microM concentration but was completely inhibited by trehalose and maltose. The inhibitor constant, Ki, of trehalose for inhibiting maltose uptake was 21 nM. In contrast, the ability of maltose to inhibit the uptake of trehalose was not equally strong. With 20 nM [14C]trehalose as the substrate, a 10-fold excess of maltose was necessary to inhibit uptake to 50%. However, full inhibition was observed at 2 microM maltose. The detergent-solubilized membranes of trehalose-induced cells contained a high-affinity binding protein for maltose and trehalose, with an M(r) of 48,000, that exhibited the same substrate specificity as the transport system found in whole cells. We conclude that maltose and trehalose are transported by the same high-affinity membrane-associated system. This represents the first report on sugar transport in any hyperthermophilic archaeon.

  18. High affinity nanobodies against human epidermal growth factor receptor selected on cells by E. coli display.

    PubMed

    Salema, Valencio; Mañas, Carmen; Cerdán, Lidia; Piñero-Lambea, Carlos; Marín, Elvira; Roovers, Rob C; Van Bergen En Henegouwen, Paul M P; Fernández, Luis Ángel

    2016-10-01

    Most therapeutic antibodies (Abs) target cell surface proteins on tumor and immune cells. Cloning of Ab gene libraries in E. coli and their display on bacteriophages is commonly used to select novel therapeutic Abs binding target antigens, either purified or expressed on cells. However, the sticky nature of bacteriophages renders phage display selections on cells challenging. We previously reported an E. coli display system for expression of VHHs (i.e., nanobodies, Nbs) on the surface of bacteria and selection of high-affinity clones by magnetic cell sorting (MACS). Here, we demonstrate that E. coli display is also an attractive method for isolation of Nbs against cell surface antigens, such as the epidermal growth factor receptor (EGFR), upon direct selection and screening of Ab libraries on live cells. We employ a whole cell-based strategy using a VHH library obtained by immunization with human tumor cells over-expressing EGFR (i.e., A431), and selection of bacterial clones bound to murine fibroblast NIH-3T3 cells transfected with human EGFR, after depletion of non-specific clones on untransfected cells. This strategy resulted in the isolation of high-affinity Nbs binding distinct epitopes of EGFR, including Nbs competing with the ligand, EGF, as characterized by flow cytometry of bacteria displaying the Nbs and binding assays with purified Nbs using surface plasmon resonance. Hence, our study demonstrates that E. coli display of VHH libraries and selection on cells enables efficient isolation and characterization of high-affinity Nbs against cell surface antigens.

  19. Binding and structural diversity among high-affinity monoclonal anti-digoxin antibodies.

    PubMed

    Mudgett-Hunter, M; Anderson, W; Haber, E; Margolies, M N

    1985-04-01

    High-affinity monoclonal antibodies specific for the cardiac glycoside digoxin provide a useful system for the study of structure-function relationships between antibody combining site and specific antigenic determinants. Fifteen high-affinity monoclonal anti-digoxin antibodies were produced when spleen cells from A/J mice immunized with digoxin coupled to human serum albumin (Dig-HSA) were fused with the non-secreting murine myeloma Sp2/0 cell line. Each subcloned hybridoma antibody was analyzed for affinity and specificity for structurally related cardiac glycosides by a radioimmunoassay based on the adsorption of free [3H]digoxin to dextran-coated charcoal. All of the anti-digoxin hybridoma proteins demonstrated high affinity constants ranging from 10(9) to 10(12) M-1. Using seven different analogs of digoxin, binding specificities of the monoclonal antibodies were assessed by inhibition radioimmunoassay. The 15 hybridomas produced from fusions involving five mice could be divided into eight sets on the basis of these binding specificities. Certain antibodies exhibit a preference for the aglycone portion of digoxin, while others are more specific for the tridigitoxose sugar moiety of digoxin. Monoclonal antibody H- and L-chains were subjected to N-terminal amino acid sequence analysis. The antibodies may be divided into several sequence homology sets for both H- and L-chains. In most instances, homologous heavy chains are associated with a set of homologous light chains. Homologous partial sequences, however, do not correlate with similar antigenic specificities and affinities for digoxin. Thus the fine specificity for antigen is not dependent on VH- and VL-encoded sequences alone. These data illustrate the broad diversity of the elicited response to a single hapten, even in inbred mice.

  20. Relations between high-affinity binding sites of markers for binding regions on human serum albumin.

    PubMed Central

    Kragh-Hansen, U

    1985-01-01

    Binding of warfarin, digitoxin, diazepam, salicylate and Phenol Red, individually or in different pair combinations, to defatted human serum albumin at ligand/protein molar ratios less than 1:1 was studied at pH 7.0. The binding was determined by ultrafiltration. Some of the experiments were repeated with the use of equilibrium dialysis in order to strengthen the results. Irrespective of the method used, all ligands bind to one high-affinity binding site with an association constant in the range 10(4)-10(6) M-1. High-affinity binding of the following pair of ligands took place independently: warfarin-Phenol Red, warfarin-diazepam, warfarin-digitoxin and digitoxin-diazepam. Simultaneous binding of warfarin and salicylate led to a mutual decrease in binding of one another, as did simultaneous binding of digitoxin and Phenol Red. Both effects could be accounted for by a coupling constant. The coupling constant is the factor by which the primary association constants are affected; in these examples of anti-co-operativity the factor has a value between 0 and 1. In the first example it was calculated to be 0.8 and in the latter 0.5. Finally, digitoxin and salicylate were found to compete for a common high-affinity binding site. The present findings support the proposal of four separate primary binding sites for warfarin, digitoxin (and salicylate), diazepam and Phenol Red. An attempt to correlate this partial binding model for serum albumin with other models in the literature is made. PMID:3977850

  1. High-affinity maltose/trehalose transport system in the hyperthermophilic archaeon Thermococcus litoralis.

    PubMed Central

    Xavier, K B; Martins, L O; Peist, R; Kossmann, M; Boos, W; Santos, H

    1996-01-01

    The hyperthermophilic marine archaeon Thermococcus litoralis exhibits high-affinity transport activity for maltose and trehalose at 85 degrees C. The K(m) for maltose transport was 22 nM, and that for trehalose was 17 nM. In cells that had been grown on peptone plus yeast extract, the Vmax for maltose uptake ranged from 3.2 to 7.5 nmol/min/mg of protein in different cell cultures. Cells grown in peptone without yeast extract did not show significant maltose or trehalose uptake. We found that the compound in yeast extract responsible for the induction of the maltose and trehalose transport system was trehalose. [14C]maltose uptake at 100 nM was not significantly inhibited by glucose, sucrose, or maltotriose at a 100 microM concentration but was completely inhibited by trehalose and maltose. The inhibitor constant, Ki, of trehalose for inhibiting maltose uptake was 21 nM. In contrast, the ability of maltose to inhibit the uptake of trehalose was not equally strong. With 20 nM [14C]trehalose as the substrate, a 10-fold excess of maltose was necessary to inhibit uptake to 50%. However, full inhibition was observed at 2 microM maltose. The detergent-solubilized membranes of trehalose-induced cells contained a high-affinity binding protein for maltose and trehalose, with an M(r) of 48,000, that exhibited the same substrate specificity as the transport system found in whole cells. We conclude that maltose and trehalose are transported by the same high-affinity membrane-associated system. This represents the first report on sugar transport in any hyperthermophilic archaeon. PMID:8759837

  2. The Phn system of Mycobacterium smegmatis: a second high-affinity ABC-transporter for phosphate.

    PubMed

    Gebhard, Susanne; Tran, Sieu L; Cook, Gregory M

    2006-11-01

    Uptake of inorganic phosphate, an essential but often limiting nutrient, in bacteria is usually accomplished by the high-affinity ABC-transport system Pst. Pathogenic species of mycobacteria contain several copies of the genes encoding the Pst system (pstSCAB), and two of the encoded proteins, PstS1 and PstS2, have been shown to be virulence factors in Mycobacterium tuberculosis. The fast-growing Mycobacterium smegmatis contains only a single copy of the pst operon. This study reports the biochemical and molecular characterization of a second high-affinity phosphate transport system, designated Phn. The Phn system is encoded by a three-gene operon that constitutes the components of a putative ABC-type phosphonate/phosphate transport system. Expression studies using phnD- and pstS-lacZ transcriptional fusions showed that both operons were induced when the culture entered phosphate limitation, indicating a role for both systems in phosphate uptake at low extracellular concentrations. Deletion mutants in either phnD or pstS failed to grow in minimal medium with a 10 mM phosphate concentration, while the isogenic wild-type strain mc(2)155 grew at micromolar phosphate concentrations. Analysis of the kinetics of phosphate transport in the wild-type and mutant strains led to the proposal that the Phn and Pst systems are both high-affinity phosphate transporters with similar affinities for phosphate (i.e. apparent K(m) values between 40 and 90 muM P(i)). The Phn system of M. smegmatis appears to be unique in that, unlike previously identified Phn systems, it does not recognize phosphonates or phosphite as substrates.

  3. Rational development of high-affinity T-cell receptor-like antibodies

    PubMed Central

    Stewart-Jones, Guillaume; Wadle, Andreas; Hombach, Anja; Shenderov, Eugene; Held, Gerhard; Fischer, Eliane; Kleber, Sascha; Nuber, Natko; Stenner-Liewen, Frank; Bauer, Stefan; McMichael, Andrew; Knuth, Alexander; Abken, Hinrich; Hombach, Andreas A.; Cerundolo, Vincenzo; Jones, E. Yvonne; Renner, Christoph

    2009-01-01

    T-cell interaction with a target cell is a key event in the adaptive immune response and primarily driven by T-cell receptor (TCR) recognition of peptide-MHC (pMHC) complexes. TCR avidity for a given pMHC is determined by number of MHC molecules, availability of coreceptors, and TCR affinity for MHC or peptide, respectively, with peptide recognition being the most important factor to confer target specificity. Here we present high-resolution crystal structures of 2 Fab antibodies in complex with the immunodominant NY-ESO-1157–165 peptide analogue (SLLMWITQV) presented by HLA-A*0201 and compare them with a TCR recognizing the same pMHC. Binding to the central methionine-tryptophan peptide motif and orientation of binding were almost identical for Fabs and TCR. As the MW “peg” dominates the contacts between Fab and peptide, we estimated the contributions of individual amino acids between the Fab and peptide to provide the rational basis for a peptide-focused second-generation, high-affinity antibody library. The final Fab candidate achieved better peptide binding by 2 light-chain mutations, giving a 20-fold affinity improvement to 2–4 nM, exceeding the affinity of the TCR by 1,000-fold. The high-affinity Fab when grafted as recombinant TCR on T cells conferred specific killing of HLA-A*0201/NY-ESO-1157–165 target cells. In summary, we prove that affinity maturation of antibodies mimicking a TCR is possible and provide a strategy for engineering high-affinity antibodies that can be used in targeting specific pMHC complexes for diagnostic and therapeutic purposes. PMID:19307587

  4. High affinity nanobodies against human epidermal growth factor receptor selected on cells by E. coli display

    PubMed Central

    Salema, Valencio; Mañas, Carmen; Cerdán, Lidia; Piñero-Lambea, Carlos; Marín, Elvira; Roovers, Rob C.; Van Bergen en Henegouwen, Paul M.P.; Fernández, Luis Ángel

    2016-01-01

    ABSTRACT Most therapeutic antibodies (Abs) target cell surface proteins on tumor and immune cells. Cloning of Ab gene libraries in E. coli and their display on bacteriophages is commonly used to select novel therapeutic Abs binding target antigens, either purified or expressed on cells. However, the sticky nature of bacteriophages renders phage display selections on cells challenging. We previously reported an E. coli display system for expression of VHHs (i.e., nanobodies, Nbs) on the surface of bacteria and selection of high-affinity clones by magnetic cell sorting (MACS). Here, we demonstrate that E. coli display is also an attractive method for isolation of Nbs against cell surface antigens, such as the epidermal growth factor receptor (EGFR), upon direct selection and screening of Ab libraries on live cells. We employ a whole cell-based strategy using a VHH library obtained by immunization with human tumor cells over-expressing EGFR (i.e., A431), and selection of bacterial clones bound to murine fibroblast NIH-3T3 cells transfected with human EGFR, after depletion of non-specific clones on untransfected cells. This strategy resulted in the isolation of high-affinity Nbs binding distinct epitopes of EGFR, including Nbs competing with the ligand, EGF, as characterized by flow cytometry of bacteria displaying the Nbs and binding assays with purified Nbs using surface plasmon resonance. Hence, our study demonstrates that E. coli display of VHH libraries and selection on cells enables efficient isolation and characterization of high-affinity Nbs against cell surface antigens. PMID:27472381

  5. High-affinity antibodies to the 1,4-dihydropyridine Ca2+-channel blockers

    SciTech Connect

    Campbell, K.P.; Sharp, A.; Strom, M.; Kahl, S.D.

    1986-05-01

    Antibodies with high affinity and specificity for the 1,4-dihydropyridine Ca2+-channel blockers have been produced in rabbits by immunization with dihydropyridine-protein conjugates. Anti-dihydropyridine antibodies were found to specifically bind (/sup 3/H)nitrendipine, (/sup 3/H)-nimodipine, (/sup 3/H)nisoldipine, and (/sup 3/H)PN 200-110 (all 1,4-dihydropyridine Ca2+-channel blockers) with high affinity, while (/sup 3/H)verapamil, (/sup 3/H)diltiazem, and (/sup 3/H)trifluoperazine were not recognized. The average dissociation constant of the (/sup 3/H)nitrendipine-antibody complex was 0.06 (+/- 0.02) X 10(-9) M for an antiserum studied in detail and ranged from 0.01 to 0.24 X 10(-9) M for all antisera. Inhibition of (/sup 3/H)nitrendipine binding was specific for the 1,4-dihydropyridine Ca2+-channel modifiers and the concentrations required for half-maximal inhibition ranged between 0.25 and 0.90 nM. Structurally unrelated Ca2+-channel blockers, calmodulin antagonists, inactive metabolites of nitrendipine, and UV-inactivated nisoldipine did not modify (/sup 3/H)nitrendipine binding to the anti-dihydropyridine antibodies. Dihydropyridines without a bulky substituent in the 4-position of the heterocycle were able to displace (/sup 3/H)nitrendipine binding, but the concentrations required for half-maximal inhibition were greater than 800 nM. In summary, anti-dihydropyridine antibodies have been shown to have high affinity and specificity for the 1,4-dihydropyridine Ca2+-channel blockers and to exhibit dihydropyridine binding properties similar to the membrane receptor for the 1,4-dihydropyridine Ca2+-channel blockers.

  6. Pre-Camp Checklists.

    ERIC Educational Resources Information Center

    Camping Magazine, 1995

    1995-01-01

    Provides checklists to prepare for camp opening. Covers such areas as health histories of all campers and staff, staff credentials, condition of facilities and equipment, staff training, horse stables and equipment, and safety of swimming areas. (LP)

  7. Astro Camp Counselors

    NASA Image and Video Library

    2007-06-08

    Barbara Marino (left), Stennis Space Center education technology specialist, shows Astro Camp Counselor Beverly Fitzsimmons a LEGO model during a teambuilding exercise May 29 at SSC's North Gate computer lab as a part of the counselors' `new hire' orientation.

  8. Pre-Camp Checklists.

    ERIC Educational Resources Information Center

    Camping Magazine, 1995

    1995-01-01

    Provides checklists to prepare for camp opening. Covers such areas as health histories of all campers and staff, staff credentials, condition of facilities and equipment, staff training, horse stables and equipment, and safety of swimming areas. (LP)

  9. Hitler's Death Camps.

    ERIC Educational Resources Information Center

    Wieser, Paul

    1995-01-01

    Presents a high school lesson on Hitler's death camps and the widespread policy of brutality and oppression against European Jews. Includes student objectives, instructional procedures, and a chart listing the value of used clothing taken from the Jews. (CFR)

  10. Camping in the Snow.

    ERIC Educational Resources Information Center

    Brown, Constance

    1979-01-01

    Describes the experience of winter snow camping. Provides suggestions for shelter, snow kitchens, fires and stoves, cooking, latrines, sleeping warm, dehydration prevention, and clothing. Illustrated with full color photographs. (MA)

  11. Hitler's Death Camps.

    ERIC Educational Resources Information Center

    Wieser, Paul

    1995-01-01

    Presents a high school lesson on Hitler's death camps and the widespread policy of brutality and oppression against European Jews. Includes student objectives, instructional procedures, and a chart listing the value of used clothing taken from the Jews. (CFR)

  12. High-affinity benzodiazepine receptor ligands among benzodiazepines and betacarbolines with different intrinsic activity

    SciTech Connect

    Yliniemelae, A.; Gynther, J. ); Konschin, H.; Tylli, H. ); Rouvinen, J. )

    1989-01-01

    Structural and electrostatic features of diazepam, flumazenil, and methyl betacarboline-3-carboxylate (BCCM) have been investigated using the molecular superimposition method. These high-affinity benzodiazepine (BZ) receptor ligands are structurally unrelated and they have different intrinsic activity. These ligands are superimposed in such a way that common structural and electrostatic features essential for the high receptor binding affinity overlap. In addition to this binding pharmacophore, there are roughly three separate binding zones in the BZ receptor, one for each class of ligands. The intrinsic activity of BZ receptor ligands depends on the molecular structures and the way the ligand approaches the receptor.

  13. Aminopyrazoles with high affinity for the human neuropeptide Y5 receptor.

    PubMed

    Kordik, C P; Luo, C; Zanoni, B C; Dax, S L; McNally, J J; Lovenberg, T W; Wilson, S J; Reitz, A B

    2001-09-03

    1,3-Disubstituted-5-aminopyrazoles were prepared based on a lead compound found through high-throughput screening of our corporate compound library in an assay measuring affinity for the human neuropeptide Y5 receptor. The target compounds were prepared by cyclization of alpha-cyanoketones with appropriate hydrazines, followed by reduction and coupling to various sulfonamido-carboxylic acids. Several of these arylpyrazoles (e.g., 19 and 45) displayed high affinity for the human NPY Y5 receptor (<20nM IC(50)s).

  14. Astro STARS Camp

    NASA Image and Video Library

    2012-06-15

    Summer is a time of educational activity at Stennis Space Center. In June 2012, 25 young people age 13-15 attended the annual Astro STARS (Spaceflight, Technology, Astronomy and Robotics at Stennis) camp at the rocket engine test facility. During the five-day camp, participants engaged in hands-on experiences in a variety of areas, including engineering and robotics. On the final day, campers launched model rockets they had assembled.

  15. Astro STARS Camp

    NASA Image and Video Library

    2011-06-28

    Tom Nicolaides, an aerospace technologist in the Engineering & Test Directorate at Stennis Space Center, looks on as 2011 Astro STARS participants take turns gazing at the sun through a special telescope. The sun-gazing activity was part of the Astro STARS (Spaceflight, Technology, Astronomy & Robotics at Stennis) camp for 13-to-15-year-olds June 27 - July 1. The weeklong science and technology camp is held each year onsite at the rocket engine test facility.

  16. The HXT2 gene of Saccharomyces cerevisiae is required for high-affinity glucose transport.

    PubMed Central

    Kruckeberg, A L; Bisson, L F

    1990-01-01

    The HXT2 gene of the yeast Saccharomyces cerevisiae was identified on the basis of its ability to complement the defect in glucose transport of a snf3 mutant when present on the multicopy plasmid pSC2. Analysis of the DNA sequence of HXT2 revealed an open reading frame of 541 codons, capable of encoding a protein of Mr 59,840. The predicted protein displayed high sequence and structural homology to a large family of procaryotic and eucaryotic sugar transporters. These proteins have 12 highly hydrophobic regions that could form transmembrane domains; the spacing of these putative transmembrane domains is also highly conserved. Several amino acid motifs characteristic of this sugar transporter family are also present in the HXT2 protein. An hxt2 null mutant strain lacked a significant component of high-affinity glucose transport when under derepressing (low-glucose) conditions. However, the hxt2 null mutation did not incur a major growth defect on glucose-containing media. Genetic and biochemical analyses suggest that wild-type levels of high-affinity glucose transport require the products of both the HXT2 and SNF3 genes; these genes are not linked. Low-stringency Southern blot analysis revealed a number of other sequences that cross-hybridize with HXT2, suggesting that S. cerevisiae possesses a large family of sugar transporter genes. Images PMID:2233722

  17. Only high-affinity receptors for interleukin 2 mediate internalization of ligand

    SciTech Connect

    Weissman, A.M.; Harford, J.B.; Svetlik, P.B.; Leonard, W.L.; Depper, J.M.; Waldmann, T.A.; Greene, W.C.; Klausner, R.D.

    1986-03-01

    Interleukin 2 (IL-2) receptors are expressed on activated T cells and in select T-cell leukemias. Recently, it has been demonstrated that at least two classes of receptor for IL-2 exist with markedly different affinities for ligand. All known biological actions of IL-2 have been correlated with occupancy of high-affinity sites; the function of the low-affinity sites remains unknown. Receptor-mediated endocytosis is the primary means of internalization of cell-surface receptors and their ligands. The internalization of IL-2 bound to high- and low-affinity receptor sites was studied in a human T-cell lymphotrophic virus type 1 (HTLV-1)-infected human T-cell leukemia cell line and in a cloned murine cytotoxic T-cell line (CTLL). Internalization of IL-2 occurred only when bound to high-affinity sites. In addition, an anti-receptor antibody (anti-Tac), which binds equally well to high- and low-affinity sites, demonstrated no detectable internalization. The implications of these findings as they relate to IL-2 receptor structure and function are discussed.

  18. Discovery of Compounds that Positively Modulate the High Affinity Choline Transporter

    PubMed Central

    Choudhary, Parul; Armstrong, Emma J.; Jorgensen, Csilla C.; Piotrowski, Mary; Barthmes, Maria; Torella, Rubben; Johnston, Sarah E.; Maruyama, Yuya; Janiszewski, John S.; Storer, R. Ian; Skerratt, Sarah E.; Benn, Caroline L.

    2017-01-01

    Cholinergic hypofunction is associated with decreased attention and cognitive deficits in the central nervous system in addition to compromised motor function. Consequently, stimulation of cholinergic neurotransmission is a rational therapeutic approach for the potential treatment of a variety of neurological conditions. High affinity choline uptake (HACU) into acetylcholine (ACh)-synthesizing neurons is critically mediated by the sodium- and pH-dependent high-affinity choline transporter (CHT, encoded by the SLC5A7 gene). This transporter is comparatively well-characterized but otherwise unexplored as a potential drug target. We therefore sought to identify small molecules that would enable testing of the hypothesis that positive modulation of CHT mediated transport would enhance activity-dependent cholinergic signaling. We utilized existing and novel screening techniques for their ability to reveal both positive and negative modulation of CHT using literature tools. A screening campaign was initiated with a bespoke compound library comprising both the Pfizer Chemogenomic Library (CGL) of 2,753 molecules designed specifically to help enable the elucidation of new mechanisms in phenotypic screens and 887 compounds from a virtual screening campaign to select molecules with field-based similarities to reported negative and positive allosteric modulators. We identified a number of previously unknown active and structurally distinct molecules that could be used as tools to further explore CHT biology or as a starting point for further medicinal chemistry. PMID:28289374

  19. Acylated heptapeptide binds albumin with high affinity and application as tag furnishes long-acting peptides

    NASA Astrophysics Data System (ADS)

    Zorzi, Alessandro; Middendorp, Simon J.; Wilbs, Jonas; Deyle, Kaycie; Heinis, Christian

    2017-07-01

    The rapid renal clearance of peptides in vivo limits this attractive platform for the treatment of a broad range of diseases that require prolonged drug half-lives. An intriguing approach for extending peptide circulation times works through a `piggy-back' strategy in which peptides bind via a ligand to the long-lived serum protein albumin. In accordance with this strategy, we developed an easily synthesized albumin-binding ligand based on a peptide-fatty acid chimera that has a high affinity for human albumin (Kd=39 nM). This ligand prolongs the elimination half-life of cyclic peptides in rats 25-fold to over seven hours. Conjugation to a peptide factor XII inhibitor developed for anti-thrombotic therapy extends the half-life from 13 minutes to over five hours, inhibiting coagulation for eight hours in rabbits. This high-affinity albumin ligand could potentially extend the half-life of peptides in human to several days, substantially broadening the application range of peptides as therapeutics.

  20. High affinity binding site-mediated prevention of chemical absorption across the gastrointestinal tract.

    PubMed

    Rasmussen, M V; Barker, T T; Silbart, L K

    2001-12-15

    Preventing mucosal absorption of low-molecular weight compounds such as carcinogens, toxins and drugs could help prevent many diseases. To characterize the effects of dose and timing on high-affinity binding site mediated sequestration of specific chemical ligands in the gastrointestinal tract, avidin was perorally-administered to mice either prior to or mixed with 3H-biotin. Avidin enhanced fecal 3H-biotin excretion in a dose-dependent manner, consistent with the accepted mechanism of egg white-induced biotin deficiency syndrome. Avidin administration up to 4 h before 3H-biotin administration also enhanced fecal 3H-biotin excretion. Activated charcoal (AC) reduced 3H-biotin absorption when mixed with 3H-biotin before ingestion, but was ineffective when ingested prior to 3H-biotin. These studies suggest that ingestion of high-affinity protein binding sites can establish an absorptive barrier at the gastrointestinal mucosa to prevent the uptake of unwanted low molecular-weight chemicals.

  1. Rac recruits high-affinity integrin alphavbeta3 to lamellipodia in endothelial cell migration.

    PubMed

    Kiosses, W B; Shattil, S J; Pampori, N; Schwartz, M A

    2001-03-01

    Integrin alphavbeta3 has an important role in the proliferation, survival, invasion and migration of vascular endothelial cells. Like other integrins, alphavbeta3 can exist in different functional states with respect to ligand binding. These changes involve both affinity modulation, by which conformational changes in the integrin heterodimer govern affinity for individual extracellular matrix proteins, and avidity modulation, by which changes in lateral mobility and integrin clustering affect the binding of cells to multivalent matrices. Here we have used an engineered monoclonal antibody Fab (antigen-binding fragment) named WOW-1, which binds to activated integrins alphavbeta3 and alphavbeta5 from several species, to investigate the role of alphavbeta3 activation in endothelial cell behaviour. Because WOW-1 is monovalent, it is insensitive to changes in integrin clustering and therefore reports only changes in affinity. WOW-1 contains an RGD tract in its variable region and binds only to unoccupied, high-affinity integrins. By using WOW-1, we have identified the selective recruitment of high-affinity integrins as a mechanism by which lamellipodia promote formation of new adhesions at the leading edge in cell migration.

  2. A high affinity monoclonal antibody recognizing the light chain of human coagulating factor VII.

    PubMed

    Sarial, Sheila; Asadi, Farzad; Jeddi-Tehrani, Mahmood; Hadavi, Reza; Bayat, Ali Ahmad; Mahmoudian, Jafar; Taghizadeh-Jahed, Masoud; Shokri, Fazel; Rabbani, Hodjattallah

    2012-12-01

    Factor VII (FVII) is a serine protease-coagulating element responsible for the initiation of an extrinsic pathway of clot formation. Here we generated and characterized a high affinity monoclonal antibody that specifically recognizes human FVII. Recombinant human FVII (rh-FVII) was used for the production of a monoclonal antibody using BALB/c mice. The specificity of the antibody was determined by Western blot using plasma samples from human, mouse, sheep, goat, bovine, rabbit, and rat. Furthermore, the antibody was used to detect transiently expressed rh-FVII in BHK21 cell line using Western blot and sandwich ELISA. A mouse IgG1 (kappa chain) monoclonal antibody clone 1F1-B11 was produced against rh-FVII. The affinity constant (K(aff)) of the antibody was calculated to be 6.4×10(10) M(-1). The antibody could specifically recognize an epitope on the light chain of hFVII, with no reactivity with factor VII from several other animals. In addition, transiently expressed rh-FVII in BHK21 cells was recognized by 1F1-B11. The high affinity as well as the specificity of 1F1-B11 for hFVII will facilitate the affinity purification of hFVII and also production of FVII deficient plasma and minimizes the risk of bovine FVII contamination when fetal bovine serum-supplemented media are used for production and subsequent purification of rh-FVII.

  3. The AVR4 elicitor protein of Cladosporium fulvum binds to fungal components with high affinity.

    PubMed

    Westerink, Nienke; Roth, Ronelle; Van den Burg, Harrold A; De Wit, Pierre J G M; Joosten, Matthieu H A J

    2002-12-01

    The interaction between tomato and the fungal pathogen Cladosporium fulvum complies with the gene-for-gene system. Strains of C. fulvum that produce race-specific elicitor AVR4 induce a hypersensitive response, leading to resistance, in tomato plants that carry the Cf-4 resistance gene. The mechanism of AVR4 perception was examined by performing binding studies with 125I-AVR4 on microsomal membranes of tomato plants. We identified an AVR4 high-affinity binding site (KD = 0.05 nM) which exhibited all the characteristics expected for ligand-receptor interactions, such as saturability, reversibility, and specificity. Surprisingly, the AVR4 high-affinity binding site appeared to originate from fungi present on infected tomato plants rather than from the tomato plants themselves. Detailed analysis showed that this fungus-derived, AVR4-specific binding site is heat- and proteinase K-resistant. Affinity crosslinking demonstrated that AVR4 specifically binds to a component of approximately 75 kDa that is of fungal origin. Our data suggest that binding of AVR4 to a fungal component or components is related to the intrinsic virulence function of AVR4 for C. fulvum.

  4. Cationic polymer brush-modified cellulose nanocrystals for high-affinity virus binding.

    PubMed

    Rosilo, Henna; McKee, Jason R; Kontturi, Eero; Koho, Tiia; Hytönen, Vesa P; Ikkala, Olli; Kostiainen, Mauri A

    2014-10-21

    Surfaces capable of high-affinity binding of biomolecules are required in several biotechnological applications, such as purification, transfection, and sensing. Therein, the rod-shaped, colloidal cellulose nanocrystals (CNCs) are appealing due to their large surface area available for functionalization. In order to exploit electrostatic binding, their intrinsically anionic surfaces have to be cationized as biological supramolecules are predominantly anionic. Here we present a facile way to prepare cationic CNCs by surface-initiated atom-transfer radical polymerization of poly(N,N-dimethylaminoethyl methacrylate) and subsequent quaternization of the polymer pendant amino groups. The cationic polymer brush-modified CNCs maintained excellent dispersibility and colloidal stability in water and showed a ζ-potential of +38 mV. Dynamic light scattering and electron microscopy showed that the modified CNCs electrostatically bind cowpea chlorotic mottle virus and norovirus-like particles with high affinity. Addition of only a few weight percent of the modified CNCs in water dispersions sufficed to fully bind the virus capsids to form micrometer-sized assemblies. This enabled the concentration and extraction of the virus particles from solution by low-speed centrifugation. These results show the feasibility of the modified CNCs in virus binding and concentrating, and pave the way for their use as transduction enhancers for viral delivery applications.

  5. Purification of high affinity benzodiazepine receptor binding site fragments from rat brain

    SciTech Connect

    Klotz, K.L.

    1984-01-01

    In central nervous system benzodiazepine recognition sites occur on neuronal cell surfaces as one member of a multireceptor complex, including recognition sites for benzodiazepines, gamma aminobutyric acid (GABA), barbiturates and a chloride ionophore. During photoaffinity labelling, the benzodiazepine agonist, /sup 3/H-flunitrazepam, is irreversibly bound to central benzodiazepine high affinity recognition sites in the presence of ultraviolet light. In these studies a /sup 3/H-flunitrazepam radiolabel was used to track the isolation and purification of high affinity agonist binding site fragments from membrane-bound benzodiazepine receptor in rat brain. The authors present a method for limited proteolysis of /sup 3/H-flunitrazepam photoaffinity labeled rat brain membranes, generating photolabeled benzodiazepine receptor fragments containing the agonist binding site. Using trypsin chymotrypsin A/sub 4/, or a combination of these two proteases, they have demonstrated the extent and time course for partial digestion of benzodiazepine receptor, yielding photolabeled receptor binding site fragments. These photolabeled receptor fragments have been further purified on the basis of size, using ultrafiltration, gel permeation chromatography, and sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) as well as on the basis of hydrophobicity, using a high performance liquid chromatography (HPLC) precolumn, several HPLC elution schemes, and two different HPLC column types. Using these procedures, they have purified three photolabeled benzodiazepine receptor fragments containing the agonist binding site which appear to have a molecular weight of less than 2000 daltons each.

  6. Substrate-induced internalization of the high-affinity choline transporter.

    PubMed

    Okuda, Takashi; Konishi, Asami; Misawa, Hidemi; Haga, Tatsuya

    2011-10-19

    Cholinergic neurons are endowed with a high-affinity choline uptake system for efficient synthesis of acetylcholine at the presynaptic terminals. The high-affinity choline transporter CHT1 is responsible for choline uptake, the rate-limiting step in acetylcholine synthesis. However, endogenous physiological factors that affect CHT1 expression or function and consequently regulate the acetylcholine synthesis rate are essentially unknown. Here we demonstrate that extracellular substrate decreases the cell-surface expression of CHT1 in rat brain synaptosomes, primary cultures from the basal forebrain, and mammalian cell lines transfected with CHT1. Extracellular choline rapidly decreases cell-surface CHT1 expression by accelerating its internalization, a process that is mediated by a dynamin-dependent endocytosis pathway in HEK293 cells. Specific inhibitor hemicholinium-3 decreases the constitutive internalization rate and thereby increases cell-surface CHT1 expression. We also demonstrate that the constitutive internalization of CHT1 depends on extracellular pH in cultured cells. Our results collectively suggest that the internalization of CHT1 is induced by extracellular substrate, providing a novel feedback mechanism for the regulation of acetylcholine synthesis at the cholinergic presynaptic terminals.

  7. Ontogeny of high-affinity GABA and benzodiazepine receptors in the rat cerebellum: an autoradiographic study.

    PubMed

    Palacios, J M; Kuhar, M J

    1981-11-01

    High-affinity GABA and benzodiazepine receptors were localized by light microscopic autoradiography in the developing rat cerebellum. [3H]muscimol was used for the labeling of GABA receptors and [3H]flunitrazepam for benzodiazepine receptors. Very low densities of GABA sites were found during the first postnatal week. GABA receptors start increasing linearly at the end of the second week up to adult levels around the fourth postnatal week. The increase in receptor density is concentrated in the developing granule cell layer. Benzodiazepine receptors are present at birth and increases in the density of receptors were observed already during the first postnatal week. Receptor concentrations reached adult values around the third to fourth weeks postnatally. The increase in benzodiazepine receptors in concentrated in the growing molecular layer with little change in the granule cell layer. The immature cell of the external granule layer were characterized by the absence of receptor sites. At least partial association of high-affinity GABA receptors with granule cells and benzodiazepine receptor with Purkinje cell dendrites is suggested by these developmental profiles.

  8. The human organic cation transporter OCT1 mediates high affinity uptake of the anticancer drug daunorubicin

    PubMed Central

    Andreev, Emil; Brosseau, Nicolas; Carmona, Euridice; Mes-Masson, Anne-Marie; Ramotar, Dindial

    2016-01-01

    Anthracyclines such as daunorubicin are anticancer agents that are transported into cells, and exert cytotoxicity by blocking DNA metabolism. Although there is evidence for active uptake of anthracyclines into cells, the specific transporter involved in this process has not been identified. Using the high-grade serous ovarian cancer cell line TOV2223G, we show that OCT1 mediated the high affinity (Km ~ 5 μM) uptake of daunorubicin into the cells, and that micromolar amounts of choline completely abolished the drug entry. OCT1 downregulation by shRNA impaired daunorubicin uptake into the TOV2223G cells, and these cells were significantly more resistant to the drug in comparison to the control shRNA. Transfection of HEK293T cells, which accommodated the ectopic expression of OCT1, with a plasmid expressing OCT1-EYFP showed that the transporter was predominantly localized to the plasma membrane. These transfected cells exhibited an increase in the uptake of daunorubicin in comparison to control cells transfected with an empty EYFP vector. Furthermore, a variant of OCT1, OCT1-D474C-EYFP, failed to enhance daunorubicin uptake. This is the first report demonstrating that human OCT1 is involved in the high affinity transport of anthracyclines. We postulate that OCT1 defects may contribute to the resistance of cancer cells treated with anthracyclines. PMID:26861753

  9. Competition STD NMR for the detection of high-affinity ligands and NMR-based screening.

    PubMed

    Wang, Yu-Sen; Liu, Dingjiang; Wyss, Daniel F

    2004-06-01

    The reported competition STD NMR method combines saturation transfer difference (STD) NMR with competition binding experiments to allow the detection of high-affinity ligands that undergo slow chemical exchange on the NMR time-scale. With this technique, the presence of a competing high-affinity ligand in the compound mixture can be detected by the disappearance or reduction of the STD signals of a low-affinity indicator ligand. This is demonstrated on a BACE1 (beta-site amyloid precursor protein cleaving enzyme 1) protein-inhibitor system. This method can also be used to derive an approximate value, or a lower limit, for the dissociation constant of the potential ligand based on the reduction of the signal intensity of the STD indicator, which is illustrated on an HSA (human serum albumin) model system. This leads to important applications of the competition STD NMR method for lead discovery: it can be used (i) for compound library screening against a broad range of drug targets to identify both high- and low-affinity ligands and (ii) to rank order analogs rapidly and derive structure-activity relationships, which are used to optimize these NMR hits into viable drug leads. Copyright 2004 John Wiley & Sons, Ltd.

  10. Selecting highly affine and well-expressed TCRs for gene therapy of melanoma.

    PubMed

    Jorritsma, Annelies; Gomez-Eerland, Raquel; Dokter, Maarten; van de Kasteele, Willeke; Zoet, Yvonne M; Doxiadis, Ilias I N; Rufer, Nathalie; Romero, Pedro; Morgan, Richard A; Schumacher, Ton N M; Haanen, John B A G

    2007-11-15

    A recent phase 1 trial has demonstrated that the generation of tumor-reactive T lymphocytes by transfer of specific T-cell receptor (TCR) genes into autologous lymphocytes is feasible. However, compared with results obtained by infusion of tumor-infiltrating lymphocytes, the response rate observed in this first TCR gene therapy trial is low. One strategy that is likely to enhance the success rate of TCR gene therapy is the use of tumor-reactive TCRs with a higher capacity for tumor cell recognition. We therefore sought to develop standardized procedures for the selection of well-expressed, high-affinity, and safe human TCRs. Here we show that TCR surface expression can be improved by modification of TCR alpha and beta sequences and that such improvement has a marked effect on the in vivo function of TCR gene-modified T cells. From a panel of human, melanoma-reactive TCRs we subsequently selected the TCR with the highest affinity. Furthermore, a generally applicable assay was used to assess the lack of alloreactivity of this TCR against a large series of common human leukocyte antigen alleles. The procedures described in this study should be of general value for the selection of well- and stably expressed, high-affinity, and safe human TCRs for subsequent clinical testing.

  11. Pharmacological characterization of a high-affinity p-tyramine transporter in rat brain synaptosomes

    PubMed Central

    Berry, Mark D.; Hart, Shannon; Pryor, Anthony R.; Hunter, Samantha; Gardiner, Danielle

    2016-01-01

    p-Tyramine is an archetypal member of the endogenous family of monoamines known as trace amines, and is one of the endogenous agonists for trace amine-associated receptor (TAAR)1. While much work has focused on the function of TAAR1, very little is known about the regulation of the endogenous agonists. We have previously reported that p-tyramine readily crosses lipid bilayers and that its release from synaptosomes is non-exocytotic. Such release, however, showed characteristics of modification by one or more transporters. Here we provide the first characterization of such a transporter. Using frontal cortical and striatal synaptosomes we show that p-tyramine passage across synaptosome membranes is not modified by selective inhibition of either the dopamine, noradrenaline or 5-HT transporters. In contrast, inhibition of uptake-2 transporters significantly slowed p-tyramine re-uptake. Using inhibitors of varying selectivity, we identify Organic Cation Transporter 2 (OCT2; SLC22A2) as mediating high affinity uptake of p-tyramine at physiologically relevant concentrations. Further, we confirm the presence of OCT2 protein in synaptosomes. These results provide the first identification of a high affinity neuronal transporter for p-tyramine, and also confirm the recently described localization of OCT2 in pre-synaptic terminals. PMID:27901065

  12. AtKUP1: an Arabidopsis gene encoding high-affinity potassium transport activity.

    PubMed Central

    Kim, E J; Kwak, J M; Uozumi, N; Schroeder, J I

    1998-01-01

    Because plants grow under many different types of soil and environmental conditions, we investigated the hypothesis that multiple pathways for K+ uptake exist in plants. We have identified a new family of potassium transporters from Arabidopsis by searching for homologous sequences among the expressed sequence tags of the GenBank database. The deduced amino acid sequences of AtKUP (for Arabidopsis thaliana K+ uptake transporter) cDNAs are highly homologous to the non-plant Kup and HAK1 potassium transporters from Escherichia coli and Schwanniomyces occidentalis, respectively. Interestingly, AtKUP1 and AtKUP2 are able to complement the potassium transport deficiency of an E. coli triple mutant. In addition, transgenic Arabidopsis suspension cells overexpressing AtKUP1 showed increased Rb+ uptake at micromolar concentrations with an apparent K(m) of approximately 22 microM, indicating that AtKUP1 encodes a high-affinity potassium uptake activity in vivo. A small, low-affinity Rb+ uptake component was also detected in AtKUP1-expressing cells. RNA gel blot analysis showed that the various members of the AtKUP family have distinct patterns of expression, with AtKUP3 transcript levels being strongly induced by K+ starvation. It is proposed that plants contain multiple potassium transporters for high-affinity uptake and that the AtKUP family may provide important components of high- and low-affinity K+ nutrition and uptake into various plant cell types. PMID:9477571

  13. High-affinity interactions of ligands at recombinant Guinea pig 5HT7 receptors

    NASA Astrophysics Data System (ADS)

    Wilcox, R. E.; Ragan, J. E.; Pearlman, R. S.; Brusniak, M. Y.-. K.; Eglen, R. M.; Bonhaus, D. W.; Tenner, T. E., Jr.; Miller, J. D.

    2001-10-01

    The serotonin 5HT7 receptor has been implicated in numerous physiological and pathological processes from circadian rhythms [1] to depression and schizophrenia. Clonal cell lines heterologously expressing recombinant receptors offer good models for understanding drug-receptor interactions and development of quantitative structure-activity relationships (QSAR). Comparative Molecular Field Analysis (CoMFA) is an important modern QSAR procedure that relates the steric and electrostatic fields of a set of aligned compounds to affinity. Here, we utilized CoMFA to predict affinity for a number of high-affinity ligands at the recombinant guinea pig 5HT7 receptor. Using R-lisuride as the template, a final CoMFA model was derived using procedures similar to those of our recent papers [2, 3, 4] The final cross-validated model accounted for >85% of the variance in the compound affinity data, while the final non-cross validated model accounted for >99% of the variance. Model evaluation was done using cross-validation methods with groups of 5 ligands. Twenty cross-validation runs yielded an average predictive r2(q2) of 0.779 ± 0.015 (range: 0.669-0.867). Furthermore, 3D-chemical database search queries derived from the model yielded hit lists of promising agents with high structural similarity to the template. Together, these results suggest a possible basis for high-affinity drug action at 5HT7 receptors.

  14. Mechanism of high affinity inhibition of the human urate transporter URAT1

    PubMed Central

    Tan, Philip K.; Ostertag, Traci M.; Miner, Jeffrey N.

    2016-01-01

    Gout is caused by elevated serum urate levels, which can be treated using inhibitors of the uric acid transporter, URAT1. We exploited affinity differences between the human and rat transporters to map inhibitor binding sites in URAT1. Human-rat transporter chimeras revealed that human URAT1 serine-35, phenylalanine-365 and isoleucine-481 are necessary and sufficient to provide up to a 100-fold increase in affinity for inhibitors. Moreover, serine-35 and phenylalanine-365 are important for high-affinity interaction with the substrate urate. A novel URAT1 binding assay provides support for direct interaction with these amino acids; thus, current clinically important URAT1 inhibitors likely bind the same site in URAT1. A structural model suggests that these three URAT1 residues are in close proximity potentially projecting within the channel. Our results indicate that amino acids from several transmembrane segments functionally cooperate to form a high-affinity URAT1 inhibitor binding site that, when occupied, prevents substrate interactions. PMID:27713539

  15. Characterization of the Saccharomyces cerevisiae high affinity copper transporter Ctr3.

    PubMed

    Pena, M M; Puig, S; Thiele, D J

    2000-10-27

    Copper is an essential nutrient required for the activity of a number of enzymes with diverse biological roles. In the bakers' yeast Saccharomyces cerevisiae, copper is transported into cells by two high affinity copper transport proteins, Ctr1 and Ctr3. Although Ctr1 and Ctr3 are functionally redundant, they bear little homology at the amino acid sequence level. In this report, we characterize Ctr3 with respect to its localization, assembly, and post-transcriptional regulation. Ctr3 is an integral membrane protein that assembles as a trimer to form a competent copper uptake permease at the plasma membrane. Whereas the CTR1 and CTR3 genes are similarly regulated at the transcriptional level in response to copper, post-transcriptional regulation of these proteins is distinct. Unlike Ctr1, the Ctr3 transporter is neither regulated at the level of protein degradation nor endocytosis as a function of elevated copper levels. Our studies suggest that Ctr3 constitutes a fundamental module found in all eukaryotic high affinity copper transporters to date, which is sufficient for copper uptake but lacks elements for post-transcriptional regulation by copper.

  16. High affinity binding of (/sup 3/H)cocaine to rat liver microsomes

    SciTech Connect

    El-Maghrabi, E.A.; Calligaro, D.O.; Eldefrawi, M.E.

    1988-01-01

    )/sup 3/H)cocaine bound reversible, with high affinity and stereospecificity to rat liver microsomes. Little binding was detected in the lysosomal, mitochondrial and nuclear fractions. The binding kinetics were slow and the kinetically calculated K/sub D/ was 2 nM. Induction of mixed function oxidases by phenobarbital did not produce significant change in (/sup 3/H)cocaine binding. On the other hand, chronic administration of cocaine reduced (/sup 3/H)cocaine binding drastically. Neither treatment affected the affinity of the liver binding protein for cocaine. Microsomes from mouse and human livers had less cocaine-binding protein and lower affinity for cocaine than those from rat liver. Binding of (/sup 3/H)cocaine to rat liver microsomes was insensitive to monovalent cations and > 10 fold less sensitive to biogenic amines than the cocaine receptor in rat striatum. However, the liver protein had higher affinity for cocaine and metabolites except for norcocaine. Amine uptake inhibitors displaced (/sup 3/H)cocaine binding to liver with a different rank order of potency than their displacement of (/sup 3/H)cocaine binding to striatum. This high affinity (/sup 3/H)cocaine binding protein in liver is not likely to be monooxygenase, but may have a role in cocaine-induced hepatotoxicity

  17. Humanization of high-affinity antibodies targeting glypican-3 in hepatocellular carcinoma

    PubMed Central

    Zhang, Yi-Fan; Ho, Mitchell

    2016-01-01

    Glypican-3 (GPC3) is a cell-surface heparan sulfate proteoglycan highly expressed in hepatocellular carcinoma (HCC). We have generated a group of high-affinity mouse monoclonal antibodies targeting GPC3. Here, we report the humanization and testing of these antibodies for clinical development. We compared the affinity and cytotoxicity of recombinant immunotoxins containing mouse single-chain variable regions fused with a Pseudomonas toxin. To humanize the mouse Fvs, we grafted the combined KABAT/IMGT complementarity determining regions (CDR) into a human IgG germline framework. Interestingly, we found that the proline at position 41, a non-CDR residue in heavy chain variable regions (VH), is important for humanization of mouse antibodies. We also showed that two humanized anti-GPC3 antibodies (hYP7 and hYP9.1b) in the IgG format induced antibody-dependent cell-mediated cytotoxicity and complement-dependent-cytotoxicity in GPC3-positive cancer cells. The hYP7 antibody was tested and showed inhibition of HCC xenograft tumor growth in nude mice. This study successfully humanizes and validates high affinity anti-GPC3 antibodies and sets a foundation for future development of these antibodies in various clinical formats in the treatment of liver cancer. PMID:27667400

  18. Is zucchini a phosphodiesterase or a ribonuclease?

    PubMed

    Nureki, Osamu

    2014-01-01

    Zucchini (Zuc), a member of the phospholipase D (PLD) superfamily, is essential for the primary PIWI-interacting RNA (piRNA) biogenesis and the suppression of transposon expression, which are crucial for the genome integrity of germline cells. However, it has been ambiguous whether Zuc acts as a phosphodiesterase to produce phosphatidic acid (PA), the lipid signaling molecule, or as a nuclease. The recent three papers describing the crystal structures and functional analyses of fly and mouse Zuc proteins have elucidated that Zuc is a PLD family single-strand ribonuclease, not a phosphodiesterase, and functions in the maturation of primary piRNAs. This review will discuss in detail how the crystal structures clearly predict the function of Zuc, which is subsequently demonstrated by biochemical analysis to conclude the previous controversial discussion on the real function of Zuc.

  19. A molecular recognizing system of serotonin in rat fetal axonal growth cones: uptake and high affinity binding.

    PubMed

    Mercado, R; Hernández, J

    1992-09-18

    Axonal growth cone particles (AGCP) isolated from prenatal and postnatal rat brain had different high-affinity 5-HT uptake characteristics. In postnatal AGCP the uptake behaves as in the adult rat brain, while in the prenatal AGCP the uptake characteristics seem to be in a transitional stage. Also in prenatal AGCP we observed specific, high-affinity 5-HT binding sites. These results support the idea of an important role for 5-HT during axogenesis.

  20. MRP4 and CFTR in the regulation of cAMP and β-adrenergic contraction in cardiac myocytes.

    PubMed

    Sellers, Zachary M; Naren, Anjaparavanda P; Xiang, Yang; Best, Philip M

    2012-04-15

    Spatiotemporal regulation of cAMP in cardiac myocytes is integral to regulating the diverse functions downstream of β-adrenergic stimulation. The activities of cAMP phosphodiesterases modulate critical and well-studied cellular processes. Recently, in epithelial and smooth muscle cells, it was found that the multi-drug resistant protein 4 (MRP4) acts as a cAMP efflux pump to regulate intracellular cAMP levels and alter effector function, including activation of the cAMP-stimulated Cl(-) channel, CFTR (cystic fibrosis transmembrane conductance regulator). In the current study we investigated the potential role of MRP4 in regulating intracellular cAMP and β-adrenergic stimulated contraction rate in cardiac myocytes. Cultured neonatal ventricular myocytes were used for all experiments. In addition to wildtype mice, β(1)-, β(2)-, and β(1)/β(2)-adrenoceptor, and CFTR knockout mice were used. MRP4 expression was probed via Western blot, intracellular cAMP was measured by fluorescence resonance energy transfer, while the functional role of MRP4 was assayed via monitoring of isoproterenol-stimulated contraction rate. We found that MRP4 is expressed in mouse neonatal ventricular myocytes. A pharmacological inhibitor of MRP4, MK571, potentiated submaximal isoproterenol-stimulated cAMP accumulation and cardiomyocyte contraction rate via β(1)-adrenoceptors. CFTR expression was critical for submaximal isoproterenol-stimulated contraction rate. Interestingly, MRP4-dependent changes in contraction rate were CFTR-dependent, however, PDE4-dependent potentiation of contraction rate was CFTR-independent. We have shown, for the first time, a role for MRP4 in the regulation of cAMP in cardiac myocytes and involvement of CFTR in β-adrenergic stimulated contraction. Together with phosphodiesterases, MRP4 must be considered when examining cAMP regulation in cardiac myocytes. Copyright © 2012 Elsevier B.V. All rights reserved.

  1. MRP4 and CFTR in the regulation of cAMP and β-adrenergic contraction in cardiac myocytes

    PubMed Central

    Sellers, Zachary M.; Naren, Anjaparavanda P.; Xiang, Yang; Best, Philip M.

    2012-01-01

    Spatiotemporal regulation of cAMP in cardiac myocytes is integral to regulating the diverse functions downstream of β-adrenergic stimulation. The activities of cAMP phosphodiesterases modulate critical and well-studied cellular processes. Recently, in epithelial and smooth muscle cells, it was found that the multi-drug resistant protein 4 (MRP4) acts as a cAMP efflux pump to regulate intracellular cAMP levels and alter effector function, including activation of the cAMP-stimulated Cl− channel, CFTR (cystic fibrosis transmembrane conductance regulator). In the current study we investigated the potential role of MRP4 in regulating intracellular cAMP and β-adrenergic stimulated contraction rate in cardiac myocytes. Cultured neonatal ventricular myocytes were used for all experiments. In addition to wildtype mice, β1-, β2-, β1/β2-adrenoceptor, and CFTR knockout mice were used. MRP4 expression was probed via Western blot, intracellular cAMP was measured by fluorescence resonance energy transfer, while the functional role of MRP4 was assayed via monitoring of isoproterenol-stimulated contraction rate. We found that MRP4 is expressed in mouse neonatal ventricular myocytes. A pharmacological inhibitor of MRP4, MK571, potentiated submaximal isoproterenol-stimulated cAMP accumulation and cardiomyocyte contraction rate via β1-adrenoceptors. CFTR expression was critical for submaximal isoproterenol-stimulated contraction rate. Interestingly, MRP4-dependent changes in contraction rate were CFTR-dependent, however, PDE4-dependent potentiation of contraction rate was CFTR-independent. We have shown, for the first time, a role for MRP4 in the regulation of cAMP in cardiac myocytes and involvement of CFTR in β-adrenergic stimulated contraction. Together with phosphodiesterases, MRP4 must be considered when examining cAMP regulation in cardiac myocytes. PMID:22381067

  2. B cell differentiation factor-induced B cell maturation: regulation via reduction in cAMP.

    PubMed

    Huang, R; Cioffi, J; Berg, K; London, R; Cidon, M; Maayani, S; Mayer, L

    1995-04-15

    We have previously described a novel human B cell differentiation factor (BCDF), 446-BCDF, that is distinct biochemically and functionally from other cytokines. Since signal transduction pathways involved in human B cell differentiation have been incompletely studied and are poorly understood, we assessed the effects of 446-BCDF on various intracellular second messenger systems. After exposure of B cells to 446-BCDF, intracellular cAMP concentration started to decrease at 5 min and was significantly lower at 30 min and reached the lowest level at 4 hr. In most cases, cAMP concentrations returned toward baseline by 24 hr. A cAMP analog (dibutyryl cAMP), a stimulator of adenyl cyclase (forskolin), and phosphodiesterase inhibitors (aminophylline and IBMX) which inhibited the 446-BCDF-induced decrease in intracellular cAMP, inhibited 446-BCDF-induced B cell differentiation, suggesting that the fall in intracellular cAMP was a critical event in this process. To understand the mechanism involved in the reduction of cAMP, B cells were treated with pertussis toxin (PTX), a Gi protein inhibitor. Pertussis toxin blocked 446-BCDF-induced B cell differentiation as well, suggesting that 446-BCDF may function by stimulation of a Gi-linked receptor resulting in the inhibition of adenylate cyclase with a consequent reduction in cAMP. Other cytokines known to promote Ig secretion (IL2 and IL6) also caused a reduction in cAMP, suggesting that this pathway may be generally important in B cell differentiation. Taken together, these data suggest that at least one pathway of terminal maturation in B cells may involve the reduction of intracellular cAMP.

  3. Putative M2 muscarinic receptors of rat heart have high affinity for organophosphorus anticholinesterases

    SciTech Connect

    Silveira, C.L.; Eldefrawi, A.T.; Eldefrawi, M.E. )

    1990-05-01

    The M2 subtype of muscarinic receptor is predominant in heart, and such receptors were reported to be located in muscles as well as in presynaptic cholinergic and adrenergic nerve terminals. Muscarinic receptors of rat heart were identified by the high affinity binding of the agonist (+)-(3H)cis-methyldioxolane ((3H)CD), which has been used to label a high affinity population of M2 receptors. A single population of sites was detected and (3H)CD binding was sensitive to the M2 antagonist himbacine but much less so to pirenzepine, the M1 antagonist. These cardiac receptors had different sensitivities to NiCl2 and N-ethylmaleimide from brain muscarinic receptors, that were also labeled with (3H)CD and considered to be of the M2 subtype. Up to 70% of the (3H)CD-labeled cardiac receptors had high affinities for several organophosphate (OP) anticholinesterases. (3H)CD binding was inhibited by the nerve agents soman, VX, sarin, and tabun, with K0.5 values of 0.8, 2, 20, and 50 nM, respectively. It was also inhibited by echothiophate and paraoxon with K0.5 values of 100 and 300 nM, respectively. The apparent competitive nature of inhibition of (3H)CD binding by both sarin and paraoxon suggests that the OPs bind to the acetylcholine binding site of the muscarinic receptor. Other OP insecticides had lower potencies, inhibiting less than 50% of 5 nM (3H)CD binding by 1 microM of EPN, coumaphos, dioxathion, dichlorvos, or chlorpyriphos. There was poor correlation between the potencies of the OPs in reversibly inhibiting (3H)CD binding, and their anticholinesterase activities and toxicities. Acetylcholinesterases are the primary targets for these OP compounds because of the irreversible nature of their inhibition, which results in building of acetylcholine concentrations that activate muscarinic and nicotinic receptors and desensitize them, thereby inhibiting respiration.

  4. Role of phosphodiesterase and adenylate cyclase isozymes in murine colonic glucagon-like peptide 1 secreting cells

    PubMed Central

    Friedlander, Ronn S; Moss, Catherine E; Mace, Jessica; Parker, Helen E; Tolhurst, Gwen; Habib, Abdella M; Wachten, Sebastian; Cooper, Dermot M; Gribble, Fiona M; Reimann, Frank

    2011-01-01

    BACKGROUND AND PURPOSE Glucagon-like peptide-1 (GLP-1) is secreted from enteroendocrine L-cells after food intake. Increasing GLP-1 signalling either through inhibition of the GLP-1 degrading enzyme dipeptidyl-peptidase IV or injection of GLP-1-mimetics has recently been successfully introduced for the treatment of type 2 diabetes. Boosting secretion from the L-cell has so far not been exploited, due to our incomplete understanding of L-cell physiology. Elevation of cyclic adenosine monophosphate (cAMP) has been shown to be a strong stimulus for GLP-1 secretion and here we investigate the activities of adenylate cyclase (AC) and phosphodiesterase (PDE) isozymes likely to shape cAMP responses in L-cells. EXPERIMENTAL APPROACH Expression of AC and PDE isoforms was quantified by RT-PCR. Single cell responses to stimulation or inhibition of AC and PDE isoforms were monitored with real-time cAMP probes. GLP-1 secretion was assessed by elisa. KEY RESULTS Quantitative PCR identified expression of protein kinase C- and Ca2+-activated ACs, corresponding with phorbolester and cytosolic Ca2+-stimulated cAMP elevation. Inhibition of PDE2, 3 and 4 were found to stimulate GLP-1 secretion from murine L-cells in primary culture. This corresponded with cAMP elevations monitored with a plasma membrane targeted cAMP probe. Inhibition of PDE3 but not PDE2 was further shown to prevent GLP-1 secretion in response to guanylin, a peptide secreted into the gut lumen, which had not previously been implicated in L-cell secretion. CONCLUSIONS AND IMPLICATIONS Our results reveal several mechanisms shaping cAMP responses in GLP-1 secreting cells, with some of the molecular components specifically expressed in L-cells when compared with their epithelial neighbours, thus opening new strategies for targeting these cells therapeutically. PMID:21054345

  5. Intrinsic sex-specific differences in microvascular endothelial cell phosphodiesterases.

    PubMed

    Wang, Jianjie; Bingaman, Susan; Huxley, Virginia H

    2010-04-01

    The importance of gonadal hormones in the regulation of vascular function has been documented. An alternate and essential contribution of the sex chromosomes to sex differences in vascular function is poorly understood. We reported previously sex differences in microvessel permeability (P(s)) responses to adenosine that were mediated by the cAMP signaling pathway (Wang J, PhD thesis, 2005; Wang J and Huxley V, Proceedings of the VIII World Congress of Microcirculation, 2007; Wang J and Huxley VH, Am J Physiol Heart Circ Physiol 291: H3094-H3105, 2006). The two cyclic nucleotides, cAMP and cGMP, central to the regulation of vascular barrier integrity, are hydrolyzed by phosphodiesterases (PDE). We hypothesized that microvascular endothelial cells (EC) would retain intrinsic and inheritable sexually dimorphic genes with respect to the PDEs modulating EC barrier function. Primary cultured microvascular EC from skeletal muscles isolated from male and female rats, respectively, were used. SRY (a sex-determining region Y gene) mRNA expression was observed exclusively in male, not female, cells. The predominant isoform among PDE1-5, present in both XY and XX EC, was PDE4. Expression mRNA levels of PDE1A (male > female) and PDE3B (male < female) were sex dependent; PDE2A, PDE4D, and PDE5A were sex independent. Barrier function, P(s), was determined from measures of albumin flux across confluent primary cultured microvessel XY and XX EC monolayers. Consistent with intact in situ microvessels, basal monolayer P(s) did not differ between XY (1.7 +/- 0.2 x 10(-6) cm/s; n = 8) and XX (1.8 +/- 0.1 x 10(-6) cm/s; n = 10) EC. Cilostazol, a PDE3 inhibitor, reduced (11%, P < 0.05) P(s) in XX, not XY, cells. These findings demonstrate the presence and maintenance of intrinsic sex-related differences in gene expression and cellular phenotype by microvascular EC in a gonadal-hormone-free environment. Furthermore, intrinsic cell-sex likely contributes significantly to sexual dimorphism

  6. Intrinsic sex-specific differences in microvascular endothelial cell phosphodiesterases

    PubMed Central

    Bingaman, Susan; Huxley, Virginia H.

    2010-01-01

    The importance of gonadal hormones in the regulation of vascular function has been documented. An alternate and essential contribution of the sex chromosomes to sex differences in vascular function is poorly understood. We reported previously sex differences in microvessel permeability (Ps) responses to adenosine that were mediated by the cAMP signaling pathway (Wang J, PhD thesis, 2005; Wang J and Huxley V, Proceedings of the VIII World Congress of Microcirculation, 2007; Wang J and Huxley VH, Am J Physiol Heart Circ Physiol 291: H3094–H3105, 2006). The two cyclic nucleotides, cAMP and cGMP, central to the regulation of vascular barrier integrity, are hydrolyzed by phosphodiesterases (PDE). We hypothesized that microvascular endothelial cells (EC) would retain intrinsic and inheritable sexually dimorphic genes with respect to the PDEs modulating EC barrier function. Primary cultured microvascular EC from skeletal muscles isolated from male and female rats, respectively, were used. SRY (a sex-determining region Y gene) mRNA expression was observed exclusively in male, not female, cells. The predominant isoform among PDE1–5, present in both XY and XX EC, was PDE4. Expression mRNA levels of PDE1A (male > female) and PDE3B (male < female) were sex dependent; PDE2A, PDE4D, and PDE5A were sex independent. Barrier function, Ps, was determined from measures of albumin flux across confluent primary cultured microvessel XY and XX EC monolayers. Consistent with intact in situ microvessels, basal monolayer Ps did not differ between XY (1.7 ± 0.2 × 10−6 cm/s; n = 8) and XX (1.8 ± 0.1 × 10−6 cm/s; n = 10) EC. Cilostazol, a PDE3 inhibitor, reduced (11%, P < 0.05) Ps in XX, not XY, cells. These findings demonstrate the presence and maintenance of intrinsic sex-related differences in gene expression and cellular phenotype by microvascular EC in a gonadal-hormone-free environment. Furthermore, intrinsic cell-sex likely contributes significantly to sexual dimorphism in

  7. Cyclic nucleotide phosphodiesterases (PDEs): coincidence detectors acting to spatially and temporally integrate cyclic nucleotide and non-cyclic nucleotide signals.

    PubMed

    Maurice, Donald H; Wilson, Lindsay S; Rampersad, Sarah N; Hubert, Fabien; Truong, Tammy; Kaczmarek, Milosz; Brzezinska, Paulina; Freitag, Silja I; Umana, M Bibiana; Wudwud, Alie

    2014-04-01

    The cyclic nucleotide second messengers cAMP and cGMP each affect virtually all cellular processes. Although these hydrophilic small molecules readily diffuse throughout cells, it is remarkable that their ability to activate their multiple intracellular effectors is spatially and temporally selective. Studies have identified a critical role for compartmentation of the enzymes which hydrolyse and metabolically inactivate these second messengers, the PDEs (cyclic nucleotide phosphodiesterases), in this specificity. In the present article, we describe several examples from our work in which compartmentation of selected cAMP- or cGMP-hydrolysing PDEs co-ordinate selective activation of cyclic nucleotide effectors, and, as a result, selectively affect cellular functions. It is our belief that therapeutic strategies aimed at targeting PDEs within these compartments will allow greater selectivity than those directed at inhibiting these enzymes throughout the cells.

  8. Pinealectomy increases ouabain high-affinity binding sites and dissociation constant in rat cerebral cortex.

    PubMed

    Acuña Castroviejo, D; del Aguila, C M; Fernández, B; Gomar, M D; Castillo, J L

    1991-06-24

    The effect of the pineal gland on the ouabain high-affinity binding sites (Kd = 3.1 +/- 0.4 nM, Bmax = 246.4 +/- 18.4 fmol/mg protein) in rat cerebral cortex was studied. Pinealectomy increased Bmax (940.7 +/- 42.8 fmol/mg protein) and Kd (7.6 +/- 1.5 nM) while melatonin injection (100 micrograms/kg b.wt.) counteracted these effects, restoring kinetic parameters (Kd = 1.9 +/- 0.05 nM; Bmax = 262.2 +/- 29.6 fmol/mg prot) to control values. Melatonin activity on ouabain binding in vitro did not depend upon a direct effect on the binding sites themselves. However, in competition experiments, melatonin increased binding affinity of ouabain as shown by the decreased IC50 values.

  9. Quantitation of the P2Y1 Receptor with a High Affinity Radiolabeled Antagonist

    PubMed Central

    Waldo, Gary L.; Corbitt, James; Boyer, José L.; Ravi, Gnana; Kim, Hak Sung; Ji, Xiao-Duo; Lacy, James; Jacobson, Kenneth A.; Harden, T. Kendall

    2015-01-01

    2-Chloro-N6-methyl-(N)-methanocarba-2′-deoxyadenosine-3′,5′-bisphosphate (MRS2279) was developed previously as a selective high-affinity, non-nucleotide P2Y1 receptor (P2Y1-R) antagonist (J Med Chem 43:829–842, 2002; Br J Pharmacol 135:2004–2010, 2002). We have taken advantage of the N6-methyl substitution in the adenine base to incorporate [3H]methylamine into the synthesis of [3H]MRS2279 to high (89 Ci/mmol) specific radioactivity and have used this molecule as a radioligand for the P2Y1-R. [3H]MRS2279 bound to membranes from Sf9 insect cells expressing recombinant human P2Y1-R but not to membranes from wild-type Sf9 cells or Sf9 cells expressing high levels of recombinant P2Y2 or P2Y12 receptors. Equilibrium binding of [3H]MRS2279 to P2Y1-R expressed in Sf9 membranes was with a high affinity (Kd = 8 nM) essentially identical to the apparent affinity of MRS2279 determined previously in studies of P2Y1-R–promoted inositol phosphate accumulation or platelet aggregation. A kinetically derived Kd calculated from independent determinations of the rate constants of association (7.15 × 107 M−1 min−1) and dissociation (0.72 min−1) of [3H]MRS2279 also was in good agreement with the Kd derived from equilibrium binding studies. Competition binding assays with [3H]MRS2279 and P2Y1-R expressing Sf9 cell membranes revealed Ki values for the P2Y1-R antagonists MRS2279 (Ki = 13 nM), N6-methyl-2′-deoxyadenosine-3′,5′-bisphosphate (MRS2179; Ki = 84 nM), adenosine-3′, 5′-bisphosphate (Ki = 900 nM), and pyridoxal phosphate-6-azophenyl-2′,4′-disulfonic acid (Ki = 6 µM) that were in good agreement with antagonist activities of these molecules previously determined at the P2Y1-R in intact tissues. Moreover, [3H]MRS2279 also bound with high affinity (Kd = 4–8 nM) to Chinese hamster ovary (CHO) or 1321N1 human astrocytoma cells stably expressing the human P2Y1-R, but specific binding was not observed in wild-type CHO or 1321N1 cells. [3H]MRS2279 bound

  10. Practical strategies for the evaluation of high-affinity protein/nucleic acid interactions

    PubMed Central

    Altschuler, Sarah E.; Lewis, Karen A.; Wuttke, Deborah S.

    2014-01-01

    The quantitative evaluation of binding interactions between proteins and nucleic acids is highly sensitive to a variety of experimental conditions. Optimization of these conditions is critical for obtaining high quality, reproducible data, particularly in the context of very high affinity interactions. Here, we discuss the practical considerations involved in optimizing the apparent binding constant of an interaction as measured by two common quantitative assays, electrophoretic mobility shift assay and double-filter binding when measuring extremely tight protein/nucleic acid interactions with sub-nanomolar binding affinities. We include specific examples from two telomere end-binding protein systems, Schizo -saccharomyces pombe Pot1 and Saccharomyces cerevisiae Cdc13, to demonstrate potential experimental pitfalls and some useful strategies for optimization. PMID:25197549

  11. Glycan-based high-affinity ligands for toxins and pathogen receptors.

    PubMed

    Kulkarni, Ashish A; Weiss, Alison A; Iyer, Suri S

    2010-03-01

    Glycans decorate over 95% of the mammalian cell surface in the form of glycolipids and glycoproteins. Several toxins and pathogens bind to these glycans to enter the cells. Understanding the fundamentals of the complex interplay between microbial pathogens and their glycan receptors at the molecular level could lead to the development of novel therapeutics and diagnostics. Using Shiga toxin and influenza virus as examples, we describe the complex biological interface between host glycans and these infectious agents, and recent strategies to develop glycan-based high-affinity ligands. These molecules are expected to ultimately be incorporated into diagnostics and therapeutics, and can be used as probes to study important biological processes. Additionally, by focusing on the specific glycans that microbial pathogens target, we can begin to decipher the "glycocode" and how these glycans participate in normal and aberrant cellular communication.

  12. Functional transformations of bile acid transporters induced by high-affinity macromolecules

    PubMed Central

    Al-Hilal, Taslim A.; Chung, Seung Woo; Alam, Farzana; Park, Jooho; Lee, Kyung Eun; Jeon, Hyesung; Kim, Kwangmeyung; Kwon, Ick Chan; Kim, In-San; Kim, Sang Yoon; Byun, Youngro

    2014-01-01

    Apical sodium-dependent bile acid transporters (ASBT) are the intestinal transporters that form intermediate complexes with substrates and its conformational change drives the movement of substrates across the cell membrane. However, membrane-based intestinal transporters are confined to the transport of only small molecular substrates. Here, we propose a new strategy that uses high-affinity binding macromolecular substrates to functionally transform the membrane transporters so that they behave like receptors, ultimately allowing the apical-basal transport of bound macromolecules. Bile acid based macromolecular substrates were synthesized and allowed to interact with ASBT. ASBT/macromolecular substrate complexes were rapidly internalized in vesicles, localized in early endosomes, dissociated and escaped the vesicular transport while binding of cytoplasmic ileal bile acid binding proteins cause exocytosis of macromolecules and prevented entry into lysosomes. This newly found transformation process of ASBT suggests a new transport mechanism that could aid in further utilization of ASBT to mediate oral macromolecular drug delivery. PMID:24566561

  13. Preorganized Peptide Scaffolds as Mimics of Phosphorylated Proteins Binding Sites with a High Affinity for Uranyl.

    PubMed

    Starck, Matthieu; Sisommay, Nathalie; Laporte, Fanny A; Oros, Stéphane; Lebrun, Colette; Delangle, Pascale

    2015-12-07

    Cyclic peptides with two phosphoserines and two glutamic acids were developed to mimic high-affinity binding sites for uranyl found in proteins such as osteopontin, which is believed to be a privileged target of this ion in vivo. These peptides adopt a β-sheet structure that allows the coordination of the latter amino acid side chains in the equatorial plane of the dioxo uranyl cation. Complementary spectroscopic and analytical methods revealed that these cyclic peptides are efficient uranyl chelating peptides with a large contribution from the phosphorylated residues. The conditional affinity constants were measured by following fluorescence tryptophan quenching and are larger than 10(10) at physiological pH. These compounds are therefore promising models for understanding uranyl chelation by proteins, which is relevant to this actinide ion toxicity.

  14. Neutrophil recruitment limited by high-affinity bent β2 integrin binding ligand in cis

    PubMed Central

    Fan, Zhichao; McArdle, Sara; Marki, Alex; Mikulski, Zbigniew; Gutierrez, Edgar; Engelhardt, Britta; Deutsch, Urban; Ginsberg, Mark; Groisman, Alex; Ley, Klaus

    2016-01-01

    Neutrophils are essential for innate immunity and inflammation and many neutrophil functions are β2 integrin-dependent. Integrins can extend (E+) and acquire a high-affinity conformation with an ‘open' headpiece (H+). The canonical switchblade model of integrin activation proposes that the E+ conformation precedes H+, and the two are believed to be structurally linked. Here we show, using high-resolution quantitative dynamic footprinting (qDF) microscopy combined with a homogenous conformation-reporter binding assay in a microfluidic device, that a substantial fraction of β2 integrins on human neutrophils acquire an unexpected E−H+ conformation. E−H+ β2 integrins bind intercellular adhesion molecules (ICAMs) in cis, which inhibits leukocyte adhesion in vitro and in vivo. This endogenous anti-inflammatory mechanism inhibits neutrophil aggregation, accumulation and inflammation. PMID:27578049

  15. Effects of anticonvulsants in vivo on high affinity choline uptake in vitro in mouse hippocampal synaptosomes.

    PubMed Central

    Miller, J. A.; Richter, J. A.

    1985-01-01

    The effects of several anticonvulsant drugs on sodium-dependent high affinity choline uptake (HACU) in mouse hippocampal synaptosomes was investigated. HACU was measured in vitro after in vivo administration of the drug to mice. HACU was inhibited by drugs which have in common the ability to facilitate gamma-aminobutyric acid (GABA) transmission, pentobarbitone, phenobarbitone, barbitone, diazepam, chloridiazepoxide, and valproic acid. Dose-response relationships were determined for these drugs and the drugs' potencies at inhibiting HACU correlated well with their anticonvulsant potencies. Clonazepam, ethosuximide, carbamazepine, and barbituric acid had no effect on HACU in the doses used while phenytoin and trimethadione stimulated HACU. These results suggest that certain anticonvulsants may elicit a part of their anticonvulsant activity by modulating cholinergic neurones. This effect may be mediated through a GABA mechanism. PMID:3978310

  16. Identification of Nucleic Acid High Affinity Binding Sequences of Proteins by SELEX.

    PubMed

    Bouvet, Philippe

    2015-01-01

    A technique is described for the identification of nucleic acid sequences bound with high affinity by proteins or by other molecules suitable for a partitioning assay. Here, a histidine-tagged protein is allowed to interact with a pool of nucleic acids and the protein-nucleic acid complexes formed are retained on a Ni-NTA matrix. Nucleic acids with a low level of recognition by the protein are washed away. The pool of recovered nucleic acids is amplified by the polymerase chain reaction and is submitted to further rounds of selection. Each round of selection increases the proportion of sequences that are avidly bound by the protein of interest. The cloning and sequencing of these sequences finally completes their identification.

  17. Identification of nucleic acid high-affinity binding sequences of proteins by SELEX.

    PubMed

    Bouvet, Philippe

    2009-01-01

    A technique is described for the identification of nucleic acid sequences bound with high affinity by proteins or by other molecules suitable for a partitioning assay. Here, a histidine-tagged protein is allowed to interact with a pool of nucleic acids and the protein-nucleic acid complexes formed are retained on a Ni-NTA matrix. Nucleic acids with a low level of recognition by the protein are washed away. The pool of recovered nucleic acids is amplified by the polymerase chain reaction and is submitted to further rounds of selection. Each round of selection increases the proportion of sequences that are avidly bound by the protein of interest. The cloning and sequencing of these sequences finally completes their identification.

  18. Protein unfolding as a switch from self-recognition to high-affinity client binding

    PubMed Central

    Groitl, Bastian; Horowitz, Scott; Makepeace, Karl A. T.; Petrotchenko, Evgeniy V.; Borchers, Christoph H.; Reichmann, Dana; Bardwell, James C. A.; Jakob, Ursula

    2016-01-01

    Stress-specific activation of the chaperone Hsp33 requires the unfolding of a central linker region. This activation mechanism suggests an intriguing functional relationship between the chaperone's own partial unfolding and its ability to bind other partially folded client proteins. However, identifying where Hsp33 binds its clients has remained a major gap in our understanding of Hsp33's working mechanism. By using site-specific Fluorine-19 nuclear magnetic resonance experiments guided by in vivo crosslinking studies, we now reveal that the partial unfolding of Hsp33's linker region facilitates client binding to an amphipathic docking surface on Hsp33. Furthermore, our results provide experimental evidence for the direct involvement of conditionally disordered regions in unfolded protein binding. The observed structural similarities between Hsp33's own metastable linker region and client proteins present a possible model for how Hsp33 uses protein unfolding as a switch from self-recognition to high-affinity client binding. PMID:26787517

  19. A linker peptide with high affinity towards silica-containing materials.

    PubMed

    Sunna, Anwar; Chi, Fei; Bergquist, Peter L

    2013-06-25

    A peptide sequence with affinity to silica-containing materials was fused to a truncated form of Streptococcus strain G148 Protein G. The resulting recombinant Linker-Protein G (LPG) was produced in Escherichia coli and purified to apparent homogeneity. It displayed high affinity towards two natural clinoptilolite zeolites. The LPG also displayed high binding affinity towards commercial-grade synthetic zeolite, silica and silica-containing materials. A commercial sample of the truncated Protein G and a basic protein, both without the linker, did not bind to natural or synthetic zeolites or silica. We conclude that the zeolite-binding affinity is mediated by the linker peptide sequence. As a consequence, these data may imply that the binding affinity is directed to the SiO2 component rather than to the atomic orientation on the zeolite crystal surface as previously assumed.

  20. Mu/sub 1/: A very high affinity subtype of enkephalin binding sites in rat brain

    SciTech Connect

    Lutz, R.A.; Cruciani, R.A.; Munson, P.J.; Rodbard, D.

    1985-06-10

    Displacement studies of (/sup 3/H)-(D-Ala/sup 2/-MePhe/sup 4/-Gly-ol/sup 5/)-enkephalin ((/sup 3/H)-DAGO) and (/sup 3/H)-(D-Ala/sup 2/-D-Leu/sup 5/)-enkephalin ((/sup 3/H)-DADL) by the corresponding unlabeled ligands show that there are at least three classes of sites which bind these enkephalin analogs with high affinity. Using computer modeling, the introduction of the third site significantly improved the goodness of fit in ten consecutive experiments. These sites appear to correspond to the ..mu.., delta, and ..mu../sub 1/ sites, with mean dissociation constants of 11, 1.3 and 0.9 nM for DADL and 2.5, 300 and 0.3 nM for DAGO, respectively. 15 reference, 3 figures, 1 table.

  1. A complex water network contributes to high-affinity binding in an antibody-antigen interface.

    PubMed

    Marino, S F; Olal, D; Daumke, O

    2016-03-01

    This data article presents an analysis of structural water molecules in the high affinity interaction between a potent tumor growth inhibiting antibody (fragment), J22.9-xi, and the tumor marker antigen CD269 (B cell maturation antigen, BCMA). The 1.89 Å X-ray crystal structure shows exquisite details of the binding interface between the two molecules, which comprises relatively few, mostly hydrophobic, direct contacts but many indirect interactions over solvent waters. These are partly or wholly buried in, and therefore part of, the interface. A partial description of the structure is included in an article on the tumor inhibiting effects of the antibody: "Potent anti-tumor response by targeting B cell maturation antigen (BCMA) in a mouse model of multiple myeloma", Mol. Oncol. 9 (7) (2015) pp. 1348-58.

  2. Cationic polymer brush-modified cellulose nanocrystals for high-affinity virus binding

    NASA Astrophysics Data System (ADS)

    Rosilo, Henna; McKee, Jason R.; Kontturi, Eero; Koho, Tiia; Hytönen, Vesa P.; Ikkala, Olli; Kostiainen, Mauri A.

    2014-09-01

    Surfaces capable of high-affinity binding of biomolecules are required in several biotechnological applications, such as purification, transfection, and sensing. Therein, the rod-shaped, colloidal cellulose nanocrystals (CNCs) are appealing due to their large surface area available for functionalization. In order to exploit electrostatic binding, their intrinsically anionic surfaces have to be cationized as biological supramolecules are predominantly anionic. Here we present a facile way to prepare cationic CNCs by surface-initiated atom-transfer radical polymerization of poly(N,N-dimethylaminoethyl methacrylate) and subsequent quaternization of the polymer pendant amino groups. The cationic polymer brush-modified CNCs maintained excellent dispersibility and colloidal stability in water and showed a ζ-potential of +38 mV. Dynamic light scattering and electron microscopy showed that the modified CNCs electrostatically bind cowpea chlorotic mottle virus and norovirus-like particles with high affinity. Addition of only a few weight percent of the modified CNCs in water dispersions sufficed to fully bind the virus capsids to form micrometer-sized assemblies. This enabled the concentration and extraction of the virus particles from solution by low-speed centrifugation. These results show the feasibility of the modified CNCs in virus binding and concentrating, and pave the way for their use as transduction enhancers for viral delivery applications.Surfaces capable of high-affinity binding of biomolecules are required in several biotechnological applications, such as purification, transfection, and sensing. Therein, the rod-shaped, colloidal cellulose nanocrystals (CNCs) are appealing due to their large surface area available for functionalization. In order to exploit electrostatic binding, their intrinsically anionic surfaces have to be cationized as biological supramolecules are predominantly anionic. Here we present a facile way to prepare cationic CNCs by surface

  3. Functional transformations of bile acid transporters induced by high-affinity macromolecules.

    PubMed

    Al-Hilal, Taslim A; Chung, Seung Woo; Alam, Farzana; Park, Jooho; Lee, Kyung Eun; Jeon, Hyesung; Kim, Kwangmeyung; Kwon, Ick Chan; Kim, In-San; Kim, Sang Yoon; Byun, Youngro

    2014-02-25

    Apical sodium-dependent bile acid transporters (ASBT) are the intestinal transporters that form intermediate complexes with substrates and its conformational change drives the movement of substrates across the cell membrane. However, membrane-based intestinal transporters are confined to the transport of only small molecular substrates. Here, we propose a new strategy that uses high-affinity binding macromolecular substrates to functionally transform the membrane transporters so that they behave like receptors, ultimately allowing the apical-basal transport of bound macromolecules. Bile acid based macromolecular substrates were synthesized and allowed to interact with ASBT. ASBT/macromolecular substrate complexes were rapidly internalized in vesicles, localized in early endosomes, dissociated and escaped the vesicular transport while binding of cytoplasmic ileal bile acid binding proteins cause exocytosis of macromolecules and prevented entry into lysosomes. This newly found transformation process of ASBT suggests a new transport mechanism that could aid in further utilization of ASBT to mediate oral macromolecular drug delivery.

  4. Experimental conditions can obscure the second high-affinity site in LeuT.

    PubMed

    Quick, Matthias; Shi, Lei; Zehnpfennig, Britta; Weinstein, Harel; Javitch, Jonathan A

    2012-01-15

    Neurotransmitter:Na(+) symporters (NSSs), the targets of antidepressants and psychostimulants, recapture neurotransmitters from the synapse in a Na(+)-dependent symport mechanism. The crystal structure of the NSS homolog LeuT from Aquifex aeolicus revealed one leucine substrate in an occluded, centrally located (S1) binding site next to two Na(+) ions. Computational studies combined with binding and flux experiments identified a second substrate (S2) site and a molecular mechanism of Na(+)-substrate symport that depends upon the allosteric interaction of substrate molecules in the two high-affinity sites. Here we show that the S2 site, which has not yet been identified by crystallographic approaches, can be blocked during preparation of detergent-solubilized LeuT, thereby obscuring its crucial role in Na(+)-coupled symport. This finding points to the need for caution in selecting experimental environments in which the properties and mechanistic features of membrane proteins can be delineated.

  5. High affinity binding of [3H]-tyramine in the central nervous system.

    PubMed Central

    Vaccari, A.

    1986-01-01

    Optimum assay conditions for the association of [3H]-para-tyramine [( 3H]-pTA) with rat brain membranes were characterized, and a saturable, reversible, drug-specific, and high affinity binding mechanism for this trace amine was revealed. The binding capacity (Bmax) for [3H]-pTA in the corpus striatum was approximately 30 times higher than that in the cerebellum, with similar dissociation constants (KD). The binding process of [3H]-pTA involved the dopamine system, inasmuch as (a) highest binding capacity was associated with dopamine-rich regions; (b) dopamine and pTA equally displaced specifically bound [3H]-pTA; (c) there was a severe loss in striatal binding capacity for [3H]-pTA and, reportedly, for [3H]-dopamine, following unilateral nigrostriatal lesion; (d) acute in vivo reserpine treatment markedly decreased the density of [3H]-pTA and, reportedly, of [3H]-dopamine binding sites. In competition experiments [3H]-pTA binding sites, though displaying nanomolar affinity for dopamine, revealed micromolar affinities for the dopamine agonists apomorphine and pergolide, and for several dopamine antagonists, while having very high affinity for reserpine, a marker for the catecholamine transporter in synaptic vesicles. The binding process of [3H]-pTA was both energy-dependent (ouabain-sensitive), and ATP-Mg2+-insensitive; furthermore, the potencies of various drugs in competing for [3H]-pTA binding to rat striatal membranes correlated well (r = 0.96) with their reported potencies in inhibiting [3H]-dopamine uptake into striatal synaptosomes. It is concluded that [3H]-pTA binds at a site located on/within synaptic vesicles where it is involved in the transport mechanism of dopamine. PMID:3801770

  6. Concurrent low- and high-affinity sulfate reduction kinetics in marine sediment

    NASA Astrophysics Data System (ADS)

    Harder Tarpgaard, Irene; Røy, Hans; Jørgensen, Bo Barker

    Bacterial sulfate reduction in marine sediments generally occurs in the presence of high millimolar concentrations of sulfate. Published data indicate that low sulfate concentrations may limit sulfate reduction rates below 0.2-2 mM. Yet, high sulfate reduction rates occur in the 1-100 μM range in freshwater sediments and at the sulfate-methane transition in marine sediments. Through a combination of 35S-tracer experiments, including initial velocity experiments and time course experiments, we searched for different sulfate affinities in the mixed community of sulfate reducers in a marine sediment. We supported the radiotracer experiments with a highly sensitive ion chromatographic technique for sulfate with a detection limit of 0.15 μM SO 42- in marine pore water. Our results showed that high and low affinities for sulfate co-occur and that the applied experimental approach may determine the observed apparent half saturation constant, Km. Our experimental and model data both show that sulfate reduction in the studied marine sediment could be explained by two dominating affinities for sulfate: a low affinity with a mean half saturation constant, Km, of 430 μM SO 42- and a high affinity with a mean Km of 2.6 μM SO 42-. The high-affinity sulfate reduction was thermodynamically un-constrained down to <1 μM SO 42-, both in our experiments and under in situ conditions. The reduction of radio-labeled sulfate was partly reversible due to concurrent re-oxidation of sulfide by Fe(III) and possibly due to a reversibility of the enzymatic pathway of sulfate reduction. A literature survey of apparent Km values for sediments and pure cultures is presented and discussed.

  7. High affinity ATP/ADP analogues as new tools for studying CFTR gating

    PubMed Central

    Zhou, Zhen; Wang, Xiaohui; Li, Min; Sohma, Yoshiro; Zou, Xiaoqin; Hwang, Tzyh-Chang

    2005-01-01

    Previous studies using non-hydrolysable ATP analogues and hydrolysis-deficient cystic fibrosis transmembrane conductance regulator (CFTR) mutants have indicated that ATP hydrolysis precedes channel closing. Our recent data suggest that ATP binding is also important in modulating the closing rate. This latter hypothesis predicts that ATP analogues with higher binding affinities should stabilize the open state more than ATP. Here we explore the possibility of using N6-modified ATP/ADP analogues as high-affinity ligands for CFTR gating, since these analogues have been shown to be more potent than native ATP/ADP in other ATP-binding proteins. Among the three N6-modified ATP analogues tested, N6-(2-phenylethyl)-ATP (P-ATP) was the most potent, with a K½ of 1.6 ± 0.4 μm (>50-fold more potent than ATP). The maximal open probability (Po) in the presence of P-ATP was ∼30% higher than that of ATP, indicating that P-ATP also has a higher efficacy than ATP. Single-channel kinetic analysis showed that as [P-ATP] was increased, the opening rate increased, whereas the closing rate decreased. The fact that these two kinetic parameters have different sensitivities to changes of [P-ATP] suggests an involvement of two different ATP-binding sites, a high-affinity site modulating channel closing and a low affinity site controlling channel opening. The effect of P-ATP on the stability of open states was more evident when ATP hydrolysis was abolished, either by mutating the nucleotide-binding domain 2 (NBD2) Walker B glutamate (i.e. E1371) or by using the non-hydrolysable ATP analogue AMP-PNP. Similar strategies to develop nucleotide analogues with a modified adenine ring could be valuable for future studies of CFTR gating. PMID:16223764

  8. Low- and high-affinity transport systems for citric acid in the yeast Candida utilis.

    PubMed Central

    Cássio, F; Leáo, C

    1991-01-01

    Citric acid-grown cells of the yeast Candida utilis induced two transport systems for citric acid, presumably a proton symport and a facilitated diffusion system for the charged and the undissociated forms of the acid, respectively. Both systems could be observed simultaneously when the transport was measured at 25 degrees C with labelled citric acid at pH 3.5 with the following kinetic parameters: for the low-affinity system, Vmax, 1.14 nmol of undissociated citric acid s-1 mg (dry weight) of cells-1, and Km, 0.59 mM undissociated acid; for the high-affinity system, Vmax, 0.38 nmol of citrate s-1 mg (dry weight) of cells-1, and Km, 0.056 mM citrate. At high pH values (above 5.0), the low-affinity system was absent or not measurable. The two transport systems exhibited different substrate specificities. Isocitric acid was a competitive inhibitor of citric acid for the high-affinity system, suggesting that these tricarboxylic acids used the same transport system, while aconitic, tricarballylic, trimesic, and hemimellitic acids were not competitive inhibitors. With respect to the low-affinity system, isocitric acid, L-lactic acid, and L-malic acid were competitive inhibitors, suggesting that all of these mono-, di-, and tricarboxylic acids used the same low-affinity transport system. The two transport systems were repressed by glucose, and as a consequence diauxic growth was observed. Both systems were inducible, and not only citric acid but also lactic acid and malic acid may induce those transport systems. The induction of both systems was not dependent on the relative concentration of the anionic form(s) and of undissociated citric acid in the culture medium.(ABSTRACT TRUNCATED AT 250 WORDS) PMID:1664712

  9. Use of fluorescence-detected sedimentation velocity to study high-affinity protein interactions.

    PubMed

    Chaturvedi, Sumit K; Ma, Jia; Zhao, Huaying; Schuck, Peter

    2017-09-01

    Sedimentation velocity (SV) analytical ultracentrifugation (AUC) is a classic technique for the real-time observation of free macromolecular migration in solution driven by centrifugal force. This enables the analysis of macromolecular mass, shape, size distribution, and interactions. Although traditionally limited to determination of the sedimentation coefficient and binding affinity of proteins in the micromolar range, the implementation of modern detection and data analysis techniques has resulted in marked improvements in detection sensitivity and size resolution during the past decades. Fluorescence optical detection now permits the detection of recombinant proteins with fluorescence excitation at 488 or 561 nm at low picomolar concentrations, allowing for the study of high-affinity protein self-association and hetero-association. Compared with other popular techniques for measuring high-affinity protein-protein interactions, such as biosensing or calorimetry, the high size resolution of complexes at picomolar concentrations obtained with SV offers a distinct advantage in sensitivity and flexibility of the application. Here, we present a basic protocol for carrying out fluorescence-detected SV experiments and the determination of the size distribution and affinity of protein-antibody complexes with picomolar KD values. Using an EGFP-nanobody interaction as a model, this protocol describes sample preparation, ultracentrifugation, data acquisition, and data analysis. A variation of the protocol applying traditional absorbance or an interference optical system can be used for protein-protein interactions in the micromolar KD value range. Sedimentation experiments typically take ∼3 h of preparation and 6-12 h of run time, followed by data analysis (typically taking 1-3 h).

  10. Neurotensin decreases high affinity [3H]-ouabain binding to cerebral cortex membranes.

    PubMed

    Rosin, Carina; Ordieres, María Graciela López; Arnaiz, Georgina Rodríguez de Lores

    2011-12-10

    Previous work from this laboratory showed the ability of neurotensin to inhibit synaptosomal membrane Na(+), K(+)-ATPase activity, the effect being blocked by SR 48692, a non-peptidic antagonist for high affinity neurotensin receptor (NTS1) [López Ordieres and Rodríguez de Lores Arnaiz 2000; 2001]. To further study neurotensin interaction with Na(+), K(+)-ATPase, peptide effect on high affinity [(3)H]-ouabain binding was studied in cerebral cortex membranes. It was observed that neurotensin modified binding in a dose-dependent manner, leading to 80% decrease with 1 × 10(-4)M concentration. On the other hand, the single addition of 1 × 10(-6)M, 1 × 10(-5)M and 1 × 10(-4)M SR 48692 (Sanofi-Aventis, U.S., Inc.) decreased [(3)H]-ouabain binding (in %) to 87 ± 16; 74 ± 16 and 34 ± 17, respectively. Simultaneous addition of neurotensin and SR 48692 led to additive or synergic effects. Partial NTS2 agonist levocabastine inhibited [(3)H]-ouabain binding likewise. Saturation assays followed by Scatchard analyses showed that neurotensin increased K(d) value whereas failed to modify B(max) value, indicating a competitive type interaction of the peptide at Na(+), K(+)-ATPase ouabain site. At variance, SR 48692 decreased B(max) value whereas it did not modify K(d) value. [(3)H]-ouabain binding was also studied in cerebral cortex membranes obtained from rats injected i. p. 30 min earlier with 100 μg and 250 μg/kg SR 48692. It was observed that the 250 μg/kg SR 48692 dose led to 19% decrease in basal [(3)H]-ouabain binding. After SR 48692 treatments, addition of 1 × 10(-6)M led to additive or synergic effect. Results suggested that [(3)H]-ouabain binding inhibition by neurotensin hardly involves NTS1 receptor.

  11. A selective, high affinity 5-HT 2B receptor antagonist inhibits visceral hypersensitivity in rats.

    PubMed

    Ohashi-Doi, K; Himaki, D; Nagao, K; Kawai, M; Gale, J D; Furness, J B; Kurebayashi, Y

    2010-02-01

    RS-127445 is a selective, high affinity 5-HT(2B)receptor antagonist. We investigated whether 5-HT(2B)receptor antagonists can reduce colonic visceral hypersensitivity caused by restraint stress or by proximal colonic inflammation. Visceral hypersensitivity was induced in rats by either restraint stress or injection of 2, 4, 6-trinitrobenzene sulfonic acid (TNBS) into the proximal colon. Restraint stress produced a significant increase in numbers of abdominal contractions evoked by colorectal distension (CRD), measured as a quantitative index of visceral nociception in rats. Seven days after TNBS injection, the pain threshold to CRD at the non-inflamed distal colon, that was determined as the minimum pressure required to evoke abdominal cramp, was significantly decreased. The effect of RS-127445 on visceral hypersensitivity was assessed in either naïve or TNBS-treated rats. Oral administration of a selective, high affinity 5-HT(2B)receptor antagonist, RS-127445, significantly inhibited visceral hypersensitivity provoked by restraint stress (35 to 74% inhibition at 1 to 10 mg kg(-1)). Oral RS-127445 produced a significant suppression of TNBS-induced visceral hypersensitivity (15 to 62% inhibition at 3 to 30 mg kg(-1)), although it was without significant effect on the visceral nociceptive threshold of naïve rats. RS-127445 (1 to 30 mg kg(-1), p.o.) also dose-dependently reduced the restraint stress-induced defecation in naïve and TNBS-treated rats. These results suggest that 5-HT(2B)receptors are involved in signaling from the colon in rats in which there is visceral hypersensitivity and that a selective 5-HT(2B)receptor antagonist could have therapeutic potential for the treatment of gut disorders characterized by visceral hypersensitivity.

  12. Enhanced Local Disorder in a Clinically Elusive von Willebrand Factor Provokes High-Affinity Platelet Clumping.

    PubMed

    Tischer, Alexander; Machha, Venkata R; Frontroth, Juan P; Brehm, Maria A; Obser, Tobias; Schneppenheim, Reinhard; Mayne, Leland; Walter Englander, S; Auton, Matthew

    2017-07-07

    Mutation of the cysteines forming the disulfide loop of the platelet GPIbα adhesive A1 domain of von Willebrand factor (VWF) causes quantitative VWF deficiencies in the blood and von Willebrand disease. We report two cases of transient severe thrombocytopenia induced by DDAVP treatment. Cys1272Trp and Cys1458Tyr mutations identified by genetic sequencing implicate an abnormal gain-of-function phenotype, evidenced by thrombocytopenia, which quickly relapses back to normal platelet counts and deficient plasma VWF. Using surface plasmon resonance, analytical rheology, and hydrogen-deuterium exchange mass spectrometry (HXMS), we decipher mechanisms of A1-GPIbα-mediated platelet adhesion and resolve dynamic secondary structure elements that regulate the binding pathway. Constrained by the disulfide, conformational selection between weak and tight binding states of A1 takes precedence and drives normal platelet adhesion to VWF. Less restrained through mutation, loss of the disulfide preferentially diverts binding through an induced-fit disease pathway enabling high-affinity GPIbα binding and firm platelet adhesion to a partially disordered A1 domain. HXMS reveals a dynamic asymmetry of flexible and ordered regions common to both variants, indicating that the partially disordered A1 lacking the disulfide retains native-like structural dynamics. Both binding mechanisms share common structural and thermodynamic properties, but the enhanced local disorder in the disease state perpetuates high-affinity platelet agglutination, characteristic of type 2B VWD, upon DDAVP-stimulated secretion of VWF leading to transient thrombocytopenia and a subsequent deficiency of plasma VWF, characteristic of type 2A VWD. Copyright © 2017 Elsevier Ltd. All rights reserved.

  13. The integration of genomic and structural information in the development of high affinity plasmepsin inhibitors.

    PubMed

    Nezami, Azin; Freire, Ernesto

    2002-12-04

    The plasmepsins are key enzymes in the life cycle of the Plasmodium parasites responsible for malaria. Since plasmepsin inhibition leads to parasite death, these enzymes have been acknowledged to be important targets for the development of new antimalarial drugs. The development of effective plasmepsin inhibitors, however, is compounded by their genomic diversity which gives rise not to a unique target for drug development but to a family of closely related targets. Successful drugs will have to inhibit not one but several related enzymes with high affinity. Structure-based drug design against heterogeneous targets requires a departure from the classic 'lock-and-key' paradigm that leads to the development of conformationally constrained molecules aimed at a single target. Drug molecules designed along those principles are usually rigid and unable to adapt to target variations arising from naturally occurring genetic polymorphisms or drug-induced resistant mutations. Heterogeneous targets need adaptive drug molecules, characterised by the presence of flexible elements at specific locations that sustain a viable binding affinity against existing or expected polymorphisms. Adaptive ligands have characteristic thermodynamic signatures that distinguish them from their rigid counterparts. This realisation has led to the development of rigorous thermodynamic design guidelines that take advantage of correlations between the structure of lead compounds and the enthalpic and entropic components of the binding affinity. In this paper, we discuss the application of the thermodynamic approach to the development of high affinity (K(i) - pM) plasmepsin inhibitors. In particular, a family of allophenylnorstatine-based compounds is evaluated for their potential to inhibit a wide spectrum of plasmepsins.

  14. Kinetics and autoradiography of high affinity uptake of serotonin by primary astrocyte cultures

    SciTech Connect

    Katz, D.M.; Kimelberg, H.K.

    1985-07-01

    Primary astrocyte cultures prepared from the cerebral cortices of neonatal rats showed significant accumulation of serotonin (5-hydroxytryptamine; (/sup 3/H)-5-HT). At concentrations in the range of 0.01 to 0.7 microM (/sup 3/H)-5-HT, this uptake was 50 to 85% Na+ dependent and gave a Km of 0.40 +/- 0.11 microM (/sup 3/H)-5-HT and a Vmax of 6.42 +/- 0.85 (+/- SEM) pmol of (/sup 3/H)-5-HT/mg of protein/4 min for the Na+-dependent component. In the absence of Na+ the uptake was nonsaturable. Omission of the monoamine oxidase inhibitor pargyline markedly reduced the Na+-dependent component of (/sup 3/H)-5-HT uptake but had a negligible effect on the Na+-independent component. This suggest significant oxidative deamination of serotonin after it has been taken up by the high affinity system, followed by release of its metabolite. The authors estimated that this system enabled the cells to concentrate (/sup 3/H)-5-HT up to 44-fold at an external (/sup 3/H)-5-HT concentration of 10(-7) M. Inhibition of (/sup 3/H)-5-HT uptake by a number of clinically effective antidepressants was also consistent with a specific high affinity uptake mechanism for 5-HT, the order of effectiveness of inhibition being chlorimipramine greater than fluoxetine greater than imipramine = amitriptyline greater than desmethylimipramine greater than iprindole greater than mianserin. Uptake of (/sup 3/H)-5-HT was dependent on the presence of Cl- as well as Na+ in the medium, and the effect of omission of both ions was nonadditive. Varying the concentration of K+ in the media from 1 to 50 mM had a limited effect on (/sup 3/H)-5-HT uptake.

  15. Selection of DNA aptamers against epidermal growth factor receptor with high affinity and specificity

    SciTech Connect

    Wang, Deng-Liang; Song, Yan-Ling; Zhu, Zhi; Li, Xi-Lan; Zou, Yuan; Yang, Hai-Tao; Wang, Jiang-Jie; Yao, Pei-Sen; Pan, Ru-Jun; Yang, Chaoyong James; Kang, De-Zhi

    2014-10-31

    Highlights: • This is the first report of DNA aptamer against EGFR in vitro. • Aptamer can bind targets with high affinity and selectivity. • DNA aptamers are more stable, cheap and efficient than RNA aptamers. • Our selected DNA aptamer against EGFR has high affinity with K{sub d} 56 ± 7.3 nM. • Our selected DNA aptamer against EGFR has high selectivity. - Abstract: Epidermal growth factor receptor (EGFR/HER1/c-ErbB1), is overexpressed in many solid cancers, such as epidermoid carcinomas, malignant gliomas, etc. EGFR plays roles in proliferation, invasion, angiogenesis and metastasis of malignant cancer cells and is the ideal antigen for clinical applications in cancer detection, imaging and therapy. Aptamers, the output of the systematic evolution of ligands by exponential enrichment (SELEX), are DNA/RNA oligonucleotides which can bind protein and other substances with specificity. RNA aptamers are undesirable due to their instability and high cost of production. Conversely, DNA aptamers have aroused researcher’s attention because they are easily synthesized, stable, selective, have high binding affinity and are cost-effective to produce. In this study, we have successfully identified DNA aptamers with high binding affinity and selectivity to EGFR. The aptamer named TuTu22 with K{sub d} 56 ± 7.3 nM was chosen from the identified DNA aptamers for further study. Flow cytometry analysis results indicated that the TuTu22 aptamer was able to specifically recognize a variety of cancer cells expressing EGFR but did not bind to the EGFR-negative cells. With all of the aforementioned advantages, the DNA aptamers reported here against cancer biomarker EGFR will facilitate the development of novel targeted cancer detection, imaging and therapy.

  16. Basic Camp Management: An Introduction to Camp Administration. Second Edition.

    ERIC Educational Resources Information Center

    Ball, Armand; Ball, Beverly

    From its perspective of experience in the field, this book offers practical and specific guidance for successfully managing an organized summer camp. It may also serve as a beginning orientation for potential camp directors, a quick reference for experienced directors, or a text for entry level courses in practical camp administration. This second…

  17. Basic Camp Management: An Introduction to Camp Administration. Second Edition.

    ERIC Educational Resources Information Center

    Ball, Armand; Ball, Beverly

    From its perspective of experience in the field, this book offers practical and specific guidance for successfully managing an organized summer camp. It may also serve as a beginning orientation for potential camp directors, a quick reference for experienced directors, or a text for entry level courses in practical camp administration. This second…

  18. How Green Is Camping? Environmental Stewardship in North Carolina Camps.

    ERIC Educational Resources Information Center

    Warren, Roger; Bingham, Cindy

    1994-01-01

    A survey of 47 residential camps in North Carolina revealed that most camps had written environmental objectives, practiced recycling, attempted to reduce water use and energy consumption, practiced low-impact camping, included environmental issues in staff training, and provided environmental education to campers. Includes survey questions. (LP)

  19. How Green Is Camping? Environmental Stewardship in North Carolina Camps.

    ERIC Educational Resources Information Center

    Warren, Roger; Bingham, Cindy

    1994-01-01

    A survey of 47 residential camps in North Carolina revealed that most camps had written environmental objectives, practiced recycling, attempted to reduce water use and energy consumption, practiced low-impact camping, included environmental issues in staff training, and provided environmental education to campers. Includes survey questions. (LP)

  20. Active site similarity between human and Plasmodium falciparum phosphodiesterases: considerations for antimalarial drug design

    NASA Astrophysics Data System (ADS)

    Howard, Brittany L.; Thompson, Philip E.; Manallack, David T.

    2011-08-01

    The similarity between Plasmodium falciparum phosphodiesterase enzymes ( PfPDEs) and their human counterparts have been examined and human PDE9A was found to be a suitable template for the construction of homology models for each of the four PfPDE isoforms. In contrast, the architecture of the active sites of each model was most similar to human PDE1. Molecular docking was able to model cyclic guanosine monophosphate (cGMP) substrate binding in each case but a docking mode supporting cyclic adenosine monophosphate (cAMP) binding could not be found. Anticipating the potential of PfPDE inhibitors as anti-malarial drugs, a range of reported PDE inhibitors including zaprinast and sildenafil were docked into the model of PfPDEα. The results were consistent with their reported biological activities, and the potential of PDE1/9 inhibitor analogues was also supported by docking.

  1. ABCD of the phosphodiesterase family: interaction and differential activity in COPD

    PubMed Central

    Halpin, David MG

    2008-01-01

    Phosphodiesterases (PDEs) are important enzymes that hydrolyze the cyclic nucleotides adenosine 3′5′-cyclic monophosphate (cAMP) and guanosine 3′5′-cyclic mono-phosphate (cGMP) to their inactive 5′ monophosphates. They are highly conserved across species and as well as their role in signal termination, they also have a vital role in intracellular localization of cyclic nucleotide signaling and integration of the cyclic nucleotide pathways with other signaling pathways. Because of their pivotal role in intracellular signaling, they are now of considerable interest as therapeutic targets in a wide variety diseases, including COPD where PDE inhibitors may have bronchodilator, anti-inflammatory and pulmonary vasodilator actions. This review examines the diversity and cellular localization of the isoforms of PDE, the known and speculative relevance of this to the treatment of COPD, and the range of PDE inhibitors in development together with a discussion of their possible role in treating COPD. PMID:19281073

  2. 36 CFR 13.1222 - Camping.

    Code of Federal Regulations, 2010 CFR

    2010-07-01

    ... Camp Campground and other designated areas. (b) Camping in Brooks Camp Campground for more than a total... site in the Brooks Camp Campground while in operation as a designated fee area is prohibited....

  3. 36 CFR 13.1222 - Camping.

    Code of Federal Regulations, 2014 CFR

    2014-07-01

    ... Camp Campground and other designated areas. (b) Camping in Brooks Camp Campground for more than a total... site in the Brooks Camp Campground while in operation as a designated fee area is prohibited....

  4. 36 CFR 13.1222 - Camping.

    Code of Federal Regulations, 2013 CFR

    2013-07-01

    ... Camp Campground and other designated areas. (b) Camping in Brooks Camp Campground for more than a total... site in the Brooks Camp Campground while in operation as a designated fee area is prohibited....

  5. 36 CFR 13.1222 - Camping.

    Code of Federal Regulations, 2011 CFR

    2011-07-01

    ... Camp Campground and other designated areas. (b) Camping in Brooks Camp Campground for more than a total... site in the Brooks Camp Campground while in operation as a designated fee area is prohibited....

  6. 36 CFR 13.1222 - Camping.

    Code of Federal Regulations, 2012 CFR

    2012-07-01

    ... Camp Campground and other designated areas. (b) Camping in Brooks Camp Campground for more than a total... site in the Brooks Camp Campground while in operation as a designated fee area is prohibited....

  7. Toward the identification of the cardiac cGMP inhibited-phosphodiesterase catalytic site

    NASA Astrophysics Data System (ADS)

    Fossa, Paola; Boggia, Raffaella; Mosti, Luisa

    1998-07-01

    Cyclic nucleotide phosphodiesterases (PDEs) comprise a complex group of enzymes; five major PDE families or classes with distinctive properties have been identified. Among these a great deal of interest has recently been focused on the so called cGMP-inhibited low Km cAMP phosphodiesterase (cGI PDE) or PDE III. A number of positive inotropic agents, including the well-known milrinone, display a specific inhibition of PDE III as primary mechanism of action. Recent studies have been carried out to develop a pharmacophore model of the PDE III active site. We therefore performed molecular modelling and 3D-SAR studies so as to better define structural requirements for potent and selective enzymatic inhibition. The DISCO (DIStance COmparison) strategy has been applied on a set of compounds taken from literature and a milrinone analogue previously synthesized by us, all of which are characterized by a marked inotropic effect but with varying degrees of enzyme selectivity. A common pharmacophoric model was derived, validated and considered as starting point to perform a 3D-SAR study using the GRID force field and PCA (Principal Component Analysis) with the aim of rationally designing more selective inhibitors. This paper presents the results of this theoretical approach.

  8. Chronic Cognitive Dysfunction after Traumatic Brain Injury Is Improved with a Phosphodiesterase 4B Inhibitor

    PubMed Central

    Titus, David J.; Wilson, Nicole M.; Freund, Julie E.; Carballosa, Melissa M.; Sikah, Kevin E.; Furones, Concepcion; Dietrich, W. Dalton; Gurney, Mark E.

    2016-01-01

    Learning and memory impairments are common in traumatic brain injury (TBI) survivors. However, there are no effective treatments to improve TBI-induced learning and memory impairments. TBI results in decreased cAMP signaling and reduced cAMP-response-element binding protein (CREB) activation, a critical pathway involved in learning and memory. TBI also acutely upregulates phosphodiesterase 4B2 (PDE4B2), which terminates cAMP signaling by hydrolyzing cAMP. We hypothesized that a subtype-selective PDE4B inhibitor could reverse the learning deficits induced by TBI. To test this hypothesis, adult male Sprague-Dawley rats received sham surgery or moderate parasagittal fluid-percussion brain injury. At 3 months postsurgery, animals were administered a selective PDE4B inhibitor or vehicle before cue and contextual fear conditioning, water maze training and a spatial working memory task. Treatment with the PDE4B inhibitor significantly reversed the TBI-induced deficits in cue and contextual fear conditioning and water maze retention. To further understand the underlying mechanisms of these memory impairments, we examined hippocampal long-term potentiation (LTP). TBI resulted in a significant reduction in basal synaptic transmission and impaired expression of LTP. Treatment with the PDE4B inhibitor significantly reduced the deficits in basal synaptic transmission and rescued LTP expression. The PDE4B inhibitor reduced tumor necrosis factor-α levels and increased phosphorylated CREB levels after TBI, suggesting that this drug inhibited molecular pathways in the brain known to be regulated by PDE4B. These results suggest that a subtype-selective PDE4B inhibitor is a potential therapeutic to reverse chronic learning and memory dysfunction and deficits in hippocampal synaptic plasticity following TBI. SIGNIFICANCE STATEMENT Currently, there are an estimated 3.2–5.3 million individuals living with disabilities from traumatic brain injury (TBI) in the United States, and 8 of

  9. Chronic Cognitive Dysfunction after Traumatic Brain Injury Is Improved with a Phosphodiesterase 4B Inhibitor.

    PubMed

    Titus, David J; Wilson, Nicole M; Freund, Julie E; Carballosa, Melissa M; Sikah, Kevin E; Furones, Concepcion; Dietrich, W Dalton; Gurney, Mark E; Atkins, Coleen M

    2016-07-06

    Learning and memory impairments are common in traumatic brain injury (TBI) survivors. However, there are no effective treatments to improve TBI-induced learning and memory impairments. TBI results in decreased cAMP signaling and reduced cAMP-response-element binding protein (CREB) activation, a critical pathway involved in learning and memory. TBI also acutely upregulates phosphodiesterase 4B2 (PDE4B2), which terminates cAMP signaling by hydrolyzing cAMP. We hypothesized that a subtype-selective PDE4B inhibitor could reverse the learning deficits induced by TBI. To test this hypothesis, adult male Sprague-Dawley rats received sham surgery or moderate parasagittal fluid-percussion brain injury. At 3 months postsurgery, animals were administered a selective PDE4B inhibitor or vehicle before cue and contextual fear conditioning, water maze training and a spatial working memory task. Treatment with the PDE4B inhibitor significantly reversed the TBI-induced deficits in cue and contextual fear conditioning and water maze retention. To further understand the underlying mechanisms of these memory impairments, we examined hippocampal long-term potentiation (LTP). TBI resulted in a significant reduction in basal synaptic transmission and impaired expression of LTP. Treatment with the PDE4B inhibitor significantly reduced the deficits in basal synaptic transmission and rescued LTP expression. The PDE4B inhibitor reduced tumor necrosis factor-α levels and increased phosphorylated CREB levels after TBI, suggesting that this drug inhibited molecular pathways in the brain known to be regulated by PDE4B. These results suggest that a subtype-selective PDE4B inhibitor is a potential therapeutic to reverse chronic learning and memory dysfunction and deficits in hippocampal synaptic plasticity following TBI. Currently, there are an estimated 3.2-5.3 million individuals living with disabilities from traumatic brain injury (TBI) in the United States, and 8 of 10 of these individuals

  10. In vivo model with targeted cAMP biosensor reveals changes in receptor-microdomain communication in cardiac disease.

    PubMed

    Sprenger, Julia U; Perera, Ruwan K; Steinbrecher, Julia H; Lehnart, Stephan E; Maier, Lars S; Hasenfuss, Gerd; Nikolaev, Viacheslav O

    2015-04-28

    3',5'-cyclic adenosine monophosphate (cAMP) is an ubiquitous second messenger that regulates physiological functions by acting in distinct subcellular microdomains. Although several targeted cAMP biosensors are developed and used in single cells, it is unclear whether such biosensors can be successfully applied in vivo, especially in the context of disease. Here, we describe a transgenic mouse model expressing a targeted cAMP sensor and analyse microdomain-specific second messenger dynamics in the vicinity of the sarcoplasmic/endoplasmic reticulum calcium ATPase (SERCA). We demonstrate the biocompatibility of this targeted sensor and its potential for real-time monitoring of compartmentalized cAMP signalling in adult cardiomyocytes isolated from a healthy mouse heart and from an in vivo cardiac disease model. In particular, we uncover the existence of a phosphodiesterase-dependent receptor-microdomain communication, which is affected in hypertrophy, resulting in reduced β-adrenergic receptor-cAMP signalling to SERCA.

  11. Regulatory mechanisms of cAMP levels as a multiple target for antiplatelet activity and less bleeding risk.

    PubMed

    Fuentes, Eduardo; Palomo, Iván

    2014-08-01

    Platelet activation is a critical component of atherothrombosis. The multiple pathways of platelet activation limit the effect of specific receptor/pathway inhibitors, resulting in limited clinical efficacy. Recent research has confirmed that combination therapy results in enhanced antithrombotic efficacy without increasing bleeding risk. In this way, the best-known inhibitor and turn off signaling in platelet activation is cAMP. In this article we discuss the mechanisms of regulation of intraplatelet cAMP levels, a) platelet-dependent pathway: Gi/Gs protein-coupled receptors, phosphodiesterase inhibition and activation of PPARs and b) platelet-independent pathway: inhibition of adenosine uptake by erythrocytes. With respect to the association between intraplatelet cAMP levels and bleeding risk it is possible to establish that compounds/drugs with pleitropic effect for increased intraplatelet cAMP level could have an antithrombotic activity with less risk of bleeding.

  12. ERK regulation of phosphodiesterase 4 enhances dopamine-stimulated AMPA receptor membrane insertion.

    PubMed

    Song, Roy S; Massenburg, Ben; Wenderski, Wendy; Jayaraman, Vino; Thompson, Lauren; Neves, Susana R

    2013-09-17

    AMPA-type glutamate receptor (AMPAR) trafficking is essential for modulating synaptic transmission strength. Prior studies that have characterized signaling pathways underlying AMPAR trafficking have identified the cAMP/PKA-mediated phosphorylation of GluA1, an AMPAR subunit, as a key step in the membrane insertion of AMPAR. Inhibition of ERK impairs AMPAR membrane insertion, but the mechanism by which ERK exerts its effect is unknown. Dopamine, an activator of both PKA and ERK, induces AMPAR insertion, but the relationship between the two protein kinases in the process is not understood. We used a combination of computational modeling and live cell imaging to determine the relationship between ERK and PKA in AMPAR insertion. We developed a dynamical model to study the effects of phosphodiesterase 4 (PDE4), a cAMP phosphodiesterase that is phosphorylated and inhibited by ERK, on the membrane insertion of AMPAR. The model predicted that PKA could be a downstream effector of ERK in regulating AMPAR insertion. We experimentally tested the model predictions and found that dopamine-induced ERK phosphorylates and inhibits PDE4. This regulation results in increased cAMP levels and PKA-mediated phosphorylation of DARPP-32 and GluA1, leading to increased GluA1 trafficking to the membrane. These findings provide unique insight into an unanticipated network topology in which ERK uses PDE4 to regulate PKA output during dopamine signaling. The combination of dynamical models and experiments has helped us unravel the complex interactions between two protein kinase pathways in regulating a fundamental molecular process underlying synaptic plasticity.

  13. ERK regulation of phosphodiesterase 4 enhances dopamine-stimulated AMPA receptor membrane insertion

    PubMed Central

    Song, Roy S.; Massenburg, Ben; Wenderski, Wendy; Jayaraman, Vino; Thompson, Lauren; Neves, Susana R.

    2013-01-01

    AMPA-type glutamate receptor (AMPAR) trafficking is essential for modulating synaptic transmission strength. Prior studies that have characterized signaling pathways underlying AMPAR trafficking have identified the cAMP/PKA-mediated phosphorylation of GluA1, an AMPAR subunit, as a key step in the membrane insertion of AMPAR. Inhibition of ERK impairs AMPAR membrane insertion, but the mechanism by which ERK exerts its effect is unknown. Dopamine, an activator of both PKA and ERK, induces AMPAR insertion, but the relationship between the two protein kinases in the process is not understood. We used a combination of computational modeling and live cell imaging to determine the relationship between ERK and PKA in AMPAR insertion. We developed a dynamical model to study the effects of phosphodiesterase 4 (PDE4), a cAMP phosphodiesterase that is phosphorylated and inhibited by ERK, on the membrane insertion of AMPAR. The model predicted that PKA could be a downstream effector of ERK in regulating AMPAR insertion. We experimentally tested the model predictions and found that dopamine-induced ERK phosphorylates and inhibits PDE4. This regulation results in increased cAMP levels and PKA-mediated phosphorylation of DARPP-32 and GluA1, leading to increased GluA1 trafficking to the membrane. These findings provide unique insight into an unanticipated network topology in which ERK uses PDE4 to regulate PKA output during dopamine signaling. The combination of dynamical models and experiments has helped us unravel the complex interactions between two protein kinase pathways in regulating a fundamental molecular process underlying synaptic plasticity. PMID:23986500

  14. Astro Camp 2000 Rocketry Exercise

    NASA Image and Video Library

    2000-06-23

    Children at Astro Camp at the John C. Stennis Space Center in Hancock County, Miss., launch rockets as one of their activities in the weeklong camp. Each week during the summer, approximately 30 children ages 9-12 from across Mississippi and Louisiana spend a week learning about space flight. Astro Camp Saturday offers a condensed version of Astro Camp on the third Saturday of each month from January through May 2001.

  15. Phosphodiesterases reduce spontaneous sinoatrial beating but not the 'fight or flight' tachycardia elicited by agonists through Gs-protein-coupled receptors.

    PubMed

    Kaumann, Alberto J

    2011-07-01

    Cyclic AMP (cAMP) steers the generation of basal heart beat in the sinoatrial node. It also induces sinoatrial tachycardia and increased cardiac force, elicited through activation of Gs-protein-coupled receptors (GsPCRs). Phosphodiesterases (PDEs) hydrolyse cAMP. In the heart mainly PDE3 and PDE4 would be expected to limit those functions, and the PDE isoenzymes do indeed reduce basal sinoatrial beating rate and blunt the positive inotropic effects of agonists, mediated by GsPCRs. By contrast, recent evidence shows that GsPCR-mediated sinoatrial tachycardia is not controlled by PDE1-5. A PDE-resistant cAMP pool in sinoatrial cells, generated through activation of GsPCRs, including β(1)- and β(2)-adrenoceptors, appears to guarantee unrestrained tachycardia during fight or flight stress.

  16. Overexpression of phosphodiesterase-4 subtypes involved in surgery-induced neuroinflammation and cognitive dysfunction in mice.

    PubMed

    Wang, Wei; Zhang, Xiao-Ying; Feng, Ze-Guo; Wang, Dong-Xin; Zhang, Hao; Sui, Bo; Zhang, Yong-Yi; Zhao, Wei-Xing; Fu, Qiang; Xu, Zhi-Peng; Mi, Wei-Dong

    2017-02-21

    Postoperative cognitive dysfunction (POCD) is characterized by cognitive impairments in patients after surgery. Hippocampal neuroinflammation induced by surgery is highly associated with POCD. Phosphodiesterase-4 (PDE4) is an enzyme that specifically hydrolyses cyclic adenosine monophosphate (cAMP), which plays an important role during neuroinflammation and the process of learning and memory. However, the role of PDE4 in the development of POCD remains unclear. Male 14-month-old C57BL/6 mice received carotid artery exposure to mimic POCD. First, we evaluated cognitive performance by a Morris water maze (MWM) and fear conditioning system (FCS) test after surgery. The expression of PDE4 subtypes, pro-inflammatory cytokines, p-CREB and PSD95 as well as cAMP levels were investigated. Then, we used rolipram, a PDE4 inhibitor, to block the effects of PDE4. The cognitive performance of the mice and the expression of PDE4 subtypes, pro-inflammatory cytokines, p-CREB and PSD95 as well as cAMP levels were examined again. Mice displayed learning and memory impairment, overexpression of PDE4B and PDE4D, elevation of pro-inflammatory cytokines, and reduction in the expression of p-CREB, PSD95 and cAMP levels after surgery. The expression of PDE4B and PDE4D in the hippocampus decreased following blocking of PDE4 by rolipram. Meanwhile, rolipram attenuated the cognitive impairment and the elevation of pro-inflammatory cytokines induced by surgery. Moreover, rolipram reversed the reduction of p-CREB and PSD95. These results indicate that PDE4 subtype overexpression may be involved in the development of surgery-induced cognitive dysfunction in mice.

  17. Profiling of functional phosphodiesterase in mesangial cells using a CRE-SEAP-based reporting system.

    PubMed

    Zhu, Ying; Yao, Jian; Meng, Yiman; Kasai, Ayumi; Hiramatsu, Nobuhiko; Hayakawa, Kunihiro; Miida, Takashi; Takeda, Masayuki; Okada, Masahiko; Kitamura, Masanori

    2006-07-01

    1. Phosphodiesterases (PDEs) are critically implicated in the regulation of mesangial cell function, but profile of functional PDEs in mesangial cells is still unclear. In this study, we investigated roles of individual PDEs in the regulation of mesangial cell behavior by the cAMP pathway. 2. Reporter mesangial cells that express secreted alkaline phosphatase (SEAP) under the control of the cAMP response element (CRE) were exposed to selective PDE inhibitors in the presence or absence of cAMP, and activity of CRE, expression of CRE-regulated protein, mitogenesis and cell survival were examined. 3. Exposure of reporter cells to cAMP-elevating agents resulted in time- and concentration-dependent activation of CRE. Treatment of the cells with any PDE inhibitors alone did not induce CRE activation. Under stimulation with 8-bromo-cAMP or 8-bromo-cGMP, however, inhibitors of PDE2, PDE3, PDE4 and PDE5 enhanced activation of CRE. Inhibition of PDE1 or PDE6 did not affect the CRE activation. 4. Among different combinations tested, only inhibitors of PDE3 and PDE4 cooperatively increased the level of intracellular cAMP, activity of protein kinase A, activation of CRE, and CRE-regulated protein, connexin43. 5. Concomitant inhibition of PDE3 and PDE4 attenuated mitogen-induced activation of extracellular signal-regulated kinases and cell proliferation. Under serum deprivation, combinational inhibition of PDE3 and PDE4 exclusively caused activation of caspase-3 and apoptosis. 6. The present data elucidated that PDE3 and PDE4 play critical roles in the regulation of mesangial cell function. PDE3 and PDE4 were identified as the novel, antiapoptotic machinery that supports survival of mesangial cells.

  18. Profiling of functional phosphodiesterase in mesangial cells using a CRE-SEAP-based reporting system

    PubMed Central

    Zhu, Ying; Yao, Jian; Meng, Yiman; Kasai, Ayumi; Hiramatsu, Nobuhiko; Hayakawa, Kunihiro; Miida, Takashi; Takeda, Masayuki; Okada, Masahiko; Kitamura, Masanori

    2006-01-01

    Phosphodiesterases (PDEs) are critically implicated in the regulation of mesangial cell function, but profile of functional PDEs in mesangial cells is still unclear. In this study, we investigated roles of individual PDEs in the regulation of mesangial cell behavior by the cAMP pathway. Reporter mesangial cells that express secreted alkaline phosphatase (SEAP) under the control of the cAMP response element (CRE) were exposed to selective PDE inhibitors in the presence or absence of cAMP, and activity of CRE, expression of CRE-regulated protein, mitogenesis and cell survival were examined. Exposure of reporter cells to cAMP-elevating agents resulted in time- and concentration-dependent activation of CRE. Treatment of the cells with any PDE inhibitors alone did not induce CRE activation. Under stimulation with 8-bromo-cAMP or 8-bromo-cGMP, however, inhibitors of PDE2, PDE3, PDE4 and PDE5 enhanced activation of CRE. Inhibition of PDE1 or PDE6 did not affect the CRE activation. Among different combinations tested, only inhibitors of PDE3 and PDE4 cooperatively increased the level of intracellular cAMP, activity of protein kinase A, activation of CRE, and CRE-regulated protein, connexin43. Concomitant inhibition of PDE3 and PDE4 attenuated mitogen-induced activation of extracellular signal-regulated kinases and cell proliferation. Under serum deprivation, combinational inhibition of PDE3 and PDE4 exclusively caused activation of caspase-3 and apoptosis. The present data elucidated that PDE3 and PDE4 play critical roles in the regulation of mesangial cell function. PDE3 and PDE4 were identified as the novel, antiapoptotic machinery that supports survival of mesangial cells. PMID:16751794

  19. Children's Camps in the Adirondacks.

    ERIC Educational Resources Information Center

    Bond, Hallie E.

    2003-01-01

    In the late 19th century, camps in the Adirondacks responded to concerns that the American character was softening. Much camping philosophy came from the progressive movement in education. Aspects of Indian culture were adopted because they seemed to fit naturally in the Adirondacks, and children loved them. Adirondack camps have always been…

  20. Saving the Camping Experience.

    ERIC Educational Resources Information Center

    Davies, Jean S.

    1989-01-01

    Discusses developmental encroachment on isolated caves near Pittsford, Vermont. Describes cooperative effort by recreational camps and other agencies to acquire land surrounding cave entrances for conservation. Details successes and hurdles of land acquisition. Describes ongoing work by local campers to maintain and enjoy caves and property. (TES)

  1. Camping and Outdoor Education.

    ERIC Educational Resources Information Center

    Bucher, Charles A.

    Outdoor education has become an integral part of the curriculum in a number of schools across the nation. Outdoor education activities can be readily integrated into physical education, recreation, and adult education programs, as well as science, mathematics, and related fields. Camping and outdoor education should become a part of each child's…

  2. A Flying Summer Camp

    ERIC Educational Resources Information Center

    Mercurio, Frank X.

    1975-01-01

    Describes a five-day summer camp which provided 12 children, ages 9-14, with a complete flying experience. The training consisted of ground school and one hour actual flying time, including the basics of aircraft control and a flight prepared and executed by the students. (MLH)

  3. Recycling at Camp.

    ERIC Educational Resources Information Center

    Cummins, William M.

    1988-01-01

    Outlines a Michigan summer camp's efforts to reduce solid waste disposal by recycling cardboard, tin, glass, aluminum, and plastic milk containers. Points out variables affecting the success of such efforts. Discusses Michigan state funding for the development of recycling programs. (SV)

  4. Camp Animal Crackers.

    ERIC Educational Resources Information Center

    McMillan, Erin; And Others

    This guide contains profiles of 16 activities for young children to participate in while attending camp. Each profile contains the theme of the activity, a list of materials needs, a description of the activity itself, and an explanation of the teacher's role in the activity. The activities focus on: (1) songs and dances; (2) dramatic play; (3)…

  5. Alaska's Logging Camp School.

    ERIC Educational Resources Information Center

    Millward, Robert E.

    1999-01-01

    A visit to Ketchikan, Alaska, reveals a floating, one-teacher logging-camp school that uses multiage grouping and interdisciplinary teaching. There are 10 students. The school gym and playground, bunkhouse, fuel tanks, mess hall, and students' homes bob up and down and are often moved to other sites. (MLH)

  6. Saving the Camping Experience.

    ERIC Educational Resources Information Center

    Davies, Jean S.

    1989-01-01

    Discusses developmental encroachment on isolated caves near Pittsford, Vermont. Describes cooperative effort by recreational camps and other agencies to acquire land surrounding cave entrances for conservation. Details successes and hurdles of land acquisition. Describes ongoing work by local campers to maintain and enjoy caves and property. (TES)

  7. Recycling at Camp.

    ERIC Educational Resources Information Center

    Cummins, William M.

    1988-01-01

    Outlines a Michigan summer camp's efforts to reduce solid waste disposal by recycling cardboard, tin, glass, aluminum, and plastic milk containers. Points out variables affecting the success of such efforts. Discusses Michigan state funding for the development of recycling programs. (SV)

  8. Spatiotemporal regulation of cAMP signaling controls the human trophoblast fusion

    PubMed Central

    Gerbaud, Pascale; Taskén, Kjetil; Pidoux, Guillaume

    2015-01-01

    During human placentation, mononuclear cytotrophoblasts fuse to form multinucleated syncytia ensuring hormonal production and nutrient exchanges between the maternal and fetal circulation. Syncytial formation is essential for the maintenance of pregnancy and for fetal growth. The cAMP signaling pathway is the major route to trigger trophoblast fusion and its activation results in phosphorylation of specific intracellular target proteins, in transcription of fusogenic genes and assembly of macromolecular protein complexes constituting the fusogenic machinery at the plasma membrane. Specificity in cAMP signaling is ensured by generation of localized pools of cAMP controlled by cAMP phosphodiesterases (PDEs) and by discrete spatial and temporal activation of protein kinase A (PKA) in supramolecular signaling clusters inside the cell organized by A-kinase-anchoring proteins (AKAPs) and by organization of signal termination by protein phosphatases (PPs). Here we present original observations on the available components of the cAMP signaling pathway in the human placenta including PKA, PDE, and PP isoforms as well as AKAPs. We continue to discuss the current knowledge of the spatiotemporal regulation of cAMP signaling triggering trophoblast fusion. PMID:26441659

  9. Aip regulates cAMP signalling and GH secretion in GH3 cells.

    PubMed

    Formosa, R; Xuereb-Anastasi, A; Vassallo, J

    2013-08-01

    Mutations in the aryl hydrocarbon receptor-interacting protein (AIP) gene have been linked to predisposition to pituitary adenomas. However, the mechanism by which this occurs remains unknown. AIP interacts with a number of interesting proteins, including members of the cAMP signalling pathway that has been shown to be consistently altered in pituitary tumours. The functional role of Aip was investigated using both over-expression and knock down of Aip in GH3 cells. cAMP signalling and its downstream effectors, including GH secretion, were then investigated. cAMP signalling was analysed using cAMP assays, cAMP-response element-promoter luciferase reporter assays, real-time PCR and finally secreted GH quantification. Over-expression of wild-type (WT)-Aip reduced forskolin-induced cAMP signalling at the total cAMP level, luciferase reporter activity and target gene expression, when compared with empty vector and the non-functional R304X mutant. Additionally, GH secretion was reduced in WT-Aip over-expressing GH3 cells treated with forskolin. Knock down of endogenous Aip resulted in increased cAMP signalling but a decrease in GH secretion was also noted. Inhibition of phosphodiesterase activity using general and selective inhibitors did not completely ablate the effect of Aip on forskolin-augmented cAMP signalling. A mechanism by which Aip acts as a tumour suppressor, by maintaining a low cAMP signalling and concentration, is suggested. Mutations of Aip render the protein incapable of such activity. This effect appears not to be mediated by the AIP-PDE interaction, suggesting the involvement of other interacting partners in mediating this outcome.

  10. UCR1C is a novel activator of phosphodiesterase 4 (PDE4) long isoforms and attenuates cardiomyocyte hypertrophy

    PubMed Central

    Wang, Li; Burmeister, Brian T.; Johnson, Keven R.; Baillie, George S.; Karginov, Andrei V.; Skidgel, Randal A.; O’Bryan, John P.; Carnegie, Graeme K.

    2015-01-01

    Hypertrophy increases the risk of heart failure and arrhythmia. Prevention or reversal of the maladaptive hypertrophic phenotype has thus been proposed to treat heart failure. Chronic β-adrenergic receptor (β-AR) stimulation induces cardiomyocyte hypertrophy by elevating 3′, 5′-cyclic adenosine monophosphate (cAMP) levels and activating downstream effectors such protein kinase A (PKA). Conversely, hydrolysis of cAMP by phosphodiesterases (PDEs) spatiotemporally restricts cAMP signaling. Here, we demonstrate that PDE4, but not PDE3, is critical in regulating cardiomyocyte hypertrophy, and may represent a potential target for preventing maladaptive hypertrophy. We identify a sequence within the upstream conserved region 1 of PDE4D, termed UCR1C, as a novel activator of PDE4 long isoforms. UCR1C activates PDE4 in complex with A-Kinase anchoring protein (AKAP)-Lbc resulting in decreased PKA signaling facilitated by AKAP-Lbc. Expression of UCR1C in cardiomyocytes inhibits hypertrophy in response to chronic β-AR stimulation. This effect is partially due to inhibition of nuclear PKA activity, which decreases phosphorylation of the transcription factor cAMP response element-binding protein (CREB). In conclusion, PDE4 activation by UCR1C attenuates cardiomyocyte hypertrophy by specifically inhibiting nuclear PKA activity. PMID:25683917

  11. [Phosphodiesterase 3 mediates cross-talk between the protein kinase- and cGMP- dependent pathways and cyclic AMP metabolism].

    PubMed

    Makuch, Edyta; Matuszyk, Janusz

    2012-07-20

    PDE3 is a dual-substrate phosphodiesterase responsible for hydrolyzing both cAMP and cGMP whilst being simultaneously inhibited by cGMP. This feature is related to presence of the 44 amino acid insert in the catalytic domain, which determines the mechanism of introduction of the cyclic nucleotide into the catalytic pocket of the enzyme. Once bound in the catalytic site cGMP results in steric hindrance for cAMP to enter the site. The regulatory domain of PDE3 consists of two hydrophobic regions: NHR1 and NHR2. Their presence defines the enzyme's intracellular localization, thus determining its participation in particular signaling cascades. Due to the properties of PDE3 this enzyme has exceptional importance for the cross-talk between cAMP-dependent signaling and other cascades. There are two different mechanisms of action of PDE3 enzymes in cell signaling pathways. In many signaling cascades assembly of a signalosome is necessary for phosphorylation and activation of the PDE3 proteins. In response to certain hormones and growth factors, PDE3 merges the metabolism of cAMP with protein kinase-dependent signaling pathways. PDE3 also controls the level of cAMP with regard to the alternating concentration of cGMP. This effect occurs in signaling cascades activated by natriuretic peptide.

  12. Regulation of high affinity iron uptake in the yeast Saccharomyces cerevisiae. Role of dioxygen and Fe.

    PubMed

    Hassett, R F; Romeo, A M; Kosman, D J

    1998-03-27

    High affinity iron uptake in Saccharomyces cerevisiae requires a metal reductase, a multicopper ferroxidase, and an iron permease. Fet3, the apparent ferroxidase, is proposed to facilitate iron uptake by catalyzing the oxidation of reductase-generated Fe(II) to Fe(III) by O2; in this model, Fe(III) is the substrate for the iron permease, encoded by FTR1 (Kaplan, J., and O'Halloran, T. V. (1996) Science 271, 1510-1512). We show here that dioxygen also plays an essential role in the expression of these iron uptake activities. Cells grown anaerobically exhibited no Fe(III) reductase or high affinity iron uptake activity, even if assayed for these activities under air. Northern blot analysis showed that the amount of those mRNAs encoding proteins associated with this uptake was repressed in anaerobic cultures but was rapidly induced by exposure of the culture to dioxygen. The anaerobic repression was reduced in cells expressing an iron-independent form of the trans-activator, Aft1, a protein that regulates the expression of these proteins. Thus, the effect of oxygenation on this expression appeared due at least in part to the state or distribution of iron in the cells. In support of this hypothesis, the membrane-permeant Fe(II) chelator, 2, 2'-bipyridyl, in contrast to the impermeant chelator bathophenanthroline disulfonate, caused a strong and rapid induction of these transcripts under anaerobic conditions. An increase in the steady-state levels of iron-regulated transcripts upon oxygenation or 2,2'-bipyridyl addition occurred within 5 min, indicating that a relatively small, labile intracellular pool of Fe(II) regulates the expression of these activities. The strength of the anaerobic repression was dependent on the low affinity, Fe(II)-specific iron transporter, encoded by FET4, suggesting that this Fe(II) pool was linked in part to iron brought into the cell via Fet4 protein. The data suggest a model in which dioxygen directly or indirectly modulates the Fe

  13. Melatonin Administration Alters Nicotine Preference Consumption via Signaling Through High-Affinity Melatonin Receptors

    PubMed Central

    Horton, William J.; Gissel, Hannah J.; Saboy, Jennifer E.; Wright, Kenneth P.; Stitzel, Jerry A.

    2015-01-01

    Rationale While it is known that tobacco use varies across the 24-hour day, the time-of-day effects are poorly understood. Findings from several previous studies indicate a potential role for melatonin in these time-of-day effects; however the specific underlying mechanisms have not been well characterized. Understanding of these mechanisms may lead to potential novel smoking cessation treatments. Objective Examine the role of melatonin and melatonin receptors in nicotine free choice consumption Methods A two-bottle oral nicotine choice paradigm was utilized with melatonin supplementation in melatonin deficient mice (C57BL/6J) or without melatonin supplementation in mice proficient at melatonin synthesis (C3H/Ibg) compared to melatonin proficient mice lacking both or one of the high affinity melatonin receptors (MT1 and MT2; double null mutant DM, or MT1 or MT2). Preference for bitter and sweet tastants also was assessed in wild type and MT1 and MT2 DM mice. Finally, home cage locomotor monitoring was performed to determine the effect of melatonin administration on activity patterns. Results Supplemental melatonin in drinking water significantly reduced free-choice nicotine consumption in C57BL/6J mice, which do not produce endogenous melatonin, while not altering activity patterns. Independently, genetic deletion of both MT1 and MT2 receptors in a melatonin proficient mouse strain (C3H) resulted in significantly more nicotine consumption than controls. However single genetic deletion of either the MT1 or MT2 receptor alone did not result in increased nicotine consumption. Deletion of MT1 and MT2 did not impact taste preference. Conclusions This study demonstrates that nicotine consumption can be affected by exogenous or endogenous melatonin and requires at least one of the high-affinity melatonin receptors. The fact that expression of either the MT1 or MT2 melatonin receptor is sufficient to maintain lower nicotine consumption suggests functional overlap and

  14. Cyclic adenosine 3',5'-monophosphate levels and activities of adenylate cyclase and cyclic adenosine 3',5'-monophosphate phosphodiesterase in Pseudomonas and Bacteroides.

    PubMed Central

    Siegel, L S; Hylemon, P B; Phibbs, P V

    1977-01-01

    A modified Gilman assay was used to determine the concentrations of cyclic adenosine 3',5'-monophosphate (cAMP) in rapidly filtered cells and in the culture filtrates of Pseudomonas aeruginosa, Escherichia coli K-12, and Bacteroides fragilis. In P. aeruginosa cultures, levels of cAMP in the filtrate increased with the culture absorbance (3.5 to 19.8 X 10(-9) M) but did not vary significantly with the carbon source used to support growth. Intracellular concentrations (0.8 to 3.2 X 10(-5) M) were substantially higher and did not vary appreciably during growth or with carbon source. Sodium cAMP (5 mM) failed to reverse the catabolite repression of inducible glucose-6-phosphate dehydrogenase (EC 1.1.1.49) synthesis caused by the addition of 10 mM succinate. Exogenous cAMP also had no discernible effect on the catabolite repression control of inducible mannitol dehydrogenase (EC 1.1.1.67). P. aeruginosa was found to contain both soluble cAMP phosphodiesterase (EC 3.1.4.17) and membrane-associated adenylate cyclase (EC 4.6.1.1) activity, and these were compared to the activities detected in crude extracts of E. coli. B. fragilis crude cell extracts contain neither of these enzyme activities, and little or no cAMP was detected in cells or culture filtrates of this anaerobic bacterium. PMID:187575

  15. Identification and Localization of the Cyclic Nucleotide Phosphodiesterase 10A in Bovine Testis and Mature Spermatozoa

    PubMed Central

    Goupil, Serge; Maréchal, Loïze; El Hajj, Hassan; Tremblay, Marie-Ève; Richard, François J.

    2016-01-01

    In mammals, adenosine 3’, 5’-cyclic monophosphate (cAMP) is known to play highly important roles in sperm motility and acrosomal exocytosis. It is known to act through protein phosphorylation via PRKA and through the activation of guanine nucleotide exchange factors like EPAC. Sperm intracellular cAMP levels depend on the activity of adenylyl cyclases, mostly SACY, though transmembrane-containing adenylyl cyclases are also present, and on the activity of cyclic nucleotide phosphodiesterases (PDE) whose role is to degrade cAMP into 5’-AMP. The PDE superfamily is subdivided into 11 families (PDE1 to 11), which act on either cAMP or cGMP, or on both cAMP and cGMP although with different enzymatic properties. PDE10, which is more effective on cAMP than cGMP, has been known for almost 15 years and is mostly studied in the brain where it is associated with neurological disorders. Although a high level of PDE10A gene expression is observed in the testis, information on the identity of the isoforms or on the cell type that express the PDE10 protein is lacking. The objective of this study was to identify the PDE10A isoforms expressed in the testis and germ cells, and to determine the presence and localization of PDE10A in mature spermatozoa. As a sub-objective, since PDE10A transcript variants were reported strictly through analyses of bovine genomic sequence, we also wanted to determine the nucleotide and amino acid sequences by experimental evidence. Using RT-PCR, 5’- and 3’-RACE approaches we clearly show that PDE10A transcript variants X3 and X5 are expressed in bovine testis as well as in primary spermatocytes and spermatids. We also reveal using a combination of immunological techniques and proteomics analytical tools that the PDE10A isoform X4 is present in the area of the developing acrosome of spermatids and of the acrosome of mature spermatozoa. PMID:27548062

  16. Roles of phosphodiesterases in the regulation of the cardiac cyclic nucleotide cross-talk signaling network.

    PubMed

    Zhao, Claire Y; Greenstein, Joseph L; Winslow, Raimond L

    2016-02-01

    The balanced signaling between the two cyclic nucleotides (cNs) cAMP and cGMP plays a critical role in regulating cardiac contractility. Their degradation is controlled by distinctly regulated phosphodiesterase isoenzymes (PDEs), which in turn are also regulated by these cNs. As a result, PDEs facilitate communication between the β-adrenergic and Nitric Oxide (NO)/cGMP/Protein Kinase G (PKG) signaling pathways, which regulate the synthesis of cAMP and cGMP respectively. The phenomena in which the cAMP and cGMP pathways influence the dynamics of each other are collectively referred to as cN cross-talk. However, the cross-talk response and the individual roles of each PDE isoenzyme in shaping this response remain to be fully characterized. We have developed a computational model of the cN cross-talk network that mechanistically integrates the β-adrenergic and NO/cGMP/PKG pathways via regulation of PDEs by both cNs. The individual model components and the integrated network model replicate experimentally observed activation-response relationships and temporal dynamics. The model predicts that, due to compensatory interactions between PDEs, NO stimulation in the presence of sub-maximal β-adrenergic stimulation results in an increase in cytosolic cAMP accumulation and corresponding increases in PKA-I and PKA-II activation; however, the potentiation is small in magnitude compared to that of NO activation of the NO/cGMP/PKG pathway. In a reciprocal manner, β-adrenergic stimulation in the presence of sub-maximal NO stimulation results in modest cGMP elevation and corresponding increase in PKG activation. In addition, we demonstrate that PDE2 hydrolyzes increasing amounts of cAMP with increasing levels of β-adrenergic stimulation, and hydrolyzes increasing amounts of cGMP with decreasing levels of NO stimulation. Finally, we show that PDE2 compensates for inhibition of PDE5 both in terms of cGMP and cAMP dynamics, leading to cGMP elevation and increased PKG activation

  17. New Synthesis and Tritium Labeling of a Selective Ligand for Studying High-affinity γ-Hydroxybutyrate (GHB) Binding Sites

    PubMed Central

    Vogensen, Stine B.; Marek, Aleš; Bay, Tina; Wellendorph, Petrine; Kehler, Jan; Bundgaard, Christoffer; Frølund, Bente; Pedersen, Martin H.F.; Clausen, Rasmus P.

    2013-01-01

    3-Hydroxycyclopent-1-enecarboxylic acid (HOCPCA, 1) is a potent ligand for the high-affinity GHB binding sites in the CNS. An improved synthesis of 1 together with a very efficient synthesis of [3H]-1 is described. The radiosynthesis employs in situ generated lithium trimethoxyborotritide. Screening of 1 against different CNS targets establishes a high selectivity and we demonstrate in vivo brain penetration. In vitro characterization of [3H]-1 binding shows high specificity to the high-affinity GHB binding sites. PMID:24053696

  18. Phosphodiesterase activity is a novel property of alkaline phosphatase from osseous plate.

    PubMed Central

    Rezende, A A; Pizauro, J M; Ciancaglini, P; Leone, F A

    1994-01-01

    Phosphodiesterase activity is a novel property of the still-enigmatic alkaline phosphatase from osseous plate. Bis-(p-nitrophenyl) phosphate was hydrolysed at both pH 7.5 and 9.4 with an apparent dissociation constant (K0.5) of 1.9 mM and 3.9 mM respectively. The hydrolysis of p-nitrophenyl-5'-thymidine phosphate followed hyberbolic kinetics with a K0.5 of 500 microM. For p-nitrophenyl phenylphosphonate, site-site interactions [Hill coefficient (h) = 1.3] were observed in the range between 0.2 and 100 microM, and K0.5 was 32.8 mM. The hydrolysis of cyclic AMP by the enzyme followed more complex kinetics, showing site-site interactions (h = 1.7) and K0.5 = 300 microM for high-affinity sites. The low-affinity sites, representing 85% of total activity, also showed site-site interactions (h = 3.8) and a K0.5 of about 22 mM. ATP and cyclic AMP were competitive inhibitors of bis-(p-nitrophenyl) phosphatase activity of the enzyme and Ki values (25 mM and 0.6 mM for cyclic AMP and ATP respectively) very close to those of the K0.5 (22 mM and 0.7 mM for cyclic AMP and ATP respectively), determined by direct assay, indicated that a single catalytic site was responsible for the hydrolysis of both substrates. Non-denaturing PAGE of detergent-solubilized enzyme showed coincident bands on the gel for phosphomonohydrolase and phosphodiesterase activities. Additional evidence for a single catalytic site was the similar pKa values (8.5 and 9.7) found for the two ionizing groups participating in the hydrolysis of bis-(p-nitrophenyl) phosphate and p-nitrophenyl phosphate. The alkaline apparent pH optima, the requirement for bivalent metal ions and the inhibition by methylxanthines, amrinone and amiloride demonstrated that rat osseous-plate alkaline phosphatase was a type I phosphodiesterase. Considering that there is still confusion as to which is the physiological substrate for the enzyme, the present results describing a novel property for this enzyme could be of relevance in

  19. Restricted spatial expression of a high-affinity phosphate transporter in potato roots.

    PubMed

    Gordon-Weeks, Ruth; Tong, Yiping; Davies, T G Emyr; Leggewie, Georg

    2003-08-01

    Phosphorus deficiency limits plant growth, and high-affinity phosphate transporters, of the Pht1 family, facilitate phosphate uptake and translocation. The family is subdivided into root specific, phosphate deprivation induced members and those also expressed in leaves. An antibody to StPT2, a potato root specific transporter, detected two bands (52 kDa and 30 kDa) on western blots of root plasma membrane extracts that were most intense in whole extracts from the root tip and slightly increased throughout the root in response to phosphate depletion. RT-PCR, using StPT2 specific primers, confirmed these findings. Low power confocal immunofluorescent images showed StPT2 expression mainly in the elongation zone at the root tip. By contrast, a vacuolar pyrophosphatase and a plasma membrane ATPase antibody labelled the whole root. High power images showed, by comparison with alpha-tubulin, cell wall and plasma membrane ATPase labelling, that StPT2 was in the epidermal plasma membrane and restricted to the apical surface. This is the first evidence of polar plasma membrane localisation of a plant nutrient transporter and is consistent with a role for StPT2 in phosphate capture and uptake.

  20. Molecular evolutionary analysis of the high-affinity K+ transporter gene family in angiosperms.

    PubMed

    Yang, P; Hua, C; Zhou, F; Zhang, B-J; Cai, X-N; Chen, Q-Z; Wang, R-L

    2016-07-15

    The high-affinity K(+) transporter (HKT) family comprises a group of multifunctional cation transporters widely distributed in organisms ranging from Bacteria to Eukarya. In angiosperms, the HKT family consists primarily of nine types, whose evolutionary relationships are not fully understood. The available sequences from 31 plant species were used to perform a comprehensive evolutionary analysis, including an examination of selection pressure and estimating phylogenetic tree and gene duplication events. Our results show that a gene duplication in the HKT1;5/HKT1;4 cluster might have led to the divergence of the HKT1;5 and HKT1;4 subfamilies. Additionally, maximum likelihood analysis revealed that the HKT family has undergone a strong purifying selection. An analysis of the amino acids provided strong statistical evidence for a functional divergence between subfamilies 1 and 2. Our study was the first to provide evidence of this functional divergence between these two subfamilies. Analysis of co-evolution in HKT identified 25 co-evolved groups. These findings expanded our understanding of the evolutionary mechanisms driving functional diversification of HKT proteins.

  1. Monochromatic multicomponent fluorescence sedimentation velocity for the study of high-affinity protein interactions.

    PubMed

    Zhao, Huaying; Fu, Yan; Glasser, Carla; Andrade Alba, Eric J; Mayer, Mark L; Patterson, George; Schuck, Peter

    2016-07-20

    The dynamic assembly of multi-protein complexes underlies fundamental processes in cell biology. A mechanistic understanding of assemblies requires accurate measurement of their stoichiometry, affinity and cooperativity, and frequently consideration of multiple co-existing complexes. Sedimentation velocity analytical ultracentrifugation equipped with fluorescence detection (FDS-SV) allows the characterization of protein complexes free in solution with high size resolution, at concentrations in the nanomolar and picomolar range. Here, we extend the capabilities of FDS-SV with a single excitation wavelength from single-component to multi-component detection using photoswitchable fluorescent proteins (psFPs). We exploit their characteristic quantum yield of photo-switching to imprint spatio-temporal modulations onto the sedimentation signal that reveal different psFP-tagged protein components in the mixture. This novel approach facilitates studies of heterogeneous multi-protein complexes at orders of magnitude lower concentrations and for higher-affinity systems than previously possible. Using this technique we studied high-affinity interactions between the amino-terminal domains of GluA2 and GluA3 AMPA receptors.

  2. Evidence that synaptosomal high-affinity carriers for amino acid neurotransmitters are glycosylated

    SciTech Connect

    Zaleska, M.M.; Erecinska, M.

    1987-05-01

    The effect of removal of surface sialic acid from synaptosomes on the high-affinity, Na/sup +/-dependent uptake systems for amino acid neurotransmitters was evaluated. Synaptosomes from rat forebrain were preincubated with neuraminidase from Vibrio cholerae for 20 min at 34/sup 0/. After washing and resuspension, their ability to transport /sup 14/C-GABA and the acidic amino acid, /sup 3/H-aspartate was studied. Pretreatment with neuraminidase resulted in a concentration-dependent inhibition of the uptake of both amino acids while the influx of /sup 3/H-L-leucine was unaffected. Inhibition was a function of the amount of sialic acid released from membranes. The analysis of the kinetic parameters of amino acid uptake revealed that inhibition resulted from a decrease of Vmax without any change in the Km. Neither the synaptosomal energy levels nor the internal concentration of potassium ions was affected by the pretreatment with neuraminidase. The maximum accumulation ratios for both amino acids remained largely unaltered. It is concluded that the GABA and acidic amino acid transporters are glycosylated and that sialic acid is involved in the operation of carrier proteins directly and not through modification of the driving forces responsible for amino acid transport.

  3. High-affinity interaction of tyrosine kinase inhibitors with the ABCG2 multidrug transporter.

    PubMed

    Ozvegy-Laczka, Csilla; Hegedus, Tamás; Várady, György; Ujhelly, Olga; Schuetz, John D; Váradi, András; Kéri, György; Orfi, László; Német, Katalin; Sarkadi, Balázs

    2004-06-01

    Tyrosine kinase inhibitors (TKIs) are promising new agents for specific inhibition of malignant cell growth and metastasis formation. Because most of the TKIs have to reach an intracellular target, specific membrane transporters may significantly modulate their effectiveness. In addition, the hydrophobic TKIs may interact with so-called multidrug transporters and thus alter the cellular distribution of unrelated pharmacological agents. In the present work, we show that certain TKIs, already in the clinical phase of drug development, directly interact with the ABCG2 multidrug transporter protein with a high affinity. We found that in several in vitro assay systems, STI-571 (Gleevec; imatinib mesylate), ZD1839 (Iressa; gefitinib), and N-[4-[(3-bromophenyl)amino]-6-quinazolinyl]-2-butynamide (EKI-785) interacted with ABCG2 at submicromolar concentrations, whereas other multidrug transporters, human multidrug resistance protein (P-glycoprotein, ABCB1) and human multidrug resistance protein 1 (ABCC1), showed much lower reactivity toward these agents. Low concentrations of the TKIs examined selectively modulated ABCG2-ATPase activity, inhibited ABCG2-dependent active drug extrusion, and significantly affected drug resistance patterns in cells expressing ABCG2. Our results indicate that multidrug resistance protein modulation by TKIs may be an important factor in the clinical treatment of cancer patients. These data also raise the possibility that an extrusion of TKIs by multidrug transporters, e.g., ABCG2, may be involved in tumor cell TKI resistance.

  4. High affinity truncated DNA aptamers for the development of fluorescence based progesterone biosensors.

    PubMed

    Alhadrami, Hani A; Chinnappan, Raja; Eissa, Shimaa; Rahamn, Anas Abdel; Zourob, Mohammed

    2017-02-24

    Aptamers have shown a number of potential applications in sensing and therapeutic due to the high affinity and specificity towards their target molecules. Not all the nucleotides in the full length aptamers are involved in the binding with their targets. The non-binding domain of the aptamer may affect the binding affinity of the aptamer-target complex. Mapping the aptamer binding region could increase the affinity and the specificity. In this paper, we designed aptamer-based fluorescence sensors from a truncated progesterone (P4) aptamer. Then, fluorescein and quencher labelled aptamer complementary oligonucleotide sequences were hybridized to the truncated aptamer at different sites to form duplex structures. We used fluorescence-quencher pair displacement assay upon progesterone binding for the determination of P4. One of the truncated sequences has shown high binding affinity with 16 fold increase in the dissociation constant, Kd (2.1 nM) compared to the original aptamer. The aptasensor was highly selective for P4 against similar compounds such as 17-β estradiol, bisphenol-A and vitamin D. The sensor has been applied for the detection of P4 in spiked tap water and in urine samples showing good recovery. This new developed aptamer-based fluorescence biosensor can be applied in food, pharmaceutical, and clinical industries.

  5. Identification of LAG3 high affinity aptamers by HT-SELEX and Conserved Motif Accumulation (CMA).

    PubMed

    Soldevilla, Mario Martínez; Hervas, Sandra; Villanueva, Helena; Lozano, Teresa; Rabal, Obdulia; Oyarzabal, Julen; Lasarte, Juan José; Bendandi, Maurizio; Inoges, Susana; López-Díaz de Cerio, Ascensión; Pastor, Fernando

    2017-01-01

    LAG3 receptor belongs to a family of immune-checkpoints expressed in T lymphocytes and other cells of the immune system. It plays an important role as a rheostat of the immune response. Focus on this receptor as a potential therapeutic target in cancer immunotherapy has been underscored after the success of other immune-checkpoint blockade strategies in clinical trials. LAG3 showcases the interest in the field of autoimmunity as several studies show that LAG3-targeting antibodies can also be used for the treatment of autoimmune diseases. In this work we describe the identification of a high-affinity LAG3 aptamer by High Throughput Sequencing SELEX in combination with a study of potential conserved binding modes according to sequence conservation by using 2D-structure prediction and 3D-RNA modeling using Rosetta. The aptamer with the highest accumulation of these conserved sequence motifs displays the highest affinity to LAG3 recombinant soluble proteins and binds to LAG3-expressing lymphocytes. The aptamer described herein has the potential to be used as a therapeutic agent, as it enhances the threshold of T-cell activation. Nonetheless, in future applications, it could also be engineered for treatment of autoimmune diseases by target depletion of LAG3-effector T lymphocytes.

  6. Consequences of inducing intrinsic disorder in a high-affinity protein-protein interaction.

    PubMed

    Papadakos, Grigorios; Sharma, Amit; Lancaster, Lorna E; Bowen, Rebecca; Kaminska, Renata; Leech, Andrew P; Walker, Daniel; Redfield, Christina; Kleanthous, Colin

    2015-04-29

    The kinetic and thermodynamic consequences of intrinsic disorder in protein-protein recognition are controversial. We address this by inducing one partner of the high-affinity colicin E3 rRNase domain-Im3 complex (K(d) ≈ 10(-12) M) to become an intrinsically disordered protein (IDP). Through a variety of biophysical measurements, we show that a single alanine mutation at Tyr507 within the hydrophobic core of the isolated colicin E3 rRNase domain causes the enzyme to become an IDP (E3 rRNase(IDP)). E3 rRNase(IDP) binds stoichiometrically to Im3 and forms a structure that is essentially identical to the wild-type complex. However, binding of E3 rRNase(IDP) to Im3 is 4 orders of magnitude weaker than that of the folded rRNase, with thermodynamic parameters reflecting the disorder-to-order transition on forming the complex. Critically, pre-steady-state kinetic analysis of the E3 rRNase(IDP)-Im3 complex demonstrates that the decrease in affinity is mostly accounted for by a drop in the electrostatically steered association rate. Our study shows that, notwithstanding the advantages intrinsic disorder brings to biological systems, this can come at severe kinetic and thermodynamic cost.

  7. The high-affinity HSP90-CHIP complex recognizes and selectively degrades phosphorylated tau client proteins

    PubMed Central

    Dickey, Chad A.; Kamal, Adeela; Lundgren, Karen; Klosak, Natalia; Bailey, Rachel M.; Dunmore, Judith; Ash, Peter; Shoraka, Sareh; Zlatkovic, Jelena; Eckman, Christopher B.; Patterson, Cam; Dickson, Dennis W.; Nahman, N. Stanley; Hutton, Michael; Burrows, Francis; Petrucelli, Leonard

    2007-01-01

    A primary pathologic component of Alzheimer’s disease (AD) is the formation of neurofibrillary tangles composed of hyperphosphorylated tau (p-tau). Expediting the removal of these p-tau species may be a relevant therapeutic strategy. Here we report that inhibition of Hsp90 led to decreases in p-tau levels independent of heat shock factor 1 (HSF1) activation. A critical mediator of this mechanism was carboxy terminus of Hsp70–interacting protein (CHIP), a tau ubiquitin ligase. Cochaperones were also involved in Hsp90-mediated removal of p-tau, while those of the mature Hsp90 refolding complex prevented this effect. This is the first demonstration to our knowledge that blockade of the refolding pathway promotes p-tau turnover through degradation. We also show that peripheral administration of a novel Hsp90 inhibitor promoted selective decreases in p-tau species in a mouse model of tauopathy, further suggesting a central role for the Hsp90 complex in the pathogenesis of tauopathies. When taken in the context of known high-affinity Hsp90 complexes in affected regions of the AD brain, these data implicate a central role for Hsp90 in the development of AD and other tauopathies and may provide a rationale for the development of novel Hsp90-based therapeutic strategies. PMID:17304350

  8. Definition of a high-affinity Gag recognition structure mediating packaging of a retroviral RNA genome

    PubMed Central

    Gherghe, Cristina; Lombo, Tania; Leonard, Christopher W.; Datta, Siddhartha A. K.; Bess, Julian W.; Gorelick, Robert J.; Rein, Alan; Weeks, Kevin M.

    2010-01-01

    All retroviral genomic RNAs contain a cis-acting packaging signal by which dimeric genomes are selectively packaged into nascent virions. However, it is not understood how Gag (the viral structural protein) interacts with these signals to package the genome with high selectivity. We probed the structure of murine leukemia virus RNA inside virus particles using SHAPE, a high-throughput RNA structure analysis technology. These experiments showed that NC (the nucleic acid binding domain derived from Gag) binds within the virus to the sequence UCUG-UR-UCUG. Recombinant Gag and NC proteins bound to this same RNA sequence in dimeric RNA in vitro; in all cases, interactions were strongest with the first U and final G in each UCUG element. The RNA structural context is critical: High-affinity binding requires base-paired regions flanking this motif, and two UCUG-UR-UCUG motifs are specifically exposed in the viral RNA dimer. Mutating the guanosine residues in these two motifs—only four nucleotides per genomic RNA—reduced packaging 100-fold, comparable to the level of nonspecific packaging. These results thus explain the selective packaging of dimeric RNA. This paradigm has implications for RNA recognition in general, illustrating how local context and RNA structure can create information-rich recognition signals from simple single-stranded sequence elements in large RNAs. PMID:20974908

  9. Proteomic High Affinity Zn2+ Trafficking: Where Does Metallothionein Fit in?

    PubMed Central

    Petering, David H.; Mahim, Afsana

    2017-01-01

    The cellular constitution of Zn-proteins and Zn-dependent signaling depend on the capacity of Zn2+ to find specific binding sites in the face of a plethora of other high affinity ligands. The most prominent of these is metallothionein (MT). It serves as a storage site for Zn2+ under various conditions, and has chemical properties that support a dynamic role for MT in zinc trafficking. Consistent with these characteristics, changing the availability of zinc for cells and tissues causes rapid alteration of zinc bound to MT. Nevertheless, zinc trafficking occurs in metallothionein-null animals and cells, hypothetically making use of proteomic binding sites to mediate the intracellular movements of zinc. Like metallothionein, the proteome contains a large concentration of proteins that strongly coordinate zinc. In this environment, free Zn2+ may be of little significance. Instead, this review sets forth the basis for the hypothesis that components of the proteome and MT jointly provide the platform for zinc trafficking. PMID:28629147

  10. Characterization of high affinity binding motifs for the discoidin domain receptor DDR2 in collagen.

    PubMed

    Konitsiotis, Antonios D; Raynal, Nicolas; Bihan, Dominique; Hohenester, Erhard; Farndale, Richard W; Leitinger, Birgit

    2008-03-14

    The discoidin domain receptors, DDR1 and DDR2, are receptor tyrosine kinases that are activated by native triple-helical collagen. Here we have located three specific DDR2 binding sites by screening the entire triple-helical domain of collagen II, using the Collagen II Toolkit, a set of overlapping triple-helical peptides. The peptide sequence that bound DDR2 with highest affinity interestingly contained the sequence for the high affinity binding site for von Willebrand factor in collagen III. Focusing on this sequence, we used a set of truncated and alanine-substituted peptides to characterize the sequence GVMGFO (O is hydroxyproline) as the minimal collagen sequence required for DDR2 binding. Based on a recent NMR analysis of the DDR2 collagen binding domain, we generated a model of the DDR2-collagen interaction that explains why a triple-helical conformation is required for binding. Triple-helical peptides comprising the DDR2 binding motif not only inhibited DDR2 binding to collagen II but also activated DDR2 transmembrane signaling. Thus, DDR2 activation may be effected by single triple-helices rather than fibrillar collagen.

  11. New Regulatory Roles of Galectin-3 in High-Affinity IgE Receptor Signaling.

    PubMed

    Bambouskova, Monika; Polakovicova, Iva; Halova, Ivana; Goel, Gautam; Draberova, Lubica; Bugajev, Viktor; Doan, Aivi; Utekal, Pavol; Gardet, Agnes; Xavier, Ramnik J; Draber, Petr

    2016-05-01

    Aggregation of the high-affinity receptor for IgE (FcεRI) in mast cells initiates activation events that lead to degranulation and release of inflammatory mediators. To better understand the signaling pathways and genes involved in mast cell activation, we developed a high-throughput mast cell degranulation assay suitable for RNA interference experiments using lentivirus-based short hairpin RNA (shRNA) delivery. We tested 432 shRNAs specific for 144 selected genes for effects on FcεRI-mediated mast cell degranulation and identified 15 potential regulators. In further studies, we focused on galectin-3 (Gal3), identified in this study as a negative regulator of mast cell degranulation. FcεRI-activated cells with Gal3 knockdown exhibited upregulated tyrosine phosphorylation of spleen tyrosine kinase and several other signal transduction molecules and enhanced calcium response. We show that Gal3 promotes internalization of IgE-FcεRI complexes; this may be related to our finding that Gal3 is a positive regulator of FcεRI ubiquitination. Furthermore, we found that Gal3 facilitates mast cell adhesion and motility on fibronectin but negatively regulates antigen-induced chemotaxis. The combined data indicate that Gal3 is involved in both positive and negative regulation of FcεRI-mediated signaling events in mast cells.

  12. Stable U(IV) complexes form at high-affinity mineral surface sites.

    PubMed

    Latta, Drew E; Mishra, Bhoopesh; Cook, Russell E; Kemner, Kenneth M; Boyanov, Maxim I

    2014-01-01

    Uranium (U) poses a significant contamination hazard to soils, sediments, and groundwater due to its extensive use for energy production. Despite advances in modeling the risks of this toxic and radioactive element, lack of information about the mechanisms controlling U transport hinders further improvements, particularly in reducing environments where U(IV) predominates. Here we establish that mineral surfaces can stabilize the majority of U as adsorbed U(IV) species following reduction of U(VI). Using X-ray absorption spectroscopy and electron imaging analysis, we find that at low surface loading, U(IV) forms inner-sphere complexes with two metal oxides, TiO2 (rutile) and Fe3O4 (magnetite) (at <1.3 U nm(-2) and <0.037 U nm(-2), respectively). The uraninite (UO2) form of U(IV) predominates only at higher surface loading. U(IV)-TiO2 complexes remain stable for at least 12 months, and U(IV)-Fe3O4 complexes remain stable for at least 4 months, under anoxic conditions. Adsorbed U(IV) results from U(VI) reduction by Fe(II) or by the reduced electron shuttle AH2QDS, suggesting that both abiotic and biotic reduction pathways can produce stable U(IV)-mineral complexes in the subsurface. The observed control of high-affinity mineral surface sites on U(IV) speciation helps explain the presence of nonuraninite U(IV) in sediments and has important implications for U transport modeling.

  13. Evolved Streptavidin Mutants Reveal Key Role of Loop Residue in High-affinity Binding

    SciTech Connect

    M Magalhaes; C Melo Czekster; R Guan; V Malashkevich; S Almo; M Levy

    2011-12-31

    We have performed a detailed analysis of streptavidin variants with altered specificity towards desthiobiotin. In addition to changes in key residues which widen the ligand binding pocket and accommodate the more structurally flexible desthiobiotin, the data revealed the role of a key, non-active site mutation at the base of the flexible loop (S52G) which slows dissociation of this ligand by approximately sevenfold. Our data suggest that this mutation results in the loss of a stabilizing contact which keeps this loop open and accessible in the absence of ligand. When this mutation was introduced into the wild-type protein, destabilization of the opened loop conferred a {approx}10-fold decrease in both the on-rate and off-rate for the ligand biotin-4-fluoroscein. A similar effect was observed when this mutation was added to a monomeric form of this protein. Our results provide key insight into the role of the streptavidin flexible loop in ligand binding and maintaining high affinity interactions.

  14. Expression of high affinity folate receptor in breast cancer brain metastasis.

    PubMed

    Leone, José Pablo; Bhargava, Rohit; Theisen, Brian K; Hamilton, Ronald L; Lee, Adrian V; Brufsky, Adam M

    2015-10-06

    High affinity folate receptor (HFR) can be overexpressed in breast cancer and is associated with poor prognosis, however the expression in breast cancer brain metastases (BCBM) is unknown. The aim of this study was to analyze the rate of HFR expression in BCBM and its role in the prognosis of this high-risk cohort. We analyzed 19 brain metastasis (BM) and 13 primary tumors (PT) from a total of 25 patients. HFR status was assessed by immunohistochemistry. Median follow-up was 4.2 years (range 0.6-18.5). HFR was positive in 4/19 BM (21.1%) and in 1/13 PT (7.7%). Positive samples had low H-scores (range 1-50). 56% of patients had apocrine differentiation. OS was similar between patients with positive HFR (median OS 48 months) and negative HFR (median OS 69 months) (P = 0.25); and between patients with apocrine differentiation (median OS 63 months) and those without apocrine differentiation (median OS 69 months) (P = 0.49). To the best of our knowledge, this is the first analysis of HFR expression in BCBM. While previous studies associated the presence of HFR with worse prognosis; in our cohort HFR was positive in only 21.1% of BM with low levels of positivity. Neither HFR nor apocrine features had impact in OS.

  15. Monochromatic multicomponent fluorescence sedimentation velocity for the study of high-affinity protein interactions

    PubMed Central

    Zhao, Huaying; Fu, Yan; Glasser, Carla; Andrade Alba, Eric J; Mayer, Mark L; Patterson, George; Schuck, Peter

    2016-01-01

    The dynamic assembly of multi-protein complexes underlies fundamental processes in cell biology. A mechanistic understanding of assemblies requires accurate measurement of their stoichiometry, affinity and cooperativity, and frequently consideration of multiple co-existing complexes. Sedimentation velocity analytical ultracentrifugation equipped with fluorescence detection (FDS-SV) allows the characterization of protein complexes free in solution with high size resolution, at concentrations in the nanomolar and picomolar range. Here, we extend the capabilities of FDS-SV with a single excitation wavelength from single-component to multi-component detection using photoswitchable fluorescent proteins (psFPs). We exploit their characteristic quantum yield of photo-switching to imprint spatio-temporal modulations onto the sedimentation signal that reveal different psFP-tagged protein components in the mixture. This novel approach facilitates studies of heterogeneous multi-protein complexes at orders of magnitude lower concentrations and for higher-affinity systems than previously possible. Using this technique we studied high-affinity interactions between the amino-terminal domains of GluA2 and GluA3 AMPA receptors. DOI: http://dx.doi.org/10.7554/eLife.17812.001 PMID:27436096

  16. Lymphocyte crawling and transendothelial migration require chemokine triggering of high-affinity LFA-1 integrin.

    PubMed

    Shulman, Ziv; Shinder, Vera; Klein, Eugenia; Grabovsky, Valentin; Yeger, Orna; Geron, Erez; Montresor, Alessio; Bolomini-Vittori, Matteo; Feigelson, Sara W; Kirchhausen, Tomas; Laudanna, Carlo; Shakhar, Guy; Alon, Ronen

    2009-03-20

    Endothelial chemokines are instrumental for integrin-mediated lymphocyte adhesion and transendothelial migration (TEM). By dissecting how chemokines trigger lymphocyte integrins to support shear-resistant motility on and across cytokine-stimulated endothelial barriers, we found a critical role for high-affinity (HA) LFA-1 integrin in lymphocyte crawling on activated endothelium. Endothelial-presented chemokines triggered HA-LFA-1 and adhesive filopodia at numerous submicron dots scattered underneath crawling lymphocytes. Shear forces applied to endothelial-bound lymphocytes dramatically enhanced filopodia density underneath crawling lymphocytes. A fraction of the adhesive filopodia invaded the endothelial cells prior to and during TEM and extended large subluminal leading edge containing dots of HA-LFA-1 occupied by subluminal ICAM-1. Memory T cells generated more frequent invasive filopodia and transmigrated more rapidly than their naive counterparts. We propose that shear forces exerted on HA-LFA-1 trigger adhesive and invasive filopodia at apical endothelial surfaces and thereby promote lymphocyte crawling and probing for TEM sites.

  17. Comparison of three high affinity SPECT radiotracers for the dopamine D2 receptor.

    PubMed

    al-Tikriti, M S; Baldwin, R M; Zea-Ponce, Y; Sybirska, E; Zoghbi, S S; Laruelle, M; Malison, R T; Kung, H F; Kessler, R M; Charney, D S

    1994-02-01

    The regional brain distribution and pharmacological specificity of three high affinity tracers for the dopamine (DA) D2 receptor: [123I]IBF, [123I]epidepride, and [123I]2'-ISP were assessed by SPECT imaging of non-human primates. The ratios of striatal-to-occipital activities at the time of peak striatal uptake were 2.2, 6.3 and 1.7, respectively. From the peak striatal activities, washout rates were 33, 4 and 16%/h for [123I]IBF, [123I]epidepride and [123I]2'-ISP, respectively. The reversibility of the striatal uptake of all three agents was demonstrated by the rapid displacement induced by the dopamine D2 selective antipsychotic agent raclopride, which increased washout rates to 96, 58 and 43%/h. The administration of d-amphetamine, which induces release of dopamine, had no noticeable effect on [123I]epidepride but increased the washout rate of [123I]IBF. These results suggest that, among these three agents, [123I]epidepride is the superior tracer for in vivo displacement studies because of its slow washout and high target-to-background ratios. However, for tracer kinetic modeling, [123I]IBF may be the superior agent because of its early time of peak uptake and its higher target-to-background ratios than [123I]2'-ISP.

  18. Hydroxamate Production as a High Affinity Iron Acquisition Mechanism in Paracoccidioides Spp

    PubMed Central

    Silva-Bailão, Mirelle Garcia; Bailão, Elisa Flávia Luiz Cardoso; Lechner, Beatrix Elisabeth; Gauthier, Gregory M.; Lindner, Herbert; Bailão, Alexandre Melo; Haas, Hubertus; de Almeida Soares, Célia Maria

    2014-01-01

    Iron is a micronutrient required by almost all living organisms, including fungi. Although this metal is abundant, its bioavailability is low either in aerobic environments or within mammalian hosts. As a consequence, pathogenic microorganisms evolved high affinity iron acquisition mechanisms which include the production and uptake of siderophores. Here we investigated the utilization of these molecules by species of the Paracoccidioides genus, the causative agents of a systemic mycosis. It was demonstrated that iron starvation induces the expression of Paracoccidioides ortholog genes for siderophore biosynthesis and transport. Reversed-phase HPLC analysis revealed that the fungus produces and secretes coprogen B, which generates dimerumic acid as a breakdown product. Ferricrocin and ferrichrome C were detected in Paracoccidioides as the intracellular produced siderophores. Moreover, the fungus is also able to grow in presence of siderophores as the only iron sources, demonstrating that beyond producing, Paracoccidioides is also able to utilize siderophores for growth, including the xenosiderophore ferrioxamine. Exposure to exogenous ferrioxamine and dimerumic acid increased fungus survival during co-cultivation with macrophages indicating that these molecules play a role during host-pathogen interaction. Furthermore, cross-feeding experiments revealed that Paracoccidioides siderophores promotes growth of Aspergillus nidulans strain unable to produce these iron chelators. Together, these data denote that synthesis and utilization of siderophores is a mechanism used by Paracoccidioides to surpass iron limitation. As iron paucity is found within the host, siderophore production may be related to fungus pathogenicity. PMID:25157575

  19. Coordinated transporter activity shapes high-affinity iron acquisition in cyanobacteria

    PubMed Central

    Kranzler, Chana; Lis, Hagar; Finkel, Omri M; Schmetterer, Georg; Shaked, Yeala; Keren, Nir

    2014-01-01

    Iron bioavailability limits biological activity in many aquatic and terrestrial environments. Broad scale genomic meta-analyses indicated that within a single organism, multiple iron transporters may contribute to iron acquisition. Here, we present a functional characterization of a cyanobacterial iron transport pathway that utilizes concerted transporter activities. Cyanobacteria are significant contributors to global primary productivity with high iron demands. Certain cyanobacterial species employ a siderophore-mediated uptake strategy; however, many strains possess neither siderophore biosynthesis nor siderophore transport genes. The unicellular, planktonic, freshwater cyanobacterium, Synechocystis sp. PCC 6803, employs an alternative to siderophore-based uptake-reduction of Fe(III) species before transport through the plasma membrane. In this study, we combine short-term radioactive iron uptake and reduction assays with a range of disruption mutants to generate a working model for iron reduction and uptake in Synechocystis sp. PCC 6803. We found that the Fe(II) transporter, FeoB, is the major iron transporter in this organism. In addition, we uncovered a link between a respiratory terminal oxidase (Alternate Respiratory Terminal Oxidase) and iron reduction - suggesting a coupling between these two electron transfer reactions. Furthermore, quantitative RNA transcript analysis identified a function for subunits of the Fe(III) transporter, FutABC, in modulating reductive iron uptake. Collectively, our results provide a molecular basis for a tightly coordinated, high-affinity iron transport system. PMID:24088625

  20. Off-rate screening for selection of high-affinity anti-drug antibodies.

    PubMed

    Ylera, Francisco; Harth, Stefan; Waldherr, Dirk; Frisch, Christian; Knappik, Achim

    2013-10-15

    The rapidly increasing number of therapeutic antibodies in clinical development and on the market requires corresponding detection reagents for monitoring the concentration of these drugs in patient samples and as positive controls for measurement of anti-drug antibodies. Phage display of large recombinant antibody libraries has been shown to enable the rapid development of fully human anti-idiotypic antibodies binding specifically to antibody drugs, since the in vitro panning approach allows for incorporation of suitable blockers to drive selection toward the paratope of the drug. A typical bottleneck in antibody generation projects is ranking of the many candidates obtained after panning on the basis of antibody binding strength. Ideally, such method will work without prior labeling of antigens and with crude bacterial lysates. We developed an off-rate screening method of crude Escherichia coli lysates containing monovalent Fab fragments obtained after phage display of the HuCAL PLATINUM® antibody library. We used the antibody drugs trastuzumab and cetuximab as antigen examples. Using the Octet® RED384 label-free sensor instrument we show that antibody off rates can be reliably determined in crude bacterial lysates with high throughput. We also demonstrate that the method can be applied to screening for high-affinity antibodies typically obtained after affinity maturation. Copyright © 2013 The Authors. Published by Elsevier Inc. All rights reserved.

  1. A high affinity Ca2(+)-ATPase on the surface membrane of Leishmania donovani promastigote

    SciTech Connect

    Ghosh, J.; Ray, M.; Sarkar, S.; Bhaduri, A. )

    1990-07-05

    A Ca2(+)-dependent ATP-hydrolytic activity was detected in the crude membrane ghost of the promastigote or vector form of the protozoal parasite Leishmania donovani, the pathogen responsible for kala azar. The Ca2(+)-ATPase was purified to apparent homogeneity after solubilization with deoxycholate. The enzyme consists of two subunits of Mr = 51,000 and 57,000 and has an apparent molecular weight of 215,000 +/- 12,000. The enzyme activity is exclusively dependent on Ca2+, and the pure enzyme can hydrolyze 1.6 mumol of ATP/min/mg of protein. The apparent Km for Ca2+ is 35 nM, which is further reduced to 12 nM in the presence of heterologous calmodulin. The enzyme is sensitive to vanadate, but is insensitive to oligomycin and ouabain. The enzyme is strongly associated with the plasma membrane and has its catalytic site oriented toward the cytoplasmic face. The enzyme spans across the plasma membrane as surface labeling with radioiodine shows considerable radioactivity in the completely purified enzyme. The localization and orientation of this high affinity, calmodulin-sensitive Ca2(+)-ATPase suggest some role of this enzyme in Ca2+ movement in the life cycle of this protozoal parasite.

  2. Enhanced Membrane Pore Formation through High-Affinity Targeted Antimicrobial Peptides

    PubMed Central

    Arnusch, Christopher J.; Pieters, Roland J.; Breukink, Eefjan

    2012-01-01

    Many cationic antimicrobial peptides (AMPs) target the unique lipid composition of the prokaryotic cell membrane. However, the micromolar activities common for these peptides are considered weak in comparison to nisin, which follows a targeted, pore-forming mode of action. Here we show that AMPs can be modified with a high-affinity targeting module, which enables membrane permeabilization at low concentration. Magainin 2 and a truncated peptide analog were conjugated to vancomycin using click chemistry, and could be directed towards specific membrane embedded receptors both in model membrane systems and whole cells. Compared with untargeted vesicles, a gain in permeabilization efficacy of two orders of magnitude was reached with large unilamellar vesicles that included lipid II, the target of vancomycin. The truncated vancomycin-peptide conjugate showed an increased activity against vancomycin resistant Enterococci, whereas the full-length conjugate was more active against a targeted eukaryotic cell model: lipid II containing erythrocytes. This study highlights that AMPs can be made more selective and more potent against biological membranes that contain structures that can be targeted. PMID:22768121

  3. The molecular basis of talin2’s high affinity toward β1-integrin

    PubMed Central

    Yuan, Yaxia; Li, Liqing; Zhu, Yanyan; Qi, Lei; Azizi, Latifeh; Hytönen, Vesa P.; Zhan, Chang-Guo; Huang, Cai

    2017-01-01

    Talin interacts with β-integrin tails and actin to control integrin activation, thus regulating focal adhesion dynamics and cell migration. There are two talin genes, Tln1 and Tln2, which encode talin1 and talin2, and it is generally believed that talin2 functions redundantly with talin1. However, we show here that talin2 has a higher affinity to β1-integrin tails than talin1. Mutation of talin2 S339 to leucine, which can cause Fifth Finger Camptodactyly, a human genetic disease, completely disrupted its binding to β–integrin tails. Also, substitution of talin1 C336 with Ser enhanced the affinity of talin1, whereas substitution of talin2 S339 with Cys diminished that of talin2. Further computational modeling analysis shows that talin2 S339 formed a hydrogen bond with E353, which is critical for inducing key hydrogen bonds between talin2 N326 and β1-integrin R760, and between talin2 K327 and β1-integrin D759. Mutation at any of these residues significantly diminished the interaction of talin2 with β1- integrin tails. These hydrogen bonds were not observed in talin1/β1-integrin, but did exist in talin1C336S/β1-integrin complex. These results suggest that talin2 S339 forms a hydrogen bond with E353 to mediate its high affinity to β1-integrin. PMID:28155884

  4. Osmoprotectants in Halomonas elongata: high-affinity betaine transport system and choline-betaine pathway.

    PubMed Central

    Cánovas, D; Vargas, C; Csonka, L N; Ventosa, A; Nieto, J J

    1996-01-01

    The osmoregulatory pathways of the moderately halophilic bacterium Halomonas elongata DSM 3043 have been investigated. This strain grew optimally at 1.5 to 2 M NaCl in M63 glucose-defined medium. It required at least 0.5 M NaCl for growth, which is a higher concentration than that exhibited by the H. elongata type strain ATCC 33173. Externally provided betaine, choline, or choline-O-sulfate (but not proline, ectoine, or proline betaine) enhanced the growth of H. elongata on 3 M NaCl-glucose-M63 plates, demonstrating the utilization of these compounds as osmoprotectants. Moreover, betaine and choline stimulated the growth of H. elongata DSM 3043 over the entire range of salinity, although betaine was more effective than choline at salinities below and above the optimum. We found that H. elongata DSM 3043 has at least one high-affinity transport system for betaine (K(m) = 3.06 microM and Vmax = 9.96 nmol of betaine min(-1) mg of protein(-1)). Competition assays demonstrated that proline betaine and ectoine, but not proline, choline, or choline-O-sulfate, are also transported by the betaine permease. Finally, thin-layer chromatography and 13C-nuclear magnetic resonance analysis showed that exogenous choline was taken up and transformed to betaine by H. elongata, demonstrating the existence of a choline-glycine betaine pathway in this moderately halophilic bacterium. PMID:8955405

  5. Cyclic GMP-AMP Containing Mixed Phosphodiester Linkages Is An Endogenous High Affinity Ligand for STING

    PubMed Central

    Zhang, Xu; Shi, Heping; Wu, Jiaxi; Zhang, Xuewu; Sun, Lijun; Chen, Chuo; Chen, Zhijian J.

    2013-01-01

    The presence of microbial or self DNA in the cytoplasm of mammalian cells is a danger signal detected by the DNA sensor cyclic-GMP-AMP (cGAMP) synthase (cGAS), which catalyzes the production of cGAMP that in turn serves as a second messenger to activate innate immune responses. Here we show that endogenous cGAMP in mammalian cells contains two distinct phosphodiester linkages, one between 2′-OH of GMP and 5′-phosphate of AMP, and the other between 3′-OH of AMP and 5′-phosphate of GMP. This molecule, termed 2′3′-cGAMP, is unique in that it binds to the adaptor protein STING with a much greater affinity than cGAMP molecules containing other combinations of phosphodiester linkages. The crystal structure of STING bound to 2′3′-cGAMP revealed the structural basis of this high-affinity binding and a ligand-induced conformational change in STING that may underlie its activation. PMID:23747010

  6. A 45-amino acid scaffold mined from the Protein Data Bank for high affinity ligand engineering

    PubMed Central

    Kruziki, Max A.; Bhatnagar, Sumit; Woldring, Daniel R.; Duong, Vandon T.; Hackel, Benjamin J.

    2015-01-01

    Summary Small protein ligands can provide superior physiological distribution versus antibodies and improved stability, production, and specific conjugation. Systematic evaluation of the Protein Data Bank identified a scaffold to push the limits of small size and robust evolution of stable, high-affinity ligands: 45-residue T7 phage gene 2 protein (Gp2) contains an α-helix opposite a β-sheet with two adjacent loops amenable to mutation. De novo ligand discovery from 108 mutants and directed evolution towards four targets yielded target-specific binders with affinities as strong as 200 ±100 pM, Tm’s from 65 ±3 °C to 80 ±1 °C, and retained activity after thermal denaturation. For cancer targeting, a Gp2 domain for epidermal growth factor receptor was evolved with 18 ±8 nM affinity, receptor-specific binding, and high thermal stability with refolding. The efficiency of evolving new binding function and the size, affinity, specificity, and stability of evolved domains render Gp2 a uniquely effective ligand scaffold. PMID:26165154

  7. Poly-cytosine DNA as a High-Affinity Ligand for Inorganic Nanomaterials.

    PubMed

    Lu, Chang; Huang, Zhicheng; Liu, Biwu; Liu, Yibo; Ying, Yibin; Liu, Juewen

    2017-05-22

    Attaching DNA to nanomaterials is the basis for DNA-directed assembly, sensing, and drug delivery using such hybrid materials. Poly-cytosine (poly-C) DNA is a high affinity ligand for four types of commonly used nanomaterials, including nanocarbons (graphene oxide and single-walled carbon nanotubes), transition metal dichalcogenides (MoS2 and WS2 ), metal oxides (Fe3 O4 and ZnO), and metal nanoparticles (Au and Ag). Compared to other homo-DNA sequences, poly-C DNA has the highest affinity for the first three types of materials. Using a diblock DNA containing a poly-C block to attach to surfaces, the target DNA was successfully hybridized to the other block on graphene oxide more efficiently than that containing a typical poly-A block, especially in the presence of non-specific background DNA, proteins, or surfactants. This work provides a simple solution for functionalizing nanomaterials with non-modified DNA and offers new insights into DNA biointerfaces. © 2017 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim.

  8. High Affinity Antibodies against Influenza Characterize the Plasmablast Response in SLE Patients After Vaccination

    PubMed Central

    Kaur, Kaval; Zheng, Nai-Ying; Smith, Kenneth; Huang, Min; Li, Lie; Pauli, Noel T.; Henry Dunand, Carole J.; Lee, Jane-Hwei; Morrissey, Michael; Wu, Yixuan; Joachims, Michelle L.; Munroe, Melissa E.; Lau, Denise; Qu, Xinyan; Krammer, Florian; Wrammert, Jens; Palese, Peter; Ahmed, Rafi; James, Judith A.; Wilson, Patrick C.

    2015-01-01

    Breakdown of B cell tolerance is a cardinal feature of systemic lupus erythematosus (SLE). Increased numbers of autoreactive mature naïve B cells have been described in SLE patients and autoantibodies have been shown to arise from autoreactive and non-autoreactive precursors. How these defects, in the regulation of B cell tolerance and selection, influence germinal center (GC) reactions that are directed towards foreign antigens has yet to be investigated. Here, we examined the characteristics of post-GC foreign antigen-specific B cells from SLE patients and healthy controls by analyzing monoclonal antibodies generated from plasmablasts induced specifically by influenza vaccination. We report that many of the SLE patients had anti-influenza antibodies with higher binding affinity and neutralization capacity than those from controls. Although overall frequencies of autoreactivity in the influenza-specific plasmablasts were similar for SLE patients and controls, the variable gene repertoire of influenza-specific plasmablasts from SLE patients was altered, with increased usage of JH6 and long heavy chain CDR3 segments. We found that high affinity anti-influenza antibodies generally characterize the plasmablast responses of SLE patients with low levels of autoreactivity; however, certain exceptions were noted. The high-avidity antibody responses in SLE patients may also be correlated with cytokines that are abnormally expressed in lupus. These findings provide insights into the effects of dysregulated immunity on the quality of antibody responses following influenza vaccination and further our understanding of the underlying abnormalities of lupus. PMID:25951191

  9. Sequential administration of the high affinity CXCR4 antagonist BKT140 promotes megakaryopoiesis and platelet production.

    PubMed

    Abraham, Michal; Weiss, Ido D; Wald, Hanna; Wald, Ori; Nagler, Arnon; Beider, Katia; Eizenberg, Orly; Peled, Amnon

    2013-10-01

    Platelets are the terminal differentiation product of megakaryocytes (MKs). Cytokines, such as thrombopoietin (TPO), are known to influence different steps in MK development; however, the complex differentiation and platelet localization processes are not fully understood. MKs express the receptor CXCR4 and have been shown to migrate in response to CXCL12 and to increase their platelet production. In this study, we studied the role of CXCR4 in platelet production with the high affinity CXCR4 antagonist, BKT140. Single and sequential administration of BKT140 significantly increased the number of MKs and haematopoietic progenitors (HPCs) within the bone marrow (BM). Increased megakaryopoiesis was associated with increased platelet production. Single and sequential administration of BKT140 also increased the number of HPCs in the blood. In a model of 5-fluorouracil-induced thrombocytopenia, BKT140 significantly reduced the severity and duration of thrombocytopenia and cytopenia when administered before and after chemotherapy. Our results demonstrated that the CXCR4 antagonist, BKT140, mediated unique beneficial effects by stimulating megakaryopoiesis and platelet production. These results provide evidence for the possible therapeutic use of BKT140 for modulating platelet numbers in thrombocytopenic conditions. © 2013 John Wiley & Sons Ltd.

  10. The Conundrum of the High-Affinity NGF Binding Site Formation Unveiled?

    PubMed Central

    Covaceuszach, Sonia; Konarev, Petr V.; Cassetta, Alberto; Paoletti, Francesca; Svergun, Dmitri I.; Lamba, Doriano; Cattaneo, Antonino

    2015-01-01

    The homodimer NGF (nerve growth factor) exerts its neuronal activity upon binding to either or both distinct transmembrane receptors TrkA and p75NTR. Functionally relevant interactions between NGF and these receptors have been proposed, on the basis of binding and signaling experiments. Namely, a ternary TrkA/NGF/p75NTR complex is assumed to be crucial for the formation of the so-called high-affinity NGF binding sites. However, the existence, on the cell surface, of direct extracellular interactions is still a matter of controversy. Here, supported by a small-angle x-ray scattering solution study of human NGF, we propose that it is the oligomerization state of the secreted NGF that may drive the formation of the ternary heterocomplex. Our data demonstrate the occurrence in solution of a concentration-dependent distribution of dimers and dimer of dimers. A head-to-head molecular assembly configuration of the NGF dimer of dimers has been validated. Overall, these findings prompted us to suggest a new, to our knowledge, model for the transient ternary heterocomplex, i.e., a TrkA/NGF/p75NTR ligand/receptors molecular assembly with a (2:4:2) stoichiometry. This model would neatly solve the problem posed by the unconventional orientation of p75NTR with respect to TrkA, as being found in the crystal structures of the TrkA/NGF and p75NTR/NGF complexes. PMID:25650935

  11. Selection of DNA Aptamers against Glioblastoma Cells with High Affinity and Specificity

    PubMed Central

    Kang, Dezhi; Wang, Jiangjie; Zhang, Weiyun; Song, Yanling; Li, Xilan; Zou, Yuan; Zhu, Mingtao; Zhu, Zhi; Chen, Fuyong; Yang, Chaoyong James

    2012-01-01

    Background Glioblastoma is the most common and most lethal form of brain tumor in human. Unfortunately, there is still no effective therapy to this fatal disease and the median survival is generally less than one year from the time of diagnosis. Discovery of ligands that can bind specifically to this type of tumor cells will be of great significance to develop early molecular imaging, targeted delivery and guided surgery methods to battle this type of brain tumor. Methodology/Principal Findings We discovered two target-specific aptamers named GBM128 and GBM131 against cultured human glioblastoma cell line U118-MG after 30 rounds selection by a method called cell-based Systematic Evolution of Ligands by EXponential enrichment (cell-SELEX). These two aptamers have high affinity and specificity against target glioblastoma cells. They neither recognize normal astraglial cells, nor do they recognize other normal and cancer cell lines tested. Clinical tissues were also tested and the results showed that these two aptamers can bind to different clinical glioma tissues but not normal brain tissues. More importantly, binding affinity and selectivity of these two aptamers were retained in complicated biological environment. Conclusion/Significance The selected aptamers could be used to identify specific glioblastoma biomarkers. Methods of molecular imaging, targeted drug delivery, ligand guided surgery can be further developed based on these ligands for early detection, targeted therapy, and guided surgery of glioblastoma leading to effective treatment of glioblastoma. PMID:23056171

  12. Immunological and structural characterization of a high affinity anti-fluorescein single-chain antibody.

    PubMed

    Bedzyk, W D; Weidner, K M; Denzin, L K; Johnson, L S; Hardman, K D; Pantoliano, M W; Asel, E D; Voss, E W

    1990-10-25

    Single-chain antibody of the (NH2) VL-linker-VH (COOH) design, was constructed based on prototype high affinity anti-fluorescein monoclonal antibody (mAb) 4-4-20. Purified single-chain antibody (SCA) 4-4-20/212 was studied relative to Ig mAb 4-4-20 in terms of ligand binding, kinetics, idiotypy, metatypy, and stability in denaturing agents. Ligand-binding data correlated with metatypic relatedness of the liganded site. Anti-metatypic reagents reacted preferentially with the liganded conformer of the 4-4-20 antibody active site and were unreactive with free ligand and the non-liganded (idiotypic) state. All results were consistent with the conclusion that SCA 4-4-20/212, with a 14-amino acid linker folded into a native conformational state that closely simulated the prototypical mAb. Furthermore, GndHCl unfolding and refolding studies demonstrated H and L chain variable domain intrinsic stability between SCA 4-4-20/212 and a 50 kDa antigen-binding fragment were nearly identical. This suggested CH1 and CL domain interactions may be more prevalent in V region molecular dynamics than structure.

  13. High affinity anchoring of the decoration protein pb10 onto the bacteriophage T5 capsid

    PubMed Central

    Vernhes, Emeline; Renouard, Madalena; Gilquin, Bernard; Cuniasse, Philippe; Durand, Dominique; England, Patrick; Hoos, Sylviane; Huet, Alexis; Conway, James F.; Glukhov, Anatoly; Ksenzenko, Vladimir; Jacquet, Eric; Nhiri, Naïma; Zinn-Justin, Sophie; Boulanger, Pascale

    2017-01-01

    Bacteriophage capsids constitute icosahedral shells of exceptional stability that protect the viral genome. Many capsids display on their surface decoration proteins whose structure and function remain largely unknown. The decoration protein pb10 of phage T5 binds at the centre of the 120 hexamers formed by the major capsid protein. Here we determined the 3D structure of pb10 and investigated its capsid-binding properties using NMR, SAXS, cryoEM and SPR. Pb10 consists of an α-helical capsid-binding domain and an Ig-like domain exposed to the solvent. It binds to the T5 capsid with a remarkably high affinity and its binding kinetics is characterized by a very slow dissociation rate. We propose that the conformational exchange events observed in the capsid-binding domain enable rearrangements upon binding that contribute to the quasi-irreversibility of the pb10-capsid interaction. Moreover we show that pb10 binding is a highly cooperative process, which favours immediate rebinding of newly dissociated pb10 to the 120 hexamers of the capsid protein. In extreme conditions, pb10 protects the phage from releasing its genome. We conclude that pb10 may function to reinforce the capsid thus favouring phage survival in harsh environments. PMID:28165000

  14. The high affinity iron permease is a key virulence factor required for Rhizopus oryzae pathogenesis

    PubMed Central

    Ibrahim, Ashraf S.; Gebremariam, Teclegiorgis; Lin, Lin; Luo, Guanpingsheng; Husseiny, Mohamed I.; Skory, Christopher D.; Fu, Yue; French, Samuel W.; Edwards, John E.; Spellberg, Brad

    2010-01-01

    SUMMARY Rhizopus oryzaeis the most common cause of mucormycosis, an angioinvasive fungal infection that causes more then 50% mortality rate despite first-line therapy. Clinical and animal model data clearly demonstrate that the presence of elevated available serum iron predisposes the host to mucormycosis. The high affinity iron permease gene (FTR1) is required for R. oryzae iron transport in iron-depleted environments. Here we demonstrate that FTR1 is required for full virulence of R. oryzae in mice. We show that FTR1 is expressed during infection in diabetic ketoacidotic (DKA) mice. In addition, we disrupted FTR1 by double cross-over homologous recombination, but multinucleated R. oryzae could not be forced to segregate to a homokaryotic null allele. Nevertheless, a reduction of the relative copy number of FTR1 and inhibition of FTR1 expression by RNAi compromised the ability of R. oryzae to acquire iron in vitro and reduced its virulence in DKA mice. Importantly, passive immunization with anti-Ftr1p immune sera protected DKA mice from infection with R. oryzae. Thus FTR1 is a virulence factor for R. oryzae, and anti-Ftr1p passive immunotherapy deserves further evaluation as a strategy to improve outcomes of deadly mucormycosis. PMID:20545847

  15. Cutting Edge: Resident Memory CD8 T Cells Express High-Affinity TCRs.

    PubMed

    Frost, Elizabeth L; Kersh, Anna E; Evavold, Brian D; Lukacher, Aron E

    2015-10-15

    Tissue-resident memory T (TRM) cells serve as vanguards of antimicrobial host defense in nonlymphoid tissues, particularly at barrier epithelia and in organs with nonrenewable cell types (e.g., brain). In this study, we asked whether an augmented ability to sense Ag complemented their role as early alarms of pathogen invasion. Using mouse polyomavirus, we show that brain-resident mouse polyomavirus-specific CD8 T cells, unlike memory cells in the spleen, progressively increase binding to MHC class I tetramers and CD8 coreceptor expression. Using the two-dimensional micropipette adhesion-frequency assay, we show that TRM cells in brain, as well as in kidney, express TCRs with up to 20-fold higher affinity than do splenic memory T cells, whereas effector cells express TCRs of similar high affinity in all organs. Together, these data demonstrate that TRM cells retain high TCR affinity, which endows them with the high Ag sensitivity needed for front-line defense against infectious agents. Copyright © 2015 by The American Association of Immunologists, Inc.

  16. High-affinity DNA base analogs as supramolecular, nanoscale promoters of macroscopic adhesion.

    PubMed

    Anderson, Cyrus A; Jones, Amanda R; Briggs, Ellen M; Novitsky, Eric J; Kuykendall, Darrell W; Sottos, Nancy R; Zimmerman, Steven C

    2013-05-15

    Adhesion phenomena are essential to many biological processes and to synthetic adhesives and manufactured coatings and composites. Supramolecular interactions are often implicated in various adhesion mechanisms. Recently, supramolecular building blocks, such as synthetic DNA base-pair mimics, have drawn attention in the context of molecular recognition, self-assembly, and supramolecular polymers. These reversible, hydrogen-bonding interactions have been studied extensively for their adhesive capabilities at the nano- and microscale, however, much less is known about their utility for practical adhesion in macroscopic systems. Herein, we report the preparation and evaluation of supramolecular coupling agents based on high-affinity, high-fidelity quadruple hydrogen-bonding units (e.g., DAN·DeUG, Kassoc = 10(8) M(-1) in chloroform). Macroscopic adhesion between polystyrene films and glass surfaces modified with 2,7-diamidonaphthyridine (DAN) and ureido-7-deazaguanine (DeUG) units was evaluated by mechanical testing. Structure-property relationships indicate that the designed supramolecular interaction at the nanoscale plays a key role in the observed macroscopic adhesive response. Experiments probing reversible adhesion or self-healing properties of bulk samples indicate that significant recovery of initial strength can be realized after failure but that the designed noncovalent interaction does not lead to healing during the process of adhesion loss.

  17. New Regulatory Roles of Galectin-3 in High-Affinity IgE Receptor Signaling

    PubMed Central

    Bambouskova, Monika; Polakovicova, Iva; Halova, Ivana; Goel, Gautam; Draberova, Lubica; Bugajev, Viktor; Doan, Aivi; Utekal, Pavol; Gardet, Agnes; Xavier, Ramnik J.

    2016-01-01

    Aggregation of the high-affinity receptor for IgE (FcεRI) in mast cells initiates activation events that lead to degranulation and release of inflammatory mediators. To better understand the signaling pathways and genes involved in mast cell activation, we developed a high-throughput mast cell degranulation assay suitable for RNA interference experiments using lentivirus-based short hairpin RNA (shRNA) delivery. We tested 432 shRNAs specific for 144 selected genes for effects on FcεRI-mediated mast cell degranulation and identified 15 potential regulators. In further studies, we focused on galectin-3 (Gal3), identified in this study as a negative regulator of mast cell degranulation. FcεRI-activated cells with Gal3 knockdown exhibited upregulated tyrosine phosphorylation of spleen tyrosine kinase and several other signal transduction molecules and enhanced calcium response. We show that Gal3 promotes internalization of IgE-FcεRI complexes; this may be related to our finding that Gal3 is a positive regulator of FcεRI ubiquitination. Furthermore, we found that Gal3 facilitates mast cell adhesion and motility on fibronectin but negatively regulates antigen-induced chemotaxis. The combined data indicate that Gal3 is involved in both positive and negative regulation of FcεRI-mediated signaling events in mast cells. PMID:26929198

  18. Paracetamol and cytarabine binding competition in high affinity binding sites of transporting protein

    NASA Astrophysics Data System (ADS)

    Sułkowska, A.; Bojko, B.; Równicka, J.; Sułkowski, W. W.

    2006-07-01

    Paracetamol (acetaminophen, AA) the most popular analgesic drug is commonly used in the treatment of pain in patients suffering from cancer. In our studies, we evaluated the competition in binding with serum albumin between paracetamol (AA) and cytarabine, antyleukemic drug (araC). The presence of one drug can alter the binding affinity of albumin towards the second one. Such interaction can result in changing of the free fraction of the one of these drugs in blood. Two spectroscopic methods were used to determine high affinity binding sites and the competition of the drugs. Basing on the change of the serum albumin fluorescence in the presence of either of the drugs the quenching ( KQ) constants for the araC-BSA and AA-BSA systems were calculated. Analysis of UV difference spectra allowed us to describe the changes in drug-protein complexes (araC-albumin and AA-albumin) induced by the presence of the second drug (AA and araC, respectively). The mechanism of competition between araC and AA has been proposed.

  19. Role of constraint in catalysis and high-affinity binding by proteins.

    PubMed Central

    Vanselow, Donald G

    2002-01-01

    Using a model for catalysis of a dynamic equilibrium, the role of constraint in catalysis is quantified. The intrinsic rigidity of proteins is shown to be insufficient to constrain the activated complexes of enzymes, irrespective of the mechanism. However, when minimization of the surface excess free energy of water surrounding a protein is considered, model proteins can be designed with regions of sufficient rigidity. Structures can be designed to focus surface tension or hydrophobic attraction as compressive stress. A monomeric structure has a limited ability to concentrate compressive stress and constrain activated complexes. Oligomeric or multidomain proteins, with domains surrounding a rigid core, have unlimited ability to concentrate stress, provided there are at least four domains. Under some circumstances, four is the optimum number, which could explain the frequency of tetrameric enzymes in nature. The minimum compressive stress in oligomers increases with the square of the radius. For tetramers of similar size to natural enzymes, this stress agrees reasonably well with that needed to constrain the activated complex. A similar principle applies to high affinity binding proteins. The models explain the trigonal pyramidal shape of fibroblast growth factor and provide a basis for interpretation of protein crystal structures. PMID:11964220

  20. Alterations of cortical pyramidal neurons in mice lacking high-affinity nicotinic receptors

    PubMed Central

    Ballesteros-Yáñez, Inmaculada; Benavides-Piccione, Ruth; Bourgeois, Jean-Pierre; Changeux, Jean-Pierre; DeFelipe, Javier

    2010-01-01

    The neuronal nicotinic acetylcholine receptors (nAChRs) are allosteric membrane proteins involved in multiple cognitive processes, including attention, learning, and memory. The most abundant form of heterooligomeric nAChRs in the brain contains the β2- and α4- subunits and binds nicotinic agonists with high affinity. In the present study, we investigated in the mouse the consequences of the deletion of one of the nAChR components: the β2-subunit (β2−/−) on the microanatomy of cortical pyramidal cells. Using an intracellular injection method, complete basal dendritic arbors of 650 layer III pyramidal neurons were sampled from seven cortical fields, including primary sensory, motor, and associational areas, in both β2−/− and WT animals. We observed that the pyramidal cell phenotype shows significant quantitative differences among different cortical areas in mutant and WT mice. In WT mice, the density of dendritic spines was rather similar in all cortical fields, except in the prelimbic/infralimbic cortex, where it was significantly higher. In the absence of the β2-subunit, the most significant reduction in the density of spines took place in this high-order associational field. Our data suggest that the β2-subunit is involved in the dendritic morphogenesis of pyramidal neurons and, in particular, in the circuits that contribute to the high-order functional connectivity of the cerebral cortex. PMID:20534523

  1. Peptide array-based characterization and design of ZnO-high affinity peptides.

    PubMed

    Okochi, Mina; Sugita, Tomoya; Furusawa, Seiji; Umetsu, Mitsuo; Adschiri, Tadafumi; Honda, Hiroyuki

    2010-08-15

    Peptides with both an affinity for ZnO and the ability to generate ZnO nanoparticles have attracted attention for the self-assembly and templating of nanoscale building blocks under ambient conditions with compositional uniformity. In this study, we have analyzed the specific binding sites of the ZnO-binding peptide, EAHVMHKVAPRP, which was identified using a phage display peptide library. The peptide binding assay against ZnO nanoparticles was performed using peptides synthesized on a cellulose membrane using the spot method. Using randomized rotation of amino acids in the ZnO-binding peptide, 125 spot-synthesized peptides were assayed. The peptide binding activity against ZnO nanoparticles varied greatly. This indicates that ZnO binding does not depend on total hydrophobicity or other physical parameters of these peptides, but rather that ZnO recognizes the specific amino acid alignment of these peptides. In addition, several peptides were found to show higher binding ability compared with that of the original peptides. Identification of important binding sites in the EAHVMHKVAPRP peptide was investigated by shortened, stepwise sequence from both termini. Interestingly, two ZnO-binding sites were found as 6-mer peptides: HVMHKV and HKVAPR. The peptides identified by amino acid substitution of HKVAPR were found to show high affinity and specificity for ZnO nanoparticles.

  2. Isolation and Characterization of a High Affinity Peptide Inhibitor of ClC-2 Chloride Channels*

    PubMed Central

    Thompson, Christopher H.; Olivetti, Pedro R.; Fuller, Matthew D.; Freeman, Cody S.; McMaster, Denis; French, Robert J.; Pohl, Jan; Kubanek, Julia; McCarty, Nael A.

    2009-01-01

    The ClC protein family includes voltage-gated chloride channels and chloride/proton exchangers. In eukaryotes, ClC proteins regulate membrane potential of excitable cells, contribute to epithelial transport, and aid in lysosomal acidification. Although structure/function studies of ClC proteins have been aided greatly by the available crystal structures of a bacterial ClC chloride/proton exchanger, the availability of useful pharmacological tools, such as peptide toxin inhibitors, has lagged far behind that of their cation channel counterparts. Here we report the isolation, from Leiurus quinquestriatus hebraeus venom, of a peptide toxin inhibitor of the ClC-2 chloride channel. This toxin, GaTx2, inhibits ClC-2 channels with a voltage-dependent apparent KD of ∼20 pm, making it the highest affinity inhibitor of any chloride channel. GaTx2 slows ClC-2 activation by increasing the latency to first opening by nearly 8-fold but is unable to inhibit open channels, suggesting that this toxin inhibits channel activation gating. Finally, GaTx2 specifically inhibits ClC-2 channels, showing no inhibitory effect on a battery of other major classes of chloride channels and voltage-gated potassium channels. GaTx2 is the first peptide toxin inhibitor of any ClC protein. The high affinity and specificity displayed by this toxin will make it a very powerful pharmacological tool to probe ClC-2 structure/function. PMID:19574231

  3. Specific high-affinity binding of fatty acids to epidermal cytosolic proteins

    SciTech Connect

    Raza, H.; Chung, W.L.; Mukhtar, H. )

    1991-08-01

    Cytosol from rat, mouse, and human skin or rat epidermis was incubated with (3H)arachidonic acid, (14C)retinoic acid, (14C)oleic acid, (3H)leukotriene A4, (3H)prostaglandin E2 (PGE2) or (3H) 15-hydroxyeicosatetraenoic acid (15-HETE), and protein-bound ligands were separated using Lipidex-1000 at 4C to assess the binding specificity. The binding of oleic acid and arachidonic acid with rat epidermal cytosol was rapid, saturable, and reversible. Binding of oleic acid was competed out with the simultaneous addition of other ligands and found to be in the following order: arachidonic acid greater than oleic acid greater than linoleic acid greater than lauric acid greater than leukotriene A4 greater than 15-HETE = PGE1 greater than PGE2 = PGF2. Scatchard analysis of the binding with arachidonic acid, oleic acid, and retinoic acid revealed high-affinity binding sites with the dissociation constant in the nM range. SDS-PAGE analysis of the oleic acid-bound epidermal cytosolic protein(s) revealed maximum binding at the 14.5 kDa region. The presence of the fatty acid-binding protein in epidermal cytosol and its binding to fatty acids and retinoic acid may be of significance both in the trafficking and the metabolism of fatty acids and retinoids across the skin.

  4. Conformation-Dependent High-Affinity Potent Ricin-Neutralizing Monoclonal Antibodies

    PubMed Central

    Hu, Wei-Gang; Yin, Junfei; Chau, Damon; Hu, Charles Chen; Lillico, Dustin; Yu, Justin; Negrych, Laurel M.; Cherwonogrodzky, John W.

    2013-01-01

    Ricin is a potential biothreat agent with no approved antidote available for ricin poisoning. The aim of this study was to develop potent antibody-based antiricin antidotes. Four strong ricin resistant hybridoma clones secreting antiricin monoclonal antibodies (mAbs) were developed. All four mAbs are bound to conformational epitopes of ricin toxin B (RTB) with high affinity (KD values from 2.55 to 36.27 nM). RTB not only triggers cellular uptake of ricin, but also facilitates transport of the ricin toxin A (RTA) from the endoplasmic reticulum to the cytosol, where RTA exerts its toxic activity. The four mAbs were found to have potent ricin-neutralizing capacities and synergistic effects among them as determined by an in vitro neutralization assay. In vivo protection assay demonstrated that all four mAbs had strong efficacy against ricin challenges. D9 was found to be exceptionally effective. Intraperitoneal (i.p.) administration of D9, at a dose of 5 μg, 6 weeks before or 6 hours after an i.p. challenge with 5 × LD50 of ricin was able to protect or rescue 100% of the mice, indicating that mAb D9 is an excellent candidate to be developed as a potent antidote against ricin poisoning for both prophylactic and therapeutic purposes. PMID:23484120

  5. High-affinity cannabinoid binding site in brain: A possible marijuana receptor

    SciTech Connect

    Nye, J.S.

    1988-01-01

    The mechanism by which delta{sup 9} tetrahydrocannabinol (delta{sup 9}THC), the major psychoactive component of marijuana or hashish, produces its potent psychological and physiological effects is unknown. To find receptor binding sites for THC, we designed a water-soluble analog for use as a radioligand. 5{prime}-Trimethylammonium-delta{sup 8}THC (TMA) is a positively charged analog of delta-{sup 8}THC modified on the 5{prime} carbon, a portion of the molecule not important for its psychoactivity. We have studied the binding of ({sup 3}H)-5{prime}-trimethylammonium-delta-{sup 8}THC (({sup 3}H)TMA) to rat neuronal membranes. ({sup 3}H)TMA binds saturably and reversibly to brain membranes with high affinity to apparently one class of sites. Highest binding site density occurs in brain, but several peripheral organs also display specific binding. Detergent solubilizes the sites without affecting their pharmacologial properties. Molecular sieve chromatography reveals a bimodal peak of ({sup 3}H)TMA binding activity of approximately 60,000 daltons apparent molecular weight.

  6. Characterization of a high affinity cocaine binding site in rat brain

    SciTech Connect

    Calligaro, D.; Eldefrawi, M.

    1986-03-05

    Binding of (/sup 3/H)cocaine to synaptic membranes from whole rat brain was reversible and saturable. Nonlinear regression analysis of binding isotherms indicated two binding affinities: one with k/sub d/ = 16 nM, B/sub max/ = 0.65 pmoles/mg protein and the other with K/sub d/ = 660 nM, B/sub max/ = 5.1 pmoles/mg protein. The high-affinity binding of (/sup 3/H)cocaine was sensitive to the actions of trypsin and chymotrypsin but not carboxypeptidase, and was eliminated by exposure of the membranes to 95/sup 0/C for 5 min. Specific binding at 2 nM was higher at pH 8.8 than at pH 7.0. Binding of (/sup 3/H)cocaine (15 nM) was inhibited by increasing concentrations of Na/sup +/ ions. Several cocaine analogues, neurotransmitter uptake inhibitors and local anesthetics displaced specific (/sup 3/H)cocaine binding at 2 nM with various potencies. The cocaine analogue (-)-norcocaine was the most potent (IC/sub 50/ = 10 nM), while the local anesthetic tetracaine was the least potent in inhibiting (/sup 3/H)cocaine binding. Several biogenic amine uptake inhibitors, including tricyclic antidepressants and phencyclidine, had IC/sub 50/ values below ..mu..M concentrations.

  7. Effects of lead on the kidney: Roles of high-affinity lead-binding proteins

    SciTech Connect

    Fowler, B.A. ); DuVal, G. )

    1991-02-01

    Lead-induced nephropathy produces both tubular and interstitial manifestations of cell injury, but the pathophysiology of these lesions is not completely understood. Delineation of the molecular factors underlying renal handling of lead is one of central importance in understanding the mechanisms of renal cell injury from this agent. Recent studies from this laboratory have identified several distinct high-affinity lead-binding proteins (PbBP) from rat kidney and brain that appear to play critical roles in the intracellular bioavailability of lead to several essential cellular processes in these target tissues at low dose levels. These studies have also shown that the real PbBP is selectively localized in only certain nephrons and only specific segments of the renal proximal tubule. The striking nephron and cell-type specificity of the localization reaction could result from physoiological differences in nephron functional activity or selective molecular uptake mechanisms/metabolism differences that act to define target cell populations in the kidney. In addition, other preliminary studies have shown that short-term, high-dose lead exposure produces increased excretion of this protein into the urine with concomitant decreases in renal concentrations.

  8. Conformation-dependent high-affinity potent ricin-neutralizing monoclonal antibodies.

    PubMed

    Hu, Wei-Gang; Yin, Junfei; Chau, Damon; Hu, Charles Chen; Lillico, Dustin; Yu, Justin; Negrych, Laurel M; Cherwonogrodzky, John W

    2013-01-01

    Ricin is a potential biothreat agent with no approved antidote available for ricin poisoning. The aim of this study was to develop potent antibody-based antiricin antidotes. Four strong ricin resistant hybridoma clones secreting antiricin monoclonal antibodies (mAbs) were developed. All four mAbs are bound to conformational epitopes of ricin toxin B (RTB) with high affinity (KD values from 2.55 to 36.27 nM). RTB not only triggers cellular uptake of ricin, but also facilitates transport of the ricin toxin A (RTA) from the endoplasmic reticulum to the cytosol, where RTA exerts its toxic activity. The four mAbs were found to have potent ricin-neutralizing capacities and synergistic effects among them as determined by an in vitro neutralization assay. In vivo protection assay demonstrated that all four mAbs had strong efficacy against ricin challenges. D9 was found to be exceptionally effective. Intraperitoneal (i.p.) administration of D9, at a dose of 5 μ g, 6 weeks before or 6 hours after an i.p. challenge with 5 × LD50 of ricin was able to protect or rescue 100% of the mice, indicating that mAb D9 is an excellent candidate to be developed as a potent antidote against ricin poisoning for both prophylactic and therapeutic purposes.

  9. The High-Affinity E. Coli Methionine ABC Transporter: Structure And Allosteric Regulation

    SciTech Connect

    Kadaba, N.S.; Kaiser, J.T.; Johnson, E.; Lee, A.; Rees, D.C.

    2009-05-18

    The crystal structure of the high-affinity Escherichia coli MetNI methionine uptake transporter, a member of the adenosine triphosphate (ATP)-binding cassette (ABC) family, has been solved to 3.7 angstrom resolution. The overall architecture of MetNI reveals two copies of the adenosine triphosphatase (ATPase) MetN in complex with two copies of the transmembrane domain MetI, with the transporter adopting an inward-facing conformation exhibiting widely separated nucleotide binding domains. Each MetI subunit is organized around a core of five transmembrane helices that correspond to a subset of the helices observed in the larger membrane-spanning subunits of the molybdate (ModBC) and maltose (MalFGK) ABC transporters. In addition to the conserved nucleotide binding domain of the ABC family, MetN contains a carboxyl-terminal extension with a ferredoxin-like fold previously assigned to a conserved family of regulatory ligand-binding domains. These domains separate the nucleotide binding domains and would interfere with their association required for ATP binding and hydrolysis. Methionine binds to the dimerized carboxyl-terminal domain and is shown to inhibit ATPase activity. These observations are consistent with an allosteric regulatory mechanism operating at the level of transport activity, where increased intracellular levels of the transported ligand stabilize an inward-facing, ATPase-inactive state of MetNI to inhibit further ligand translocation into the cell.

  10. Affinity Crystallography: A New Approach to Extracting High-Affinity Enzyme Inhibitors from Natural Extracts.

    PubMed

    Aguda, Adeleke H; Lavallee, Vincent; Cheng, Ping; Bott, Tina M; Meimetis, Labros G; Law, Simon; Nguyen, Nham T; Williams, David E; Kaleta, Jadwiga; Villanueva, Ivan; Davies, Julian; Andersen, Raymond J; Brayer, Gary D; Brömme, Dieter

    2016-08-26

    Natural products are an important source of novel drug scaffolds. The highly variable and unpredictable timelines associated with isolating novel compounds and elucidating their structures have led to the demise of exploring natural product extract libraries in drug discovery programs. Here we introduce affinity crystallography as a new methodology that significantly shortens the time of the hit to active structure cycle in bioactive natural product discovery research. This affinity crystallography approach is illustrated by using semipure fractions of an actinomycetes culture extract to isolate and identify a cathepsin K inhibitor and to compare the outcome with the traditional assay-guided purification/structural analysis approach. The traditional approach resulted in the identification of the known inhibitor antipain (1) and its new but lower potency dehydration product 2, while the affinity crystallography approach led to the identification of a new high-affinity inhibitor named lichostatinal (3). The structure and potency of lichostatinal (3) was verified by total synthesis and kinetic characterization. To the best of our knowledge, this is the first example of isolating and characterizing a potent enzyme inhibitor from a partially purified crude natural product extract using a protein crystallographic approach.

  11. Interprofessional Flight Camp.

    PubMed

    Alfes, Celeste M; Rowe, Amanda S

    2016-01-01

    The Dorothy Ebersbach Academic Center for Flight Nursing in Cleveland, OH, holds an annual flight camp designed for master's degree nursing students in the acute care nurse practitioner program, subspecializing in flight nursing at the Frances Payne Bolton School of Nursing at Case Western Reserve University. The weeklong interprofessional training is also open to any health care provider working in an acute care setting and focuses on critical care updates, trauma, and emergency care within the critical care transport environment. This year, 29 graduate nursing students enrolled in a master's degree program from Puerto Rico attended. Although the emergency department in Puerto Rico sees and cares for trauma patients, there is no formal trauma training program. Furthermore, the country only has 1 rotor wing air medical transport service located at the Puerto Rico Medical Center in San Juan. Flight faculty and graduate teaching assistants spent approximately 9 months planning for their participation in our 13th annual flight camp. Students from Puerto Rico were extremely pleased with the learning experiences at camp and expressed particular interest in having more training time within the helicopter flight simulator. Copyright © 2016 Air Medical Journal Associates. Published by Elsevier Inc. All rights reserved.

  12. Immunomodulatory Effects of Lippia sidoides Extract: Induction of IL-10 Through cAMP and p38 MAPK-Dependent Mechanisms

    PubMed Central

    Rajgopal, Arun; Rebhun, John F.; Burns, Charlie R.; Scholten, Jeffrey D.; Balles, John A.

    2015-01-01

    Abstract Lippia sidoides is an aromatic shrub that grows wild in the northeastern region of Brazil. In local traditional medicine, the aerial portions of this species are used as anti-infectives, antiseptics, spasmolytics, sedatives, hypotensives, and anti-inflammatory agents. In this research, we evaluate the potential immunological properties of Lippia extract through in vitro analysis of its ability to modulate intracellular cyclic adenosine monophosphate (cAMP) levels and interleukin-10 (IL-10) production. These results show that Lippia extract increases intracellular cAMP through the inhibition of phosphodiesterase activity. They also demonstrate that Lippia extract increases IL-10 production in THP-1 monocytes through both an increase in intracellular cAMP and the activation of p38 MAPK. These results suggest that the Lippia-mediated inhibition of phosphodiesterase activity and the subsequent increase in intracellular cAMP may explain some of the biological activities associated with L. sidoides. In addition, the anti-inflammatory activity of L. sidoides may also be due, in part, to its ability to induce IL-10 production through the inhibition of cyclic nucleotide-dependent phosphodiesterase activity and by its activation of the p38 MAPK pathway. PMID:25599252

  13. Immunomodulatory effects of Lippia sidoides extract: induction of IL-10 through cAMP and p38 MAPK-dependent mechanisms.

    PubMed

    Rajgopal, Arun; Rebhun, John F; Burns, Charlie R; Scholten, Jeffrey D; Balles, John A; Fast, David J

    2015-03-01

    Lippia sidoides is an aromatic shrub that grows wild in the northeastern region of Brazil. In local traditional medicine, the aerial portions of this species are used as anti-infectives, antiseptics, spasmolytics, sedatives, hypotensives, and anti-inflammatory agents. In this research, we evaluate the potential immunological properties of Lippia extract through in vitro analysis of its ability to modulate intracellular cyclic adenosine monophosphate (cAMP) levels and interleukin-10 (IL-10) production. These results show that Lippia extract increases intracellular cAMP through the inhibition of phosphodiesterase activity. They also demonstrate that Lippia extract increases IL-10 production in THP-1 monocytes through both an increase in intracellular cAMP and the activation of p38 MAPK. These results suggest that the Lippia-mediated inhibition of phosphodiesterase activity and the subsequent increase in intracellular cAMP may explain some of the biological activities associated with L. sidoides. In addition, the anti-inflammatory activity of L. sidoides may also be due, in part, to its ability to induce IL-10 production through the inhibition of cyclic nucleotide-dependent phosphodiesterase activity and by its activation of the p38 MAPK pathway.

  14. An adenylyl cyclase with a phosphodiesterase domain in basal plants with a motile sperm system

    PubMed Central

    Kasahara, Masahiro; Suetsugu, Noriyuki; Urano, Yuki; Yamamoto, Chiaki; Ohmori, Mikiya; Takada, Yuki; Okuda, Shujiro; Nishiyama, Tomoaki; Sakayama, Hidetoshi; Kohchi, Takayuki; Takahashi, Fumio

    2016-01-01

    Adenylyl cyclase (AC), which produces the signalling molecule cAMP, has numerous important cellular functions in diverse organisms from prokaryotes to eukaryotes. Here we report the identification and characterization of an AC gene from the liverwort Marchantia polymorpha. The encoded protein has both a C-terminal AC catalytic domain similar to those of class III ACs and an N-terminal cyclic nucleotide phosphodiesterase (PDE) domain that degrades cyclic nucleotides, thus we designated the gene MpCAPE (COMBINED AC with PDE). Biochemical analyses of recombinant proteins showed that MpCAPE has both AC and PDE activities. In MpCAPE-promoter-GUS lines, GUS activity was specifically detected in the male sexual organ, the antheridium, suggesting MpCAPE and thus cAMP signalling may be involved in the male reproductive process. CAPE orthologues are distributed only in basal land plants and charophytes that use motile sperm as the male gamete. CAPE is a subclass of class III AC and may be important in male organ and cell development in basal plants. PMID:27982074

  15. Modulation of Compartmentalised Cyclic Nucleotide Signalling via Local Inhibition of Phosphodiesterase Activity

    PubMed Central

    Brescia, Marcella; Zaccolo, Manuela

    2016-01-01

    Cyclic nucleotide phosphodiesterases (PDEs) are the only enzymes that degrade the cyclic nucleotides cAMP and cGMP, and play a key role in modulating the amplitude and duration of the signal delivered by these two key intracellular second messengers. Defects in cyclic nucleotide signalling are known to be involved in several pathologies. As a consequence, PDEs have long been recognized as potential drug targets, and they have been the focus of intense research for the development of therapeutic agents. A number of PDE inhibitors are currently available for the treatment of disease, including obstructive pulmonary disease, erectile dysfunction, and heart failure. However, the performance of these drugs is not always satisfactory, due to a lack of PDE-isoform specificity and their consequent adverse side effects. Recent advances in our understanding of compartmentalised cyclic nucleotide signalling and the role of PDEs in local regulation of cAMP and cGMP signals offers the opportunity for the development of novel strategies for therapeutic intervention that may overcome the current limitation of conventional PDE inhibitors. PMID:27706091

  16. Immunochemical characterization of the distinct monocyte cyclic AMP-phosphodiesterase from patients with atopic dermatitis.

    PubMed

    Chan, S C; Reifsnyder, D; Beavo, J A; Hanifin, J M

    1993-06-01

    Previous findings have suggested that the immunopathology of patients with atopic dermatitis (AD) results from altered cellular responses caused by cyclic nucleotide regulatory abnormalities. One such defect is the increased degradation of the second messenger, cyclic adenosine monophosphate (cAMP), by elevated cAMP-phosphodiesterase (PDE) activity in patients with AD. We used two monoclonal antibodies to identify the major PDE isoform in AD blood monocytes. We have also characterized the abnormal PDE activity by means of chromatofocusing and sucrose gradient centrifugation. The chromatofocusing technique allowed the separation of a PDE-containing fraction (isoelectric point = 6.1) from AD monocytes but not from normal cells. This monocyte fraction accounted for most of the elevated leukocyte-PDE activity and was a cytosolic, cAMP-specific, low Michaelis constant, calcium-calmodulin-dependent enzyme, inhibited by the cAMP-PDE inhibitor, Ro 20-1724. The majority of the PDE activity in this chromatofocused fraction was immunoadsorbed by the solid-phase immobilized antibodies against calcium-calmodulin-dependent PDE. The increased degradation of cAMP by a unique form of PDE may cause defective regulation of intracellular functions of AD monocytes, leading to the characteristic hyperreactive immune and inflammatory events. Characterization of PDE isoenzymes from different leukocyte subpopulations may allow further expansion of cell-directed therapy for inflammatory disease.

  17. The roles of phosphodiesterase 2 in the central nervous and peripheral systems.

    PubMed

    Zhang, Chong; Yu, Yingcong; Ruan, Lina; Wang, Chuang; Pan, Jianchun; Klabnik, Jonathan; Lueptow, Lindsay; Zhang, Han-Ting; O'Donnell, James M; Xu, Ying

    2015-01-01

    Phosphodiesterase 2 (PDE2) is a ubiquitous enzyme whose major role is to hydrolyze the important second messengers cyclic adenosine monophosphate (cAMP) and cyclic guanosine monophosphate (cGMP). In the central nervous system, pharmacological inhibition of PDE2 results in boosted cAMP and/or cGMP signaling, which is responsible for series of changes in protein expression relevant to psychiatric and learning and memory disorders, such as depression, anxiety, and cognition deficits in Alzheimer's disease. In the periphery, inhibition of PDE2 exhibits beneficial effects in the diseased cardiovascular system, the respiratory system, skeletal muscles and Candida albicans-caused systemic infections. Even though blood-brain barrier penetration properties and selectivity of currently available PDE2 inhibitors have hindered them from entering clinical trials, PDE2 is still of great potential therapeutic values in different categories of diseases, and there is demand for development of new generation drugs targeting PDE2 for treatment of diseases in central nervous and peripheral systems.

  18. Levodopa-induced dyskinesias are associated with transient down-regulation of cAMP and cGMP in the caudate-putamen of hemiparkinsonian rats: reduced synthesis or increased catabolism?

    PubMed

    Sancesario, Giuseppe; Morrone, Luigi Antonio; D'Angelo, Vincenza; Castelli, Valentina; Ferrazzoli, Davide; Sica, Francesco; Martorana, Alessandro; Sorge, Roberto; Cavaliere, Federica; Bernardi, Giorgio; Giorgi, Mauro

    2014-12-01

    Second messenger cAMP and cGMP represent a key step in the action of dopamine that modulates directly or indirectly their synthesis. We aimed to verify whether levodopa-induced dyskinesias are associated with changes of the time course of levodopa/dopamine stimulated cAMP and cGMP levels, and/or with changes of their catabolism by phosphodiesterase activity in rats with experimental hemiparkinsonism. Microdialysis and tissue homogenates of the striatal tissues demonstrated that extracellular and intracellular cAMP/cGMP levels were lower in dyskinetic animals during the increasing phase of dyskinesias compared to eukinetic animals, but cAMP/cGMP levels increased in dyskinetic animals during the phase of decreasing and extinction of dyskinesias. Dyskinesias and the abnormal lowering of striatal cGMP and cAMP after levodopa were prevented by pretreatment with the multipotent drug amantadine, outlining the inverse relationship of cAMP/cGMP to dyskinesias. Moreover, dyskinetic animals showed higher striatal hydrolyzing cGMP-phosphodiesterase but not hydrolyzing cAMP-phosphodiesterase activity, suggesting that low cGMP but not cAMP levels could be due to increased catabolism. However, expressions of isozyme phosphodiesterase-1B and -10A highly and specifically located in the basal ganglia were not changed after levodopa in dyskinetic and eukinetic animals: accordingly, selective inhibitors of phosphodiesterase-1B and -10A were ineffective on levodopa dyskinesias. Therefore, the isozyme(s) expressing higher cGMP-phosphodiesterase activity in the striatum of dyskinetic animal should be determined. These observations suggest that dopamine-mediated processes of synthesis and/or degradation of cAMP/cGMP could be acutely impaired in levodopa dyskinesias, opening new ways to understanding physiopathology and treatment.

  19. Loss of cAMP-specific phosphodiesterase rescues spore development in G protein mutant in dictyostelium.

    PubMed

    Schwebs, David J; Nguyen, Hoai-Nghia; Miller, Jamison A; Hadwiger, Jeffrey A

    2014-02-01

    Cyclic AMP (cAMP) is an important intracellular signaling molecule for many G protein-mediated signaling pathways but the specificity of cAMP signaling in cells with multiple signaling pathways is not well-understood. In Dictyostelium, at least two different G protein signaling pathways, mediated by the Gα2 and Gα4 subunits, are involved with cAMP accumulation, spore production, and chemotaxis and the stimulation of these pathways results in the activation of ERK2, a mitogen-activated protein kinase that can down regulate the cAMP-specific phosphodiesterase RegA. The regA gene was disrupted in gα2(−) and gα4(−) cells to determine if the absence of this phosphodiesterase rescues the development of these G protein mutants as it does for erk2(−) mutants. There gA(−) mutation had no major effects on developmental morphology but enriched the distribution of the Gα mutant cells to the prespore/prestalk border in chimeric aggregates. The loss of RegA function had no effect on Gα4- mediated folate chemotaxis. However, the regA gene disruption in gα4(−) cells, but not in gα2(−) cells, resulted in a substantial rescue and acceleration of spore production. This rescue in sporulation required cell autonomous signaling because the precocious sporulation could not be induced through intercellular signaling in chimeric aggregates. However, intercellular signals from regA(−) strains increased the expression of the prestalk gene ecmB and accelerated the vacuolization of stalk cells. Intercellular signaling from the gα4(−)regA(−) strain did not induce ecmA gene expression indicating cell-type specificity in the promotion of prestalk cell development. regA gene disruption in a Gα4(HC) (Gα4 overexpression) strain did not result in precocious sporulation or stalk cell development indicating that elevated Gα4 subunit expression can mask regA(−) associated phenotypes even when provided with wild-type intercellular signaling. These findings indicate that

  20. Day Camp Manual: A Study of Mandatory Standards and Desirable Camping Practices for Children's Day Camps. Book V. Revised 1974.

    ERIC Educational Resources Information Center

    Babcock, William

    The final book in a 5-book day camp manual is a questionnaire developed by the Ontario Camping Association to help directors of children's summer day camps check camp operations, principles, and procedures, and to help them determine how and where a camp can be improved. The questionnaire, to be completed by the director with the aid of another…

  1. Intracellular Lipid Droplets Contain Dynamic Pools of Sphingomyelin: ADRP Binds Phospholipids with High Affinity

    PubMed Central

    McIntosh, Avery L.; Storey, Stephen M.

    2011-01-01

    During the last several years, intracellular lipid droplets have become the focus of intense study. No longer an inert bystander, the lipid droplet is now known as a dynamic organelle contributing lipids to many cellular events. However, while the dynamics of cholesterol efflux from both the plasma membrane and lipid droplets have been studied, less is known regarding the efflux of sphingomyelin from these membranes. In order to address this issue, sphingomyelin efflux kinetics and binding affinities from different intracellular pools were examined. When compared to the plasma membrane, lipid droplets had a smaller exchangeable sphingomyelin efflux pool and the time required to efflux that pool was significantly shorter. Fluorescence binding assays revealed that proteins in the plasma membrane and lipid droplet pool bound sphingomyelin with high affinity. Further characterization identified adipose differentiation-related protein (ADRP) as one of the sphingomyelin binding proteins in the lipid droplet fraction and revealed that ADRP demonstrated saturable binding to 6-((N-(7-nitrobenz-2-oxa-1,3-diazol-4-yl) amino)-hexanoyl)sphingosyl-phosphocholine (NBD-sphingomyelin) and also 2-(6-(7-nitrobenz-2-oxa-1, 3-diazol-4-yl)amino)hexanoyl-1-hexadecanoyl-sn-glycero-3-phosphocholine (NBD-phosphatidylcholine) with binding affinities in the nanomolar range. Taken together, these results suggest that lipid droplet associated proteins such as ADRP may play a significant role in regulating the intracellular distribution of phospholipids and lipids in general. Overall, insights from the present work suggest new and important roles for lipid droplets and ADRP in phospholipid metabolism. PMID:20473576

  2. A functionally active presynaptic high-affinity kainate receptor in the rat hippocampal CA3 subregion.

    PubMed

    Malva, J O; Ambrósio, A F; Cunha, R A; Ribeiro, J A; Carvalho, A P; Carvalho, C M

    1995-02-09

    We studied the modulation of the intracellular free calcium concentrations ([Ca2+]i) by kainate/AMPA receptor activation in synaptosomes isolated from whole rat hippocampus, or from its CA1, CA3 or dentate gyrus subregions. The receptor was activated either by 100 microM S-alpha-amino-3-hydroxy-5-methyl-4-isoxazolopropionic acid (AMPA) (EC50 = 26.6 +/- 4.9 microM) or by 100 microM kainate (EC50 = 0.81 +/- 0.1 microM), but the effects of these agonists were not additive. The response to either AMPA or kainate was competitively inhibited by 10 microM 6-cyano-7-nitroquinoxaline-2,3-dioxine. Higher [Ca2+]i responses to 100 microM AMPA or to 100 microM kainate were observed in the CA3 subregion (43.2 +/- 2.5 nM or 42.8 +/- 2.3 nM, respectively) than in the whole hippocampus (22.4 +/- 1.1 nM or 22.4 +/- 1.6, respectively), in the CA1 subregion (26.4 +/- 1.1 nM or 26.6 +/- 2.6 nM, respectively) or in dentate gyrus (24.6 +/- 1.4 nM or 21.5 +/- 1.0 nM, respectively). These results indicate that the CA3 subregion of the hippocampus is enriched in a presynaptic high-affinity kainate receptor which modulates the [Ca2+]i in nerve terminals.

  3. Insulin Regulates the Activity of the High-Affinity Choline Transporter CHT

    PubMed Central

    Fishwick, Katherine J.; Rylett, R. Jane

    2015-01-01

    Studies in humans and animal models show that neuronal insulin resistance increases the risk of developing Alzheimer’s Disease (AD), and that insulin treatment may promote memory function. Cholinergic neurons play a critical role in cognitive and attentional processing and their dysfunction early in AD pathology may promote the progression of AD pathology. Synthesis and release of the neurotransmitter acetylcholine (ACh) is closely linked to the activity of the high-affinity choline transporter protein (CHT), but the impact of insulin receptor signaling and neuronal insulin resistance on these aspects of cholinergic function are unknown. In this study, we used differentiated SH-SY5Y cells stably-expressing CHT proteins to study the effect of insulin signaling on CHT activity and function. We find that choline uptake activity measured after acute addition of 20 nM insulin is significantly lower in cells that were grown for 24 h in media containing insulin compared to cells grown in the absence of insulin. This coincides with loss of ability to increase phospho-Protein Kinase B (PKB)/Akt levels in response to acute insulin stimulation in the chronic insulin-treated cells. Inhibition of phosphatidylinositol-4,5-bisphosphate 3-kinase (PI3-kinase) in cells significantly lowers phospho-PKB/Akt levels and decreases choline uptake activity. We show total internal reflection microscopy (TIRF) imaging of the dynamic movement of CHT proteins in live cells in response to depolarization and drug treatments. These data show that acute exposure of depolarized cells to insulin is coupled to transiently increased levels of CHT proteins at the cell surface, and that this is attenuated by chronic insulin exposure. Moreover, prolonged inhibition of PI3-kinase results in enhanced levels of CHT proteins at the cell surface by decreasing their rate of internalization. PMID:26161852

  4. Synthesis and characterization of a high affinity radioiodinated probe for the alpha 2-adrenergic receptor

    SciTech Connect

    Lanier, S.M.; Hess, H.J.; Grodski, A.; Graham, R.M.; Homcy, C.J.

    1986-03-01

    The availability of radioiodinated probes has facilitated the localization and molecular characterization of cell membrane receptors for hormones and neurotransmitters. However, such probes are not available for the study of the alpha 2-adrenergic receptor. This report describes the synthesis and characterization of functionalized derivatives of the selective alpha 2-adrenergic antagonists, rauwolscine and yohimbine, which can be radiolabeled to high specific activity with 125I. Following demethylation of rauwolscine or yohimbine, the resultant carboxylic acid derivatives were reacted with 4-aminophenethylamine to yield the respective 4-aminophenethyl carboxamides, 17 alpha-hydroxy-20 alpha-yohimban-16 beta-(N-4-amino-phenethyl)carboxamide (rau-pAPC) and 17 alpha-hydroxy-20 beta-yohimban-16 alpha-(N-4-aminophenethyl)carboxamide. In competitive inhibition studies using rat renal membranes and the radioligand (3H)rauwolscine, rau-pAPC (Ki = 11 +/- 1 nM) exhibited a 14-fold greater affinity than the corresponding yohimbine derivative (Ki = 136 +/- 45 nM). The higher affinity compound, rau-pAPC, was radioiodinated by the chloramine T method, and the product, 125I-rau-pAPC (17 alpha-hydroxy-20 alpha-yohimban-16 beta-(N-4-amino-3 -(125I)iodophenethyl)carboxamide), was purified by reverse phase HPLC to high specific activity (2175 Ci/mmol) and its binding characteristics were investigated in rat kidney membranes. Specific binding of 125I-rau-pAPC was saturable and of high affinity as determined by Scatchard analysis (KD = 1.8 +/- 0.3 nM) or from kinetic studies (KD = k2/k1 = 0.056 +/- 0.013 min-1)/4.3 +/- 0.2 X 10(7) M-1 min-1 = 1.3 +/- 0.3 nM).

  5. High affinity of acid phosphatase encoded by PHO3 gene in Saccharomyces cerevisiae for thiamin phosphates.

    PubMed

    Nosaka, K

    1990-02-09

    The enzymatic properties of acid phosphatase (orthophosphoric-monoester phosphohydrolase, EC 3.1.3.2) encoded by PHO3 gene in Saccharomyces cerevisiae, which is repressed by thiamin and has thiamin-binding activity at pH 5.0, were investigated to study physiological functions. The following results led to the conclusion that thiamin-repressible acid phosphatase physiologically catalyzes the hydrolysis of thiamin phosphates in the periplasmic space of S. cerevisiae, thus participating in utilization of the thiamin moiety of the phosphates by yeast cells: (a) thiamin-repressible acid phosphatase showed Km values of 1.6 and 1.7 microM at pH 5.0 for thiamin monophosphate and thiamin pyrophosphate, respectively. These Km values were 2-3 orders of magnitude lower than those (0.61 and 1.7 mM) for p-nitrophenyl phosphate; (b) thiamin exerted remarkable competitive inhibition in the hydrolysis of thiamin monophosphate (Ki 2.2 microM at pH 5.0), whereas the activity for p-nitrophenyl phosphate was slightly affected by thiamin; (c) the inhibitory effect of inorganic phosphate, which does not repress the thiamin-repressible enzyme, on the hydrolysis of thiamin monophosphate was much smaller than that of p-nitrophenyl phosphate. Moreover, the modification of thiamin-repressible acid phosphatase of S. cerevisiae with 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide resulted in the complete loss of thiamin-binding activity and the Km value of the modified enzyme for thiamin monophosphate increased nearly to the value of the native enzyme for p-nitrophenyl phosphate. These results also indicate that the high affinity of the thiamin-repressible acid phosphatase for thiamin phosphates is due to the thiamin-binding properties of this enzyme.

  6. Engineering of Bispecific Affinity Proteins with High Affinity for ERBB2 and Adaptable Binding to Albumin

    PubMed Central

    Nilvebrant, Johan; Åstrand, Mikael; Georgieva-Kotseva, Maria; Björnmalm, Mattias; Löfblom, John; Hober, Sophia

    2014-01-01

    The epidermal growth factor receptor 2, ERBB2, is a well-validated target for cancer diagnostics and therapy. Recent studies suggest that the over-expression of this receptor in various cancers might also be exploited for antibody-based payload delivery, e.g. antibody drug conjugates. In such strategies, the full-length antibody format is probably not required for therapeutic effect and smaller tumor-specific affinity proteins might be an alternative. However, small proteins and peptides generally suffer from fast excretion through the kidneys, and thereby require frequent administration in order to maintain a therapeutic concentration. In an attempt aimed at combining ERBB2-targeting with antibody-like pharmacokinetic properties in a small protein format, we have engineered bispecific ERBB2-binding proteins that are based on a small albumin-binding domain. Phage display selection against ERBB2 was used for identification of a lead candidate, followed by affinity maturation using second-generation libraries. Cell surface display and flow-cytometric sorting allowed stringent selection of top candidates from pools pre-enriched by phage display. Several affinity-matured molecules were shown to bind human ERBB2 with sub-nanomolar affinity while retaining the interaction with human serum albumin. Moreover, parallel selections against ERBB2 in the presence of human serum albumin identified several amino acid substitutions that dramatically modulate the albumin affinity, which could provide a convenient means to control the pharmacokinetics. The new affinity proteins competed for ERBB2-binding with the monoclonal antibody trastuzumab and recognized the native receptor on a human cancer cell line. Hence, high affinity tumor targeting and tunable albumin binding were combined in one small adaptable protein. PMID:25089830

  7. KW-3902, a selective high affinity antagonist for adenosine A1 receptors.

    PubMed Central

    Nonaka, H.; Ichimura, M.; Takeda, M.; Kanda, T.; Shimada, J.; Suzuki, F.; Kase, H.

    1996-01-01

    1. We demonstrate that 8-(noradamantan-3-yl)-1,3-dipropylxanthine (KW-3902) is a very potent and selective adenosine A1 receptor antagonist, assessed by radioligand binding and cyclic AMP response in cells. 2. In rat forebrain adenosine A1 receptors labelled with [3H]-cyclohexyladenosine (CHA), KW-3902 had a Ki value of 0.19 nM, whereas it showed a Ki value of 170 nM in rat striatal A2A receptors labelled with [3H]-2-[p-(2-carboxyethyl)-phenethylamino]-5'-N-ethylcarboxamidoad enosine (CGS21680), indicating 890 fold A1 receptor selectivity versus the A2A receptor. KW-3902 at 10 microM showed no effect on recombinant rat A3 receptors expressed on CHO cells. 3. Saturation studies with [3H]-KW-3902 revealed that it bound with high affinity (Kd = 77 pM) and limited capacity (Bmax = 470 fmol mg-1 of protein) to a single class of recognition sites. A high positive correlation was observed between the pharmacological profile of adenosine ligands inhibiting the binding of [3H]-KW-3902 and that of [3H]-CHA. 4. KW-3902 showed potent A1 antagonism against the inhibition of forskolin-induced cyclic AMP accumulation in DDT1 MF-2 cells by the A1-selective agonist, cyclopentyladenosine with a dissociation constant (KB value) of 0.34 nM. KW-3902 antagonized 5'-N-ethylcarboxamidoadenosine-elicited cyclic AMP accumulation via A2B receptors with a KB value of 52 nM. 5. KW-3902 exhibited marked species-dependent differences in the binding affinities. The highest affinity was for the rat A1 receptor (ki = 0.19 nM) and these values for guinea-pig and dog A1 receptors were 1.3 and 10 nM, respectively. PMID:8732272

  8. Sugar-binding proteins from fish: selection of high affinity "lambodies" that recognize biomedically relevant glycans.

    PubMed

    Hong, Xia; Ma, Mark Z; Gildersleeve, Jeffrey C; Chowdhury, Sudipa; Barchi, Joseph J; Mariuzza, Roy A; Murphy, Michael B; Mao, Li; Pancer, Zeev

    2013-01-18

    Glycan-binding proteins are important for a wide variety of basic research and clinical applications, but proteins with high affinity and selectivity for carbohydrates are difficult to obtain. Here we describe a facile and cost-effective strategy to generate monoclonal lamprey antibodies, called lambodies, that target glycan determinants. We screened a library of yeast surface-displayed (YSD) lamprey variable lymphocyte receptors (VLR) for clones that can selectively bind various biomedically important glycotopes. These glycoconjugates included tumor-associated carbohydrate antigens (Tn and TFα), Lewis antigens (LeA and LeX), N-glycolylneuraminic acid, targets of broadly neutralizing HIV antibodies (poly-Man9 and the HIV gp120), and the glycoproteins asialo-ovine submaxillary mucin (aOSM) and asialo-human glycophorin A (aGPA). We isolated clones that bind each of these targets in a glycan-dependent manner and with very strong binding constants, for example, 6.2 nM for Man9 and 44.7 nM for gp120, determined by surface plasmon resonance (SPR). One particular lambody, VLRB.aGPA.23, was shown by glycan array analysis to be selective for the blood group H type 3 trisaccharide (BG-H3, Fucα1-2Galβ1-3GalNAcα), aGPA, and TFα (Galβ1-3GalNAcα), with affinity constants of 0.2, 1, and 8 nM, respectively. In human tissue microarrays this lambody selectively detected cancer-associated carbohydrate antigens in 14 different types of cancers. It stained 27% of non-small cell lung cancer (NSCLC) samples in a pattern that correlated with poor patient survival. Lambodies with exquisite affinity and selectivity for glycans may find myriad uses in glycobiology and biomedical research.

  9. Molecular basis for the high-affinity binding and stabilization of firefly luciferase by PTC124

    SciTech Connect

    Auld, Douglas S.; Lovell, Scott; Thorne, Natasha; Lea, Wendy A.; Maloney, David J.; Shen, Min; Rai, Ganesha; Battaile, Kevin P.; Thomas, Craig J.; Simeonov, Anton; Hanzlik, Robert P.; Inglese, James

    2010-04-07

    Firefly luciferase (FLuc), an ATP-dependent bioluminescent reporter enzyme, is broadly used in chemical biology and drug discovery assays. PTC124 Ataluren; (3-[5-(2-fluorophenyl)-1,2,4-oxadiazol-3-yl]benzoic acid) discovered in an FLuc-based assay targeting nonsense codon suppression, is an unusually potent FLuc-inhibitor. Paradoxically, PTC124 and related analogs increase cellular FLuc activity levels by posttranslational stabilization. In this study, we show that FLuc inhibition and stabilization is the result of an inhibitory product formed during the FLuc-catalyzed reaction between its natural substrate, ATP, and PTC124. A 2.0 {angstrom} cocrystal structure revealed the inhibitor to be the acyl-AMP mixed-anhydride adduct PTC124-AMP, which was subsequently synthesized and shown to be a high-affinity multisubstrate adduct inhibitor (MAI; KD = 120 pM) of FLuc. Biochemical assays, liquid chromatography/mass spectrometry, and near-attack conformer modeling demonstrate that formation of this novel MAI is absolutely dependent upon the precise positioning and reactivity of a key meta-carboxylate of PTC124 within the FLuc active site. We also demonstrate that the inhibitory activity of PTC124-AMP is relieved by free coenzyme A, a component present at high concentrations in luciferase detection reagents used for cell-based assays. This explains why PTC124 can appear to increase, instead of inhibit, FLuc activity in cell-based reporter gene assays. To our knowledge, this is an unusual example in which the 'off-target' effect of a small molecule is mediated by an MAI mechanism.

  10. Early Signs of Pathological Cognitive Aging in Mice Lacking High-Affinity Nicotinic Receptors.

    PubMed

    Konsolaki, Eleni; Tsakanikas, Panagiotis; Polissidis, Alexia V; Stamatakis, Antonios; Skaliora, Irini

    2016-01-01

    In order to address pathological cognitive decline effectively, it is critical to adopt early preventive measures in individuals considered at risk. It is therefore essential to develop approaches that identify such individuals before the onset of irreversible dementia. A deficient cholinergic system has been consistently implicated as one of the main factors associated with a heightened vulnerability to the aging process. In the present study we used mice lacking high affinity nicotinic receptors (β2-/-), which have been proposed as an animal model of accelerated/premature cognitive aging. Our aim was to identify behavioral signs that could serve as indicators or predictors of impending cognitive decline. We used test batteries in order to assess cognitive functions and additional tasks to investigate spontaneous behaviors, such as species-specific activities and exploration/locomotion in a novel environment. Our data confirm the hypothesis that β2-/- animals exhibit age-related cognitive impairments in spatial learning. In addition, they document age-related deficits in other areas, such as recognition memory, burrowing and nesting building, thereby extending the validity of this animal model for the study of pathological aging. Finally, our data reveal deficits in spontaneous behavior and habituation processes that precede the onset of cognitive decline and could therefore be useful as a non-invasive behavioral screen for identifying animals at risk. To our knowledge, this is the first study to perform an extensive behavioral assessment of an animal model of premature cognitive aging, and our results suggest that β2-nAChR dependent cognitive deterioration progressively evolves from initial subtle behavioral changes to global dementia due to the combined effect of the neuropathology and aging.

  11. High affinity group III mGluRs regulate mossy fiber input to CA3 interneurons.

    PubMed

    Cosgrove, Kathleen E; Meriney, Stephen D; Barrionuevo, Germán

    2011-12-01

    Stratum lacunosum-moleculare interneurons (L-Mi) in hippocampal area CA3 target the apical dendrite of pyramidal cells providing feedforward inhibition. Here we report that selective activation of group III metabotropic glutamate receptors (mGluRs) 4/8 with L(+)-2-amino-4-phosphnobytyric acid (L-AP4; 10 μM) decreased the probability of glutamate release from the mossy fiber (MF) terminals synapsing onto L-Mi. Consistent with this interpretation, application of L-AP4 in the presence of 3 mM strontium decreased the frequency of asynchronous MF EPSCs in L-Mi. Furthermore, the dose response curve showed that L-AP4 at 400 μM produced no further decrease in MF EPSC amplitude compared with 20 μM L-AP4, indicating the lack of mGluRs 7 at these MF terminals. We also found that one mechanism of mGluRs 4/8-mediated inhibition of release is linked to N-type voltage gated calcium channels at MF terminals. Application of the group III mGluR antagonist MSOP (100 μM) demonstrated that mGluRs 4/8 are neither tonically active nor activated by low and moderate frequencies of activity. However, trains of stimuli to the MF at 20 and 40 Hz delivered during the application of MSOP revealed a relief of inhibition of transmitter release and an increase in the overall probability of action potential firing in the postsynaptic L-Mi. Interestingly, the time to first action potential was significantly shorter in the presence of MSOP, indicating that mGluR 4/8 activation delays L-Mi firing in response to MF activity. Taken together, our data demonstrate that the timing and probability of action potentials in L-Mi evoked by MF synaptic input is regulated by the activation of presynaptic high affinity group III mGluRs.

  12. Water channel in the binding site of a high affinity anti-methotrexate antibody.

    PubMed

    Gayda, Susan; Longenecker, Kenton L; Manoj, Sharmila; Judge, Russell A; Saldana, Sylvia C; Ruan, Qiaoqiao; Swift, Kerry M; Tetin, Sergey Y

    2014-06-17

    In the present study, we report the structure of the free and drug-bound Fab fragment of a high affinity anti-methotrexate antibody and perform a thermodynamic analysis of the binding process. The anti-methotrexate Fab fragment features a remarkably rigid tunnel-like binding site that extends into a water channel serving as a specialized route to move solvent out and into the site upon ligand binding and dissociation. This new finding in antibody structure-function relationships directly relates to the fast association (1 × 10⁷ M⁻¹ s⁻¹) and slow dissociation (4 × 10⁻⁵ s⁻¹) rates determined for mAb ADD056, resulting in a very strong binding with a K(D) ~ 3.6 pM at 20 °C. As follows from the X-ray data analysis, the methotrexate-antibody complex is stabilized by an extended network of hydrogen bonds and stacking interactions. The analysis also shows structural involvement of the CDR H3 in formation of the water channel revealing another important role of this hypervariable region. This suggests a new direction in natural affinity maturation and opens a new possibility in antibody engineering. Methotrexate is a widely used therapeutic agent for many malignant diseases and inflammatory disorders. Unfortunately, it may also interfere with central aspects of metabolism and thereby cause inevitable side effects. Therefore, methotrexate therapy requires careful monitoring of drug blood levels, which is traditionally done by immunoassays. An understanding of the structure-function properties of antibodies selected for drug monitoring substantiates the performance and robustness of such tests.

  13. High-affinity uptake of gamma-aminobutyric acid in cultured glial and neuronal cells.

    PubMed

    Balcar, V J; Mark, J; Borg, J; Mandel, P

    1979-06-01

    Both glial and neuronal cells maintained in primary culture were found to accumulate [3H]GABA by an efficient "high-affinity" uptake system (apparent Km = 9 muM, Vmax = 0.018 and 0.584 nmol/mg/min, respectively) which required sodium ions and was inhibited by 1 mM ouabain. Strychnine and parachloromercuriphenylsulfonate (pCS) (both at 1mM) also strongly inhibited uptake of [3H]GABA, but metabolic inhibitors (2,4-dinitrophenol, potassium cyanide, and malonate) were without effect. Only three structural analogs of GABA (nipecotate, beta-alanine, and 2,4-diaminobutyrate) inhibited uptake of [3H]GABA, while several other compounds with structural similarities to GABA (e.g. glycine, L-proline, and taurine) did not interact with the system. The kinetic studies indicated presence of a second uptake (Km = 92 muM, Vmax = 0.124 nmol/mg/min) in the primary cultures containing predominantly glioblasts. On the other hand, only one of the neuronal cell lines transformed by simian virus SV40 appeared to accumulate [3H]GABA against a concentration gradient. Apparent Km of this uptake was relatively high (819 muM), and it was only weakly inhibited by 1 mM ouabain and 1 mM pCS. The structural specificity also differed from that of the uptake observed in the primary cultures. Significantly, non of the nontransformed continuous cell lines of either tumoral (glioma, C6; neuroblastoma, M1; M1NN) or normal (NN;I6) origin actively accumulated [3H]GABA. It is suggested that for the neurochemical studies related to GABA and requiring homogeneous cell populations, the primary cultures offer a better experimental model than the continuous cell lines.

  14. Early Signs of Pathological Cognitive Aging in Mice Lacking High-Affinity Nicotinic Receptors

    PubMed Central

    Konsolaki, Eleni; Tsakanikas, Panagiotis; Polissidis, Alexia V.; Stamatakis, Antonios; Skaliora, Irini

    2016-01-01

    In order to address pathological cognitive decline effectively, it is critical to adopt early preventive measures in individuals considered at risk. It is therefore essential to develop approaches that identify such individuals before the onset of irreversible dementia. A deficient cholinergic system has been consistently implicated as one of the main factors associated with a heightened vulnerability to the aging process. In the present study we used mice lacking high affinity nicotinic receptors (β2-/-), which have been proposed as an animal model of accelerated/premature cognitive aging. Our aim was to identify behavioral signs that could serve as indicators or predictors of impending cognitive decline. We used test batteries in order to assess cognitive functions and additional tasks to investigate spontaneous behaviors, such as species-specific activities and exploration/locomotion in a novel environment. Our data confirm the hypothesis that β2-/- animals exhibit age-related cognitive impairments in spatial learning. In addition, they document age-related deficits in other areas, such as recognition memory, burrowing and nesting building, thereby extending the validity of this animal model for the study of pathological aging. Finally, our data reveal deficits in spontaneous behavior and habituation processes that precede the onset of cognitive decline and could therefore be useful as a non-invasive behavioral screen for identifying animals at risk. To our knowledge, this is the first study to perform an extensive behavioral assessment of an animal model of premature cognitive aging, and our results suggest that β2-nAChR dependent cognitive deterioration progressively evolves from initial subtle behavioral changes to global dementia due to the combined effect of the neuropathology and aging. PMID:27199738

  15. Devices and approaches for generating specific high-affinity nucleic acid aptamers

    NASA Astrophysics Data System (ADS)

    Szeto, Kylan; Craighead, Harold G.

    2014-09-01

    High-affinity and highly specific antibody proteins have played a critical role in biological imaging, medical diagnostics, and therapeutics. Recently, a new class of molecules called aptamers has emerged as an alternative to antibodies. Aptamers are short nucleic acid molecules that can be generated and synthesized in vitro to bind to virtually any target in a wide range of environments. They are, in principal, less expensive and more reproducible than antibodies, and their versatility creates possibilities for new technologies. Aptamers are generated using libraries of nucleic acid molecules with random sequences that are subjected to affinity selections for binding to specific target molecules. This is commonly done through a process called Systematic Evolution of Ligands by EXponential enrichment, in which target-bound nucleic acids are isolated from the pool, amplified to high copy numbers, and then reselected against the desired target. This iterative process is continued until the highest affinity nucleic acid sequences dominate the enriched pool. Traditional selections require a dozen or more laborious cycles to isolate strongly binding aptamers, which can take months to complete and consume large quantities of reagents. However, new devices and insights from engineering and the physical sciences have contributed to a reduction in the time and effort needed to generate aptamers. As the demand for these new molecules increases, more efficient and sensitive selection technologies will be needed. These new technologies will need to use smaller samples, exploit a wider range of chemistries and techniques for manipulating binding, and integrate and automate the selection steps. Here, we review new methods and technologies that are being developed towards this goal, and we discuss their roles in accelerating the availability of novel aptamers.

  16. In vitro selection, characterization, and biosensing application of high-affinity cylindrospermopsin-targeting aptamers.

    PubMed

    Elshafey, Reda; Siaj, Mohamed; Zourob, Mohammed

    2014-09-16

    Contamination of freshwater with cyanotoxin cylindrospermopsin (CYN) represents a significant global concern for public health. The sensitive detection of CYN is necessary to effectively manage and control the treatment of water resources. Here we report a novel, highly sensitive label-free aptasensor for CYN analysis, using aptamers as specific receptors. We have selected the DNA aptamers from a diverse random library using the in vitro screening SELEX approach. The aptamers exhibited high affinity for CYN with Kd of nanomolar range. One aptamer exhibited conformational change upon CYN recognition (CD analysis) and was used to fabricate the label-free impedimetric aptasensor for CYN. A self-assembled monolayer from a disulfide-derivatized aptamer was formed on a gold electrode to fabricate the aptasensor. Upon CYN capturing to the aptasensor surface, a marked drop in the electron transfer resistance was obtained, which was used as the principle of detection of CYN. This resulted from the aptamer's conformational change induced by CYN recognition. The present aptasensor could detect CYN with the limit of detection as low as 100 pM and a wide linear range of 0.1 to 80 nM. When mounted on the gold surface, the aptamer exhibited a lower dissociation constant for CYN than that observed in the fluorescence assay, implying that the anchoring of the aptamer on the Au surface improved its affinity to CYN. Moreover, the aptasensor showed high specificity toward other coexistent cyanobacterial toxins of microcystin-LR and Anatoxin-a. Further biosensor designs will be generated using those aptamers for simple and sensitive CYN monitoring.

  17. High affinity nanobodies against the Trypanosome brucei VSG are potent trypanolytic agents that block endocytosis.

    PubMed

    Stijlemans, Benoît; Caljon, Guy; Natesan, Senthil Kumar A; Saerens, Dirk; Conrath, Katja; Pérez-Morga, David; Skepper, Jeremy N; Nikolaou, Alexandros; Brys, Lea; Pays, Etienne; Magez, Stefan; Field, Mark C; De Baetselier, Patrick; Muyldermans, Serge

    2011-06-01

    The African trypanosome Trypanosoma brucei, which persists within the bloodstream of the mammalian host, has evolved potent mechanisms for immune evasion. Specifically, antigenic variation of the variant-specific surface glycoprotein (VSG) and a highly active endocytosis and recycling of the surface coat efficiently delay killing mediated by anti-VSG antibodies. Consequently, conventional VSG-specific intact immunoglobulins are non-trypanocidal in the absence of complement. In sharp contrast, monovalent antigen-binding fragments, including 15 kDa nanobodies (Nb) derived from camelid heavy-chain antibodies (HCAbs) recognizing variant-specific VSG epitopes, efficiently lyse trypanosomes both in vitro and in vivo. This Nb-mediated lysis is preceded by very rapid immobilisation of the parasites, massive enlargement of the flagellar pocket and major blockade of endocytosis. This is accompanied by severe metabolic perturbations reflected by reduced intracellular ATP-levels and loss of mitochondrial membrane potential, culminating in cell death. Modification of anti-VSG Nbs through site-directed mutagenesis and by reconstitution into HCAbs, combined with unveiling of trypanolytic activity from intact immunoglobulins by papain proteolysis, demonstrates that the trypanolytic activity of Nbs and Fabs requires low molecular weight, monovalency and high affinity. We propose that the generation of low molecular weight VSG-specific trypanolytic nanobodies that impede endocytosis offers a new opportunity for developing novel trypanosomiasis therapeutics. In addition, these data suggest that the antigen-binding domain of an anti-microbial antibody harbours biological functionality that is latent in the intact immunoglobulin and is revealed only upon release of the antigen-binding fragment.

  18. RNA sequence and secondary structure participate in high-affinity CsrA-RNA interaction.

    PubMed

    Dubey, Ashok K; Baker, Carol S; Romeo, Tony; Babitzke, Paul

    2005-10-01

    The global Csr regulatory system controls bacterial gene expression post-transcriptionally. CsrA of Escherichia coli is an RNA binding protein that plays a central role in repressing several stationary phase processes and activating certain exponential phase functions. CsrA regulates translation initiation of several genes by binding to the mRNA leaders and blocking ribosome binding. CsrB and CsrC are noncoding regulatory RNAs that are capable of sequestering CsrA and antagonizing its activity. Each of the known target transcripts contains multiple CsrA binding sites, although considerable sequence variation exists among these RNA targets, with GGA being the most highly conserved element. High-affinity RNA ligands containing single CsrA binding sites were identified from a combinatorial library using systematic evolution of ligands by exponential enrichment (SELEX). The SELEX-derived consensus was determined as RUACARGGAUGU, with the ACA and GGA motifs being 100% conserved and the GU sequence being present in all but one ligand. The majority (51/55) of the RNAs contained GGA in the loop of a hairpin within the most stable predicted structure, an arrangement similar to several natural CsrA binding sites. Strikingly, the identity of several nucleotides that were predicted to form base pairs in each stem were 100% conserved, suggesting that primary sequence information was embedded within the base-paired region. The affinity of CsrA for several selected ligands was measured using quantitative gel mobility shift assays. A mutational analysis of one selected ligand confirmed that the conserved ACA, GGA, and GU residues were critical for CsrA binding and that RNA secondary structure participates in CsrA-RNA recognition.

  19. High-affinity σ1 protein agonist reduces clinical and pathological signs of experimental autoimmune encephalomyelitis

    PubMed Central

    Oxombre, B; Lee-Chang, C; Duhamel, A; Toussaint, M; Giroux, M; Donnier-Maréchal, M; Carato, P; Lefranc, D; Zéphir, H; Prin, L; Melnyk, P; Vermersch, P

    2015-01-01

    Background and Purpose Selective agonists of the sigma-1 receptor (σ1 protein) are generally reported to protect against neuronal damage and modulate oligodendrocyte differentiation. Human and rodent lymphocytes possess saturable, high-affinity binding sites for compounds binding to the σ1 protein and potential immunomodulatory properties have been described for σ1 protein ligands. Experimental autoimmune encephalomyelitis (EAE) is recognized as a valuable model of the inflammatory aspects of multiple sclerosis (MS). Here, we have assessed the role of a σ1 protein agonist, containing the tetrahydroisoquinoline-hydantoin structure, in EAE. Experimental Approach EAE was induced in SJL/J female mice by active immunization with myelin proteolipid protein (PLP)139–151 peptide. The σ1 protein agonist was injected i.p. at the time of immunization (day 0). Disease severity was assessed clinically and by histopathological evaluation of the CNS. Phenotyping of B-cell subsets and regulatory T-cells were performed by flow cytometry in spleen and cervical lymph nodes. Key Results Prophylactic treatment of EAE mice with the σ1 protein agonist prevented mononuclear cell accumulation and demyelination in brain and spinal cord and increased T2 B-cells and regulatory T-cells, resulting in an overall reduction in the clinical progression of EAE. Conclusions and Implications This σ1 protein agonist, containing the tetrahydroisoquinoline-hydantoin structure, decreased the magnitude of inflammation in EAE. This effect was associated with increased proportions of B-cell subsets and regulatory T-cells with potential immunoregulatory functions. Targeting of the σ1 protein might thus provide new therapeutic opportunities in MS. PMID:25521311

  20. [3H]-lifarizine, a high affinity probe for inactivated sodium channels.

    PubMed Central

    MacKinnon, A. C.; Wyatt, K. M.; McGivern, J. G.; Sheridan, R. D.; Brown, C. M.

    1995-01-01

    1. [3H]-lifarizine bound saturably and reversibly to an apparently homogeneous class of high affinity sites in rat cerebrocortical membranes (Kd = 10.7 +/- 2.9 nM; Bmax = 5.10 +/- 1.43 pmol mg-1 protein). 2. The binding of [3H]-lifarizine was unaffected by sodium channel toxins binding to site 1 (tetrodotoxin), site 3 (alpha-scorpion venom) or site 5 (brevetoxin), Furthermore, lifarizine at concentrations up to 10 microM had no effect on [3H]-saxitoxin (STX) binding to toxin site 1. Lifarizine displaced [3H]-batrachotoxinin-A 20-alpha-benzoate (BTX) binding with moderate affinity (pIC50 7.31 +/- 0.24) indicating an interaction with toxin site 2. However, lifarizine accelerated the dissociation of [3H]-BTX and decreased both the affinity and density of sites labelled by [3H]-BTX, suggesting an allosteric interaction with toxin site 2. 3. The binding of [3H]-lifarizine was voltage-sensitive, binding to membranes with higher affinity than to synaptosomes (pIC50 for cold lifarizine = 7.99 +/- 0.09 in membranes and 6.68 +/- 0.14 in synaptosomes). Depolarization of synaptosomes with 130 mM KCl increased the affinity of lifarizine almost 10 fold (pIC50 = 7.86 +/- 0.25). This suggests that lifarizine binds selectively to inactivated sodium channels which predominate both in the membrane preparation and in the depolarized synaptosomal preparation. 4. There was negligible [3H]-lifarizine and [3H]-BTX binding to solubilized sodium channels, although [3H]-STX binding was retained under these conditions.(ABSTRACT TRUNCATED AT 250 WORDS) PMID:7582509

  1. Targeting protein-protein interactions with trimeric ligands: high affinity inhibitors of the MAGUK protein family.

    PubMed

    Nissen, Klaus B; Haugaard-Kedström, Linda M; Wilbek, Theis S; Nielsen, Line S; Åberg, Emma; Kristensen, Anders S; Bach, Anders; Jemth, Per; Strømgaard, Kristian

    2015-01-01

    PDZ domains in general, and those of PSD-95 in particular, are emerging as promising drug targets for diseases such as ischemic stroke. We have previously shown that dimeric ligands that simultaneously target PDZ1 and PDZ2 of PSD-95 are highly potent inhibitors of PSD-95. However, PSD-95 and the related MAGUK proteins contain three consecutive PDZ domains, hence we envisioned that targeting all three PDZ domains simultaneously would lead to more potent and potentially more specific interactions with the MAGUK proteins. Here we describe the design, synthesis and characterization of a series of trimeric ligands targeting all three PDZ domains of PSD-95 and the related MAGUK proteins, PSD-93, SAP-97 and SAP-102. Using our dimeric ligands targeting the PDZ1-2 tandem as starting point, we designed novel trimeric ligands by introducing a PDZ3-binding peptide moiety via a cysteine-derivatized NPEG linker. The trimeric ligands generally displayed increased affinities compared to the dimeric ligands in fluorescence polarization binding experiments and optimized trimeric ligands showed low nanomolar inhibition towards the four MAGUK proteins, thus being the most potent inhibitors described. Kinetic experiments using stopped-flow spectrometry showed that the increase in affinity is caused by a decrease in the dissociation rate of the trimeric ligand as compared to the dimeric ligands, likely reflecting the lower probability of simultaneous dissociation of all three PDZ ligands. Thus, we have provided novel inhibitors of the MAGUK proteins with exceptionally high affinity, which can be used to further elucidate the therapeutic potential of these proteins.

  2. Targeting Protein-Protein Interactions with Trimeric Ligands: High Affinity Inhibitors of the MAGUK Protein Family

    PubMed Central

    Nissen, Klaus B.; Haugaard-Kedström, Linda M.; Wilbek, Theis S.; Nielsen, Line S.; Åberg, Emma; Kristensen, Anders S.; Bach, Anders; Jemth, Per; Strømgaard, Kristian

    2015-01-01

    PDZ domains in general, and those of PSD-95 in particular, are emerging as promising drug targets for diseases such as ischemic stroke. We have previously shown that dimeric ligands that simultaneously target PDZ1 and PDZ2 of PSD-95 are highly potent inhibitors of PSD-95. However, PSD-95 and the related MAGUK proteins contain three consecutive PDZ domains, hence we envisioned that targeting all three PDZ domains simultaneously would lead to more potent and potentially more specific interactions with the MAGUK proteins. Here we describe the design, synthesis and characterization of a series of trimeric ligands targeting all three PDZ domains of PSD-95 and the related MAGUK proteins, PSD-93, SAP-97 and SAP-102. Using our dimeric ligands targeting the PDZ1-2 tandem as starting point, we designed novel trimeric ligands by introducing a PDZ3-binding peptide moiety via a cysteine-derivatized NPEG linker. The trimeric ligands generally displayed increased affinities compared to the dimeric ligands in fluorescence polarization binding experiments and optimized trimeric ligands showed low nanomolar inhibition towards the four MAGUK proteins, thus being the most potent inhibitors described. Kinetic experiments using stopped-flow spectrometry showed that the increase in affinity is caused by a decrease in the dissociation rate of the trimeric ligand as compared to the dimeric ligands, likely reflecting the lower probability of simultaneous dissociation of all three PDZ ligands. Thus, we have provided novel inhibitors of the MAGUK proteins with exceptionally high affinity, which can be used to further elucidate the therapeutic potential of these proteins. PMID:25658767

  3. Can we accurately quantify nanoparticle associated proteins when constructing high-affinity MRI molecular imaging probes?

    PubMed

    Rimkus, Gabriella; Bremer-Streck, Sibylle; Grüttner, Cordula; Kaiser, Werner Alois; Hilger, Ingrid

    2011-01-01

    Targeted magnetic resonance contrast agents (e.g. iron oxide nanoparticles) have the potential to become highly selective imaging tools. In this context, quantification of the coupled amount of protein is essential for the design of antibody- or antibody fragment-conjugated nanoparticles. Nevertheless, the presence of magnetic iron oxide nanoparticles is still an unsolved problem for this task. The aim of the present work was to clarify whether proteins can be reliably quantified directly in the presence of magnetic iron oxide nanoparticles without the use of fluorescence or radioactivity. Protein quantification via Bradford was not influenced by the presence of magnetic iron oxide nanoparticles (0-17.2 mmol Fe l(-1) ). Instead, bicinchoninic acid based assay was, indeed, distinctly affected by the presence of nanoparticle-iron in suspension (0.1-17.2 mmol Fe l(-1) ), although the influence was linear. This observation allowed for adequate mathematical corrections with known iron content of a given nanoparticle. The applicability of our approach was demonstrated by the determination of bovine serum albumin (BSA) content coupled to dextrane-coated magnetic nanoparticles, which was found with the QuantiPro Bicinchoninic acid assay to be of 1.5 ± 0.2 µg BSA per 1 mg nanoparticle. Both Bradford and bicinchoninic acid assay protein assays allow for direct quantification of proteins in the presence of iron oxide containing magnetic nanoparticles, without the need for the introduction of radioactivity or fluorescence modules. Thus in future it should be possible to make more precise estimations about the coupled protein amount in high-affinity targeted MRI probes for the identification of specific molecules in living organisms, an aspect which is lacking in corresponding works published so far. Additionally, the present protein coupling procedures can be drastically improved by our proposed protein quantification method.

  4. In silico analysis of high affinity potassium transporter (HKT) isoforms in different plants.

    PubMed

    Zamani Babgohari, Mahbobeh; Ebrahimie, Esmaeil; Niazi, Ali

    2014-01-01

    High affinity potassium transporters (HKTs) are located in the plasma membrane of the vessels and have significant influence on salt tolerance in some plants. They exclude Na(+) from the parenchyma cells to reduce Na(+) concentration. Despite many studies, the underlying regulatory mechanisms and the exact functions of HKTs within different genomic backgrounds are relatively unknown. In this study, various bioinformatics techniques, including promoter analysis, identification of HKT-surrounding genes, and construction of gene networks, were applied to investigate the HKT regulatory mechanism. Promoter analysis showed that rice HKTs carry ABA response elements. Additionally, jasmonic acid response elements were detected on promoter region of TmHKT1;5. In silico synteny highlighted several unknown and new loci near rice, Arabidopsis thaliana and Physcomitrella patent HKTs, which may play a significant role in salt stress tolerance in concert with HKTs. Gene network prediction unravelled that crosstalk between jasmonate and ethylene reduces AtHKT1;1 expression. Furthermore, antiporter and transferase proteins were found in AtHKT1;1 gene network. Interestingly, regulatory elements on the promoter region of HKT in wild genotype (TmHKT1;5) were more frequent and variable than the ones in cultivated wheat (TaHKT1;5) which provides the possibility of rapid response and better understanding of environmental conditions for wild genotype. Detecting ABA and jasmonic acid response elements on promoter regions of HKTs provide valuable clues on underlying regulatory mechanisms of HKTs. In silico synteny and pathway discovery indicated several candidates which act in concert with HKTs in stress condition. We highlighted different arrangement of regulatory elements on promoter region of wild wheat (TmHKT1;5) compared to bread wheat (TaHKT1;5) in this study.

  5. A novel somatostatin conjugate with a high affinity to all five somatostatin receptor subtypes.

    PubMed

    Wulbrand, Ulrich; Feldman, Martin; Pfestroff, Andreas; Fehman, Hans-Cristophe; Du, Jin; Hiltunen, Jukka; Marquez, Marcela; Arnold, Rudolf; Westlin, Jan-Erik; Nilsson, Sten; Holmberg, Anders R

    2002-02-15

    Somatostatin receptors (SRS, five subtypes) are expressed in a variety of human tumors, including most tumors of neuroendocrine origin, breast tumors, certain brain tumors, renal cell tumors, lymphomas, and prostate cancer. Somatostatin (SMS) triggers cytostatic and cytotoxic effects and has a general inhibitory effect on secretion mediated through its interaction with SRS. That is the basis for its use in the treatment of SRS-positive tumors. Radiolabeled SMS analogs can also be used for systemic radiotherapy and for diagnostic investigations. Sms-14 was conjugated to a periodate-activated dextran70 (mean molecular weight, 70 kD) by reductive amination. The human tumor cell line LCC-18, from a neuroendocrine colonic tumor, was used for stable transfection with each SRS gene separately; transfection was achieved with the expression system TETon (Clontech, Palo Alto, CA). Clones were selected by culturing with G418 and hygromycin B, and positive clones were identified by reverse transcriptase-polymerase chain reaction and binding of iodine-125-labeled SMS-14. The binding affinity for each SRS subtype was then determined for the SMS-dextran conjugate (with SMS-14 used as a positive control). Sms-dextran70 showed high affinity binding to all five receptor subtypes. The IC50 values were between 3 and 80 nM. This conjugate has a long circulation half-life (i.e., approximately 27 hours after subcutaneous administration in mice) and, with high SRS pan-affinity demonstrated in this study, it has potential in the therapy of SRS-positive tumors. Currently, the clinical significance of SMS-dextran70 is being explored in a clinical Phase I-II study of patients with hormone-refractory prostate cancer. The outcome of this study will be reported when it is available. Copyright 2002 American Cancer Society.

  6. High affinity receptors for vasoactive intestinal peptide on a human glioma cell line

    SciTech Connect

    Nielsen, F.C.; Gammeltoft, S.; Westermark, B.; Fahrenkrug, J. )

    1990-11-01

    Vasoactive intestinal peptide (VIP) bound with high affinity (Kd 0.13 nmol/l) to receptors on the human glioma cell line U-343 MG Cl 2:6. The receptors bound the related peptides helodermin, PHM and secretin with 10, 400 and 5000 times lower affinity, respectively. Deamidated VIP (VIP-COOH) and (des-His1)VIP bound with 10 and 100 times lower affinity. The fragment VIP(7-28) displaced 25% of the receptor-bound {sup 125}I-VIP whereas VIP(16-28) and VIP(1-22-NH2) were inactive. The binding of {sup 125}I-VIP could be completely inhibited by 10 mumol/l of the antagonists (N-Ac-Tyr1,D-Phe2)GRF(1-29)-NH2, (pCl-D-Phe6,Leu17)VIP and VIP(10-28); in contrast, the antagonist L-8-K was inactive. Affinity labeling showed that VIP bound to proteins with Mr's of 75 kDa, 66 kDa and 50 kDa, respectively. Following binding, the peptide was rapidly internalized, and at steady-state only 20% of cell-associated {sup 125}I-VIP was bound to receptors on the cell surface. The internalized {sup 125}I-VIP was completely degraded to {sup 125}I-tyrosine which was released from the cells. Degradation of internalized {sup 125}I-VIP was significantly reduced by chloroquine phenanthroline and pepstatin-A. Surface binding and internalization of {sup 125}I-VIP was increased 3 times by phenanthroline, and pepstatin-A caused a 5 times increase in surface binding. Chloroquine reduced surface-bound {sup 125}I-VIP, but caused retention of internalized {sup 125}I-VIP.

  7. High Affinity Transport of CO2 in the Cyanobacterium Synechococcus UTEX 625 1

    PubMed Central

    Espie, George S.; Miller, Anthony G.; Canvin, David T.

    1991-01-01

    The active transport of CO2 in Synechococcus UTEX 625 was measured by mass spectrometry under conditions that preclude HCO3− transport. The substrate concentration required to give one half the maximum rate for whole cell CO2 transport was determined to be 0.4 ± 0.2 micromolar (mean ± standard deviation; n = 7) with a range between 0.2 and 0.66 micromolar. The maximum rates of CO2 transport ranged between 400 and 735 micromoles per milligram of chlorophyll per hour with an average rate of 522 for seven experiments. This rate of transport was about three times greater than the dissolved inorganic carbon saturated rate of photosynthetic O2 evolution observed under these conditions. The initial rate of chlorophyll a fluorescence quenching was highly correlated with the initial rate of CO2 transport (correlation coefficient = 0.98) and could be used as an indirect method to detect CO2 transport and calculate the substrate concentration required to give one half the maximum rate of transport. Little, if any, inhibition of CO2 transport was caused by HCO3− or by Na+-dependent HCO3− transport. However, 12CO2 readily interfered with 13CO2 transport. CO2 transport and Na+-dependent HCO3− transport are separate, independent processes and the high affinity CO2 transporter is not only responsible for the initial transport of CO2 into the cell but also for scavenging any CO2 that may leak from the cell during ongoing photosynthesis. PMID:16668535

  8. Predictive QSAR modeling of phosphodiesterase 4 inhibitors.

    PubMed

    Kovalishyn, Vasyl; Tanchuk, Vsevolod; Charochkina, Larisa; Semenuta, Ivan; Prokopenko, Volodymyr

    2012-02-01

    A series of diverse organic compounds, phosphodiesterase type 4 (PDE-4) inhibitors, have been modeled using a QSAR-based approach. 48 QSAR models were compared by following the same procedure with different combinations of descriptors and machine learning methods. QSAR methodologies used random forests and associative neural networks. The predictive ability of the models was tested through leave-one-out cross-validation, giving a Q² = 0.66-0.78 for regression models and total accuracies Ac=0.85-0.91 for classification models. Predictions for the external evaluation sets obtained accuracies in the range of 0.82-0.88 (for active/inactive classifications) and Q² = 0.62-0.76 for regressions. The method showed itself to be a potential tool for estimation of IC₅₀ of new drug-like candidates at early stages of drug development. Copyright © 2011 Elsevier Inc. All rights reserved.

  9. Advances in targeting cyclic nucleotide phosphodiesterases

    PubMed Central

    Maurice, Donald H.; Ke, Hengming; Ahmad, Faiyaz; Wang, Yousheng; Chung, Jay; Manganiello, Vincent C.

    2014-01-01

    Cyclic nucleotide phosphodiesterases (PDEs) catalyse the hydrolysis of cyclic AMP and cyclic GMP, thereby regulating the intracellular concentrations of these cyclic nucleotides, their signalling pathways and, consequently, myriad biological responses in health and disease. Currently, a small number of PDE inhibitors are used clinically for treating the pathophysiological dysregulation of cyclic nucleotide signalling in several disorders, including erectile dysfunction, pulmonary hypertension, acute refractory cardiac failure, intermittent claudication and chronic obstructive pulmonary disease. However, pharmaceutical interest in PDEs has been reignited by the increasing understanding of the roles of individual PDEs in regulating the subcellular compartmentalization of specific cyclic nucleotide signalling pathways, by the structure-based design of novel specific inhibitors and by the development of more sophisticated strategies to target individual PDE variants. PMID:24687066

  10. Biochemical characterization of high-affinity 3H-opioid binding. Further evidence for Mu1 sites

    SciTech Connect

    Nishimura, S.L.; Recht, L.D.; Pasternak, G.W.

    1984-01-01

    In saturation studies with (/sup 3/H)dihydromorphine, unlabeled D-Ala2-D-Leu5-enkephalin (1 nM) inhibited the high-affinity binding component far more potently than the lower-affinity one. Similarly, morphine (1 nM) inhibited the higher-affinity binding of /sup 3/H-D-Ala2-D-Leu5-enkephalin to a greater extent than its lower-affinity binding component, consistent with a common high-affinity binding site for opiates and enkephalins. Treatment of tissue with either trypsin (1 microgram/ml) or N-ethylmaleimide (25 microM) effectively eliminated the high-affinity binding component of a series of /sup 3/H-opiates and opioid peptides. Competition studies following both treatments were consistent with a common high-affinity binding site. Both treatments also eliminated the ability of low morphine concentrations (less than 1 nM) to inhibit /sup 3/H-D-Ala2-D-Leu5-enkephalin binding and of low D-Ala2-D-Leu5-enkephalin concentrations (less than 1 nM) to inhibit (/sup 3/H)dihydromorphine binding. Protection experiments examining N-ethylmaleimide (25 microM) inhibition of (/sup 3/H)dihydromorphine binding showed significant protection (p less than 0.002) by both unlabeled D-Ala2-D-Leu5-enkephalin and morphine (both at 1 nM). When studied together, both naloxonazine and N-ethylmaleimide inhibited (/sup 3/H)dihydromorphine binding to a similar extent. Equally important, tissue previously treated with naloxonazine was far less sensitive to N-ethylmaleimide than was untreated control tissue, consistent with the possibility that both treatments affected the same site. Together, these results support the concept of a common high-affinity binding site for opiates and opioid peptides.

  11. Specific high-affinity binding of high density lipoproteins to cultured human skin fibroblasts and arterial smooth muscle cells.

    PubMed

    Biesbroeck, R; Oram, J F; Albers, J J; Bierman, E L

    1983-03-01

    Binding of human high density lipoproteins (HDL, d = 1.063-1.21) to cultured human fibroblasts and human arterial smooth muscle cells was studied using HDL subjected to heparin-agarose affinity chromatography to remove apoprotein (apo) E and B. Saturation curves for binding of apo E-free 125I-HDL showed at least two components: low-affinity nonsaturable binding and high-affinity binding that saturated at approximately 20 micrograms HDL protein/ml. Scatchard analysis of high-affinity binding of apo E-free 125I-HDL to normal fibroblasts yielded plots that were significantly linear, indicative of a single class of binding sites. Saturation curves for binding of both 125I-HDL3 (d = 1.125-1.21) and apo E-free 125I-HDL to low density lipoprotein (LDL) receptor-negative fibroblasts also showed high-affinity binding that yielded linear Scatchard plots. On a total protein basis, HDL2 (d = 1.063-1.10), HDL3 and very high density lipoproteins (VHDL, d = 1.21-1.25) competed as effectively as apo E-free HDL for binding of apo E-free 125I-HDL to normal fibroblasts. Also, HDL2, HDL3, and VHDL competed similarly for binding of 125I-HDL3 to LDL receptor-negative fibroblasts. In contrast, LDL was a weak competitor for HDL binding. These results indicate that both human fibroblasts and arterial smooth muscle cells possess specific high affinity HDL binding sites. As indicated by enhanced LDL binding and degradation and increased sterol synthesis, apo E-free HDL3 promoted cholesterol efflux from fibroblasts. These effects also saturated at HDL3 concentrations of 20 micrograms/ml, suggesting that promotion of cholesterol efflux by HDL is mediated by binding to the high-affinity cell surface sites.

  12. Inter-residue coupling contributes to high-affinity subtype-selective binding of α-bungarotoxin to nicotinic receptors

    SciTech Connect

    Sine, Steven M.; Huang, Sun; Li, Shu-Xing; daCosta, Corrie J. B.; Chen, Lin

    2013-09-01

    The crystal structure of a pentameric α7 ligand-binding domain chimaera with bound α-btx (α-bungarotoxin) showed that of the five conserved aromatic residues in α7, only Tyr184 in loop C of the ligand-binding site was required for high-affinity binding. To determine whether the contribution of Tyr184 depends on local residues, we generated mutations in an α7/5HT3A (5-hydroxytryptamine type 3A) receptor chimaera, individually and in pairs, and measured 125I-labelled α-btx binding. The results show that mutations of individual residues near Tyr184 do not affect α-btx affinity, but pairwise mutations decrease affinity in an energetically coupled manner. Kinetic measurements show that the affinity decreases arise through increases in the α-btx dissociation rate with little change in the association rate. Replacing loop C in α7 with loop C from the α-btx-insensitive α2 or α3 subunits abolishes high-affinity α-btx binding, but preserves acetylcholine-elicited single channel currents. However, in both the α2 and α3 construct, mutating either residue that flanks Tyr184 to its α7 counterpart restores high-affinity α-btx binding. Analogously, in α7, mutating both residues that flank Tyr184 to the α2 or α3 counterparts abolishes high-affinity α-btx binding. Thus interaction between Tyr184 and local residues contributes to high-affinity subtype-selective α-btx binding.

  13. Inter-residue coupling contributes to high-affinity subtype-selective binding of α-bungarotoxin to nicotinic receptors

    PubMed Central

    Sine, Steven M.; Huang, Sun; Li, Shu-Xing; daCOSTA, Corrie J. B.; Chen, Lin

    2014-01-01

    The crystal structure of a pentameric α7 ligand-binding domain chimaera with bound α-btx (α-bungarotoxin) showed that of the five conserved aromatic residues in α7, only Tyr184 in loop C of the ligand-binding site was required for high-affinity binding. To determine whether the contribution of Tyr184 depends on local residues, we generated mutations in an α7/5HT3A (5-hydroxytryptamine type 3A) receptor chimaera, individually and in pairs, and measured 125I-labelled α-btx binding. The results show that mutations of individual residues near Tyr184 do not affect α-btx affinity, but pairwise mutations decrease affinity in an energetically coupled manner. Kinetic measurements show that the affinity decreases arise through increases in the α-btx dissociation rate with little change in the association rate. Replacing loop C in α7 with loop C from the α-btx-insensitive α2 or α3 subunits abolishes high-affinity α-btx binding, but preserves acetylcholine-elicited single channel currents. However, in both the α2 and α3 construct, mutating either residue that flanks Tyr184 to its α7 counterpart restores high-affinity α-btx binding. Analogously, in α7, mutating both residues that flank Tyr184 to the α2 or α3 counterparts abolishes high-affinity α-btx binding. Thus interaction between Tyr184 and local residues contributes to high-affinity subtype-selective α-btx binding. PMID:23802200

  14. cAMP and Mitochondria

    PubMed Central

    Valsecchi, Federica; Ramos-Espiritu, Lavoisier S.; Buck, Jochen; Levin, Lonny R.

    2013-01-01

    Phosphorylation of mitochondrial proteins has emerged as a major regulatory mechanism for metabolic adaptation. cAMP signaling and PKA phosphorylation of mitochondrial proteins have just started to be investigated, and the presence of cAMP-generating enzymes and PKA inside mitochondria is still controversial. Here, we discuss the role of cAMP in regulating mitochondrial bioenergetics through protein phosphorylation and the evidence for soluble adenylyl cyclase as the source of cAMP inside mitochondria. PMID:23636265

  15. Foreign Language Camps at the College Level.

    ERIC Educational Resources Information Center

    Griswold, Jean S.

    Information on Colorado State University's foreign language camps for college students is presented. Advantages of the following two models for camps are identified: a single language camp, and a combination multi-language camp with four languages (Spanish, French, German, and Chinese). Features of the camps include: speaking in the foreign…

  16. Attenuation of Inhibitory Prostaglandin E2 Signaling in Human Lung Fibroblasts Is Mediated by Phosphodiesterase 4

    PubMed Central

    Michalski, Joel; Kanaji, Nobuhiro; Liu, Xiangde; Nogel, Steve; Wang, Xingqi; Basma, Hesham; Nakanishi, Masanori; Sato, Tadashi; Gunji, Yoko; Fahrid, Maha; Nelson, Amy; Muller, Kai-Christian; Holz, Olaf; Magnussen, Helgo; Rabe, Klaus F.; Toews, Myron L.

    2012-01-01

    The etiology of chronic obstructive pulmonary disease (COPD) is complex and involves an aberrant inflammatory response. Prostaglandin (PG)E2 is elevated in COPD, is a key modulator of lung fibroblast functions, and may influence COPD progression. Most studies evaluating the effects of PGE2 on lung fibroblasts have used acute exposures. The current study evaluated whether longer-term exposure would induce attenuation of PGE2 signaling as part of an autoregulatory pathway. Human fetal lung fibroblasts were pretreated with PGE2 for 24 hours, and migration and cAMP accumulation in response to acute stimulation with PGE2 were assessed. Fibroblasts from adults with and without COPD were pretreated, and migration was assessed. PGE2 pretreatment attenuated subsequent PGE2-mediated inhibition of chemotaxis and cAMP stimulation. This attenuation was predominantly due to an increase in phosphodiesterase (PDE)4-mediated degradation of cAMP rather than to decreased activation of PGE2 receptors (receptor desensitization). Albuterol- and iloprost-mediated signaling were also attenuated after PGE2 pretreatment, suggesting that activation of PDE4 was able to broadly modulate multiple cAMP-coupled pathways. Lung fibroblasts from adult control subjects pretreated with PGE2 also developed attenuation of PGE2-mediated inhibition of chemotaxis. In contrast, fibroblasts obtained from patients with COPD maintained inhibitory PGE2 signaling after PGE2 pretreatment. These data identify a PDE4-mediated attenuation of PGE2 inhibitory signaling in normal fibroblasts that appears to be altered in COPD fibroblasts. These alterations may contribute to COPD pathogenesis and could provide novel therapeutic targets. PMID:23043089

  17. The Function of Vascular Smooth Muscle Phosphodiesterase III is Preserved in Healthy Human Aging

    PubMed Central

    Elvebak, Rachel L.; Eisenach, John H.; Joyner, Michael J.; Nicholson, Wayne T.

    2010-01-01

    Abstract Phosphodiesterase (PDE) III is an enzyme in vascular smooth muscle that metabolizes cyclic adenosine monophosphate (cAMP). Milrinone inhibits PDE III, increasing the availability of cAMP. Cyclic guanosine monophosphate (cGMP), which is regulated by nitric oxide (NO), also inhibits PDE III. The endothelial NO component of prostacyclin (PGI2)‐mediated vasodilation is reduced in aging. This study investigated if PGI2‐mediated vasodilation during concomitant inhibition of endothelial NO and smooth muscle PDE III is affected by healthy aging. PDE III was inhibited with milrinone in 10 older subjects and 10 young matched controls while simultaneously infusing NG‐monomethyl‐l‐arginine acetate (l‐NMMA) to remove the confounding inhibitory effects of cGMP on PDE III. Incremental doses of PGI2 and sodium nitroprusside (SNP) were administered to the brachial artery during separate trials. l‐NMMA decreased baseline blood flow similarly, and the addition of milrinone increased baseline blood flow similarly in both groups. The forearm blood flow responses to PGI2 were similar between groups (younger: 7.62 ± 0.72; older: 6.88 ± 0.81 mL•dL−1 FAV•min−1 at the highest dose of PGI2). SNP responses were also similar. This study suggests that the vasodilator pathway associated with PDE III function, the bioavailability of cAMP, and the interaction with cGMP may be preserved in healthy aging. Clin Trans Sci 2010; Volume 3: 239–242. PMID:21500398

  18. Resveratrol and curcumin enhance pancreatic β-cell function by inhibiting phosphodiesterase activity.

    PubMed

    Rouse, Michael; Younès, Antoine; Egan, Josephine M

    2014-11-01

    Resveratrol (RES) and curcumin (CUR) are polyphenols that are found in fruits and turmeric, and possess medicinal properties that are beneficial in various diseases, such as heart disease, cancer, and type 2 diabetes mellitus (T2DM). Results from recent studies have indicated that their therapeutic properties can be attributed to their anti-inflammatory effects. Owing to reports stating that they protect against β-cell dysfunction, we studied their mechanism(s) of action in β-cells. In T2DM, cAMP plays a critical role in glucose- and incretin-stimulated insulin secretion as well as overall pancreatic β-cell health. A potential therapeutic target in the management of T2DM lies in regulating the activity of phosphodiesterases (PDEs), which degrade cAMP. Both RES and CUR have been reported to act as PDE inhibitors in various cell types, but it remains unknown if they do so in pancreatic β-cells. In our current study, we found that both RES (0.1-10 μmol/l) and CUR (1-100 pmol/l)-regulated insulin secretion under glucose-stimulated conditions. Additionally, treating β-cell lines and human islets with these polyphenols led to increased intracellular cAMP levels in a manner similar to 3-isobutyl-1-methylxanthine, a classic PDE inhibitor. When we investigated the effects of RES and CUR on PDEs, we found that treatment significantly downregulated the mRNA expression of most of the 11 PDE isozymes, including PDE3B, PDE8A, and PDE10A, which have been linked previously to regulation of insulin secretion in islets. Furthermore, RES and CUR inhibited PDE activity in a dose-dependent manner in β-cell lines and human islets. Collectively, we demonstrate a novel role for natural-occurring polyphenols as PDE inhibitors that enhance pancreatic β-cell function. © 2014 The authors.

  19. Astro Camp Goes to Florida

    NASA Image and Video Library

    2007-08-08

    Katie Craig, daughter of former Stennis Space Center Deputy Director Mark Craig, launches a 'balloon rocket' with the help of Rebecca Compretta, Astro Camp coordinator at SSC. SSC took Astro Camp on the road to Florida this week to engage children and their parents during activities surrounding the Aug. 8 launch of Space Shuttle Endeavour on NASA's STS-118 mission to the International Space Station. Astro Camp is SSC's popular space camp program designed to inspire and educate students using science and math principles.

  20. Evidence that cyclic AMP phosphodiesterase inhibitors suppress interleukin-2 release from murine splenocytes by interacting with a ‘low-affinity' phosphodiesterase 4 conformer

    PubMed Central

    Souness, John E; Houghton, Clare; Sardar, Nughat; Withnall, Michael T

    1997-01-01

    We have investigated the suppressive effects of rolipram, RP 73401 (piclamilast) and other structurally diverse inhibitors of cyclic AMP-specific phosphodiesterase 4 (PDE4) on interleukin (IL)-2 generation from Balb/c mouse splenocytes exposed to the superantigen, Staphylococcocal enterotoxin-A (Staph. A). The purpose was to determine whether their potencies are more closely correlated with inhibition of PDE4 from CTLL cells, against which rolipram displays weak potency (low-affinity PDE4), or displacement of [3H]-(±)-rolipram from its high-affinity binding site (HARBS) in mouse brain cytosol. RP 73401 (IC50 0.46±0.07 nM, n=4) was a very potent inhibitor of Staph. A-induced IL-2 release from Balb/c mouse splenocytes, being >1100 fold more potent than (±)-rolipram (IC50 540±67 nM, n=3). A close correlation (r=0.95) was observed between suppression of IL-2 release by PDE inhibitors and inhibition of PDE4. In contrast, little correlation (r=0.39) was observed between suppression of IL-2 release and their affinities for the high-affinity rolipram binding site (HARBS). RP 73401 only inhibited partially (30–40%) Staph. A-induced incorporation of [3H]-thymidine into splenocyte DNA. The PDE3 inhibitor, siguazodan (10 μM), had little or no effect on IL-2 release or DNA synthesis. This concentration of siguazodan did not enhance the inhibitory action of RP 73401 on IL-2 release but potentiated its effect on DNA synthesis, increasing potency and efficacy. Staph. A-induced DNA synthesis was only partially inhibited by anti-IL-2 neutralizing antibody, whereas dexamethazone (100 nM) and cyclosporine A (100 nM) completely blocked the response. RP 73401 (IC50 6.3±1.9 nM, n=4) was 140 fold more potent than rolipram (IC50 900±300 nM, n=3) in inhibiting Staph. A-induced [3H]-thymidine incorporation into splenocyte DNA. The results implicate a low-affinity form of PDE4 in the suppression of Staph. A-induced IL-2 release from murine splenocytes by PDE inhibitors

  1. Renal Epithelial Cyst Formation and Enlargement in vitro: Dependence on cAMP

    NASA Astrophysics Data System (ADS)

    Mangoo-Karim, Roberto; Uchic, Marie; Lechene, Claude; Grantham, Jared J.

    1989-08-01

    Cysts, a common abnormality of kidneys, are collections of urine-like fluid enclosed by a continuous layer of epithelial cells. Renal cysts derive from nephrons and collecting ducts and progressively enlarge as a consequence of epithelial proliferation and transepithelial fluid secretion. The initiation of cyst formation and the factors that control cyst enlargement are unknown. We used an in vitro model of renal cysts to explore the role of the cAMP signal transduction system in the formation and expansion of cysts. MDCK cells, cultured in hydrated-collagen gel, produced polarized monolayered epithelial cysts when intracellular cAMP was increased by prostaglandin E1, arginine vasopressin, cholera toxin, forskolin, or 8-bromoadenosine 3',5'-cyclic monophosphate. All agonists were potentiated by 3-isobutyl-1-methylxanthine, a nucleotide phosphodiesterase inhibitor. The cell proliferation component of cyst enlargement was accelerated by cAMP agonists, as shown by the increased growth of MDCK cells in subconfluent monolayers. The fluid secretion component, reflected by the transepithelial movement of fluid across polarized monolayers of MDCK cells grown on permeable supports, was stimulated by cAMP agonists in the basolateral medium. Chloride levels were higher in the cyst fluid and the secreted fluid than in the bathing medium. We conclude that the development of MDCK cysts is dependent on cAMP. This signal transduction system may be an important modulator of epithelial cell proliferation and transepithelial fluid secretion in the kidney.

  2. Eviprostat Activates cAMP Signaling Pathway and Suppresses Bladder Smooth Muscle Cell Proliferation

    PubMed Central

    Li, Kai; Yao, Jian; Chi, Yuan; Sawada, Norifumi; Araki, Isao; Kitamura, Masanori; Takeda, Masayuki

    2013-01-01

    Eviprostat is a popular phytotherapeutic agent for the treatment of lower urinary tract symptoms (LUTS). At present, the signaling mechanisms underlying its therapeutic effects are still poorly understood. Given that cAMP has been reported to suppress cell hyperplasia and hypertrophy in various pathological situations, we asked whether the effect of Eviprostat could be ascribed to the activation of the cAMP signaling pathway. In the study, exposure of cAMP response element (CRE)-secreted alkaline phosphatase (SEAP) (CRE-SEAP)-reporter cells to Eviprostat elevated SEAP secretion, which was associated with an increased phosphorylation of vasodilator-stimulated phosphoprotein (VASP) and cAMP-response element-binding protein (CREB), as well as enhanced expression of CRE-regulated protein connexin43, indicating an activation of the cAMP signaling pathway. Consistent with these observations, Eviprostat-induced expression of Cx43 was abolished in the presence of adenylyl cyclase inhibitor SQ22536 or PKA inhibitor H89, whereas it was mimicked by adenylyl cyclase activator, forskolin. Further analysis demonstrated that Eviprostat significantly potentiated the effect of phosphodiesterase 3 (PDE3) inhibitor, but not that of PDE4 inhibitor, on CRE activation. Moreover, Eviprostat suppressed PDGF-induced activation of ERK and Akt and inhibited cell proliferation and hillock formation in both mesangial cells and bladder smooth muscle cells. Collectively, activation of the cAMP signaling pathway could be an important mechanism by which Eviprostat exerts its therapeutic effects for LUTS. PMID:23743824

  3. cAMP and Schwann cells promote axonal growth and functional recovery after spinal cord injury.

    PubMed

    Pearse, Damien D; Pereira, Francisco C; Marcillo, Alexander E; Bates, Margaret L; Berrocal, Yerko A; Filbin, Marie T; Bunge, Mary Bartlett

    2004-06-01

    Central neurons regenerate axons if a permissive environment is provided; after spinal cord injury, however, inhibitory molecules are present that make the local environment nonpermissive. A promising new strategy for inducing neurons to overcome inhibitory signals is to activate cAMP signaling. Here we show that cAMP levels fall in the rostral spinal cord, sensorimotor cortex and brainstem after spinal cord contusion. Inhibition of cAMP hydrolysis by the phosphodiesterase IV inhibitor rolipram prevents this decrease and when combined with Schwann cell grafts promotes significant supraspinal and proprioceptive axon sparing and myelination. Furthermore, combining rolipram with an injection of db-cAMP near the graft not only prevents the drop in cAMP levels but increases them above those in uninjured controls. This further enhances axonal sparing and myelination, promotes growth of serotonergic fibers into and beyond grafts, and significantly improves locomotion. These findings show that cAMP levels are key for protection, growth and myelination of injured CNS axons in vivo and recovery of function.

  4. No ordinary boot camp.

    PubMed

    Tichy, N M

    2001-04-01

    Many companies now run boot camps--comprehensive orientation programs designed to help new hires hit the ground running. They're intense and intimidating, and new employees emerge from them with strong bonds to other recruits and to the organization. But at Trilogy, organizational consultant Noel Tichy discovered one program that's a breed apart. In this article, Tichy gives us a detailed tour of Trilogy's boot camp, Trilogy University, to demonstrate why it's so different--and so effective. Like the best boot camps, it serves as an immersion in both the technical skills new recruits will need for their jobs and Trilogy's corporate culture, which emphasizes risk-taking, teamwork, humility, and a strong customer focus. But this is a new-employee orientation session that's so fundamental to the company as a whole that it's presided over by the CEO and top corporate executives for fully six months of the year. Why? In two three-month sessions, these top executives hone their own strategic thinking about the company as they decide what to teach the new recruits each session. They also find the company's next generation of new products as they judge the innovative ideas the recruits are tasked with developing--making the program Trilogy's main R&D engine. And they pull the company's rising technical stars into mentoring roles for the new recruits, helping to build the next generation of top leadership. After spending months on-site studying Trilogy University, Tichy came away highly impressed by the power of the virtuous teaching cycle the program has set in motion. Leaders of the organization are learning from recruits at the same time that the recruits are learning from the leaders. It's a model, he argues, that other companies would do well to emulate.

  5. Newly synthesized cAMP is integrated at a membrane protein complex signalosome to ensure receptor response specificity.

    PubMed

    Guinzberg, Raquel; Díaz-Cruz, Antonio; Acosta-Trujillo, Carlos; Vilchis-Landeros, María Magdalena; Vázquez-Meza, Héctor; Lozano-Flores, Carlos; Chiquete-Felix, Natalia; Varela-Echavarría, Alfredo; Uribe-Carvajal, Salvador; Riveros-Rosas, Héctor; Piña, Enrique

    2017-01-01

    Spatiotemporal regulation of cAMP within the cell is required to achieve receptor-specific responses. The mechanism through which the cell selects a specific response to newly synthesized cAMP is not fully understood. In hepatocyte plasma membranes, we identified two functional and independent cAMP-responsive signaling protein macrocomplexes that produce, use, degrade, and regulate their own nondiffusible (sequestered) cAMP pool to achieve their specific responses. Each complex responds to the stimulation of an adenosine G protein-coupled receptor (Ado-GPCR), bound to either A2A or A2B , but not simultaneously to both. Each isoprotein involved in each signaling cascade was identified by measuring changes in cAMP levels after receptor activation, and its participation was confirmed by antibody-mediated inactivation. A2A -Ado-GPCR selective stimulation activates adenylyl cyclase 6 (AC6), which is bound to AKAP79/150, to synthesize cAMP which is used by two other AKAP79/150-tethered proteins: protein kinase A (PKA) and phosphodiesterase 3A (PDE3A). In contrast, A2B -Ado-GPCR stimulation activates D-AKAP2-attached AC5 to generate cAMP, which is channeled to two other D-AKAP2-tethered proteins: guanine-nucleotide exchange factor 2 (Epac2) and PDE3B. In both cases, prior activation of PKA or Epac2 with selective cAMP analogs prevents de novo cAMP synthesis. In addition, we show that cAMP does not diffuse between these protein macrocomplexes or 'signalosomes'. Evidence of coimmunoprecipitation and colocalization of some proteins belonging to each signalosome is presented. Each signalosome constitutes a minimal functional signaling unit with its own machinery to synthesize and regulate a sequestered cAMP pool. Thus, each signalosome is devoted to ensure the transmission of a unique and unequivocal message through the cell.

  6. Cellular mechanisms underlying prostaglandin-induced transient cAMP signals near the plasma membrane of HEK-293 cells

    PubMed Central

    Rich, Thomas; Xin, Wenkuan; Mehats, Céline; Hassell, Kathryn; Piggott, Leslie; Le, Xuan; Karpen, Jeffrey; Conti, Marco

    2007-01-01

    We have previously used cyclic nucleotide-gated (CNG) channels as sensors to measure cAMP signals in human embryonic kidney (HEK)-293 cells. We found that prostaglandin E1 (PGE1) triggered transient increases in cAMP concentration near the plasma membrane, whereas total cAMP levels rose to a steady plateau over the same time course. In addition, we presented evidence that the decline in the near-membrane cAMP levels was due primarily to a PGE1-induced stimulation of phosphodiesterase (PDE) activity, and that the differences between near-membrane and total cAMP levels were largely due to diffusional barriers and differential PDE activity. Here, we examine the mechanisms regulating transient, near-membrane cAMP signals. We observed that 5-min stimulation of HEK-293 cells with prostaglandins triggered a two- to threefold increase in PDE4 activity. Extracellular application of H89 (a PKA inhibitor) inhibited stimulation of PDE4 activity. Similarly, when we used CNG channels to monitor cAMP signals we found that both extracellular and intracellular (via the whole-cell patch pipette) application of H89, or the highly selective PKA inhibitor, PKI, prevented the decline in prostaglandin-induced responses. Following pretreatment with rolipram (a PDE4 inhibitor), H89 had little or no effect on near-membrane or total cAMP levels. Furthermore, disrupting the subcellular localization of PKA with the A-kinase anchoring protein (AKAP) disruptor Ht31 prevented the decline in the transient response. Based on these data we developed a plausible kinetic model that describes prostaglandin-induced cAMP signals. This model has allowed us to quantitatively demonstrate the importance of PKA-mediated stimulation of PDE4 activity in shaping near-membrane cAMP signals. PMID:16899551

  7. The Camp Health Manual. An Excellent Reference Written Especially for Organized Camps. Revised.

    ERIC Educational Resources Information Center

    Goldring, David; Middelkamp, J. Neal

    This book is a guide to the diagnosis and care of sick children in organized camping situations. This book presents health care information for the management of medical and surgical problems by the camp counselor, camp director, camp nurse, and camp physician. The chapters are: (1) Camp Standards; (2) The Infirmary; (3) Infirmary Supplies; (4)…

  8. The Camp Health Manual. An Excellent Reference Written Especially for Organized Camps. Revised.

    ERIC Educational Resources Information Center

    Goldring, David; Middelkamp, J. Neal

    This book is a guide to the diagnosis and care of sick children in organized camping situations. This book presents health care information for the management of medical and surgical problems by the camp counselor, camp director, camp nurse, and camp physician. The chapters are: (1) Camp Standards; (2) The Infirmary; (3) Infirmary Supplies; (4)…

  9. Long-term niacin treatment induces insulin resistance and adrenergic responsiveness in adipocytes by adaptive downregulation of phosphodiesterase 3B.

    PubMed

    Heemskerk, Mattijs M; van den Berg, Sjoerd A A; Pronk, Amanda C M; van Klinken, Jan-Bert; Boon, Mariëtte R; Havekes, Louis M; Rensen, Patrick C N; van Dijk, Ko Willems; van Harmelen, Vanessa

    2014-04-01

    The lipid-lowering effect of niacin has been attributed to the inhibition of cAMP production in adipocytes, thereby inhibiting intracellular lipolysis and release of nonesterified fatty acids (NEFA) to the circulation. However, long-term niacin treatment leads to a normalization of plasma NEFA levels and induces insulin resistance, for which the underlying mechanisms are poorly understood. The current study addressed the effects of long-term niacin treatment on insulin-mediated inhibition of adipocyte lipolysis and focused on the regulation of cAMP levels. APOE*3-Leiden.CETP transgenic mice treated with niacin for 15 wk were subjected to an insulin tolerance test and showed whole body insulin resistance. Similarly, adipocytes isolated from niacin-treated mice were insulin resistant and, interestingly, exhibited an increased response to cAMP stimulation by 8Br-cAMP, β1- and β2-adrenergic stimulation. Gene expression analysis of the insulin and β-adrenergic pathways in adipose tissue indicated that all genes were downregulated, including the gene encoding the cAMP-degrading enzyme phosphodiesterase 3B (PDE3B). In line with this, we showed that insulin induced a lower PDE3B response in adipocytes isolated from niacin-treated mice. Inhibiting PDE3B with cilostazol increased lipolytic responsiveness to cAMP stimulation in adipocytes. These data show that long-term niacin treatment leads to a downregulation of PDE3B in adipocytes, which could explain part of the observed insulin resistance and the increased responsiveness to cAMP stimulation.

  10. Long-term niacin treatment induces insulin resistance and adrenergic responsiveness in adipocytes by adaptive downregulation of phosphodiesterase 3B

    PubMed Central

    Heemskerk, Mattijs M.; van den Berg, Sjoerd A. A.; Pronk, Amanda C. M.; van Klinken, Jan-Bert; Boon, Mariëtte R.; Havekes, Louis M.; Rensen, Patrick C. N.; van Dijk, Ko Willems

    2014-01-01

    The lipid-lowering effect of niacin has been attributed to the inhibition of cAMP production in adipocytes, thereby inhibiting intracellular lipolysis and release of nonesterified fatty acids (NEFA) to the circulation. However, long-term niacin treatment leads to a normalization of plasma NEFA levels and induces insulin resistance, for which the underlying mechanisms are poorly understood. The current study addressed the effects of long-term niacin treatment on insulin-mediated inhibition of adipocyte lipolysis and focused on the regulation of cAMP levels. APOE*3-Leiden.CETP transgenic mice treated with niacin for 15 wk were subjected to an insulin tolerance test and showed whole body insulin resistance. Similarly, adipocytes isolated from niacin-treated mice were insulin resistant and, interestingly, exhibited an increased response to cAMP stimulation by 8Br-cAMP, β1- and β2-adrenergic stimulation. Gene expression analysis of the insulin and β-adrenergic pathways in adipose tissue indicated that all genes were downregulated, including the gene encoding the cAMP-degrading enzyme phosphodiesterase 3B (PDE3B). In line with this, we showed that insulin induced a lower PDE3B response in adipocytes isolated from niacin-treated mice. Inhibiting PDE3B with cilostazol increased lipolytic responsiveness to cAMP stimulation in adipocytes. These data show that long-term niacin treatment leads to a downregulation of PDE3B in adipocytes, which could explain part of the observed insulin resistance and the increased responsiveness to cAMP stimulation. PMID:24473440

  11. Summer Camp as Therapeutic Context: The Camp Logan Program.

    ERIC Educational Resources Information Center

    McCammon, Susan; And Others

    These symposium papers describe various aspects of the Camp Logan, South Carolina, program, a therapeutic summer residential program for children, ages 8-14, who have significant behavior problems. The philosophy and advantages of the therapeutic camping model are discussed, e.g., structure during the summer, controlled though informal…

  12. Blending Technology with Camp Tradition: Technology Can Simplify Camp Operations.

    ERIC Educational Resources Information Center

    Salzman, Jeff

    2000-01-01

    Discusses uses of technology appropriate for camps, which are service organizations based on building relationships. Describes relationship marketing and how it can be enhanced through use of Web sites, interactive brochures, and client databases. Outlines other technology uses at camp: automated dispensing of medications, satellite tracking of…

  13. A JUNIOR HIGH SCHOOL ORIENTATION CAMP, A COORDINATED CAMPING EXPERIENCE.

    ERIC Educational Resources Information Center

    NEALE, DANIEL; AND OTHERS

    A 2-WEEK SUMMER CAMPING PROGRAM WAS OFFERED TO 61 DISADVANTAGED STUDENTS ABOUT TO ENTER LINCOLN JUNIOR HIGH IN MINNEAPOLIS, MINNESOTA. THE PROGRAM'S PRIMARY PURPOSE WAS TO EASE THE STUDENT'S TRANSITION INTO JUNIOR HIGH SCHOOL. THROUGH CAMPING ACTIVITIES AND SCHOOL ORIENTATION CLASSES CONDUCTED BY THE LINCOLN STAFF, CAMPERS WOULD BECOME ACQUAINTED…

  14. Camp Director Education Curriculum Guide. Camp Administration Series.

    ERIC Educational Resources Information Center

    Stein, Sue, Ed.

    Part of Project STRETCH, a special personnel preparation grant, this guide contains 13 units on the practical and philosophical areas practitioners, educators, and consumers believed should be included in a basic course for administration of an organized camp: growth and development special populations, camp director's role, philosophy and…

  15. Camp Greentop's Adventure Camp: We Ain't No Rudypoo's.

    ERIC Educational Resources Information Center

    Groff, Diane; Albright, Brian; Purvis, Katie; Creamer, Justin; Pease, Alicia

    2002-01-01

    A day-by-day account describes Camp Greentop's first 5-day adventure camping trip, which was attended by five individuals with disabilities and their counselors. The first day was spent in games and initiatives designed to develop communication, teamwork, and dependability. Other days were devoted to hiking, rock climbing, and whitewater rafting.…

  16. Structural characterization of a high affinity mononuclear site in the copper(II)-α-synuclein complex.

    PubMed

    Bortolus, Marco; Bisaglia, Marco; Zoleo, Alfonso; Fittipaldi, Maria; Benfatto, Maurizio; Bubacco, Luigi; Maniero, Anna Lisa

    2010-12-29

    Human α-Synuclein (aS), a 140 amino acid protein, is the main constituent of Lewy bodies, the cytoplasmatic deposits found in the brains of Parkinson's disease patients, where it is present in an aggregated, fibrillar form. Recent studies have shown that aS is a metal binding protein. Moreover, heavy metal ions, in particular divalent copper, accelerate the aggregation process of the protein. In this work, we investigated the high affinity binding mode of truncated aS (1-99) (aS99) with Cu(II), in a stoichiometric ratio, to elucidate the residues involved in the binding site and the role of copper ions in the protein oligomerization. We used Electron Paramagnetic Resonance spectroscopy on the Cu(II)-aS99 complex at pH 6.5, performing both multifrequency continuous wave experiments and pulsed experiments at X-band. The comparison of 9.5 and 95 GHz data showed that at this pH only one binding mode is present. To identify the nature of the ligands, we performed Electron Spin Echo Envelope Modulation, Hyperfine Sublevel Correlation Spectroscopy, and pulsed Davies Electron-Nuclear Double Resonance (Davies-ENDOR) experiments. We determined that the EPR parameters are typical of a type-II copper complex, in a slightly distorted square planar geometry. Combining the results from the different pulsed techniques, we obtained that the equatorial coordination is {N(Im), N(-), H(2)O, O}, where N(im) is the imino nitrogen of His50, N(-) a deprotonated amido backbone nitrogen that we attribute to His50, H(2)O an exchangeable water molecule, and O an unidentified oxygen ligand. Moreover, we propose that the free amino terminus (Met1) participates in the complex as an axial ligand. The MXAN analysis of the XAS k-edge absorption data allowed us to independently validate the structural features proposed on the basis of the magnetic parameters of the Cu(II)-aS99 complex and then to further refine the quality of the proposed structural model.

  17. Characterization of high affinity (/sup 3/H)triazolam binding in rat brain

    SciTech Connect

    Earle, M.; Concas, A.; Yamamura, H.I.

    1986-03-01

    The hypnotic Triazolam (TZ), a triazolo (1,4)-benzodiazepine, displays a short physiological half life and has been used for the treatment of insomnia related to anxiety states. Specific binding properties of this recently tritiated TZ were characterized. The authors major objectives were the direct measurement of the temperature dependence and the GABA effect on (/sup 3/H)TZ binding. Saturation studies showed a shift to lower affinity at 37/sup 0/C (K/sub d/ = 0.25 +/- 0.01 nM at O/sup 0/C; K/sub d/ = 1.46 +/- 0.03 nM at 37/sup 0/C) while the B/sub max/ values remained unchanged (1003 +/- 37 fmoles/mg prot. at 0/sup 0/C and 1001 +/- 43 fmoles/mg prot. at 37/sup 0/C). Inhibition studies showed that (/sup 3/H)TZ binding displayed no GABA shift at 0/sup 0/C(K/sub i/ 0.37 +/- 0.03 nM/- GABA and K/sub i/ = 0.55 +/- 0.13 nM/+GABA) but a nearly two-fold shift was apparent at 37/sup 0/C (K/sub i/ = 2.92 +/- 0.2 nM/-GABA; K/sub i/ = 1.37 +/- 0.11 mM/+GABA). These results were also confirmed by saturation studies in the presence or absence of GABA showing a shift to higher affinity in the presence of GABA only at 37/sup 0/C. In Ro 15-1788/(/sup 3/H)TZ competition experiments the presence of GABA did not affect the inhibitory potency of Ro 15-1788 on (/sup 3/H)TZ binding at both temperatures. In conclusion (/sup 3/H)TZ binding showed an extremely high affinity for benzodiazepine receptors. In contrast to reported literature, the findings suggest that TZ interacts with benzodiazepine receptors similar to other benzodiazepine agonists.

  18. Developing High-Affinity Protein Capture Agents and Nanotechnology-Based Platforms for In Vitro Diagnostics

    NASA Astrophysics Data System (ADS)

    Rohde, Rosemary Dyane

    In this thesis, I describe projects that were aimed at improving ways to capture proteins for clinical diagnostics. Nanoelectronic sensors, such as silicon nanowires (SiNWs), can provide label-free quantitative measurements of protein biomarkers in real time. One technical challenge for SiNWs is to develop chemistry that can be applied for selectively encoding the nanowire surfaces with capture agents, thus making them sensors that have selectivity for specific proteins. Furthermore, because of the nature of how the sensor works, it is desirable to achieve this spatially selective chemical functionalization without having the silicon undergo oxidation. This method is described here and provides a general platform that can incorporate organic and biological molecules on Si (111) with minimal oxidation of the silicon surface. The development of these devices is, in part, driven by early diagnosis, treatment, monitoring, and personalized medicine---all of which are increasingly requiring quantitative, rapid, and multiparameter measurements. To begin achieving this goal, a large number of protein biomarkers need to be captured and quantitatively measured to create a diagnostic panel. One of the greatest challenges towards making protein-biomarker-based in vitro diagnostics inexpensive involves developing capture agents to detect the proteins. A major thrust of this thesis is to develop multi-valent, high-affinity and high-selectivity protein capture agents using in situ click chemistry. In situ click chemistry is a tool that utilizes the protein itself to catalyze the formation of a biligand from individual azide and alkyne ligands that are co-localized. Large one-bead one-compound (OBOC) libraries of peptides are used to form the body of these ligands, also providing high chemical diversity with minimal synthetic effort. This process can be repeated to identify a triligand, tetraligand, and so forth. Moreover, the resulting multiligand protein capture agents can be

  19. A new therapeutic approach to erectile dysfunction: urotensin-II receptor high affinity agonist ligands.

    PubMed

    di Villa Bianca, Roberta d'Emmanuele; Mitidieri, Emma; Donnarumma, Erminia; Fusco, Ferdinando; Longo, Nicola; Rosa, Giuseppe De; Novellino, Ettore; Grieco, Paolo; Mirone, Vincenzo; Cirino, Giuseppe; Sorrentino, Raffaella

    2015-01-01

    Urotensin-II (U-II) is a cyclic peptide that acts through a G protein-coupled receptor (urotensin-II receptor [UTR]) mainly involved in cardiovascular function in humans. The urotensinergic system is also implicated in the urogenital tract. Indeed, U-II relaxes human corpus cavernosum strips and causes an increase in intracavernous pressure (ICP) in rats. In light of this, the U-II/UTR pathway can be considered a new target for the treatment of erectile dysfunction. On this hypothesis, herein we report on two new UTR high affinity-agonists, P5U (H-Asp-c[Pen-Phe-Trp-Lys-Tyr-Cys]-Val-OH) and UPG84(H-Asp-c[Pen-Phe-DTrp-Orn-(pNH 2 ) Phe-Cys]-Val-OH). The effects of P5U and UPG84 were each compared separately with U-II by monitoring the ICP in anesthetized rats. Intracavernous injection of U-II (0.03-1 nmol), P5U (0.03-1 nmol) or UPG84 (0.03-1 nmol) caused an increase in ICP. P5U, in particular, elicited a significant increase in ICP as compared to U-II. The observed effect by using P5U at a dose of 0.1 nmol per rat was comparable to the effect elicited by U-II at a dose of 0.3 nmol. Moreover, UPG84 at the lowest dose (0.03 nmol) showed an effect similar to the highest dose of U-II (1 nmol). Furthermore, UPG84 was found to be more effective than P5U. Indeed, while the lowest dose of P5U (0.03 nmol) did not affect the ICP, UPG84, at the same dose, induced a prominent penile erection in rat. These compounds did not modify the blood pressure, which indicates a good safety profile. In conclusion, UPG84 and P5U may open new perspectives for the management of erectile dysfunction.

  20. High-affinity VEGF antagonists by oligomerization of a minimal sequence VEGF-binding domain.

    PubMed

    Stefano, James E; Bird, Julie; Kyazike, Josephine; Cheng, Anthony Wai-Ming; Boudanova, Ekaterina; Dwyer, Markryan; Hou, Lihui; Qiu, Huawei; Matthews, Gloria; O'Callaghan, Michael; Pan, Clark Q

    2012-12-19

    Vascular endothelial growth factor (VEGF) neutralizing antagonists including antibodies or receptor extracellular domain Fc fusions have been applied clinically to control angiogenesis in cancer, wet age-related macular degeneration, and edema. We report here the generation of high-affinity VEGF-binding domains by chemical linkage of the second domain of the VEGF receptor Flt-1 (D2) in several configurations. Recombinant D2 was expressed with a 13 a.a. C-terminal tag, including a C-terminal cysteine to enable its dimerization by disulfide bond formation or by attachment to divalent PEGs and oligomerization by coupling to multivalent PEGs. Disulfide-linked dimers produced by Cu(2+) oxidation of the free-thiol form of the protein demonstrated picomolar affinity for VEGF in solution, comparable to that of a D2-Fc fusion (sFLT01) and ~50-fold higher than monomeric D2, suggesting the 26 a.a. tag length between the two D2 domains permits simultaneous interaction of both faces of the VEGF homodimer. Extending the separation between the D2 domains by short PEG spacers from 0.35 kD to 5 kD produced a modest ~2-fold increase in affinity over the disulfide, thus defining the optimal distance between the two D2 domains for maximum affinity. By surface plasmon resonance (SPR), a larger (~5-fold) increase in affinity was observed by conjugation of the D2 monomer to the termini of 4-arm PEG, and yielding a product with a larger hydrodynamic radius than sFLT01. The higher affinity displayed by these D2 PEG tetramers than either D2 dimer or sFLT01 was largely a consequence of a slower rate of dissociation, suggesting the simultaneous binding by these tetramers to neighboring surface-bound VEGF. Finally, disulfide-linked D2 dimers showed a greater resistance to autocatalytic fragmentation than sFLT01 under elevated temperature stress, indicating such minimum-sequence constructs may be better suited for sustained-release formulations. Therefore, these constructs represent novel Fc

  1. Identification of a high-affinity ligand that exhibits complete aryl hydrocarbon receptor antagonism.

    PubMed

    Smith, Kayla J; Murray, Iain A; Tanos, Rachel; Tellew, John; Boitano, Anthony E; Bisson, William H; Kolluri, Siva K; Cooke, Michael P; Perdew, Gary H

    2011-07-01

    The biological functions of the aryl hydrocarbon receptor (AHR) can be delineated into dioxin response element (DRE)-dependent or -independent activities. Ligands exhibiting either full or partial agonist activity, e.g., 2,3,7,8-tetrachlorodibenzo-p-dioxin and α-naphthoflavone, have been demonstrated to potentiate both DRE-dependent and -independent AHR function. In contrast, the recently identified selective AHR modulators (SAhRMs), e.g., 1-allyl-3-(3,4-dimethoxyphenyl)-7-(trifluoromethyl)-1H-indazole (SGA360), bias AHR toward DRE-independent functionality while displaying antagonism with regard to ligand-induced DRE-dependent transcription. Recent studies have expanded the physiological role of AHR to include modulation of hematopoietic progenitor expansion and immunoregulation. It remains to be established whether such physiological roles are mediated through DRE-dependent or -independent pathways. Here, we present evidence for a third class of AHR ligand, "pure" or complete antagonists with the capacity to suppress both DRE-dependent and -independent AHR functions, which may facilitate dissection of physiological AHR function with regard to DRE or non-DRE-mediated signaling. Competitive ligand binding assays together with in silico modeling identify N-(2-(1H-indol-3-yl)ethyl)-9-isopropyl-2-(5-methylpyridin-3-yl)-9H-purin-6-amine (GNF351) as a high-affinity AHR ligand. DRE-dependent reporter assays, in conjunction with quantitative polymerase chain reaction analysis of AHR targets, reveal GNF351 as a potent AHR antagonist that demonstrates efficacy in the nanomolar range. Furthermore, unlike many currently used AHR antagonists, e.g., α-naphthoflavone, GNF351 is devoid of partial agonist potential. It is noteworthy that in a model of AHR-mediated DRE-independent function, i.e., suppression of cytokine-induced acute-phase gene expression, GNF351 has the capacity to antagonize agonist and SAhRM-mediated suppression of SAA1. Such data indicate that GNF351 is a

  2. Inhibition of the high affinity choline transporter enhances hyperalgesia in a rat model of chronic pancreatitis.

    PubMed

    Luo, Dan; Chen, Lei; Yu, Baoping

    2017-06-17

    The mechanisms underlying chronic and persistent pain associated with chronic pancreatitis (CP) are not completely understood. The cholinergic system is one of the major neural pathways of the pancreas. Meanwhile, this system plays an important role in chronic pain. We hypothesized that the high affinity choline transporter CHT1, which is a main determinant of cholinergic signaling capacity, is involved in regulating pain associated with CP. CP was induced by intraductal injection of 2% trinitrobenzene sulfonic acid (TNBS) in Sprague-Dawley rats. Pathological examination was used to evaluate the inflammation of pancreas and hyperalgesia was assessed by measuring the number of withdrawal events evoked by application of the von Frey filaments. CHT1 expression in pancreas-specific dorsal root ganglia (DRGs) was assessed through immunohistochemistry and western blotting. We also intraperitoneally injected the rats with hemicholinium-3 (HC-3, a specific inhibitor of CHT1). Then we observed its effects on the visceral hyperalgesia induced by CP, and on the acetylcholine (ACh) levels in the DRGs through using an acetylcholine/acetylcholinesterase assay kit. Signs of CP were observed 21 days after TNBS injection. Rats subjected to TNBS infusions had increased sensitivity to mechanical stimulation of the abdomen. CHT1-immunoreactive cells were increased in the DRGs from rats with CP compared to naive or sham rats. Western blots indicated that CHT1 expression was significantly up-regulated in TNBS-treated rats when compared to naive or sham-operated rats at all time points following surgery. In the TNBS group, CHT1 expression was higher on day 28 than on day 7 or day 14, but there was no statistical difference in CHT1 expression on day 28 vs. day 21. Treatment with HC-3 (60 μg/kg, 80 μg/kg, or 100 μg/kg) markedly enhanced the mechanical hyperalgesia and reduced ACh levels in a dose-dependent manner in rats with CP. We report for the first time that CHT1 may be involved

  3. Biochemical identification of base and phosphate contacts between Fis and a high affinity DNA binding site

    PubMed Central

    Shao, Yongping; Feldman-Cohen, Leah S.; Osuna, Robert

    2008-01-01

    SUMMARY Fis is a nucleoid-associated protein in E. coli and other bacteria that stimulates certain site-specific DNA recombination events, alters DNA topology, and serves as a global gene regulator. DNA binding is central to the functions of Fis and involves a helix-turn-helix DNA binding motif located in the carboxy-terminal region. Specific DNA binding is observed at a number of sites exhibiting poorly related sequences. Such interactions require four critical base pairs positioned −7, −3, +3, and +7 nucleotides relative to the central nucleotide of a 15 bp core-binding site. To further understand how Fis interacts with DNA, we identified the positions of fourteen DNA phosphates (based on ethylation interference assays) that are required for Fis binding. These are the 5’ phosphates of the nucleotides at positions −8, −7, −6, +1, +2, +3, and +4 relative to the central nucleotide on both DNA strands. Another five phosphates located in the flanking regions from positions +10 through +14 can serve as additional contact sites. Using a combination of biochemical approaches and various mutant Fis proteins, we probed possible interactions between several key Fis residues and DNA bases or phosphates within a high-affinity binding site. We provide evidence in support of interactions between the R85 Fis residue and a highly conserved guanine at position −7, and between T87 and the critical base pairs at −3 and +3. In addition, we present evidence in support of interactions between N84 and the phosphate 5’ to the base at +4, between R89 and the −7 phosphate, between T87 and the +3 and +4 phosphates, and between K90 and the +3 phosphate. This work provides functional evidence for some of the most critical interactions between Fis and DNA required for a high binding affinity and demonstrates the large contribution made by numerous phosphates to the stability of the Fis-DNA complex. PMID:18514225

  4. High-Affinity Functional Fluorescent Ligands for Human β-Adrenoceptors.

    PubMed

    Mitronova, Gyuzel Y; Lukinavičius, Gražvydas; Butkevich, Alexey N; Kohl, Tobias; Belov, Vladimir N; Lehnart, Stephan E; Hell, Stefan W

    2017-09-26

    Visualization of the G-protein coupled receptor (GPCR) is of great importance for studying its function in a native cell. We have synthesized a series of red-emitting fluorescent probes targeting β-adrenergic receptor (βAR) that are compatible with confocal and Stimulated Emission Depletion (STED) microscopy as well as with Time-Resolved Fluorescence Resonance Energy Transfer (TR-FRET) binding assay in living cells. The probe based on the agonist BI-167107 and fluorescent dye KK114 demonstrates nanomolar binding affinity and up to nine-fold β2AR selectivity over β1AR. Carazolol-derived probes are fluorogenic and allow no-wash imaging experiments. STED microscopy of β2ARs stained at the native expression level on pancreatic CAPAN cells provides two-fold improvement in lateral optical resolution over confocal mode and reveals the formation of receptor microdomains. These probes retain their functional (agonist or antagonist) properties, allowing simultaneous modulation of cyclic adenosine monophosphate (cAMP) levels and receptor internalization as well as imaging receptor localization.

  5. Living with Cancer at Camp Rainbow.

    ERIC Educational Resources Information Center

    Williams, Wayne; Breitenstein, Donna

    1988-01-01

    Describes the camping experience at Camp Rainbow (Arkansas), specifically designed for children with cancer and their siblings. Discusses the emotional impact of childhood cancer on patients, parents, and siblings, and suggests positive outcomes of camp participation. Includes eight references. (SV)

  6. Living with Cancer at Camp Rainbow.

    ERIC Educational Resources Information Center

    Williams, Wayne; Breitenstein, Donna

    1988-01-01

    Describes the camping experience at Camp Rainbow (Arkansas), specifically designed for children with cancer and their siblings. Discusses the emotional impact of childhood cancer on patients, parents, and siblings, and suggests positive outcomes of camp participation. Includes eight references. (SV)

  7. Involvement of phosphodiesterase 4 in beta-adrenoceptor agonist-induced amylase release in parotid acinar cells.

    PubMed

    Satoh, Keitaro; Guo, Ming-Yu; Sairenji, Nakayasu

    2009-06-01

    beta-Adrenoceptor activation increases intracellular cAMP levels and consequently induces exocytotic amylase release in parotid acinar cells. Phosphodiesterase (PDE) catalyses the hydrolysis of cAMP, which terminates the downstream signaling of this second messenger. We investigated the involvement of PDE4, a cAMP-PDE, in beta-adrenoceptor agonist-induced amylase release in mouse, rat and rabbit parotid acinar cells by using the specific PDE4 inhibitor rolipram. cAMP-PDE activity was detected in mouse, rat and rabbit parotid acinar cells. In the presence of rolipram, cAMP-PDE activity was reduced by about 31%, 38% and 33% in mouse, rat and rabbit parotid acinar cells, respectively. The increase in cAMP levels induced by the beta-adrenoceptor agonist isoproterenol was enhanced in the presence of rolipram in mouse, rat and rabbit parotid acinar cells. Isoproterenol-induced amylase release, but not constitutive amylase release, was also enhanced in the presence of rolipram in mouse, rat and rabbit parotid acinar cells. These results suggest that the rolipram-sensitive cAMP-PDE, PDE4, is involved in beta-adrenoceptor agonist-induced amylase release in parotid acinar cells.

  8. In vitro toxicity of mercury, cadmium, and arsenic to platelet aggregation: influence of adenylate cyclase and phosphodiesterase activity.

    PubMed

    Kumar, S V; Bhattacharya, S

    2000-01-01

    In vitro effect of mercury (Hg2+), cadmium (Cd2+), and arsenic (As3+) on adenylate cyclase (AC) and phosphodiesterase (PDE) activity in relation to platelet aggregation (PA) was studied in rats. Cd(2+) significantly elevated cAMP (p < 0.005) in a dose-dependent (5, 10 and 20 pmoles) manner while Hg(2+) and As(3+) significantly reduced the cAMP level (p < 0.01 and p < 0.005, respectively). Our studies further reveal that Hg21 and As(3+) inhibit AC and stimulate PDE activity with a concomitant increase in the rate of PA. On the other hand, Cd(2+) stimulates AC and inhibits PDE activity with a decrease in the rate of PA. The present investigation suggests that cellular cAMP is a regulatory molecule in the event of PA and the disruption of its homeostasis is directly correlated to xenobiotic effects on PA. It is concluded that other than divalent heavy metal cations, As(3+) appears to be one of the most toxic xenobiotics to platelet function.

  9. The effect of phosphodiesterase inhibitors on the extinction of cocaine-induced conditioned place preference in mice.

    PubMed

    Liddie, Shervin; Anderson, Karen L; Paz, Andres; Itzhak, Yossef

    2012-10-01

    Several phosphodiesterase inhibitors (PDEis) improve cognition, suggesting that an increase in brain cAMP and cGMP facilitates learning and memory. Since extinction of drug-seeking behavior requires associative learning, consolidation and formation of new memory, the present study investigated the efficacy of three different PDEis in the extinction of cocaine-induced conditioned place preference (CPP) in B6129S mice. Mice were conditioned by escalating doses of cocaine which was resistant to extinction by free exploration. Immediately following each extinction session mice received (a) saline/vehicle, (b) rolipram (PDE4 inhibitor), (c) BAY-73-6691 (PDE9 inhibitor) or (d) papaverine (PDE10A inhibitor). Mice that received saline/vehicle during extinction training showed no reduction in CPP for >10 days. BAY-73-6691 (a) dose-dependently increased cGMP in hippocampus and amygdala, (b) significantly facilitated extinction and (c) diminished the reinstatement of cocaine CPP. Rolipram, which selectively increased brain cAMP levels, and papaverine which caused increases in both cAMP and cGMP levels, had no significant effect on the extinction of cocaine CPP. The results suggest that increase in hippocampal and amygdalar cGMP levels via blockade of PDE9 has a prominent role in the consolidation of extinction learning.

  10. AMPK antagonizes hepatic glucagon-stimulated cyclic AMP signalling via phosphorylation-induced activation of cyclic nucleotide phosphodiesterase 4B

    PubMed Central

    Johanns, M.; Lai, Y.-C.; Hsu, M.-F.; Jacobs, R.; Vertommen, D.; Van Sande, J.; Dumont, J. E.; Woods, A.; Carling, D.; Hue, L.; Viollet, B.; Foretz, M; Rider, M H

    2016-01-01

    Biguanides such as metformin have previously been shown to antagonize hepatic glucagon-stimulated cyclic AMP (cAMP) signalling independently of AMP-activated protein kinase (AMPK) via direct inhibition of adenylate cyclase by AMP. Here we show that incubation of hepatocytes with the small-molecule AMPK activator 991 decreases glucagon-stimulated cAMP accumulation, cAMP-dependent protein kinase (PKA) activity and downstream PKA target phosphorylation. Moreover, incubation of hepatocytes with 991 increases the Vmax of cyclic nucleotide phosphodiesterase 4B (PDE4B) without affecting intracellular adenine nucleotide concentrations. The effects of 991 to decrease glucagon-stimulated cAMP concentrations and activate PDE4B are lost in hepatocytes deleted for both catalytic subunits of AMPK. PDE4B is phosphorylated by AMPK at three sites, and by site-directed mutagenesis, Ser304 phosphorylation is important for activation. In conclusion, we provide a new mechanism by which AMPK antagonizes hepatic glucagon signalling via phosphorylation-induced PDE4B activation. PMID:26952277

  11. Phosphodiesterase 2 negatively regulates adenosine-induced transcription of the tyrosine hydroxylase gene in PC12 rat pheochromocytoma cells.

    PubMed

    Makuch, Edyta; Kuropatwa, Marianna; Kurowska, Ewa; Ciekot, Jaroslaw; Klopotowska, Dagmara; Matuszyk, Janusz

    2014-07-05

    Adenosine induces expression of the tyrosine hydroxylase (TH) gene in PC12 cells. However, it is suggested that atrial natriuretic peptide (ANP) inhibits expression of this gene. Using real-time PCR and luciferase reporter assays we found that ANP significantly decreases the adenosine-induced transcription of the TH gene. Results of measurements of cyclic nucleotide concentrations indicated that ANP-induced accumulation of cGMP inhibits the adenosine-induced increase in cAMP level. Using selective phosphodiesterase 2 (PDE2) inhibitors and a synthetic cGMP analog activating PDE2, we found that PDE2 is involved in coupling the ANP-triggered signal to the cAMP metabolism. We have established that ANP-induced elevated levels of cGMP as well as cGMP analog stimulate hydrolytic activity of PDE2, leading to inhibition of adenosine-induced transcription of the TH gene. We conclude that ANP mediates negative regulation of TH gene expression via stimulation of PDE2-dependent cAMP breakdown in PC12 cells.

  12. Trends in developed forest camping

    Treesearch

    Barry A. Garst; Dan R. Williams; Joseph W. Roggenbuck

    2012-01-01

    Over the past 40 years, the number of forest campers has grown from 13 million in the 1960s to approximately 56 million in 2000 (table 4.6). Camping is now one of the more common ways that Americans spend time in the outdoors, with over one-fourth of the U.S. population participating in some form of camping.

  13. Encountering Child Abuse at Camp.

    ERIC Educational Resources Information Center

    Durall, John K.

    1997-01-01

    Defines child abuse, including the three categories: physical, sexual, and psychological. Presents characteristics and behaviors of each type of abuse, and long-term effects. Discusses how to handle abuse that occurs at camp, and the effects on the camp. Sidebars present abuse statistics, 15 activities that promote psychological wellness, and 8…

  14. Explaining the Value of Camp.

    ERIC Educational Resources Information Center

    Chenery, Mary Faeth

    1994-01-01

    Overviews the philosophy and theory of camp experiences and discusses the special benefits of camps, including experiences that can lead to significant life-changing outcomes, sound educational goals, a sense of psychological and physical safety, and helping children to deal with social problems such as the degradation of the environment and human…

  15. Kids Camping Takes the Challenge.

    ERIC Educational Resources Information Center

    James, Vickie L.; Hohnbaum, Claudia

    2002-01-01

    A Wisconsin Girl Scout camp integrated The Healthy Kids Challenge into its program. The camp evaluated policies related to meals, snacks, physical activities, team building, and self-esteem. Staff inservice training resulted in healthier meals on the same budget and developed ownership of the program. Campers and families had opportunities to…

  16. Local BLyS production by T follicular cells mediates retention of high affinity B cells during affinity maturation

    PubMed Central

    Goenka, Radhika; Matthews, Andrew H.; Zhang, Bochao; O’Neill, Patrick J.; Scholz, Jean L.; Migone, Thi-Sau; Leonard, Warren J.; Stohl, William; Hershberg, Uri

    2014-01-01

    We have assessed the role of B lymphocyte stimulator (BLyS) and its receptors in the germinal center (GC) reaction and affinity maturation. Despite ample BLyS retention on B cells in follicular (FO) regions, the GC microenvironment lacks substantial BLyS. This reflects IL-21–mediated down-regulation of the BLyS receptor TACI (transmembrane activator and calcium modulator and cyclophilin ligand interactor) on GC B cells, thus limiting their capacity for BLyS binding and retention. Within the GC, FO helper T cells (TFH cells) provide a local source of BLyS. Whereas T cell–derived BLyS is dispensable for normal GC cellularity and somatic hypermutation, it is required for the efficient selection of high affinity GC B cell clones. These findings suggest that during affinity maturation, high affinity clones rely on TFH-derived BLyS for their persistence. PMID:24367004

  17. High Affinity Binding of Chp1 Chromodomain to K9 Methylated Histone H3 is Required to Establish Centromeric Hterochromatin

    SciTech Connect

    Schalch, T.; Job, G; Noffsinger, V; Shanker, S; Kuscu, C; Joshua-Tor, L; Partridge, J

    2009-01-01

    In fission yeast, assembly of centromeric heterochromatin requires the RITS complex, which consists of Ago1, Tas3, Chp1, and siRNAs derived from centromeric repeats. Recruitment of RITS to centromeres has been proposed to depend on siRNA-dependent targeting of Ago1 to centromeric sequences. Previously, we demonstrated that methylated lysine 9 of histone H3 (H3K9me) acts upstream of siRNAs during heterochromatin establishment. Our crystal structure of Chp1's chromodomain in complex with a trimethylated lysine 9 H3 peptide reveals extensive sites of contact that contribute to Chp1's high-affinity binding. We found that this high-affinity binding is critical for the efficient establishment of centromeric heterochromatin, but preassembled heterochromatin can be maintained when Chp1's affinity for H3K9me is greatly reduced.

  18. Complementary DNA display selection of high-affinity peptides binding the vacuolating toxin (VacA) of Helicobacter pylori.

    PubMed

    Hayakawa, Yumiko; Matsuno, Mitsuhiro; Tanaka, Makoto; Wada, Akihiro; Kitamura, Koichiro; Takei, Osamu; Sasaki, Ryuzo; Mizukami, Tamio; Hasegawa, Makoto

    2015-09-01

    Artificial peptides designed for molecular recognition of a bacterial toxin have been developed. Vacuolating cytotoxin A protein (VacA) is a major virulence factor of Helicobacter pylori, a gram-negative microaerophilic bacterium inhabiting the upper gastrointestinal tract, particularly the stomach. This study attempted to identify specific peptide sequences with high affinity for VacA using systematic directed evolution in vitro, a cDNA display method. A surface plasmon resonance-based biosensor and fluorescence correlation spectroscopy to examine binding of peptides with VacA identified a peptide (GRVNQRL) with high affinity. Cyclization of the peptide by attaching cysteine residues to both termini improved its binding affinity to VacA, with a dissociation constant (Kd ) of 58 nm. This study describes a new strategy for the development of artificial functional peptides, which are promising materials in biochemical analyses and medical applications. Copyright © 2015 European Peptide Society and John Wiley & Sons, Ltd.

  19. 36 CFR 13.25 - Camping.

    Code of Federal Regulations, 2013 CFR

    2013-07-01

    ... impacting park resources. (c) Designated campgrounds. Except at designated campgrounds, camping is... camping in designated campgrounds. Violating restrictions, terms, and conditions is prohibited....

  20. 36 CFR 13.25 - Camping.

    Code of Federal Regulations, 2011 CFR

    2011-07-01

    ... impacting park resources. (c) Designated campgrounds. Except at designated campgrounds, camping is... camping in designated campgrounds. Violating restrictions, terms, and conditions is prohibited....

  1. 36 CFR 13.25 - Camping.

    Code of Federal Regulations, 2014 CFR

    2014-07-01

    ... impacting park resources. (c) Designated campgrounds. Except at designated campgrounds, camping is... camping in designated campgrounds. Violating restrictions, terms, and conditions is prohibited....

  2. 36 CFR 13.25 - Camping.

    Code of Federal Regulations, 2012 CFR

    2012-07-01

    ... impacting park resources. (c) Designated campgrounds. Except at designated campgrounds, camping is... camping in designated campgrounds. Violating restrictions, terms, and conditions is prohibited....

  3. 36 CFR 13.25 - Camping.

    Code of Federal Regulations, 2010 CFR

    2010-07-01

    ... impacting park resources. (c) Designated campgrounds. Except at designated campgrounds, camping is... camping in designated campgrounds. Violating restrictions, terms, and conditions is prohibited....

  4. Pyrido pyrimidinones as selective agonists of the high affinity niacin receptor GPR109A: optimization of in vitro activity.

    PubMed

    Peters, Jens-Uwe; Kühne, Holger; Dehmlow, Henrietta; Grether, Uwe; Conte, Aurelia; Hainzl, Dominik; Hertel, Cornelia; Kratochwil, Nicole A; Otteneder, Michael; Narquizian, Robert; Panousis, Constantinos G; Ricklin, Fabienne; Röver, Stephan

    2010-09-15

    Pyrido pyrimidinones are selective agonists of the human high affinity niacin receptor GPR109A (HM74A). They show no activity on the highly homologous low affinity receptor GPR109B (HM74). Starting from a high throughput screening hit the in vitro activity of the pyrido pyrimidinones was significantly improved providing lead compounds suitable for further optimization. Copyright (c) 2010 Elsevier Ltd. All rights reserved.

  5. Multipurpose ligand, DAKLI (Dynorphin A-analogue Kappa LIgand), with high affinity and selectivity for dynorphin (. kappa. opioid) binding sites

    SciTech Connect

    Goldstein, A.; Nestor, J.J. Jr.; Naidu, A.; Newman, S.R. )

    1988-10-01

    The authors describe a synthetic ligand, DALKI (Dynorphin A-analogue Kappa LIgand), related to the opioid peptide dynorphin A. A single reactive amino group at the extended carboxyl terminus permits various reporter groups to be attached, such as {sup 125}I-labeled Bolton-Hunter reagent, fluorescein isothiocyanate, or biotin. These derivatives have high affinity and selectivity for the dynorphin ({kappa} opioid) receptor. An incidental finding is that untreated guinea pig brain membranes have saturable avidin binding sites.

  6. Imaging the high-affinity state of the dopamine D2 receptor in vivo: Fact or fiction?

    PubMed Central

    Skinbjerg, Mette; Sibley, David R.; Javitch, Jonathan A.; Abi-Dargham, Anissa

    2013-01-01

    Positron Emission Tomography (PET) has been used for more than three decades to image and quantify dopamine D2 receptors (D2R) in vivo with antagonist radioligands but in the recent years agonist radioligands have also been employed. In vitro competition studies have demonstrated that agonists bind to both a high and a low-affinity state of the D2Rs, of which the high affinity state reflects receptors that are coupled to G-proteins and the low-affinity state reflects receptors uncoupled from G-proteins. In contrast, antagonists bind with uniform affinity to the total pool of receptors. Results of these studies led to the proposal that D2Rs exist in high and low-affinity states for agonists in vivo and sparked the development and use of agonist radioligands for PET imaging with the primary purpose of measuring the proportion of receptors in the high-affinity (activating) state. Although several lines of research support the presence of high and low-affinity states of D2Rs and their detection by in vivo imaging paradigms, a growing body of controversial data has now called this into question. These include both in vivo and ex vivo studies of anesthesia effects, rodent models with increased proportions of high-affinity state D2Rs as well as the molecular evidence for stable receptor–G-protein complexes. In this commentary we review these data and discuss the evidence for the in vivo existence of D2Rs configured in high and low-affinity states and whether or not the high-affinity state of the D2R can, in fact, be imaged in vivo with agonist radioligands. PMID:21945484

  7. Ultraviolet light induction of skin carcinoma in the mouse; influence of cAMP modifying agents.

    PubMed

    Zajdela, F; Latarjet, R

    1978-01-01

    A short review of pathogenic factors in U.V. light skin carcinogenesis in the mouse is presented. Caffeine and theophylline applied locally during U.V. irradiation caused a 50 percent reduction of skin tumour induction in Swiss mice. These two chemicals are inhibitors of DNA postreplication repair, but they also raise the intracellular level of cyclic AMP by inhibiting cAMP phosphodiesterase with, as a consequence, a possible slowing down of cellular growth. Control experiments using three different chemicals capable of raising the cAMP level in epidermal cells gave negative results. These experimental data are compatible with our original hypothesis according to which production of skin cancers by U.V. radiation is in same way related to DNA repair which helps the cell to survive but allows or favours the occurrence of errors in cellular DNA.

  8. Deterministic mathematical models of the cAMP pathway in Saccharomyces cerevisiae.

    PubMed

    Williamson, Thomas; Schwartz, Jean-Marc; Kell, Douglas B; Stateva, Lubomira

    2009-07-16

    Cyclic adenosine monophosphate (cAMP) has a key signaling role in all eukaryotic organisms. In Saccharomyces cerevisiae, it is the second messenger in the Ras/PKA pathway which regulates nutrient sensing, stress responses, growth, cell cycle progression, morphogenesis, and cell wall biosynthesis. A stochastic model of the pathway has been reported. We have created deterministic mathematical models of the PKA module of the pathway, as well as the complete cAMP pathway. First, a simplified conceptual model was created which reproduced the dynamics of changes in cAMP levels in response to glucose addition in wild-type as well as cAMP phosphodiesterase deletion mutants. This model was used to investigate the role of the regulatory Krh proteins that had not been included previously. The Krh-containing conceptual model reproduced very well the experimental evidence supporting the role of Krh as a direct inhibitor of PKA. These results were used to develop the Complete cAMP Model. Upon simulation it illustrated several important features of the yeast cAMP pathway: Pde1p is more important than is Pde2p for controlling the cAMP levels following glucose pulses; the proportion of active PKA is not directly proportional to the cAMP level, allowing PKA to exert negative feedback; negative feedback mechanisms include activating Pde1p and deactivating Ras2 via phosphorylation of Cdc25. The Complete cAMP model is easier to simulate, and although significantly simpler than the existing stochastic one, it recreates cAMP levels and patterns of changes in cAMP levels observed experimentally in vivo in response to glucose addition in wild-type as well as representative mutant strains such as pde1Delta, pde2Delta, cyr1Delta, and others. The complete model is made available in SBML format. We suggest that the lower number of reactions and parameters makes these models suitable for integrating them with models of metabolism or of the cell cycle in S. cerevisiae. Similar models could be

  9. CANDLES, an assay for monitoring GPCR induced cAMP generation in cell cultures.

    PubMed

    Trehan, Ashutosh; Rotgers, Emmi; Coffey, Eleanor T; Huhtaniemi, Ilpo; Rivero-Müller, Adolfo

    2014-11-04

    G protein-coupled receptors (GPCRs) represent a physiologically and pharmacologically important family of receptors that upon coupling to GαS stimulate cAMP production catalyzed by adenylyl cyclase. Thus, developing assays to monitor cAMP production is crucial to screen for ligands in studies of GPCR signaling. Primary cell cultures represent a more robust model than cell lines to study GPCR signaling since they physiologically resemble the parent tissue. Current cAMP assays have two fundamental limitations: 1) absence of cAMP kinetics as competition-based assays require cell lysis and measure only a single time-point, and 2) high variation with separate samples needed to measure consecutive time points. The utility of real-time cAMP biosensors is also limited in primary cell cultures due to their poor transfection efficiency, variable expression levels and inability to select stable clones. We therefore, decided to develop an assay that can measure cAMP not only at a single time-point but the entire cAMP kinetics after GPCR activation in untransfected primary cells. CANDLES (Cyclic AMP iNdirect Detection by Light Emission from Sensor cells) assay for monitoring cAMP kinetics in cell cultures, particularly in primary cultures was developed. The assay requires co-culturing of primary cells with sensor cells that stably express a luminescent cAMP sensor. Upon GPCR activation in primary cells, cAMP is transferred to sensor cells via gap junction channels, thereby evoking a luminescent read-out. GPCR activation using primary cultures of rat cortical neurons and mouse granulosa cells was measured. Kinetic responses of different agonists to adrenergic receptors were also compared using rat cortical neurons. The assay optimization was done by varying sensor-test cell ratio, using phosphodiesterase inhibitors and testing cell-cell contact requirement. Here we present CANDLES assay based on co-culturing test cells with cAMP-detecting sensor cells. This co-culture setup

  10. Analysis of high affinity self-association by fluorescence optical sedimentation velocity analytical ultracentrifugation of labeled proteins: opportunities and limitations.

    PubMed

    Zhao, Huaying; Lomash, Suvendu; Glasser, Carla; Mayer, Mark L; Schuck, Peter

    2013-01-01

    Sedimentation velocity analytical ultracentrifugation (SV) is a powerful first-principle technique for the study of protein interactions, and allows a rigorous characterization of binding stoichiometry and affinities. A recently introduced commercial fluorescence optical detection system (FDS) permits analysis of high-affinity interactions by SV. However, for most proteins the attachment of an extrinsic fluorophore is an essential prerequisite for analysis by FDS-SV. Using the glutamate receptor GluA2 amino terminal domain as a model system for high-affinity homo-dimerization, we demonstrate how the experimental design and choice of fluorescent label can impact both the observed binding constants as well as the derived hydrodynamic parameter estimates for the monomer and dimer species. Specifically, FAM (5,6-carboxyfluorescein) was found to create different populations of artificially high-affinity and low-affinity dimers, as indicated by both FDS-SV and the kinetics of dimer dissociation studied using a bench-top fluorescence spectrometer and Förster Resonance Energy Transfer. By contrast, Dylight488 labeled GluA2, as well as GluA2 expressed as an EGFP fusion protein, yielded results consistent with estimates for unlabeled GluA2. Our study suggests considerations for the choice of labeling strategies, and highlights experimental designs that exploit specific opportunities of FDS-SV for improving the reliability of the binding isotherm analysis of interacting systems.

  11. Relations between high-affinity binding sites for L-tryptophan, diazepam, salicylate and Phenol Red on human serum albumin.

    PubMed Central

    Kragh-Hansen, U

    1983-01-01

    Binding of L-tryptophan, diazepam, salicylate and Phenol Red to defatted human serum albumin was studied by ultrafiltration at pH 7.0. All ligands bind to one high-affinity binding site with association constants of the order of 10(4)-10(5)M-1. The number of secondary binding sites was found to vary from zero to five, with association constants about 10(3)M-1. Competitive binding studies with different pairs of the ligands were performed. Binding of both ligands was determined simultaneously. L-Tryptophan and diazepam were found to compete for a common high-affinity binding site on albumin. The following combinations of ligands do not bind competitively to albumin: L-tryptophan-Phenol Red, L-tryptophan-salicylate and Phenol Red-salicylate. On the other hand, high-affinity bindings of the three ligands do not take place independently but in such a way that binding of one of the ligands results in a decrease in binding of the other ligands. The decreases in binding are reciprocal and can be accounted for by introducing a coupling constant. The magnitude of the constant is dependent on the ligands being bound. In the present study, the mutual decrease in binding was more pronounced with L-tryptophan-salicylate and Phenol Red-salicylate than with L-tryptophan-Phenol Red. PMID:6847607

  12. High affinity IgM(+) memory B cells are generated through a germinal center-dependent pathway.

    PubMed

    Hara, Yasushi; Tashiro, Yasuyuki; Murakami, Akikazu; Nishimura, Miyuki; Shimizu, Takeyuki; Kubo, Masato; Burrows, Peter D; Azuma, Takachika

    2015-12-01

    During a T cell-dependent immune response, B cells undergo clonal expansion and selection and the induction of isotype switching and somatic hypermutation (SHM). Although somatically mutated IgM(+) memory B cells have been reported, it has not been established whether they are really high affinity B cells. We tracked (4-hydroxy-3-nitrophenyl) acetyl hapten-specific GC B cells from normal immunized mice based on affinity of their B cell receptor (BCR) and performed BCR sequence analysis. SHM was evident by day 7 postimmunization and increased with time, such that high affinity IgM(+) as well as IgG(+) memory B cells continued to be generated up to day 42. In contrast, class-switch recombination (CSR) was almost completed by day 7 and then the ratio of IgG1(+)/IgM(+) GC B cells remained unchanged. Together these findings suggest that IgM(+) B cells undergo SHM in the GC to generate high affinity IgM(+) memory cells and that this process continues even after CSR is accomplished.

  13. Differences between high-affinity forskolin binding sites in dopamine-riche and other regions of rat brain

    SciTech Connect

    Poat, J.A.; Cripps, H.E.; Iversen, L.L.

    1988-05-01

    Forskolin labelled with (/sup 3/H) bound to high- and low-affinity sites in the rat brain. The high-affinity site was discretely located, with highest densities in the striatum, nucleus accumbens, olfactory tubercule, substantia nigra, hippocampus, and the molecular layers of the cerebellum. This site did not correlate well with the distribution of adenylate cyclase. The high-affinity striatal binding site may be associated with a stimulatory guanine nucleotide-binding protein. Thus, the number of sites was increased by the addition of Mg/sup 2 +/ and guanylyl imidodiphosphate. Cholera toxin stereotaxically injected into rat striatum increased the number of binding sites, and no further increase was noted following the subsequent addition of guanyl nucleotide. High-affinity forskolin binding sites in non-dopamine-rich brain areas (hippocampus and cerebullum) were modulated in a qualitatively different manner by guanyl nucleotides. In these areas the number of binding sites was significantly reduced by the addition of guanyl nucleotide. These results suggest that forskolin may have a potential role in identifying different functional/structural guanine nucleotide-binding proteins.

  14. High-Affinity Glucose Transport in Aspergillus nidulans Is Mediated by the Products of Two Related but Differentially Expressed Genes

    PubMed Central

    Ventura, Luisa; González, Ramón; Ramón, Daniel; MacCabe, Andrew P.

    2014-01-01

    Independent systems of high and low affinity effect glucose uptake in the filamentous fungus Aspergillus nidulans. Low-affinity uptake is known to be mediated by the product of the mstE gene. In the current work two genes, mstA and mstC, have been identified that encode high-affinity glucose transporter proteins. These proteins' primary structures share over 90% similarity, indicating that the corresponding genes share a common origin. Whilst the function of the paralogous proteins is little changed, they differ notably in their patterns of expression. The mstC gene is expressed during the early phases of germination and is subject to CreA-mediated carbon catabolite repression whereas mstA is expressed as a culture tends toward carbon starvation. In addition, various pieces of genetic evidence strongly support allelism of mstC and the previously described locus sorA. Overall, our data define MstC/SorA as a high-affinity glucose transporter expressed in germinating conidia, and MstA as a high-affinity glucose transporter that operates in vegetative hyphae under conditions of carbon limitation. PMID:24751997

  15. Energy-dependent dissociation of ATP from high affinity catalytic sites of beef heart mitochondrial adenosine triphosphatase

    SciTech Connect

    Penefsky, H.S.

    1985-11-05

    Incubation of (gamma-TSP)ATP with a molar excess of the membrane-bound form of mitochondrial ATPase (F1) results in binding of the bulk of the radioactive nucleotide in high affinity catalytic sites (Ka = 10(12) M-1). Subsequent initiation of respiration by addition of succinate or NADH is accompanied by a profound decrease in the affinity for ATP. About one-third of the bound radioactive ATP appears to dissociate, that is, the (gamma-TSP)ATP becomes accessible to hexokinase. The NADH-stimulated dissociation of (gamma-TSP)ATP is energy-dependent since the stimulation is inhibited by uncouplers of oxidative phosphorylation and is prevented by respiratory chain inhibitors. The rate of the energy-dependent dissociation of ATP that occurs in the presence of NADH, ADP, and Pi is commensurate with the measured initial rate of ATP synthesis in NADH-supported oxidative phosphorylation catalyzed by the same submitochondrial particles. Thus, the rate of dissociation of ATP from the high affinity catalytic site of submitochondrial particles meets the criterion of kinetic competency under the conditions of oxidative phosphorylation. These experiments provide evidence in support of the argument that energy conserved during the oxidation of substrates by the respiratory chain can be utilized to reduce the very tight binding of product ATP in high affinity catalytic sites and to promote dissociation of the nucleotide.

  16. High-affinity RNA aptamers to C-reactive protein (CRP): newly developed pre-elution methods for aptamer selection

    NASA Astrophysics Data System (ADS)

    Orito, N.; Umekage, S.; Sato, K.; Kawauchi, S.; Tanaka, H.; Sakai, E.; Tanaka, T.; Kikuchi, Y.

    2012-03-01

    We have developed a modified SELEX (systematic evolution of ligands by exponential enrichment) method to obtain RNA aptamers with high affinity to C-reactive protein (CRP). CRP is a clinical biomarker present in plasma, the level of which increases in response to infections and noninfectious inflammation. The CRP level is also an important prognostic indicator in patients with several syndromes. At present, CRP content in blood is measured immunochemically using antibodies. To develop a more sensitive method using RNA aptamers, we have attempted to obtain high-affinity RNA aptamers to CRP. We succeeded in obtaining an RNA aptamer with high affinity to CRP using a CRP-immobilized Sepharose column and pre-elution procedure. Pre-elution is a method that removes the weak binding portion from a selected RNA population by washing for a short time with buffer containing CRP. By surface plasmon-resonance (SPR) analysis, the affinity constant of this aptamer for CRP was calculated to be KD = 2.25×10-9 (M). The secondary structure, contact sites with CRP protein, and application of this aptamer will be described.

  17. Dual inhibition of human type 4 phosphodiesterase isostates by (R, R)-(+/-)-methyl 3-acetyl-4-[3-(cyclopentyloxy)-4-methoxyphenyl]-3- methyl-1-pyrrolidinecarboxylate.

    PubMed

    Tian, G; Rocque, W J; Wiseman, J S; Thompson, I Z; Holmes, W D; Domanico, P L; Stafford, J A; Feldman, P L; Luther, M A

    1998-05-12

    Purified recombinant human type 4 phosphodiesterase B2B (HSPDE4B2B) exists in both a low- and a high-affinity state that bind (R)-rolipram with Kd's of ca. 500 and 1 nM, respectively [Rocque, W. J., Tian, G., Wiseman, J. S., Holmes, W. D., Thompson, I. Z., Willard, D. H., Patel, I. R., Wisely, G. B., Clay, W. C., Kadwell, S. H., Hoffman, C. R., and Luther, M. A. (1997) Biochemistry 36, 14250-14261]. Since the tissue distribution of the two isostates may be significantly different, development of inhibitors that effectively inhibit both forms may be advantageous pharmacologically. In this study, enzyme inhibition and binding of HSPDE4B2B by (R, R)-(+/-)-methyl 3-acetyl-4-[3-(cyclopentyloxy)-4-methoxyphenyl]-3-methyl-1-pyrrolidin ecarboxylate (1), a novel inhibitor of phosphodiesterase 4 (PDE 4), were investigated. Binding experiments demonstrated high-affinity binding of 1 to HSPDE4B2B with a stoichiometry of 1:1. Inhibition of PDE activity showed only a single transition with an observed Ki similar to the apparent Kd determined by the binding experiments. Deletional mutants of HSPDE4B2B, which have been shown to bind (R)-rolipram with low affinity, were shown to interact with 1 with high affinity, indistinguishable from the results obtained with the full-length enzyme. Bound 1 was completely displaced by (R)-rolipram, and the displacement showed a biphasic transition that resembles the biphasic inhibition of HSPDE4B2B by (R)-rolipram. Theoretical analysis of the two transitions exemplified in the interaction of (R)-rolipram with HSPDE4B2B indicated that the two isostates were nonexchangeable. Phosphorylation at serines 487 and 489 on HSPDE4B2B had no effect on the stoichiometry of binding, the affinity for binding, or the inhibition of the enzyme by 1. These data further illustrate the presence of two isostates in PDE 4 as shown previously for (R)-rolipram binding and inhibition. In contrast to (R)-rolipram, where only one of the two isostates of PDE 4 binds with

  18. Detergents stabilize the conformation of phosphodiesterase 6.

    PubMed

    Baker, Bo Y; Palczewski, Krzysztof

    2011-11-08

    Membrane-bound phosphodiesterase 6 (PDE6) plays an important role in visual signal transduction by regulating cGMP levels in rod photoreceptor cells. Our understanding of PDE6 catalysis and structure suffers from inadequate characterization of the α and β subunit catalytic core, interactions of the core with two intrinsically disordered, proteolysis-prone inhibitory PDEγ (Pγ) subunits, and binding of two types of isoprenyl-binding protein δ, called PrBP/δ, to the isoprenylated C-termini of the catalytic core. Structural studies of native PDE6 have been also been hampered by the lack of a heterologous expression system for the holoenzyme. In this work, we purified PDE6 in the presence of PrBP/δ and screened for additives and detergents that selectively suppress PDE6 basal activity while sparing that of the trypsin-activated enzyme. Some detergents removed PrBP/δ from the PDE complex, separating it from the holoenzyme after PDE6 purification. Additionally, selected detergents also significantly reduced the level of dissociation of PDE6 subunits, increasing their homogeneity and stabilizing the holoenzyme by substituting for its native membrane environment.

  19. Desynchronization of Cells on the Developmental Path Triggers the Formation of Spiral Waves of cAMP during Dictyostelium Aggregation

    NASA Astrophysics Data System (ADS)

    Lauzeral, Jacques; Halloy, Jose; Goldbeter, Albert

    1997-08-01

    Whereas it is relatively easy to account for the formation of concentric (target) waves of cAMP in the course of Dictyostelium discoideum aggregation after starvation, the origin of spiral waves remains obscure. We investigate a physiologically plausible mechanism for the spontaneous formation of spiral waves of cAMP in D. discoideum. The scenario relies on the developmental path associated with the continuous changes in the activity of enzymes such as adenylate cyclase and phosphodiesterase observed during the hours that follow starvation. These changes bring the cells successively from a nonexcitable state to an excitable state in which they relay suprathreshold cAMP pulses, and then to autonomous oscillations of cAMP, before the system returns to an excitable state. By analyzing a model for cAMP signaling based on receptor desensitization, we show that the desynchronization of cells on this developmental path triggers the formation of fully developed spirals of cAMP. Developmental paths that do not correspond to the sequence of dynamic transitions no relay-relay-oscillations-relay are less able or fail to give rise to the formation of spirals.

  20. Pharmacological characterization of metabotropic glutamate receptor-mediated high-affinity GTPase activity in rat cerebral cortical membranes

    PubMed Central

    Nishi, Nobuyuki; Odagaki, Yuji; Koyama, Tsukasa

    2000-01-01

    Activation of heterotrimeric guanine nucleotide-binding regulatory proteins (G-proteins) functionally coupled to metabotropic glutamate receptors (mGluRs) was assessed by agonist-induced high-affinity GTPase (EC3.6.1.-) activity in rat cerebral cortical membranes. L-Glutamate (1 mM) stimulated high-affinity GTPase activity to the same extent throughout the incubation period up to 20 min, in a Mg2+-dependent manner. The addition of 1 mM L-glutamate augmented Vmax of the enzyme activity (1670 to 3850 pmol mg−1 protein 15 min−1) with slight increase in KM value (0.26 to 0.63 μM). The high-affinity GTPase activity was stimulated by the following compounds with a rank order of potency of (2S,2′R,3′R)-2-(2′,3′-dicarboxycyclopropyl) glycine (DCG-IV) >  (2S,1′S,2′S)-2-(carboxycyclopyropyl)glycine (L-CCG-I) > L-glutamate ≥ 2R,4R-4-aminopyrrolidine-2,4-dicarboxylate [(2R,4R)-APDC] > 1S,3R-1-aminocyclopentane-1,3-dicarboxylate [(1S,3R)-ACPD] > (S)-4-carboxy-3-hydroxyphenylglycine [(S)-4C3HPG] > (S)-3-carboxy-4-hydroxyphenylglycine [(S)-3C4HPG] > ibotenate, but not by L-(+)-2-amino-4-phosphonobutyrate (L-AP4), (RS)-3,5-dihydroxyphenylglycine [(RS)-3,5-DHPG], quisqualate, or L-serine-O-phosphate (L-SOP), indicative of involvement of group II mGluRs, in particular mGluR2. (2S)-α-Ethylglutamate (EGLU), a presumably selective antagonist against group II mGluRs, inhibited DCG-IV-stimulated high-affinity GTPase activity in a competitive manner with an apparent KB of 220 μM. L-Glutamate-stimulated activity was eliminated by pretreatment of the membranes with sulfhydryl alkylating agent N-ethylmaleimide (NEM) at 30–50 μM, indicating that G-proteins of the Gi family are involved. These results indicate that mGluR agonist-induced high-affinity GTPase activity in rat cerebral cortical membranes may be used to detect the functional interaction between group II mGluRs, in particular mGluR2, and NEM-sensitive Gi proteins. PMID:10928972

  1. IGF-I–induced Differentiation of L6 Myogenic Cells Requires the Activity of cAMP-Phosphodiesterase

    PubMed Central

    De Arcangelis, Vania; Coletti, Dario; Conti, Marco; Lagarde, Michel; Molinaro, Mario; Adamo, Sergio; Nemoz, Georges; Naro, Fabio

    2003-01-01

    Inhibition of type 4 cAMP-specific phosphodiesterase (PDE4) activity in L6-C5 and L6-E9 abolished myogenic differentiation induced by low-serum medium and IGF-I. L6-C5 cells cultured in low-serum medium displayed a PDE4 activity higher than cells cultured in serum-free medium, a condition not sufficient to induce differentiation. In the presence of serum, PDE4D3, the major isoform natively expressed in L6-C5 cells, translocated to a Triton-insoluble fraction, which increased the PDE specific activity of the fraction, and exhibited a Mr shift typical of phosphorylation of this isoform. Furthermore, serum promoted the localization of PDE4D3 to a vesicular subcellular compartment. In L6-C5 cells, IGF-I is a stronger inducer of myogenic differentiation in the presence than in absence of serum. Its ability to trigger differentiation in the absence of serum was restored by overexpressing wild-type PDE4D3, but not a phosphorylation-insensitive mutant. This finding was confirmed in single cells overexpressing a GFP-PDE4D3 fusion protein by assessing nuclear accumulation of myogenin in both L6-C5 and L6-E9. Overexpression of other PDE isoforms was less efficient, confirming that PDE4D3 is the physiologically relevant phosphodiesterase isoform in the control of myogenesis. These results show that downregulation of cAMP signaling through cAMP-phosphodiesterase stimulation is a prerequisite for induction of myogenesis. PMID:12686596

  2. IGF-I-induced differentiation of L6 myogenic cells requires the activity of cAMP-phosphodiesterase.

    PubMed

    De Arcangelis, Vania; Coletti, Dario; Conti, Marco; Lagarde, Michel; Molinaro, Mario; Adamo, Sergio; Nemoz, Georges; Naro, Fabio

    2003-04-01

    Inhibition of type 4 cAMP-specific phosphodiesterase (PDE4) activity in L6-C5 and L6-E9 abolished myogenic differentiation induced by low-serum medium and IGF-I. L6-C5 cells cultured in low-serum medium displayed a PDE4 activity higher than cells cultured in serum-free medium, a condition not sufficient to induce differentiation. In the presence of serum, PDE4D3, the major isoform natively expressed in L6-C5 cells, translocated to a Triton-insoluble fraction, which increased the PDE specific activity of the fraction, and exhibited a Mr shift typical of phosphorylation of this isoform. Furthermore, serum promoted the localization of PDE4D3 to a vesicular subcellular compartment. In L6-C5 cells, IGF-I is a stronger inducer of myogenic differentiation in the presence than in absence of serum. Its ability to trigger differentiation in the absence of serum was restored by overexpressing wild-type PDE4D3, but not a phosphorylation-insensitive mutant. This finding was confirmed in single cells overexpressing a GFP-PDE4D3 fusion protein by assessing nuclear accumulation of myogenin in both L6-C5 and L6-E9. Overexpression of other PDE isoforms was less efficient, confirming that PDE4D3 is the physiologically relevant phosphodiesterase isoform in the control of myogenesis. These results show that downregulation of cAMP signaling through cAMP-phosphodiesterase stimulation is a prerequisite for induction of myogenesis.

  3. Pharmacological profile of T-1032, a novel specific phosphodiesterase type 5 inhibitor, in isolated rat aorta and rabbit corpus cavernosum.

    PubMed

    Takagi, M; Mochida, H; Noto, T; Yano, K; Inoue, H; Ikeo, T; Kikkawa, K

    2001-01-05

    This study was designed to examine the pharmacological properties of T-1032 (methyl-2-(4-aminophenyl)-1,2-dihydro-1-oxo-7-(2-pyridinylmethoxy)-4-(3,4,5-trimethoxyphenyl)-3-isoquinoline carboxylate sulfate), a novel phosphodiesterase type 5 inhibitor, in isolated rat aorta and rabbit corpus cavernosum. T-1032 (3x10(-11) to 3x10(-7) M) caused an endothelium-dependent relaxation in the isolated rat aorta precontracted with phenylephrine, and the relaxation was accompanied by an increase in cGMP but not cAMP levels. The T-1032-induced relaxation was attenuated by N(G)-nitro-L-arginine methyl ester (L-NAME) (10(-3) M), a nitric oxide (NO) synthase inhibitor, or 1H-[1,2,4]oxadiazolo[4,3-alpha]quinoxalin-1-one (ODQ) (10(-5) M), a guanylyl cyclase inhibitor. T-1032 (10(-9), 10(-8) M) produced a potentiation of the relaxation induced by sodium nitroprusside, but not of the relaxation induced by isoproterenol. In the isolated rabbit corpus cavernosum precontracted with phenylephrine, the electrical field stimulation-induced relaxation was attenuated by treatment with tetrodotoxin (10(-6) M) as well as L-NAME (10(-4) M). The L-NAME-inhibited relaxation was restored by treatment with L-arginine (5x10(-4) M). T-1032 (10(-9) to 10(-6) M) and sildenafil (10(-9) to 10(-6) M) produced a potentiation of the electrical field stimulation-induced relaxation as well as a decrease in basal tension in a concentration-dependent manner. It was concluded that T-1032 had potentiating effects on the NO/cGMP signaling pathway in isolated tissues, probably through specific blockade of phosphodiesterase type 5. T-1032 would be a useful compound to examine the physiologic functions of phosphodiesterase type 5 in mammalian tissues.

  4. Exposure to an Extremely-Low-Frequency Magnetic Field Stimulates Adrenal Steroidogenesis via Inhibition of Phosphodiesterase Activity in a Mouse Adrenal Cell Line

    PubMed Central

    Kitaoka, Kazuyoshi; Kawata, Shiyori; Yoshida, Tomohiro; Kadoriku, Fumiya; Kitamura, Mitsuo

    2016-01-01

    Extremely low-frequency magnetic fields (ELF-MFs) are generated by power lines and household electrical devices. In the last several decades, some evidence has shown an association between ELF-MF exposure and depression and/or anxiety in epidemiological and animal studies. The mechanism underlying ELF-MF-induced depression is considered to involve adrenal steroidogenesis, which is triggered by ELF-MF exposure. However, how ELF-MFs stimulate adrenal steroidogenesis is controversial. In the current study, we investigated the effect of ELF-MF exposure on the mouse adrenal cortex-derived Y-1 cell line and the human adrenal cortex-derived H295R cell line to clarify whether the ELF-MF stimulates adrenal steroidogenesis directly. ELF-MF exposure was found to significantly stimulate adrenal steroidogenesis (p < 0.01–0.05) and the expression of adrenal steroid synthetic enzymes (p < 0.05) in Y-1 cells, but the effect was weak in H295R cells. Y-1 cells exposed to an ELF-MF showed significant decreases in phosphodiesterase activity (p < 0.05) and intracellular Ca2+ concentration (p < 0.01) and significant increases in intracellular cyclic adenosine monophosphate (cAMP) concentration (p < 0.001–0.05) and cAMP response element-binding protein phosphorylation (p < 0.05). The increase in cAMP was not inhibited by treatment with NF449, an inhibitor of the Gs alpha subunit of G protein. Our results suggest that ELF-MF exposure stimulates adrenal steroidogenesis via an increase in intracellular cAMP caused by the inhibition of phosphodiesterase activity in Y-1 cells. The same mechanism may trigger the increase in adrenal steroid secretion in mice observed in our previous study. PMID:27100201

  5. Localization and activity of tissue bound cyclic nucleotide phosphodiesterase in normal and lack of changes in psoriatic human skin.

    PubMed

    Mahrle, G; Organos, C E

    1976-12-01

    This study has been undertaken to elucidate the localization and the activity of cyclic nucleotide phosphodiesterase (PDE) in psoriatic epidermis compared to normal. The results showed that the evaluation of cytochemical methods may be difficult because of the various factors which interfere with the reaction and the considerable amount of background staining. Additionally, only the tissue bound particulate enzyme fraction may be demonstrated by cytochemical means. Nevertheless, the method did reveal that the activity of PDE, if any, is localized on the cytoplasmic membranes of the cells, independent of their origin, and not on the cell surface. Moreover, no differences were found between normal and psoriatic skin. It seems, therefore, that the intracellular degradation of cAMP remains unaltered in psoriasis.

  6. Identification in Silico and Experimental Validation of Novel Phosphodiesterase 7 Inhibitors with Efficacy in Experimental Autoimmune Encephalomyelitis Mice

    PubMed Central

    2012-01-01

    A neural network model has been developed to predict the inhibitory capacity of any chemical structure to be a phosphodiesterase 7 (PDE7) inhibitor, a new promising kind of drugs for the treatment of neurological disorders. The numerical definition of the structures was achieved using CODES program. Through the validation of this neural network model, a novel family of 5-imino-1,2,4-thiadiazoles (ITDZs) has been identified as inhibitors of PDE7. Experimental extensive biological studies have demonstrated the ability of ITDZs to inhibit PDE7 and to increase intracellular levels of cAMP. Among them, the derivative 15 showed a high in vitro potency with desirable pharmacokinetic profile (safe genotoxicity and blood brain barrier penetration). Administration of ITDZ 15 in an experimental autoimmune encephalomyelitis (EAE) mouse model results in a significant attenuation of clinical symptoms, showing the potential of ITDZs, especially compound 15, for the effective treatment of multiple sclerosis. PMID:23077723

  7. Cigarette Smoke-Induced Emphysema and Pulmonary Hypertension Can Be Prevented by Phosphodiesterase 4 and 5 Inhibition in Mice.

    PubMed

    Seimetz, Michael; Parajuli, Nirmal; Pichl, Alexandra; Bednorz, Mariola; Ghofrani, Hossein Ardeschir; Schermuly, Ralph Theo; Seeger, Werner; Grimminger, Friedrich; Weissmann, Norbert

    2015-01-01

    Chronic obstructive pulmonary disease (COPD) is a widespread disease, with no curative therapies available. Recent findings suggest a key role of NO and sGC-cGMP signaling for the pathogenesis of the disease. Previous data suggest a downregulation/inactivation of the cGMP producing soluble guanylate cyclase, and sGC stimulation prevented cigarette smoke-induced emphysema and pulmonary hypertension (PH) in mice. We thus aimed to investigate if the inhibition of the cGMP degrading phosphodiesterase (PDE)5 has similar effects. Results were compared to the effects of a PDE 4 inhibitor (cAMP elevating) and a combination of both. C57BL6/J mice were chronically exposed to cigarette smoke and in parallel either treated with Tadalafil (PDE5 inhibitor), Piclamilast (PDE4 inhibitor) or both. Functional measurements (lung compliance, hemodynamics) and structural investigations (alveolar and vascular morphometry) as well as the heart ratio were determined after 6 months of tobacco smoke exposure. In addition, the number of alveolar macrophages in the respective lungs was counted. Preventive treatment with Tadalafil, Piclamilast or a combination of both almost completely prevented the development of emphysema, the increase in lung compliance, tidal volume, structural remodeling of the lung vasculature, right ventricular systolic pressure, and right ventricular hypertrophy induced by cigarette smoke exposure. Single, but not combination treatment prevented or reduced smoke-induced increase in alveolar macrophages. Cigarette smoke-induced emphysema and PH could be prevented by inhibition of the phosphodiesterases 4 and 5 in mice.

  8. Characterization of Binding and Inhibitory Properties of TAK-063, a Novel Phosphodiesterase 10A Inhibitor

    PubMed Central

    Harada, Akina; Suzuki, Kazunori; Kamiguchi, Naomi; Miyamoto, Maki; Tohyama, Kimio; Nakashima, Kosuke; Taniguchi, Takahiko; Kimura, Haruhide

    2015-01-01

    Phosphodiesterase 10A (PDE10A) inhibition is a novel and promising approach for the treatment of central nervous system disorders such as schizophrenia and Huntington’s disease. A novel PDE10A inhibitor, TAK-063 [1-[2-fluoro-4-(1H-pyrazol-1-yl)phenyl]-5-methoxy-3-(1-phenyl-1H-pyrazol-5-yl)-pyridazin-4(1H)-one] has shown high inhibitory activity and selectivity for human recombinant PDE10A2 in vitro; the half-maximal inhibitory concentration was 0.30 nM, and selectivity over other phosphodiesterases (PDEs) was more than 15000-fold. TAK-063 at 10 µM did not show more than 50% inhibition or stimulation of 91 enzymes or receptors except for PDEs. In vitro autoradiography (ARG) studies using rat brain sections revealed that [3H]TAK-063 selectively accumulated in the caudate putamen (CPu), nucleus accumbens (NAc), globus pallidus, substantia nigra, and striatonigral projection, where PDE10A is highly expressed. This [3H]TAK-063 accumulation was almost entirely blocked by an excess amount of MP-10, a PDE10A selective inhibitor, and the accumulation was not observed in brain slices of Pde10a-knockout mice. In rat brain sections, [3H]TAK-063 bound to a single high-affinity site with mean ± SEM dissociation constants of 7.2 ± 1.2 and 2.6 ± 0.5 nM for the CPu and NAc shell, respectively. Orally administered [14C]TAK-063 selectively accumulated in PDE10A expressing brain regions in an in vivo ARG study in rats. Striatal PDE10A occupancy by TAK-063 in vivo was measured using T-773 as a tracer and a dose of 0.88 mg/kg (p.o.) was calculated to produce 50% occupancy in rats. Translational studies with TAK-063 and other PDE10A inhibitors such as those presented here will help us better understand the pharmacological profile of this class of potential central nervous system drugs. PMID:25815469

  9. The swing of it: Hammock camping.

    USGS Publications Warehouse

    Marion, Jeff

    2016-01-01

    Hammock camping is dramatically expanding along the Appalachian Trail and raising both questions and concerns among Trail land managers, club members, and backpackers. This article examines some of the advantages and disadvantages of hammock camping, including resource and social impacts. Some Leave No Trace hammock camping practices are included for those using hammocks at well-established campsites and when "pristine-site" camping.

  10. Camp Courageous of Iowa Staff Manual.

    ERIC Educational Resources Information Center

    Camp Courageous of Iowa, Monticello.

    Designed as a useful and practical tool for the staff at Camp Courageous of Iowa, a year-round residential camp serving all handicapped individuals, the manual outlines safety rules for camp activities, characteristics of the mentally and physically handicapped, and a general description of the camp and its objectives. Contents of the manual…

  11. Camp Barnabas! No Limits! No Boundaries!

    ERIC Educational Resources Information Center

    Teas, Cyndy; Robertson, Donna

    2007-01-01

    This article profiles Camp Barnabas, a non-denominational Christian camp for people with special needs located in southwest Missouri. For one week, campers have every opportunity possible to be a participant in the world around them, not just an observer. The camp's goal is to provide a typical camp experience--bugs, chants, cheers and incredible…

  12. Slave Labor Camps of the Third Reich.

    ERIC Educational Resources Information Center

    Stone, Adolf

    1983-01-01

    Describes the ground rules used by Nazi architects in choosing the sites for slave labor camps. While some, like Auschwitz, became extermination camps, others also produced armaments. One camp, Theresienstadt, became a "model" camp to show to reporters and Red Cross representatives. (CS)

  13. Self-Concept Change in Camp Staff.

    ERIC Educational Resources Information Center

    Henderson, Karla A.; Bialeschki, M. Deborah

    The 1981 study ascertained whether the self-concept of 66 camp staff from 2 Wisconsin camps changed more than a control group of 18 college students attending summer school; if differences in self-concept were based on a particular characteristic (age, gender, staff position, years at camp); and in what ways, if any, self-concept of camp staff…

  14. DAY CAMPING FOR THE MENTALLY RETARDED.

    ERIC Educational Resources Information Center

    GINGLEND, DAVID; GOULD, KAY

    EMPHASIS IS PLACED ON MENTAL HEALTH, PHYSICAL DEVELOPMENT AND COORDINATION (BOTH MOTOR AND MUSCULAR), SOCIAL ADJUSTMENT, AND LANGUAGE AND INTELLECTUAL DEVELOPMENT. SECTIONS ARE DEVOTED TO ORGANIZATION OF A DAY CAMPING PROGRAM, SELECTING THE STAFF AND THE CAMPERS, THE DAY CAMP IN OPERATION, DAY CAMPING AS A TRAINING PERIOD, CAMP RELATIONS WITH THE…

  15. Easter Seal Guide to Special Camping Programs.

    ERIC Educational Resources Information Center

    Crane, Helen B., Ed.

    Intended for organizations having or planning to establish resident resident camping programs for people with special needs, this guide supplements the American Camping Association's Standards. The philosophy, aims, and objectives of specialized camping programs are considered, and the following are discussed: administration, camp site selection,…

  16. Self-Concept Change in Camp Staff.

    ERIC Educational Resources Information Center

    Henderson, Karla A.; Bialeschki, M. Deborah

    The 1981 study ascertained whether the self-concept of 66 camp staff from 2 Wisconsin camps changed more than a control group of 18 college students attending summer school; if differences in self-concept were based on a particular characteristic (age, gender, staff position, years at camp); and in what ways, if any, self-concept of camp staff…

  17. American Camping Association Annual Report, 1999.

    ERIC Educational Resources Information Center

    American Camping Association, Martinsville, IN.

    Founded in 1910 as the Camp Directors' Association of America, the American Camping Association (ACA) is the largest organization serving the organized camping industry. Over 5,500 members come from all segments of the camp profession. This annual report for 1999 describes ACA activities in support of organizational commitments. These commitments…

  18. The effect of resveratrol on beta amyloid-induced memory impairment involves inhibition of phosphodiesterase-4 related signaling

    PubMed Central

    Wang, Gang; Chen, Ling; Pan, Xiaoyu; Chen, Jiechun; Wang, Liqun; Wang, Weijie; Cheng, Ruochuan; Wu, Fan; Feng, Xiaoqing; Yu, Yingcong; Zhang, Han-Ting; O'Donnell, James M.; Xu, Ying

    2016-01-01

    Resveratrol, a natural polyphenol found in red wine, has wide spectrum of pharmacological properties including antioxidative and antiaging activities. Beta amyloid peptides (Aβ) are known to involve cognitive impairment, neuroinflammatory and apoptotic processes in Alzheimer's disease (AD). Activation of cAMP and/or cGMP activities can improve memory performance and decrease the neuroinflammation and apoptosis. However, it remains unknown whether the memory enhancing effect of resveratrol on AD associated cognitive disorders is related to the inhibition of phosphodiesterase 4 (PDE4) subtypes and subsequent increases in intracellular cAMP and/or cGMP activities. This study investigated the effect of resveratrol on Aβ1-42-induced cognitive impairment and the participation of PDE4 subtypes related cAMP or cGMP signaling. Mice microinfused with Aβ1-42 into bilateral CA1 subregions displayed learning and memory impairment, as evidenced by reduced memory acquisition and retrieval in the water maze and retention in the passive avoidance tasks; it was also significant that neuroinflammatory and pro-apoptotic factors were increased in Aβ1-42-treated mice. Aβ1-42-treated mice also increased in PDE4A, 4B and 4D expression, and decreased in PKA level. However, PKA inhibitor H89, but not PKG inhibitor KT5823, prevented resveratrol's effects on these parameters. Resveratrol also reversed Aβ1-42-induced decreases in phosphorylated cAMP response-element binding protein (pCREB), brain derived neurotrophic factor (BDNF) and anti-apoptotic factor BCl-2 expression, which were reversed by H89. These findings suggest that resveratrol reversing Aβ-induced learning and memory disorder may involve the regulation of neuronal inflammation and apoptosis via PDE4 subtypes related cAMP-CREB-BDNF signaling. PMID:26980711

  19. Berberine attenuates cAMP-induced lipolysis via reducing the inhibition of phosphodiesterase in 3T3-L1 adipocytes.

    PubMed

    Zhou, Libin; Wang, Xiao; Yang, Ying; Wu, Ling; Li, Fengying; Zhang, Rong; Yuan, Guoyue; Wang, Ning; Chen, Mingdao; Ning, Guang

    2011-04-01

    Berberine, a hypoglycemic agent, has been shown to decrease plasma free fatty acids (FFAs) level in insulin-resistant rats. In the present study, we explored the mechanism responsible for the antilipolytic effect of berberine in 3T3-L1 adipocytes. It was shown that berberine attenuated lipolysis induced by catecholamines, cAMP-raising agents, and a hydrolyzable cAMP analog, but not by tumor necrosis factor α and a nonhydrolyzable cAMP analog. Unlike insulin, the inhibitory effect of berberine on lipolysis in response to isoproterenol was not abrogated by wortmannin, an inhibitor of phosphatidylinositol 3-kinase, but additive to that of PD98059, an extracellular signal-regulated kinase kinase inhibitor. Prior exposure of adipocytes to berberine decreased the intracellular cAMP production induced by isoproterenol, forskolin, and 3-isobutyl-1-methylxanthine (IBMX), along with hormone-sensitive lipase (HSL) Ser-563 and Ser-660 dephosphorylation, but had no effect on perilipin phosphorylation. Berberine stimulated HSL Ser-565 as well as adenosine monophosphate-activated protein kinase (AMPK) phosphorylation. However, compound C, an AMPK inhibitor, did not reverse the regulatory effect of berberine on HSL Ser-563, Ser-660, and Ser-565 phosphorylation, nor the antilipolytic effect of berberine. Knockdown of AMPK using RNA interference also failed to restore berberine-suppressed lipolysis. cAMP-raising agents increased AMPK activity, which was not additive to that of berberine. Stimulation of adipocytes with berberine increased phosphodiesterase (PDE) 3B and PDE4 activity measured by hydrolysis of (3)[H]cAMP. These results suggest that berberine exerts an antilipolytic effect mainly by reducing the inhibition of PDE, leading to a decrease in cAMP and HSL phosphorylation independent of AMPK pathway.

  20. Standards for Day and Resident Camps: The Accreditation Programs of the American Camping Association. 1990 Edition.

    ERIC Educational Resources Information Center

    American Camping Association, Martinsville, IN.

    The purpose of this manual is to educate camp directors and camp personnel regarding government-recognized standard practices and procedures followed within the camp industry. These standards also provide a basis for voluntary accreditation of camps by the American Camping Association (ACA) beyond the minimum requirements of licensing. The manual…